Mcb 130 Midterm Iii Flashcards

Front 1

Lecture 1. What are the five general properties of the cyto-skeleton?

Back 1

1) Filamentous polymers (made of monomer subunits that assemble end to end).
2) Polymerize depends on concentration of free monomers. Note the concept of the critical concentration.
3) A dynamic system (filaments are constantly assembled and disassembled, and that monomers bind to nucleotide, either GTP or ATP, which is required for assembly and nucleotide hydrolysis is required for disassembly).
4) Cytoskeletal elements have binding proteins in cells which serve a regulatory function.
5) Use energy for its dynamic property (hydrolyze ATP). Also, it has polarity (2 ends of the filament are different and have different properties).

Front 2

Lecture 1. What are the four main functions of the cytoskeleton?

Back 2

1) Structural support for the cell or parts of the cell.
2) Chromosome segregation, or segregation of genetic material and separation of the mother cell into two during mitosis.
3) Internal support and protein transport. The cytoskeleton can serve as tracks on which certain cargo proteins use for movement.
4) Cell movement. Cytoskeleton and its components have the ability to generate force. This is done largely by subunit polymerization at a surface to exert a pushing force or depolymerizing to exert a pulling force.

Front 3

Lecture 1. What are the four prokaryotic cytoskeletal filaments and what are the eukaryotic counterparts?

Back 3

1) and 2) MreB and ParM-actin.
3) FtsZ-mictotubules.
4) CreS-intermediate filaments.

Front 4

Lecture 1. What gave researchers the evidence needed to show that prokaryotic cytoskeletal components had a similar evolutionary origin as eukaryotic cytoskeletal components?

Back 4

Structural similarity of both the monomers and overall structure of the polymers themselves, which were similar enough to discount the possibility that these structures could've evolved separately.

Front 5

Lecture 1. What are the cellular functions of MreB, ParM, FtsZ, and CreS?

Back 5

1) MreB-Cell shape determination.
2) ParM-plasmid and chromosome separation.
3) FtsZ-Cytokinesis.
4) CreS-Cell shape determination.

Front 6

Lecture 1. What are the intracellular locations of ParM, FtsZ, CreS, and MreB?

Back 6

1) MreB-spiral filaments along the length of the cell.
2) ParM-filaments in cell cytosol.
3) FtsZ-ring at division site.
4) Cres-filament along the length of the cell.

Front 7

Lecture 1. Why couldn't molecular biologists use protein sequencing to determine the evolutionary relationship between the prokaryotic and eukaryotic

Back 7

Sequencing cannot be used to determine evolutionary relationships for two organisms that are so distantly related.

Front 8

Lecture 1. How were the functions of the bacterial cytoskeletal proteins determined? There are two approaches.

Back 8

1) Mutagenesis-use knockouts (needs to be temperature sensitive mutatants or else no info can be obtained) and see what happens to the cell.
2) Tag it-being able to visualize where the cytoskeletal protein goes in the cell gives good info of what it does. Follow where it goes and how it changed with the cell cycle.

Front 9

Lecture 1. What do scientists use to visualize structures that are close together? Why can't this method be used all the time?

Back 9

Electron microscopy. This method cannot be used all the time because it requires the constant bombardment of high energy electrons, which kills cells.

Front 10

Lecture 1. What are some contrast enhancing techniques used to distinguish structures in cells better?

Back 10

1) Staining.
2) Phase contrast and differential interference contrast microscopy-which are both useful for viewing live cells, because cells can be filmed over time, enabling the observation of dynamic cellular processes.
3) Florescent microscopy-use a florescent microscope to visualize cytoskeletal elements that have been tagged florescently (commonly used florescent tags include GFPs, small florescent molecules attached to small molecules that bind a protein of interest, indirect immunofluorescence).

Front 11

Lecture 1. What are the three main techniques used in florescent microscopy?

Back 11

1) GFPs.
2) Small fluorescent molecules such as rhodamine (red) and fluoroscein (green) that are attached to small molecules that bind to a protein of interest.
3) Indirect immunofluorescence-use primary antibodies that recognize a protein of interest (antigen). Then, fluorescently labeled secondary antibodies are added that bind to the primary antibody.

Front 12

Lecture 1. What kind of mutants did molecular cell biologists use to get genetic knockouts of prokaryotic cytoskeletal components?

Back 12

Temperature sensitive mutants.

Front 13

Lecture 1. What information did molecular cell biologists need, in addition to observing what happens in genetic knockouts, to confirm the tru function of each prokaryotic cytoskeletal component?

Back 13

Microscopy-seeing where the protein localizes in the cell.

Front 14

Lecture 1. How and where are cytoskeletal elements made in the cell?

Back 14

Cytoskeletal elements are made in the cytoplasm. They often need chaperone proteins to help the newly made protein fold into each kind of subunit. The chaperones are synthesized to prevent inappropriate folding.

Front 15

Lecture 1. Where is the genetic information of ParM localized in the prokaryotic cell? How exactly does MreB carry out its task of determining the shape of the cell.

Back 15

In the plasmids, not the chromosomes. MreB determines cell shape by directing cell wall synthesis.

Front 16

Lecture 1. What are the subunits of intermediate filaments?

Back 16

Nuclear lamins, vinmetin, keratin, and neurofilaments.

Front 17

Lecture 1. What are the diameters of actin, microtubules, and intermediate filaments?

Back 17

1) Actin-7 nm.
2) Microtubules-24 nm with a hollow tube.
3) Intermediate filaments-10 nm.

Front 18

Lecture 1. What are the organization schemes of actin, microtubules, and intermediate filaments in the cell?

Back 18

1) Actin-bundles, networks.
2) Microtubules-individual tubules.
3) Intermediate filaments-individual filaments.

Front 19

Lecture 1. Where are actin, microtubules, and intermediate filaments located within the cell?

Back 19

1) Actin-cytoplasm, plasma membrane, and cell junctions.
2) Microtubules-Cytoplasm, attached to centrosome.
3) Intermediate filaments-cytoplasm, cell junctions, nucleus.

Front 20

Lecture 1. What are the main functions of actin, microtubules, and intermediate filaments/

Back 20

1) Actin-locomotion, shape change, cytokinesis, and adhesion.
2) Microtubules-vesicle transport, cilia and flagella, and mitotic spindle.
3) Intermediate filaments-nuclear and cell mechanical strength, junctions.

Front 21

Lecture 1. Cellular work are a direct result of mechanical work carried out by ________ that convert energy stored in _________ into.

Back 21

Proteins such as actin and myosin which convert chemical energy stored in ATP into mechanical work.

Front 22

Lecture 2. The ATPase fold binds to ATP or ADP complexed with what?

Back 22

Salts such as Mg++.

Front 23

Lecture 2. F-actin is composed of actin subunits held together by what kinds of forces?

Back 23

Non-covalent forces (mainly ionic and hydrophobic interactions).

Front 24

Lecture 2. Do mammals have one gene that codes for all the actin or multiple genes that code for multiple actin?

Back 24

Multiple genes that code for multiple forms of actin.

Front 25

Lecture 2. How did molecular cell biologists determine the polarity in actin filaments?

Back 25

Decorating it with myosin S-1.

Front 26

Lecture 2. How does one initiate the polymerization process in actin?

Back 26

Add salts, G-actin, and ATP.

Front 27

Lecture 2. There are four ways to visualize the polymerization dynamics of actin. What are they?

Back 27

1) Measure the scattering of light (polymers scatter more light than monomers).
2) Attaching a fluorescent tag onto actin which fluoresces more brightly when incorporated into F actin.
3) Visualizing filaments using EM.
4) Sedimentation analysis-larger filaments sediments more readily than the small subunits.

Front 28

Lecture 2. AFTER initiation, what are the three steps necessary for actin polymerization?

Back 28

1) Nucleation-formation of s table seed or nucleus of actin (THREE actin monomers) that can elongate.
2) Elongation.
3) Steady state.

Front 29

Lecture 2. Why is the nucleation process slow? Is elongation fast or slow? What does the rate of elongation depend on?

Back 29

Nucleation is slow because intermediates in the pathway (a dimer of actin monomers) are unstable. Elongation is fast and is dependent on the concentration of G actin and the number of filament ends.

Front 30

Lecture 2. What is the concentration of free actin at steady state called?

Back 30

The Cc or Critical Concentration, at which subunit addition to an actin filament is balanced out by subunit loss.

Front 31

Lecture 2. Why is the steady state not associated with a state of equilibrium?

Back 31

Steady state is not a state of equilibrium because energy is still used at this state (ATP hydrolysis). In any equilibrium state, there is no energy expenditure.

Front 32

Lecture 2. At the steady state, the rate of _______ as denoted by _________ equals the rate of _________, as denoted by ____________. Hence, it is possible to write the following equation :_________.

Back 32

At steady state, the rate of subunit addition (Kon[C]) equals the rate of subunit loss (Koff). Hence, at steady state, one can write the following equation: Kon[C] = Koff.

Front 33

Lecture 2. In most conditions, actin filaments will veer towards what state?

Back 33

The steady state, and this can be achieved via polymerization or depolymerization of the existing filaments.

Front 34

Lecture 2. How can one demonstrate that the plus end of actin filaments polymerized much faster than the minus end?

Back 34

1) Decoration of actin filament with myosin S-1 to ID the plus and minus ends.
2) Visualize the polymerization processes at both ends of the filament. The plus end has a much longer addition of subunits.

Front 35

Lecture 2. What is the main reason behind the two ends of actin filaments having different dynamic properties? What happens if the actin filaments are only exposed to ADP?

Back 35

The role of ATP hydrolysis. After ATP has been hydrolyzed, there is a conformational change in the actin filament and this destabilizes the interactions within the filament. In the presence of only ADP, the critical concentrations of the plus and minus ends are the same and there would be no treadmilling.

Front 36

Lecture 2. Why would the cell invest so much ATP in actin polymerization?

Back 36

Cells require a lot of ATP because dynamic processes such as cell motility and locomotion require the rapid assembly and disassembly of filaments at different locations and at different times. The steady state created by ATP hydrolysis makes this possible as ATP-actin can easily assemble at the plus ends and ADP-actin can disassemble just as easily at the minus ends.

Front 37

Lecture 2. Can actin subunits still polymerize without ATP?

Back 37

Yes, but would require a large amount of G actin.

Front 38

Lecture 2. What do the three drugs, Phalloidin, Cytochalasin, and Latrunculin do?

Back 38

1) Phalloidin-binds at the interface between the subunits in the filaments and prevents filament depolymerization.
2) Cytochalasin-binds to the + end of actin filaments and prevents elongation. This shifts the Cc of the entire filaments to the minus end, resulting in depolymerization of the entire filament.
3) Latrunculin-binds to actin monomers and sequesters them (preventing them to polymerize).

Front 39

Lecture 2. In addition to adding myosin S-1, what did researchers use to actually VISUALIZE the polarity in actin filaments?

Back 39

EM.

Front 40

Lecture 2, which are cytochalasin and latrunculin membrane permeable or not? Are their effects on cells reversible or not?

Back 40

Both drugs are membrane permeable and their effects are reversible.

Front 41

Lecture 2. The polymerization dynamics of actin were first studied in-vitro or in-vivo?

Back 41

In-vitro.

Front 42

Lecture 2. What was one method discussed in class on how the three phases of actin polymerization can be measured?

Back 42

Pyrene actin, which is labeled version of actin and gets more fluorescent when in the F actin form. The measurement of fluorescence over time is proportional to the amount of F actin.

Front 43

Lecture 2. When [G-actin] > Cc, what will happen? What about when [G-actin] < Cc?

Back 43

When [G-actin] > Cc, actin filaments will polymerize and the opposite will happen when [G-actin] < Cc.

Front 44

Lecture 2. What are the two things happen after ATP hydrolysis on an actin filament?

Back 44

1) The polymer is destabilized.
2) The affinity of the subunits for each other is lowered.

Front 45

Lecture 3. In-vitro, researchers can control the assembly and disassembly of actin filaments via what methods? What about the control of these filaments in the cell in vivo?

Back 45

In vitro, actin filaments are controlled by the amount of salts that are added G actin. In vivo, there are actin binding proteins that control the polymerization and depolymerization of actin.

Front 46

Lecture 3. Actin binding proteins perform a variety of functions that can be put into three main categories, what are they?

-Specifically, what are the 9 different actions that actin binding proteins can perform on actin?

Back 46

1) Control polymerization/depolymerization.
2) Organize filaments.
3) Move filaments.

-Eight actions:
1) Monomer sequestering.
2) Monomer nucleating.
3) End blocking (capping).
4) Cross-linking.
5) Bundling.
6) Monomer polymerizing.
7) depolymerize.
8) Filament severing.
9) Membrane binding.

Front 47

Lecture 3. There are four steps in actin polymerization that can be controlled by actin binding proteins, what are they?

Back 47

1) Monomer availability.
2) Nucleation.
3) Elongation.
4) Depolymerization.

Front 48

Lecture 3. What are the two proteins that cells can use to sequester proteins? There is a drug that does the same thing, what is it?

Back 48

1) Thymosin Beta 4.
2) Profilin.

Drug is called ________.

Front 49

Lecture 3. How does profilin promote polymerization sometimes?

Back 49

Profilin binds opposite to the ATP binding cleft, thereby preventing the polymerization at the minus end, and only the plus end can polymerize. Profilin immediately dissociates from the filament at binding.

Front 50

Lecture 3. In in-vitro conditions, what is the rate limiting step for actin polymerization?

Back 50

Depolymerization, which gives you a pool of monomers to polymerize again.

Front 51

Lecture 3. What are the three main nucleating factors?

Back 51

1) Arp2/3 with WASP to activate it. Caps the minus end and elongated fromthe plus end. It needs a mother filament to branch off from. Arp2/3 can bind to actin because it is structurally similar.
2) F-actin.
3) Formins-binds at plus end as a "leaky" cap. It promotes nucleation as well as elongation at the plus end, by preventing capping proteins specific to the plus end to bind. However, the minus end is free to elongate as well. It is not capped as in the case with Arp2/3 and WASP.

Front 52

T/F. Profilin can prevent and promote polymerization in actin.

Back 52

T.

Front 53

Lecture 3. Typically, the Arp2/3 and WASP complex grow on which parts of the existing mother filament? (The parts with ATP actin or ADP actin?)

Back 53

The parts of the filament with ATP actin.

Front 54

Lecture 3. How exactly does profilin promote the addition of new actin subunits at the plus end?

Back 54

It promotes actin subunit at the plus end of an actin filament by opening the ATP cleft and promoting the exchange of ATP for ADP.

Front 55

Lecture 3. What proteins cap the minus end of the actin filament, what about the plus end?

Back 55

Plus end: Cap Z.
Minus end: tropomodulin.

Front 56

Lecture 3. What proteins bind to F-actin to stabilize it?

Back 56

Tropomyosin, which.is a long, snake-like protein that binds along the length of the filament to stabilize the polymerized state.

Front 57

Lecture 3. What proteins are important for the depolymerization process in actin filaments?

Back 57

1) ADF/Cofilin: binds to ADP-actin and severs the filament at that region and accelerating ADP-actin depolymerization from the minus ends.

Front 58

Lecture 3. How are ADF/Cofilins regulated in cells?

Back 58

LIM kinase.

Front 59

Lecture 3. Other than ADF/Cofilin, what is another protein that plays a role in actin depolymerization? How is this protein regulated in the cell? Name an example in which gelosin is used.

Back 59

Gelosin. It is regulated in the cell by intracellular Ca++ levels. When Ca++ levels are high, gelosin is activated and it is inactivated when intracellular Ca++ levels are low. A good example of gelosin in action is in ameboid activity. When the cytoplasm moves to the front of the cell, it is turned into a gel. The gel is disassembled and turned into sol by gelosin, largely by severing actin filaments.

Front 60

Lecture 3. What is the function of the cell cortex? Where is this region in the cell? What is it made out of?

Back 60

The function of the cell cortex is to give the cell mechanical strength and also to give it ability to form surface structures and to perform surface functions. It is made primarily of actin filaments organized into networks and bundles.

Front 61

Lecture 3. What are four examples of bundling proteins in the cell? Proteins with shorter spacer tend to bundle filaments into what arrays?

Back 61

1) Fascin.
2) Villin.
3) Fimbrin.
4) Alpha actinin.

-Shorter spacer bundling proteins often bundle actin filaments into parallel arrays.

Front 62

Lecture 3. Cross-linking proteins often have longer or shorter spacer domains compared to bundling proteins?

Back 62

Cross-linking proteins tend to have longer spacer domains.

Front 63

Lecture 3. What are 3 examples of actin cross linking proteins?

Back 63

1) Filamen.
2) Dystrophin.
3) Spectrin.

Front 64

Lecture 3. What is the function of dystrophin?

Back 64

Dystrophin function to connect the muscle cell's cytoskeleton to the plasma membrane. Dystrophin organizes actin filaments into a network and tethers this network to a glycoprotein complex at the plasma membrane. This serves to let the muscle cell withstand multiple muscle contractions.

Front 65

Lecture 3. T/F. Profilin serves a function in monomer availability by sequestering monomers as well as what other function?

Back 65

Promoting elongation of actin filaments. It promotes actin binding at the plus ends.

Front 66

Lecture 3. What are the four actin binding proteins that affect elongation of actin filaments?

Back 66

1) CapZ.
2) Tropomodulin.
3) Profilin.
4) Formin.

Front 67

Lecture 4. What are the two structures, often filled with actin filaments, that are crucial to a cell's ability to migrate forwards? What are the functions of stress fibers (also filled with actin)?

Back 67

1) Lamellipodia.
2) Filapodia.

-Stress fibers' function are related to cell adhesion and contraction.

Front 68

Lecture 4. How are actin filaments organized in stress fibers & filapodia? What about in lamellipodia? Which end of the actin filaments usually sticks to the face of protrusion?

Back 68

In Lamellipodia, actin filaments are organized in meshworks. In Filapodia and stress fibers, actin filaments are organized into parallel bundles. The plus end.

Front 69

Lecture 4. In a migrating cell, where is the nucleation taking place? How did researchers reach this conclusion?

Back 69

Nucleation of actin filaments take place near the leading edge of the cell. Researchers confirmed this by micro-injecting cells with fluorescently labeled actin, which is immediately incorporated into the filaments at the leading edge of the cell.

Front 70

Lecture 4. Actin filaments are thought to be nucleated in what fashion at the leading edge of the cell? What about in the filapodia and stress fibers?

Back 70

At the leading edge, actin filaments are thought be nucleated by Arp2/3 with WASP, which generates y branched structures that at be used to give a motile force. In filapodia and stress fibers, the actin filaments are organized in parallel bundles, and thus you would need formins, even though Arp2/3 with WASP is still required.

Front 71

Lecture 4. Behind the branched network of actin filaments near the leading edge is what? How is this formed? What proteins are necessary to form this structure?

Back 71

Immediately behind the y-branched actin filaments near the leading edge, there is a meshwork of actin filaments that are cross linked by cross linking proteins called filamen, which have two actin binding domains separated by a spacer region.

Front 72

What actin binding proteins (used for organization of actin filaments) are present in filapodia ands tress fibers?

Back 72

These are actin bundling proteins.

Stress fibers = alpha actinin.
Filapodia = fimbrin.

-However, these two bundling proteins have no specific preference for either structures.

-Both proteins have 2 actin binding regions separated by a spacer region, which is not as long or as flexible as that of filamen. Alpha actinin has a slightly longer spacer domain compared to fimbrin.

Front 73

Lecture 4. Which protein is more abundant, thymosin beta 4 or profilin?

Back 73

Thymosin Beta 4.

Front 74

Lecture 4. ADF/Cofilin binds to areas of actin filaments with ADP actin and does specifically what? How does depolymerization occur so quick after?

Back 74

After ADF/Cofilin binds to ADP actin region of actin filaments, it causes the filament to twist, decreasing the spacing in the helical structure of the actin and causing it to break. This produces a bunch of minus ends and cause them to rapidly depolymerize.

Front 75

Lecture 4. What is the surface on which the cell is crawling called?

Back 75

Substratum.

Front 76

Lecture 4. What are the four steps involved in cell migration?

Back 76

1) Protrusion.
2) Adhesion.
3) Translocation.
4) De-adhesion.

Front 77

Lecture 4. What two things are associated with the lamellipodium being able to make the plasma membrane protrude and elongate to allow cell movement?

Back 77

Actin nucleation at the leading edge and actin polymerization (elongation) are thought to be associated with the protrusion and elongation of the cell's leading edge.

Front 78

Lecture 4. What dynamic process is thought to push the leading edge of a migrating cell forward? What are the steps to this process?

Back 78

Retrograde flow. There are 7 steps.

1) Elongation of the actin filaments that have just been nucleated.
2) Growing filaments push membrane forward.
3) Capping proteins limit elongation.
4) ATP hydrolysis on the newly polymerized G actin and Pi dissociation.
5) ADF/cofilin binds and severs ADP-actin portions of actin filaments.
6) LIM kinase limits ADF/cofilin
7) Profilin promotes the exchange of ADP for ATP and the actin monomer is ready to bind to the plus end of the existing actin filament again.

Front 79

Lecture 4. How were researchers able to establish that the "comet tail" at the end of the listeria bacteria was driving its motility? How does the bacteria infect other cells and how does it evade the immune system?

Back 79

They micro-injected fluorescent actin and it is immediately incorporated into the actin tail at the end enarest the bacterium. The bacteria infects other cells via forming "microspikes" off one cell to ram its way into another cell, hoping to eventually be engulfed and taken in. It can evade the immune system because it never passes through the bloodstream, where the lymphocytes are stationed.

Front 80

Lecture 4. What are the two most important factors keep the actin monomers recycling?

Back 80

1) Arp2/3 with WASP gives polymerization at the front).
and
2) ADF/cofilin (gives depolymerization at the rear).

Front 81

Lecture 4. In actin, the ATP binding cleft is oriented towards the minus or plus end?

Back 81

Minus.

Front 82

Lecture 4. Can profilin (which can also compete with formin for binding at the plus end) compete with thymosin beta 4 for binding on the actin monomers?

Back 82

Yes.

Front 83

Lecture 4. What three factors control an actin filament's ability to elongate?

Back 83

1) End availability.
2) Nucleators.
3) Capping proteins.

Front 84

Lecture 5. What are the primary functions of myosins? What kind of enzymes are they primarily? Where do they get their energy from?

Back 84

Myosins are proteins that move along actin filaments by coupling the energy from ATP hydrolysis to conformational changes. THey are called mechanochemical enzymes. They are AKA motor proteins.

Front 85

Lecture 5. Myosins are composed of what two types of chains? What are the three main parts in the structure of the heavy chain? What does each of these three parts do?

Back 85

Myosin has heavy and light chains. The heavy chains all have a head, neck, and tail. The head has the actin binding and ATPase domain. The neck is the site of attachment for regulatory and essential light chains or calmodulin. The tail domains differ among different myosins. They determine specific properties such as what it will bind to, whether it will form dimers or monomers, and whether it will polymerize into thick filaments.

Front 86

Lecture 5. Describe where myosin II is found, its functions, and all components that make up its structure.

Back 86

Myosin II is found in both non muscle and muscle cells. It was described as one of the first motor proteins in muscle that power muscle contraction. It has 2 heavy chains, each with an essential and regulatory light chain. The tails are long alpha helices that mediate dimerization by forming coiled coil structures, and also facilitates the formation of bipolar thick filaments.

Front 87

Lecture 5. Describe the structure of myosin I (esp. the length of the tail compared to myosin I and its characteristics), and its main functions it performs for the cell.

Back 87

Myosin I has a shorter tail compared to myosin II and these tails do not facilitate the formation of dimers. Some myosin Is have two actin binding sites, whcih help mediate the movement of filaments past one another. Some have membrane binding sites that attach to vesicles or organelles. Thus myosin I is used to move organelles and other cargo along actin tracks.

Front 88

Lecture 5. Other than myosins I and II, what is the only other myosin found in eukaryots?

Back 88

Myosin V.

Front 89

Lecture 5. What does myosin V look like (dimer or monomer?) and what does it do?

Back 89

Myosin V is a dimer like myosin II and it is thought to be involved in vesicle transport like myosin I.

Front 90

Lecture 5. Myosins III, IV, and VI-XV all have what regions that are conserved and what regions that are variable?

Back 90

Head regions are conserved and tail regions are variable.

Front 91

Lecture 5. What are the consequences of mutation in myosin VI in mice and humans? What about mutations in myosin VII?

Back 91

In mice, a mutation in myosin VI would cause deafness and a mutation in myosin VII would cause deafness in addition to neurological disorders.

In humans, a mutation in myosin VI would cause unknown consequences, and a mutation in myosin VII would cause deafness and blindness.

Front 92

Lecture 5. For myosins to work, they need ______ as an energy source. To activate their ATPase activity, they need this.

Back 92

ATP. Need actin to activate their ATPase activity.

Front 93

Lecture 5. Most myosins move toward what end of the actin filament? What about myosin VI?

Back 93

Most myosins move toward the plus end of the actin filament. Only myosin VI moves toward the minus end.

Front 94

Lecture 5. Describe the steps in the myosin cross bridge cycle.

-Are the steps of this cycle debated or well established?

Back 94

1) Rigor state-ATP bind site empty and myosin is tightly bound to actin.
2) ATP binding-upon binding to ATP, myosins release themselves from actin.
3) ATP hydrolysis-this moves the head region to a new position before rebinding to the actin filament.
4) Pi release-the myosin head undergoes a second conformational change called the power stroke that makes the myosin move reltaive to the actin and restores myosin to the rigor conformation.
5) ADP release-myosin is restored to its original rigor state.

-The steps of this cycle are still debated.

Front 95

Lecture 5. How can the motor functions of myosins be demonstrated?

Back 95

Video microscopy. Myosin molecules are stuck to glass such that tail domains bind to glass and head domains are free. If fluorescent actin and ATP are added, the filaments slide along the glass MINUS END FIRST!!!! The velocity of this movement differs among different myosins.

Front 96

Lecture 5. What is the best understood model of actin-myosin based motility? What two types of muscles are striated>?

Back 96

Muscle contraction. Skeletal and cardiac muscles appear to be striated under the microscope.

Front 97

Lecture 5. What are the different levels of organization in muscle tissue?

Back 97

Largest: Myofibers (giant cells that contain several nuclei)-> myofibrils (each myofibril contains a chain of contractile units called sarcomeres) -> sarcomeres.

Front 98

Lecture 5. What is the sarcomere composed of? In particular what two types of filaments? and what does each filament compose of? What makes the polymerization seen in thick filaments possible?

Back 98

Thin and thick filaments. The think filaments are composed of actin filaments and actin binding proteins such as tyopomyosin, troponin, cap Z, tropomodulin, and nubulin. Thick filaments are composed of myosin filaments (mostly myosin II). Polymerization of the thick filaments are made possible by the coiled coil tail domains of the myosin IIs in the thick filament.

Front 99

Lecture 5. How are the thin filaments oriented in the sarcomere? (what direction does the plus end face?)

Back 99

The barbed ends (plus end) faces the Z disk.

Front 100

Lecture 5. When proteases cut myosins into ______ (a number) pieces, what are they? What is one of these fragments used to do? Can isolated myosin tail domains polymerize into filaments?

Back 100

2. The motor domains (subfragment S1, used for decorating actin filaments), and the tail domains. Yes.

Front 101

Lecture 5. Within the think filaments, the plus end of actin filaments are attached to what, and then capped by what? What two proteins are needed for the interaction between the actin filaments and this binding interaction. How is polymerization and depolymerization of the actin in thin filaments prevented?

Back 101

Capped by CapZ at the plus end and attaches to the Z disk. Alpha actinin and capZ are needed for the interaction between actin and the Z disk. Capping proteins CapZ and tropomodulin.

Front 102

Lecture 5. What do the two proteins titin and nebulin do?

Back 102

Nebulin wraps around the entire length of the actin thin filament. It acts as a molecular ruler in that it is the same length in most species as the thin filament. It determines the length of the thin filament.

Titin connects the ends of myosin thick filaments to the Z disk and extends along the filament to the M-line (half the length of the sarcomere). It is thought to function as a spring to keep the myosins centered at the sarcomere when muscle of contracted or stretched.

Front 103

Lecture 5. What does the sliding filament model of contraction say about muscle contraction?

Back 103

Sarcomere shorten during muscle contraction. This is caused by the thick and thin filaments sliding past one another with no change in the length of either filament. Interactions between myosin heads and thin filaments can be seen in EM as cross bridges. ATP hydrolysis in the cross bridge cycle moves the myosin head towards the barbed ends of the actin filaments (at the Z disk), thus shortening the entire sarcomere. This happens because this happens at both ends of the myosin thick filament.

Front 104

Lecture 5. How many light chains does each heavy chain have in myosin I and II?

Back 104

In myosin II, each heavy chain has 2 light chains and in each myosin I heav chain there are several light chains.

Front 105

Lecture 5. Describe how the myosin motility assay works.

Back 105

1) You absorb some myosin on slide.
2) Add LABELED actin and ATP.
3) Observe actin filaments.

-You will see that the actin filaments actually move along the myosin. the myosins are all moving towards the plus ends, so the minus end seems to be moving forward.

Front 106

Lecture 6. Why is it that myosin and actin and ATP in a test tube will allow myosin to bind and hydrolyze ATP while not in muscle cells?

Back 106

There are regulatory proteins called troponin and tropomyosin in muscle cells that regulate the ATPase activity of the myosin.

Front 107

Lecture 6. What does tropomyosin look like and how is it organized along the actin filament? How does it regulate myosin's interactions with actin?

Back 107

Tropomyosin is a rod shaped protein multiple tropomyosins organize themselves head to tail in a long chain along the actin filaments. In the presence of Ca++, troponin moves tropomyosin out of myosin's way so it can bind and interact with actin.

Front 108

Lecture 6. What does troponin look like and how does it regulate troponin?

Back 108

Troponin is a complex of three different proteins-troponin I, troponin C, and troponin T.

-In the presence of Ca++, troponin T and I position tropomyosin such that myosin cannot bind and interact with actin. When Ca++ is present, the Ca++ binds to troponin C, which changes the conformation and shifts troponin I and tropomyosin so that myosin can bind and move.

Front 109

Lecture 6. What molecule does troponin-C resemble? How so?

Back 109

Calmodulin, because it has 4 Ca++ binding sites.

Front 110

Lecture 6. List the steps of how a nerve can trigger muscle contraction.

Back 110

1) Nerve action potential triggers a muscle action potential that spreads down the T-tubules (membrane invaginations that extends into the cytosol of the myofiber).
2) The signal passes to the SR and this releases Ca++ from the Ca++ storing tubules in the SR.
3) The Ca++ binds to troponin, which moves tropomyosin out of myosin's way so myosin can bind and interact with actin.

Front 111

Lecture 6. How are cardiac and smooth muscle contraction regulated?

Back 111

Cardiac muscle resembles skeletal muscle in structure and is regulated similarly.

Smooth muscle is regulated also by Ca++ levels.
1) It is triggered by an increase in cytosolic Ca++, which binds and activates calmodulin.
2) Ca++-calmodulin then bind to MLCK.
3) MLCK phosphorylates the regulatory light chain, which activates myosin and allows it to bind and move along actin filaments.

Front 112

Lecture 6. True or false, smooth muscle contracts more than skeletal and cardiac muscle but to a lesser extent.

Back 112

False. Smooth muscle contract less than skeletal and cardiac muscle but to a greater extent.

Front 113

Lecture 6. Where are the I, A, and M bands in the sarcomere?

Back 113

The I bands are the actin filaments not overlapping with the myosin thick filaments. The I bands get shorter during muscle contraction.

The A bands are the the myosins themselves.

Front 114

Lecture 6. T/F. Ca++ released after a muscle cell is stimulated by nerve can also bind to myosin light chains. Is this how skeletal muscle is regulated?

Back 114

T. No, skeletal muscle is not regulated this way.

Front 115

Lecture 6. How many polypeptides are there total in myosin II? What are each of these peptides called?

Back 115

There are 6 polypeptides. 2 heavy chains, and each heavy chain binds to 2 light regulatory chains.

Front 116

Lecture 6. What muscle types have myosin II?

Back 116

Cardiac, smooth, skeletal.

Front 117

Lecture 6. When you look at the cross sections of the thick and think filaments in a sarcomere through EM, what does it look like?

Back 117

A hexogonal lattice where one thick filament is surrounded by 6 think filaments. All the myosins sticking out can potentially interact with a thin filament that surrounds it.

Front 118

Lecture 6. Within each sarcomere, you can see that the pointed ends on each side points towards where?

Back 118

The pointed ends point towards the center.

Front 119

Lecture 6. Which part of the cross bridge cycle in muscle contraction is the most hotly debated?

Back 119

The power stroke step . Some say that it happens at the same time the inorganic phosphate is released and some say it happens AFTER the phosphate is released.

Front 120

Lecture 6. How is Ca++ pumped back into the SR tubules after an action potential?

Back 120

Active transport via Ca++ pumps on the surface of the SR.

Front 121

Lecture 6. Why is smooth muscle regulated through myosin and not through actin?

Back 121

It is not possible to regulate contraction through myosin in smooth muscle as there are no troponins in smooth muscle.

Front 122

Lecture 6. How are the adjacent sarcomeres able to contract in tandem following an action potential?

Back 122

The way the muscle plasma membrane is organized gives this. There are T tubules that go into the muscle cytosol close to the SR. Thus, the action potential from the nerve depolarizes the membrane rapidly and causes the release of Ca++ from SR, and this is transient as there are Ca++ pumps that immediately pump the Ca++ back.

Front 123

Lecture 6. What are the four proteins that maintain the sarcomere structure after contraction?

Back 123

-Various actin and myosin binding proteins.

1) Titin.
2) Nebulin.
3) CapZ.
4) Tropomodulin.

Front 124

Lecture 6. What are dystrophins used for? What happens if the gene coding for it has a mutation in the gene that codes for it?

Back 124

Binding actin to the membrane. Muscle dystrophy, in which muscle degeneration occurs.

Front 125

Lecture 6. Where in the sarcomere are the titins located?

Back 125

Extends from the middle of the sarcomere in the myosin all the way to the Z-disk. There are 2 per sarcomere.

Front 126

Lecture 6. What two things are needed for muscle contraction from outside the muscle? Do dystrophins connect actins to the plasma membrane?

Back 126

ATP and Ca++.

No, dystrophins must connect with transmembrane proteins called dystrophin associated proteins first.

Front 127

Lecture 7. In addition to their roles in muscle cells, actin and myosins also carry out what function in non-muscle cells?

Back 127

1) Cell substrate attachment.
2) Cell-cell attachment.
3) Cell division.
4) Cell motility

Front 128

Lecture 7. What is the extracellular matrix made out of and what does it do? What are the proteins, specifically?

Back 128

The ECM is made out of fibrous proteins that can link cells together and regulate aspects of their development and fucntion.

-The proteins are:
1) Collagens
2) Proteoglycans
3) Fibronectin
4) Laminin

Front 129

Lecture 7. What do each of the four types of proteins in the ECM do?

Back 129

1) Collagens-glycoproteins that are highly resistant to stress and provide mechanical strength to tissues.
2) Proteoglycans-protein core COVALENTLY attached to polysaccharides. It forms a hydrate gel that fills extracellular spaces.
3) Fibronectin and lamin-bind to other ECM components and to cell surface receptors and regulate cell behavior such as motility and differentiation.

Front 130

Lecture 7. What can mutations in genes encoding ECM proteins entail?

Back 130

Diseases affecting the skin, bones, and connective tissue.

Front 131

Lecture 7. Cells interact with the ECM via what proteins? What is the structure and organization of this protein? How does it get its specificity?

Back 131

Integrins. They are composed of two mrmbrane spanning polypeptide chains (alpha and beta) that are non-covalently linked. Forming different combinations of alpha and beta integrins give integrins their specificity.

Front 132

Lecture 7. Different combinations of alpha and beta integrins bind to different ECM proteins. The site on most ECM proteins at which integrins bind is called the _______________. How did reearchers know this?

Back 132

RGD repeats. Experimental evidence for this interaction is shown when adding the inclusion of RGD peptide in cell culture media disrupts interaction of cells with a fibronectin matrix.

Front 133

Lecture 7. In cell cultures, integrins are all clustered in _______ that function as what.

Back 133

Focal adhesions that function as attachment sites to he sustratum (ECM stuff or just a glass slide).

Front 134

Lecture 7. What is on the intracellular side of focal adhesions in most cells?

Back 134

At the intracellular domain, integrins are linked to the termini of actin STRESS FIBERS (which have tropomyosin andnature mysoin II, making it contractile by nature). This is done by having integrins linked to actin binding accessory proteins, which in turn bind to actin.

Front 135

Lecture 7. How are actin stress fibers able to flatten a cell? What are the three actin binding accessory proteins that connect integrins at focal adhesions with actin stress fibers?

Back 135

Actin filaments (stress fibers) can flatten a cell by attaching to substratum via integrins and their inherent ability to contract, they can generate enough tension to flatten the cell.

The three accessory proteins are talins, vinculins, and actinins.

Front 136

Lecture 7. Other than a site of cell-surface attachment, what are focal adhesions good for?

Back 136

Site of accumulation of signaling proteins, which bind shortly after ECM binding. This allows the integrins to relay information about the ECM and the extracellular environment to the inside of the cell.

Front 137

Describe how the focal adhesions and stress fiber formation are regulated.

Back 137

Regulated by Rho. One pathway in which Rho regulates stress fiber and focal adhesion assembly:

1) Rho-GTP binds to and activates ROCK (Rho kinase).
2) ROCK phosphorylates and inactivates the myosin light chain phosphatase.
3) This leads to an increase in myosin light chain phosphorylation and myosin activity.
4) Myosin-mediated contraction then lead to integrin clustering and stress fiber assembly.
5) Rho also activates formin proteins in similar ways that CDC42 activates WASP, which nucleates actin filaments giving the stress fibers.

Front 138

Lecture 7. What processes are responsible for the following:

Back 138

1) Protrusion of lamellipodium- actin polymerization.
2) Adhesion-integrins.
3) Translocation-occurs via a dynamic network model in which myosin mediated contraction of the actin network between the junction of the cell body and lemellipodium pulls the cell body forward.
4) De-adhesion-

Front 139

Lecture 7. In addition to cell-ECM interactions, what else is necessary for the tissue formation and development? What is the primary protein involves in this process? What kind of protein is this and describe its main characteristics. How does it get its specificty ?

Back 139

Cell-cell interactions. Cadherins are primarily responsible for this. Cadherins are Ca++ binding transmembrane glycoproteins. They bind to one another via extracellullar domains on the surface of adjacent cells in a Ca++ dependent manner.

-Cadherins gain specificity by having each type of cadherins only able to bind to members of its kind, thus gives highly specific interactions between cells expressing one class of cadherins.

Front 140

Lecture 7. Where do cadherins interface with the actin cytoskeleton? What do these junctions look like? Describe its intracellular and extracellular domains.

Back 140

At cadherin junctions. These junctions encircles the cell like a belt that connects adjacent cells to one another. The intracellular domains are composed of a circumferential belt of actin that is connected to the junction via actin binding proteins called alpha and beta catenin. This circumferential belt has tropomyosin and myosin (so contractile in nature).

Extracellular domains consists of simply cadherins attaching to their own class of cadherins from other adjacent cells.

Front 141

Lecture 7. What are the two types of tissues that cadherins for cell to cell junctions in?

Back 141

Epithelial and cardiac tissue.

Front 142

Lecture 7. What kind of actin cellular structure (stress fiber, lamellipodia, filopodia) most resembles the circumferential belt of actin in the intracellular domains of cadherin junctions?

Back 142

Stress fibers.

Front 143

Lecture 7. What can tensions created by the circumferential belts of actin in the intracellular domains of cadherin junctions do for the cell? What about contraction

Back 143

Tensions may help stabilize tissue, and contraction helps to alter the shape of these tissue during development.

Front 144

Lecture 7. What are the three main functions of the cadherin junctions in cells?

Back 144

1) Cell-cell attachment with high specificty.
2) Stabilize tissue and also change shape in development.
3) Cell signaling to inside of cell via catenin proteins.

Front 145

Lecture 7. Which catenin, alpha or beta links comes first relative to the plasma membrane/cadherin junction?

Back 145

Bate catenin-which links to the alpha actinin, which links to the actin.

Front 146

Lecture 7. What is thought to have provided the force necessary for the furrow invagination seen in cytokinesis?

Back 146

A contractile ring consisting of parallel arrays of actin interspersed with myosin thick filaments, which functions to constrict the cell and divide it in two.

Front 147

Lecture 7. What two experiments show that myosin II is necessary for cytokinesis?

Back 147

1) Injecting an antibody against myosin II inactivates it and causes cytokinesis to fail and generates multi-nucleate cells.
2) Genetic knockouts of myosin II has the same effect.

Front 148

Lecture 7. How is stress fiber contractility controlled?

Back 148

Not via the actin filaments, since there are no troponins present. Tropomyosin is there only to provide structural support. There are tropomyosin, actin, and myosin. It's controlled by regulations on the myosin light chains.

Front 149

Lecture 7. What kind of signaling molecules are recruited to focal adhesions? What are some examples?

Back 149

Tyrosine kinases. Src and Fak. So focal adhasions are able to function in cell signaling and cell-surface interactions.

Front 150

Lecture 7. What kinds of things can Fak and Src affect? Give a pathway in which these signaling proteins carry out their task.

Back 150

Src and Fak can affect things like adhesion, gene expression, and locomation.

-Pathway:

1) Fak and Src recruited to focal adhesion sites and phosphorylate tyrosine residues on effector proteins.
2) Rho is phosphorylated and activated. Activated Rho is in GTP bound state, which activates a kinase called ROCK.
3) You know the rest of the pathway--> ROCK--> MLCP--> .....

-Need to know that the pathway starts at Src and Fak phosphorylating Rho to activate it, and that activation of myosin triggers interactions with actin and formation of little bipolar myosin, which allows myosin bundles to form to interact with and form stress fibers.

Front 151

Lecture 7. How can we be sure that ROCK is necessary in the formation of stress fibers in the cell?

Back 151

Use a ROCK inhibitor and see what happens.

Front 152

Lecture 7. There are two pathways in which activated Rho can lead to the formation of stress fibers, what are they?

Back 152

1) Fak and Src pathway in which the myosin regulatory light chain was phosphorylated, and promotes the formation of myosin bunndles that interact and form actin stress fibers.
2)Also depends on Rho, which activates ROCK and also activates formins, which nucleate and form the actin polymerization needed to make stress fibers. The formin pathway regulates only stress fiber formation while the other pathway regulates their formation and function (contractility).

Front 153

Lecture 7. Other than the actin polymerization and depolymerization that is helping to push the cell forwards, what else does the cell need at the rear?

Back 153

The cell needs the contractile bundles (which are parallel), which contain compressed remains of the original cross linked actin with myosin II. These bundles contract and push the cell forward like a toothpaste tube being squeezed.

Front 154

Lecture 7. What are the two gradients at the front and rear of the cell that allows it to move?

Back 154

A gradient of adhesion with greatest adhesion at the front of the cell and a gradient of traction with greatest traction at the rear of the cell.

-The traction is due to the myosin and actin. This is only possible near the rear of the cell because myosins are only located there.

Front 155

Lecture 7. How can one test the gradient of adhesion/traction model?

Back 155

All kinds of things (can add GTPase mutatants, etc.)

-In class, we discusses adding myosin inhibitor called blebbistatin, which does not affect the protrusion of the cell but affects the traction at the rear. There is no contractility, since myosin is inhibited. There is protrusion at front but its not coupled to movement.

Front 156

Lecture 7. What are the consequences of improper cell adhesion.

Back 156

A ruffle forms, a sheet of the cell forms a roll.

Front 157

Lecture 7. At the very last step of cytokinesis, the two daughter cells are still connected by a segment called the ____.

Back 157

Midbody.

Front 158

Lecture 7. Can the ECM be considered as a growth factor for the cell?

Back 158

Yes.

Front 159

Lecture 7. What are the three major proteins in a cadherin junction?

Back 159

1) Cadherins.
2) Alpha catenin.
3) Beta catenin.

Front 160

Lecture 8. IFs are important for what in cells?

Back 160

1) Cell-cell and cell-ECM attachments.
2) Cell strength.

Front 161

Lecture 8. What are 5 general properties of IFs that are important?

Back 161

1) 10 nm in diameter.
2) Very stable-need protein denaturing agents to disassemble (but only IN VITRO!!).
3) No nucleotide binding (so not dynamic).
4) Non-polar.
5) Can withstand large stretching forces.

Front 162

Lecture 8. Describe the first level of organized structure of IFs. Does this have polarity?

Back 162

-Have N and C terminus.
-Basic structure is a DIMER that consists of the head domain (N terminus), a coiled coil (mediates dimerization), then the tail domain (the C terminus).

This structure has polarity.

Front 163

Lecture 8. The first level of organization in IFs is the dimer, what is the basic level of organization, one that people usually find as monomers to IF polymers? What are the next levels of organization or IFs after this level? Does the IF (highest level of organization) have polarity? Why or why not?

Back 163

Anti-parallel tetramers, in which 2 dimers, arranged in anti-parallel fashion are put together. This structure has no polarity and is thought of as the basic unit of polymeization. After the tetramer comes the protofilaments, then the protofibril, then the actual IF-has no polarity because the basic subunit has no polarity.

Front 164

Lecture 8. Describe the five different types of IFs.

Back 164

1) Types I (acidic) and Type II (basic)- There are always heterodimers consisting of one basic and one acidic subunit. Examples are keratins of epithelial cells foun din hair, nails, skin, and those that line the interal body cavities.
2) Type III-vimentins, found in fibroblasts, WBCs, and embryonic cells, which extend throughout the cytoplasm. They are mostly homopolymers consisting of a single IF protein species-vimentin is the most widely distributed protein of this type. It extends from the nuclear envelope to the PM, probably yo position the nucleus Also examples are desmin, douns main in muscle where it links adjacent Z-disks of myofibrils and also glial fibrillary acidic proteins, found in glial cells.
3) Type IV-neurofilaments (have three types of heteropolymers NF-L, NF-H, and NF-M). They are good for structural support for specialized neual processes like axons and also controlling the axon diameter.
4) Type V-lamins, which are found only in nucleus lining the nuclear envelope. Three types exist: A, B, and C. They help maintain the structure of the nucleus and helps in the disassemble and assembly of the nucleus

Front 165

Lecture 8. What kind of disease can IFs use to Dx?

Back 165

Cancer. The tumor types are hard to detemine because the cells have changed so much. However, by looking at what types of IFs (keratins, whcih are types I and II IFs) are in the cell, one can determine where the tumor cell originated.

Front 166

Lecture 8. How are nuclear lamins different from the other types of IFs?

Back 166

1) They are more dynamic.
2) They have alonger central rod domain.
3) Contain a NLS.
4) Assemble into 2D sheet-like lattice.

Front 167

Lecture 8. What needs to happen to the mains for the disassembly of the nuclear lamina?

Back 167

Phosphorylation.

Front 168

Lecture 8. What is the organization of IFs in the cell like?

Back 168

IF binding proteins cross link Ifs into networks or bundles, which is often coincident with the microtubule cytoskeleton. They align with the microtubules and they also align with actin. How they align with the microtubules or actin determines the IF's orientation in space.

Front 169

Lecture 8. What happens to the IF networks in a cell if drugs used to disrupt the microtubule cytoskeleton is used?

Back 169

The IF networks can collapse.

Front 170

Lecture 8. What are the structures connecting the ECM and the cell called reltaed to IFs called?

Back 170

Hemidesmosomes. Just like in the case of actin, there are transmembrane integrin proteins. Inside, the integrins are connected to a plaque protein, which is connected to the IFs.

Front 171

Lecture 8. What are the structures connecting cell to cell that reltates to IFs called?

Back 171

Desmosomes. Within the space between the adjacent cells, there are the cadherins, which need Ca++ to bind to each other. Each cadherin binds to a plaque proteins which is in turn bound to IFs of that cell.

Front 172

Lecture 8. The IFs that work in desmosomes and hemidesmosomes connect the PM with what structure in the cell?

Back 172

The nucleus.

Front 173

Lecture 8. What are some of the diseases associated with IF dysfunction discussed in class? In each type of disease, state which type of IF is dysfunctional.

Back 173

1) Epidermolysis-the type I and II IFs in the epidermis of the skin having problems-skin sensitive to injury.
2) Muscular dystrophy-caused by problems in type V IFs. Mechanism is not well understood. This disease causes muscle weakness and degeneration.
3) Premature aging-problem with type V IFs-no one knows why.
4) Myopathy-type III IFs having problems. Desmins (hold sarcomeres together) are dysfunctional.

Front 174

Lecture 8. Why do cells need to use both actin and IF for cell to cell or cell to ECM attachments?

Back 174

Using actin (which often comes with myosin) gives the option of contractility and the ability to exert tension. These can exert a little bit of force to hold the tissue together.

Using IFs gives the resistance against sheer stress. They act as a really strong rope.

Front 175

Lecture 8. Can microtubules connect with IFs, what about actins?

Back 175

Both actins and microtubules can attach to IFs.

Front 176

Lecture 8. What were the two methods used to measure dynamics mentioned in the previous lectures? What is the last one mentioned in this lecture? How does it work?

Back 176

1) Photo-activation microscopy.
2) Speckle microscopy.
3) FRAP-fluorescently label the IFs (GFP-vimentin). You bleach the labeled IFs after and stop after some time and observe how long it takes bleached area to recover the fluorescence. The only way fluorescence can recover is through the polymerization of new unbleached vimentin.

-One way to measure the dynamics is using the half-life-the time it takes to recover half of the original fluorescence.

Front 177

Lecture 8. What are the half lives of actin in lamellapodia, actin in stress fibers, and IFs using FRAP?

Back 177

-Actin in lamellipodia = 30 sec.
-Actin in stress fibers = 5 min.
-IFs = 1 hour.

Front 178

Lecture 8. What happens to the IFs during mitosis? What type of dynamics is driving this transformation?

Back 178

The IFs disassemble during mitosis. This is drive by very slow, equilibrium based dynamics. No energy is spent and no nucleotide binding is present. It is thought that the mitotic kinases phosphorylate the IFs, causing the equilibrium to change and favoring the depolymerized state.

Front 179

Lecture 8. Septins were first identified on what organism? How were they ID'd? What were they later found to be a part of in the cell?

Back 179

Yeast. They were first ID's in a screen for yeast mutants tat arrest in various stages of the cell cycle. Septins were later found to be the structural components of filaments observed at the neck between mother and daughter cells right before they divide.

Front 180

Lecture 8. Mutations in what septin genes cause cell cycle arrest?

Back 180

Cdc3, Cdc10, Cdc1, and Cdc12.

Front 181

Lecture 8. What do septins associate with in mammalian cells?

Back 181

Cellular membranes and the actin and microtubules cytoskeletons.

Front 182

Lecture 8. What are the 3 physical properties of septins as discussed in class?

Back 182

1) 10 nm in diameter.
2) Bind and hydrolyze GTP.
3) Found to be associated with membranes, actin and microtubules.

Front 183

Lecture 8. What are the domains on a septin? What is the role of the GTPase domain? How does it polymerize?

Back 183

A variable extension, a GTPase domain at the N terminus, and a coiled coil domain at the C terminus. No one knows what the GTPase domain is for. Septins usully form complexes of multiple septins, which then polymerize to form filamens in vitro and in vivo.

Front 184

Lecture 8. How were researchers able to tell that septins were necessary for cytokinesis in yeast? Specifically, why are septins needed that even allows cytokinesis in the first place? In mammalian cells, are septins needed ONLY for cytokinesis? What about buds in yeats, do they require septins?

Back 184

They used temperature sensitive mutants. Using a regular septin null knockout would've killed the cell and no one would know what it was for. The TS mutant just kept on growing and growing without dividing. This gave it away that septins were essential for cytokinesis.

Septins allows the contractility generated by the actins and myosins in the contractile ring during cytokinesis. No, there are other functions for septin in mammalian cells, such as scaffolding (help recruit signaling proteins that function to regulate cytokinesis and other cellular processes) and membrane trafficking (movement and fusion of membrane vesicles).

Septins are not needed to make the bud in yeast, but are needed in assembling the contractile ring.

Front 185

Lecture 8. Are IFs ever found as monomers? How do you get IFs in vitro?

Back 185

No. As soon as the proteins are synthesized they dimerize. You can obtain IFs in vitro by purifying it and then treating it with urea.

Front 186

Lecture 8. What are the main difference between different types of IFs?

Back 186

Their N and C termini in their monomer subunits.

Front 187

Lecture 8. How can IFs undergo retrograde motion like actin?

Back 187

IFs are attached to actin, which is involved in retrograde flow.

Front 188

Lecture 8. How do lamins turn over?

Back 188

They don't turn over in interphase but they do in mitosis in which they disassmble and assemble around the daughter nuclei.

Front 189

Lecture 8. Is it better to have IFs or mutated IFs?

Back 189

You would rather have no IFs than mutated IFs because that makes the cell even more fragile, as you would need greater mechanical stress to break the cells in null mutations and minor mechanical stress to break cells in cells with mutated IFs.

Front 190

Lecture 9. What is the diameter of microtubule filaments? What is the basic subunit? What is the structure of the basic subunit? Does it bind to GTP?

Back 190

24nm. Basic subunit is the tubulin (100kD), which is a stable heterodimer consisting of alpha and beta tubulin. These alpha and beta tubulins neer separate. BOTH the alpha and beta tubulins bind GTP.

Front 191

Lecture 9. What are the critical differences between nucleotide binding in alpha and beta tubulin?

Back 191

Alpha tubulin never exchanges or hydrolyzes GTP but beta tubulin does. (GTP-> GDP = hydrolysis reaction, GDP-> GTP = nucleotide exchange).

Front 192

Lecture 9. Are there various forms of alpha and beta tubulins in mammals?

Back 192

Yes at least 6 different genes for each type of tubulin.

Front 193

Lecture 9. Microtubules are made of ____ (a number) protofilaments. They interact ____ to form what shape? How does the microtubule have polarity? Describe the polarity and any differences in dynamics it may cause.

Back 193

Microtubules are made of 13 protofilaments. They interact LATERALLY to form a cylinder. Each protofilament has intrinsic polarity and alpha and beta tubulins are in the same orientation and thus gives the entire microtubule polarity. The plus end is ringed with beta tubulin and the alpha tubulin rings the minus end. The plus end is the faster growing end.

Front 194

Lecture 9. The minus end of MTs are usually ______ and less _____ compared to the plus end.

Back 194

Capped. Less dynamic.

Front 195

Lecture 9. How can one initiate MT assembly IN VITRO? What else matters in whether polymerization occurs or not?

Back 195

To initiate assembly of MTs IN VITRO, one needs to raise the temperature to 37 degrees C and lower it to 4 degrees C to depolymerize.

-The concentrations of the free monomers also matter. If it is above the Cc (14 micromolars), then assembly will occurs, otherwise it will not.

Front 196

Lecture 9. The timecouse of MT polymerization is very similar to that of actin in that there are ____ ( a #) phases called ___, ______, and ____, provided, in in-vitro conditions, that all the necessary factors for polymerization initiation are there.

Back 196

3. Nucleation, elongation, steady state.

Front 197

Lecture 9. What is the critical concentration of tubulins for MT nucleation? This poses a problem, what is it and how do cells try to solve this problem?

Back 197

40 micromolar without any seeds. Need A LOT of MT monomers to start polymerization. Cells solve this problem by always having some seeded assembly at the centrosome.

Front 198

Lecture 9. Once you reach steady state in MT polymerization, what happens? is there treadmilling like in actin? Is there still dynamaticity?

Back 198

At SS, there is no net growth or loss in length of the MT, however, there is no treadmilling, but there is still dynamaticity.

Front 199

Lecture 9. Describe how MTs are organized in the cell.

Back 199

The minus ends are capped and embedded in the MT organizing center, AKA centrosome. The plus end faces outwards from the centrosome.

Front 200

Lecture 9. If tubulin is depolymerized by cooling cells to 4 degrees C or by adding a MT destabilizing drug, then allowed to re-polymerize by warming or washing drug out, what happens?

Back 200

New MT grows out from the centrosometo form a small star like aster. After longer periods of recovery, the normal MT distribution re-surfaces.

Front 201

Lecture 9. Describe the structure of the centrosome.

Back 201

The centrosome consists of a pair of centrioles surrounded by PCM, where the minus ends of MTs are embedded and capped.

Front 202

Lecture 9. Describe how the MT nucleation on the centrosome works. Also name the main protein that's involved.

Back 202

You need gamma-tubulin, which is functionally distinct from alpha and beta tubulin. Gamma tubulin exists on the protein complex called gamma tubulin ring complex (gamma-TURC), which forms a ring like structure on the PCM of the centrosome. This can also be seen by EM if it's purified gamma-TURC. Mechanism of nucleation is not well understood.

Front 203

Lecture 9. What are some assays people ca use to monitor the polymerization status of MTs? They are used to monitor the formation of MT polymers. What are some methods that can be used on actin that can't be used on MTs? Why not?

Back 203

1) Pelleting assay in which you separate the polymerized MT (pellet) to unpolymerized tubulin (supernatant). You compare the amount of both.
2) Light scattering assay.


-You cannot use pyrene fluorescence because you can't modify tubulin and still retain it characteristics.

-The pyrene fluorescent technique works by having it fluoresce more when it is in a hydrophobic environment (when actin is in a filament).

Front 204

Lecture 9. What is the definition of dynamic instability?

Back 204

The co-existence of MTs populations in which some are growing and some are shrinking, both from the plus end.

Front 205

Lecture 9. What is the experiment that demonstrated the dynamic instability nature of MTs?

Back 205

1) You start off with MTs that have already formed and put them in different concentrations of tubulin. Typically there are 2 concentrations, one that is close to the Cc (14 micromolars) and one that is below it.
2) At the [tubulin] = 20 micromolars > Cc solution, we would expect the amount of polymers to increase, but since [tubulin] < concentrations needed for nucleation, the # of MTs stays the same but surprisingly the length of some of the MTs shrank but much more grew giving net growth of the MTs overall.
3) At [tubulin] < Cc, there is a decrease in number of MTs over time because some MTs will have completely depolymerized. In addition, there is a net decrease in the length of MTs, however, some of the MTs actually grew.

Front 206

Lecture 9. What would happen to the MT # if we put MTs in a solution with tubulin concentrations ABOVE 40 micromolars?

Back 206

The MT # would increase because nucleation would take place.

Front 207

Lecture 9. What is the reason behind dynamic instability in MTs?

Back 207

Mainly due to the GTP binding and hydrolysis ability of beta tubulin. When you have [tubulin] far greater than the Cc, addition of new GTP-tubulin outpaces the hydrolysis of GDP-tubulin, thus maintaining the GTP cap at the plus end, which has a more favorable interactions with surrounding subunits than GDP-tubulin. However, when [tubulin] is close to the Cc, then the rate of GTP-tubulin addition might be outpaced by GTP hydrolysis. When there is not a GTP-tubulin left, it results in a splayed protofilament arrangement at the plus end. This causes the MT to shrink, until it is rescued.

Front 208

Lecture 9. Can the minus end of the MT have GTP caps? How does the hydrolysis of the GTP on beta tubulin occur shortly after the tubulin is added to the MT? What is behind the interconversions between MT growth and loss in length?

Back 208

Yes. The association of GTP subunit to the adjacent subunit that promotes GTP hydrolysis. The interconversions between a GTP and GDP cap.

Front 209

Lecture 9. If you have a [tubulin] much greater than the Cc, what will happen to MTs in terms of length change? What about when it's close to the Cc, what about when it's far below the Cc?

Back 209

-[tubulin] far above the Cc = MTs can shrink and grow but much much more growth than shrinkage.
-[tubulin] close to the Cc = you get both shrinkage and growth. Hence, if you freeze in time and compare the average length of MTs, it may be greater or less compared to what you started out with.
-[tubulin] far below Cc, you still get shrinkage and growth, but much less growth.

Front 210

Lecture 9. What is the catastrophe and rescue in MT dynamics?

Back 210

-Catastrophe is at the moment when the MT goes from growth to shrinkage. This happens only when you've lost the GTP cap.

-Rescue is at the moment when the MT goes from shrinkage to growth. This only happens after you've regained the GTP cap.

Front 211

Lecture 9. How was the structure of MT solved? What similarities does FtsZ share with MTs? Do all MTs HAVE TO HAVE 13 protofilaments?

Back 211

EM. There is no sequence similarity between FtsZ and MTs but there is structural similarity in that they both form protofilaments IN VITRO. No, some can have more or less than 13 protofilaments.

Front 212

Lecture 9. What does the structure of GDP bound beta tubulin look like? What about GTP bound beta tubulin? What can a tubulin sheet form? What happens after it forms?

Back 212

It looks bent (forms into a ring before it dissociates). GTP bound beta tubulin is straight. A tubulin sheet can form a MT, but shortly it forms GTP hydrolysis commences.

Front 213

Lecture 9. How come you would not see the simultaneous shrinkage and elongation of actin filaments then [G-actin] is close to the Cc like the dynamic instability case in MTs?

Back 213

Because actin is not always capped at the minus end, at the Cc, monomer adds at plus end and comes off at minus end at a rate such that addition rate = depolymerization rate, so length of filament doesn't change.

Front 214

Lecture 9. What is a great source of tubulin disucssed in lecture? Why did people start to believe that gamma tubulin ring complexes were the site of MT nucleation?

Back 214

Brains. The gamma tubulin ring complexes have the same diameter as most MTs.

Front 215

Lecture 9. What do the drugs, Taxol, Colchicine, and Nocodazole do?

Back 215

-Taxol binds MTs and stabilizes the polymer, preventing it from disassembly. This can block key MT events such as spindle assembly. Useful against ovarian cancer.

-Colchicine-binds to tubulin dimers such that when the dimer polymerizes, the drug prevents further polymerization onto the MT end, so this forms a GDP cap and promotes destabilization.

-Nocodazole binds to tubulin and prevents it from polymerizing.

Front 216

Lecture 10. What's all that MT in the brain doing? How do you isolate proteins in usual cases, what about for tubulin and actin?

Back 216

Usually, to isolate proteins you express it in bacteria. You can't do that for actin and tubulin, you need to get it from tissue. Actin can be obtained from rabbit muscle and tubulin can be obtaind from brains, where it is useful for transport of synaptic vesicles.

Front 217

Lecture 10. Can dynamic instability occur in vitro, in vivo, or both?

Back 217

Both.

Front 218

Lecture 10. What are the different classes of MT binding proteins? What do each of them do, and name some examples for each class.

Back 218

1) Sequestering proteins: Stathmin/Op18. Promotes catastrophe and depolymerization.
2) Nucleating proteins: gamma-TURC-nucleates at the centrosome.
3) End binding proteins AKA +TIPs: CLSAP, EB1 and CLIP-170, which bind to plus end of MTs and stabilize MTs, and help mediate its interactions with certain cellular components, such as chromosomes.
4) Severing proteins: katanin; also promotes depolymerization at both ends.
5) Depolymerization proteins: Kinesin-13 family-promotes depolymerization of plus end by binding to it and inducing protofilament curling, which destabilizes it and causes subunits to come off. This can happen even to GTP caps.

Front 219

Lecture 10. Centrosomes contain a pair of _______ surrounded by a material called ______, which has the _____ embedded in it that actually nucleates the MTs.

Back 219

Centrioles. PCM. Gamma-TURC.

Front 220

Lecture 10. What are four general properties of MT organizing proteins? What do these proteins do that make these properties made possible?

Back 220

1) Stabilize MTs-binding to their sides and preventing disassembly.
2) Enhance stability-stabilizing nuclei and facilitating nucleation.
3) Organize MTs-into bundles and other structures.
4) Mediate MT interactions with other proteins.

Front 221

Lecture 10. Proteins that help organize MTs (AKA MAPS) have what domains? Which domain determines the spacing between the bound molecules?

Back 221

-2 major domains:

1) MT binding domain which binds several tubulins at once and stabilizes the dimer.
2) Projection domain, which interact with MTs or other cellular components such as IFs.

-Projection domains determine spacing.

Front 222

Lecture 10. What are the two types of projections in neurons? What needs to happen for th MTs to be transported from the center of the cell to processes such as axons?

Back 222

Axons and dendrites. Being capped at the minus end at the centrosomes.

Front 223

Lecture 10. In the axons, the MTs are the the same or different orientations? What about in dendrites? How is the spacing in the dendrites and axons determined?

Back 223

-In axons, all the orientations of MTs are the same. In dendrites, they are mixed.
-The spacing is determined by MAPS, in dendrites, spacing is determined by MAP2.
-In axons, the spacing is determined by Tau.

Front 224

Lecture 10. Which protein has a shorter projection domain, Tau or MAP2?

Back 224

Tau has a shorter projection domain, so bundles of MTs with Tau as the MAP are more compact.

Front 225

Lecture 10. What would happen if you expressed Tau and MAP2 proteins in cells that normally don't express them?

Back 225

The cell would start forming axon like structures.

Front 226

Lecture 10. What are the two types of MT-based motor proteins? What kind of proteins are they? What do they require to work?

Back 226

1) Dynein
2) Kinesins.

-They are mechano-chemical enzymes and need ATP to work.

Front 227

Lecture 10. Kinesins were first discovered where? What was the first kinesin purified called? Give the general structure of a kinesin.

-Which end is the motor domain?

Back 227

-Giant squid axon. Kinesin-1.
-Kinesins generally have 2 heavy chains and 2 light chains. Each heavy chain has the head (ATP binding domain), neck, and coiled coil that mediates dimerization of two heavy chains.

-Two light chains at the tail, which are NOT part of the coiled coil domain-mediate the association of vesicles-binds cargo.

The head region is the motor domain.

Front 228

Lecture 10. Where are katanins located in the cell?

Back 228

Near or on centrosomes.

Front 229

Lecture 10. In the axons, where the MTs are in the same orientation, where are the plus ends facing?

Back 229

The plus ends are facing the front of the axon.

Front 230

Lecture 10. There are _____ classes of kinesins, which are divided into ______ categories, based on _________.

Back 230

14. 3. Based on on the position of the motor domain within the kinesins.

Front 231

Lecture 10. What are the three categories of kinesins? Which ends of the MT are each type directed (or move towards?)

Back 231

1) Kinesin-1, AKA N-type: the motor domain is at the N terminus. They are plus end directed.
2) C-type: the motor domain is at the C terminus. They are directed towards the minus end.
3) I-type: motor domain is in the middle. They destabilize the MTs by binding MT ends and promoting protofilament peeling and depolymerization. They don't move toward either end of the MT.

Front 232

Lecture 10. Dyneins. Describe its basic structure, which end of the MT it's directed or move towards, and the two types.

Back 232

Dyneins are always minus end directed. The structure has 2 or 3 heavy chains, a few intermediate chains and a few light chains. Two types of dyneins:

1) Ones that function in flagella.
2) Cytoplasmic dynein (found in all eukaryotic cells).

Front 233

Lecture 10. What is the dynein complex.

Back 233

Dynein complexes are huge multi-subunit proteins that co-purifies with dynein and are though to facilitate the attachment of dyneins with vesicles and organelles.

Front 234

Lecture 10. Vesicles can be transported on MTs to and back from the synapase. Forward (or anterograde movement) is driven by which MT motor protein? What about reverse (retrograde movement)?

Back 234

Kinesin (kinesin-1) drives forward movement, and dynein drives retrograde movement.

Front 235

Lecture 10. In non-neural cells, what are the kinesins and dyneins useful for? What are some examples of this? What can be done to find out about this experimentally?

Back 235

-They function to organize the components of the cell. Examples are the Golgi and ER.

-You could use experiments where the dynein are inhibited, add nocodazole to get ride of MTs, interfere with MT polymerization and you will see that the Golgi has dispersed into vesicles and not packed into stacks, and ER has collapsed.

Front 236

Lecture 10. Can microtubules be cargoes of microtubules motor proteins? Can projection domains bind to IFs?

Back 236

Yes. Yes.

Front 237

Lecture 10. Kinesins need what to activate its ATPase activity? What is the mechanism that generates their motor activity?

Back 237

Kinesins need MTs to activate ATPase activity. They go through a cross-bridge cycle similar to myosin in which ATP is hydrolyzed and the release of ADP and Pi are coupled to conformational change in head and neck domains.

Front 238

Lecture 10. What kind of experiments were used to isolate the kinesins and dyneins?

Back 238

In vitro motility assays in which cell cytoplasm was added to ATP and MTs and observed under advanced video microscopy.

Front 239

Lecture 10. How do stathmins sequester tubulins?

Back 239

It binds to two tubulin dimers and prevent dimerization.

Front 240

Lecture 10. In MT protofilaments, even when you add stabilizing agents such as taxols, what happens when you add kinesin-I?

Back 240

The ends shrivel out as kinesins I eat up both ends, but not completely because taxols are added.

Front 241

Lecture 10. In kinesin motility assays, what kind of MTs do you need to add?

Back 241

Fluorescent ones.

Front 242

Lecture 10. Describe the Kinesin cross bridge cycle steps.

Back 242

1) In the beginning, both heads on the kinesins are attached to ADP.
2) One head binds to microtubule and releases its ADP, which is exchanged for ATP.
3) The ATP exchange triggers the neck linker, which triggers to throw the other head forward to the next kinesin binding site on the MT.
4) The first head hydrolyzes ATP and releases Pi.
5) The second head releases ADP has ATP comes in, and throws the first head forward in the same fashion.
6) Cycles goes on.

Front 243

Lecture 11. What are the four things kinesins-1 and myosin II have in common?

Back 243

1) Both are plus end oriented.
2) Myosin II walks on actin and kinesins walk on MTs.
3) They belong to large gene families.
4) They both use energy in ATP, usually to drive a conformational change that translates to movement.

Front 244

Lecture 11. Where are the regions in kinesin I and myosin I that drives the conformational change and movement?

Back 244

Kinesin I: neck linker region.
Myosin I: lever arm in neck.

Front 245

Lecture 11. Answer the following questions for kinesin I and myosin I:

1) How much time do they spend on the MT/actin.
2) The two heads, do they move in a coordinated fashion?
3) Low or high processivity?

Back 245

2) The two heads on kinesin Is move in a coordinated fashion because they need to be constantly on the MT; when one head lets go the other has to immediately attach. This is not in myosins, since they are spending so little time on the actin and are attached to different actin filaments.
1) Myosins spend little time on the actin, kinesins spend a lot of time on MTs.
3) Kinesins have high processivity and myosins have low processivity.

Front 246

Lecture 11. What is the meaning of processivity?

Back 246

1) The time the motor protein spends attached to the filament.
2) How coordinated the movement of the motor protein is along the filament.

Front 247

Lecture 11. The concentration of tubulin is about _____ micromolars, which is above or below the Cc? MTs in vivo or in vitro are more dynamic and variable in dynamics, why is that?

Back 247

15 micromolars. That is above the Cc. MTs in vivo are more dynamics and variable, mainly because of MT binding proteins.

Front 248

Lecture 11. What are the parameters used to mesasure the difference in dynamics of MTs in vitro and in vivo? And what are the differences seen in each parameter for both conditions?

Back 248

1) Growth rate (Vg): Lower growth rate in vitro and larger range of growth rate in vivo.
2) Shrinkage rate (Vs): Smaller skinkage rate in vivo and larger range of rates in vivo.
3) Catastrophe frequency (fcat): Less of these events in vitro (catastrophe happens because 15 uM is above the Cc of MTs) and a wider range of number of events/min of MT assembly in vivo.
4) Rescue (Fres): There is a wider range of number of events/min of MT assembly in vivo and generally there are more of these events in vitro.
5) Turnover rate: shorter time to turnover in vivo.

-Generally, you seen more dynamaticity in in vivo conditions and more variability because mainly of the MT binding proteins in vivo.

Front 249

Lecture 11. Cell growth occurs when during the cell cycle? What about the centrosome replication?

Back 249

G2 and G1. Centrosome replication occurs in G2 and S.

Front 250

Lecture 11. In terms of the cytoskeleton, what three events occur at the entry of mitosis? Why is each step important?

Back 250

1) Chromosome condensation-chromosomes need to be packed so can be moved more easily-100 to 500 fold compaction occurs.
2) Nuclear envelope breakdown-MT is in cytoplasm, need to get access to the chromosomes.
3) Changes in MT dynamics-MT arrays changes from interphase MT to mitotic MT array.

Front 251

Lecture 11. Where do the kinetochores form in the cell? How many of these are there in each replicated chromosome? What holds the two sister chromatids together before anaphase along the chromatids and the chromatids are bound most tightly where? describe this region. What happens on the kinetochores?

Back 251

Centromeres. There are two kinetochores in each replicated chromosome, one per sister chromatid. Cohesins. Centromeres, which is a contriction that is composed of centromeric DNA. MT spindles attach to the kinetochores which allow each sister chromatid to be pulled to opposite poles later on.

Front 252

Lecture 11. What is primarily responsible for condensing the DNA into compactions? They link the coiled DNA together. What do condensins work in conjunction with? How do condensins condense DNA further?

Back 252

Condensins. Topoisomerases. They condense DNA further by binding DNA coils.

Front 253

Lecture 11. What event in the cell is primarily responsible for the breakdown of the nuclear envelope? What else contributes to the NE breakdown? There are 2 more things.

Back 253

Phosphorylation of the nuclear lamins by cyclin B and CDK1.

-Membrane vesiculations-the nuclear envelope breaks down into vesicles.

-A dynein dependent pathway. Dyneins in the cytoplasm bind to proteins in the outer nuclear envelope and it moves towards the minus ends of MTs at the centrosome, which is on the opposite side of theNE as where the dyneins bind. This rips a hole in the NE.

Front 254

Lecture 11. On the kinetochore, what three proteins exist? What is the use of these three proteins?

Back 254

1) A plus end directed MT motor called Cenp-E (a N-type kinesin).
2) A minus end directed dynein (a kinesin-13).
3) Other proteins.

-These proteins are needed for the proper attachment to the MTs in the mitotic spindle and required for proper movement.

Front 255

Lecture 11. MTs are attached to chromosomes at the plus or minus end?

Back 255

Plus end.

Front 256

Lecture 11. The interphase MT spindle is more or less dynamic than the mitotic spindle? What MT parameters would you see changing and how would each of them change? There is more catastrophe, but also more _______ that raises the amount of polymers.

Back 256

Less dynamic than mitotic spindle. The Vg and Vs would stay about the same, but the rate of catastrophe would go up and rate of rescue would go down, as seen in the difference between the Fres and Fcat in in vivo and in vitro conditions. In addition, the half life gets shorter.

-MT nucleation from the centrosomes.

Front 257

Lecture 11. Which part of mitosis does NE breakdown begin to occur?

Back 257

Prometaphase.

Front 258

Lcture 11. In interphase the MTs are longer/shorter and more stable/less stable compared to mitotic MTs. This is one of the things that happens inthe change of dynamics at the interphase-mitosis transition. In addition, what else happens in this transition? What are the two major players that regulate the changing dynamics of MTs at the interphase-mitosis transition and what do they do?

Back 258

Interphase MTs are longer and more stable than mitotic MTs. In addition to changing MT dynsmics, there is also an increase in nucleation at centrosomes. Two major regulatory players in this process are:

1) Kinesin-13: de-stabilizes the MTs.
2) MAPs: stabilizes the MTs. It's activity goes down in M phase of cell cycle.

Front 259

Lecture 11. Kinesin-13s are always active or just more active at the interphase-mitosis transition when the dynamics of MTs change?

Back 259

Kinesin-13s are always active, but they only have The MTs to act upon when the catastrphe rate goes up after the dynamics of MTs change at the transition.

Front 260

Lecture 11. What are the two spindle assembly mechanisms?

Back 260

1) The centrosome pathway-AKA the "search and capture" pathway.
2) The chromosomes pathway.

Front 261

Lecture 11. How are the two centrosome, which form by duplication and originally on one pole of the cell, move to opposite poles?

Back 261

The two centrosomes separate at early M phase an nucleates an array of MTs called an aster. These asters move even further apart, and nucleat MTs that grow and shrink, hoping to hit a kinetochore.

Front 262

Leture 11. Describe the search and capture pathway of spindle formation. What are two pieces of evidence for this pathway?

Back 262

The dynamic MTs are searching from the outside in for kinetochores, in the process, they are randomly assembling and shrinking. Once MTs hit kinetochores, they stabilize. Eventually, you get a mitotic spindle. Both kinetochores on a chromosome don't get hit at once by the MTs from opposite poles.

-Evidence #1: on video.
-Evidence #2: The knowledge that kinetochores can attach to MTs.

Front 263

Lecture 11. Describe the chromosome pathway of spindle formation and proof that it exists.

Back 263

This pathway occurs in the absence of centrosomes. The spindle is organized by the chromatin in chromosomes. This was demonstrated by reconstituting spindle assembly in cytoplasmic extracts of frog eggs arrested in M phase, which do not have centrosomes. If you added beads coated with DNA, you will see chromatin form around them and induce a bipolar mitotic spindle, showing that mitotic chromatin is enough to direct spindle assembly, EVEN IN THE ABSENCE OF CENTROMERES AND KINETOCHORES.

Front 264

Lecture 11. Are centromeres needed for spindle assembly in the chromosome pathway? What is the DNA that lies beneath the centromeres? What's inside the centromere?

Back 264

NO!! Centromere. Centroles

Front 265

Lecture 11. What three events are occuring at Prophase?

Back 265

1) Chromosome condensation.
2) Interphase MTs disassemble near the end.
3) Centrosomes separate and mitotic spindle begins to form.

Front 266

Lecture 11. What two events happen at prometaphase?

Back 266

1) Nuclear envelope breakdown.
2) Assuming the centrosome exist, mitotic spindle goes in and attaches to kinetochores.

Front 267

Lecture 11. What are astral and polar MTs?

Back 267

-Polar MTs are those MTs within the spindle that don't attach to kinetochores.
-Astral MTs are those MTs not even in the mitotic spindle.

Front 268

Lecture 11. What are the two types of movements that can be seen in anaphase?

Back 268

1) Anaphase A: the kinetochore MTs shorten and chromosomes move towards the polars.
2) Anaphase B: the polar MTs elongate, increasing the separation of the two poles and chromosomes being pulled apart.

Front 269

Lecture 11. What events happen at telophase?

Back 269

1) Kinetochore MTs disappear.
2) A new NE forms around the chromosomes of each daughter cell.
3) Chromosomes de-condense.

Front 270

Lecture 11. What happens during cytokinesis? There are 4.

Back 270

1) Cytoplasm is divided by cleavage, WHICH STARTS DURING ANAPHASE.
2) Contracile ring causes the plasma membrane to form a cleavage furrow, which deepens until the midbody is encountered, which is the remnant of the spindle overlapping from each daughter cell.
3) Re-formation of interphase MT spindle.
4) Nucleolus reforms.

Front 271

Lecture 12. What are the two main steps in the chromosome pathway of MT spindle assembly?

Back 271

1) MT assembly around chromatin.
2) MT organization into a bipolar array through the activity of motor proteins.

Front 272

Lecture 12. What mediates the MT assembly around the chromatin?

Back 272

Ran-GTPase, which cycles between GTP bound and GDP bound states.

-The Ran-GEF AKA RCC1 binds to chromatin and Ran-GAP is in the cytoplasm, and this generates a gradient of Ran-GEFs and GAPs as well as Ran-GTPs and Ran-GDPs. Ran-GTP binds to importins, which helps it release its cargo, then when you get out into the cytoplasm, where you have Ran-GDP, the importin binds to the cargo, inhibiting the cargo. Some of the cargos are MT nucleating and stabilizing proteins, which function in polymerizing the MTs around chromatin.

Front 273

Lecture 12. How is the second step of the chromosome pathway to spindle assembly brought about?

Back 273

MT organization into a bipolar array: you need motor proteins.

Front 274

Lecture 12. What part of the chromosome does RCC1 bind to?

Back 274

Nucleosomal proteins.

Front 275

Lecture 12. In cells without centrosomes, where are the spindle cargo during interphase? Mitosis? There is a shift. How does this shift promote spindle assembly?

Back 275

In interphase, the spindle cargos are in the nucleus. In mitosis, the NE breaks down and the cargoes are released in the cytoplasm through Ran-GTP binding to importin B near the chromatin, which then promotes spindle assembly.

Front 276

Lecture 12. How does the Ran-GTP move from where it was to regions near Ran-GAPs?

Back 276

Diffusion.

Front 277

Lecture 12. What causes the reversal of direction of transport of MT stabilization/polymerization factors in interphase and mitosis? From nucleus to cytoplasm in interphase and from cytoplasm to nucleus in mitosis?

Back 277

A reversal of Ran-GTP/GDP and Ran-GEF/GAP gradients in the two stages of the cell cycle. Also, when the NE breaks down, there is a huge increase of Ran-GEFs in the nucleus (RCC1 protein), which causes cargo release right there at the nucleus, where they was originally bound to importin B and inhibited (because no Ran-GTP was present).

Front 278

Lecture 12. Describe the second step of spindle formation (MT organization into bipolar array) in the absence of centrosomes (chromosome pathway).

Back 278

1) The Ran-GTPs stimulate nucleation and MT assembly.
2) Need motor proteins to organize MTs, which bind now to MTs formed.
3) Bundling and sorting of MTs, which is nucleated by kinesin 5.
4) Focusing of the two poles done by dynein or kinesin 14, which are both MINUS end orientated.
5) Maintaining the connections between the MTsand chromatin is done by kinesin 10, which is localized to the chromatin.

Front 279

What is the defining difference between a spindle array that has a centrosome and one without? What are the common features? There are 4.

Back 279

The presence of astral MTs, which always and only accompanies spindles with centrosomes.

Common features:
1)Bipolarity of spindle to generate two sets of chromosomes.
2) Anti-parallel MTs that overlap one another at spindle equator.
3) Minus ends of MTs are all at poles.
4) Plus ends of all MTs face chromosomes but not all MTs interact with them.

Front 280

Lecture 12. Are kinesin-14s plus or minus end oriented. What about kinesin-10s?

Back 280

Kinesin-14s are minus end. Kinesin-10s are plus end oriented.

Front 281

Lecture 12. Kinesin 5s are often depicted as a complex of how man subunits? What kind of domains do each subunit serve as? and this can bind to how many MTs and do what with them? How does it do this? What is it important for?

Back 281

Kinesin-5s are often depicted as tetramers, which can bind to MTs and bundle and SORT them. All four sununits act as motor domains to move MTs. It does this by first localizing to the middle region of the cell where the polar MTs are and 2 motor domains bind to the polar MT from one pole and the other 2 motor domains bind to the polar MT from the other pole. It does this by being a positive oriented motor protein, thus pushing the negative ends of polar MTs from different poles out and plus ends close to each other. This generates the anti-parallel array of MTs at spindle. Kinesin-5s are important for mediating the interaction of overlapping antiparallel MTs from one another (from both poles) by bundling them (no need to sort) in the overlapping zone. It also pushes the poles apart.

Front 282

Lecture 12. What is the force generated by kinesin-5 balance by? How is this done and why is it necessary? What is this motor good for?

Back 282

Kinesin-14. They are all C-type kinesins, so they are minus end directed. They are located at the overlapping zone of the polar MTs from both poles. They tend to pull the poles closer to each other and this is necessary because it prevents the spindle from elongating too much. This motor is also good for focusing the spindles poles (because it can cross link MTs) and maintain the connection between spindle MTs and centrosomes.

Front 283

Lecture 12. Describe the basic structure of kinesin-14.

Back 283

Kinesin-14 has a MT binding domain and a motor domain.

Front 284

Lecture 12. What are the two possible orientations with which kinesin-5s can bind to two different MTs?

Back 284

1) + -, - +.
2) + -, + -.

They ultimately give different structures when kinesin-5 is finished with its work

Front 285

Leture 12. Kinesin-14 are _____ end oriented, and kinesin-10 are ______ end oriented. What does kinesin-10s do?

Back 285

Kinesin-14 are minus end oriented and kinesin-10 are plus end oriented. Kinesin 10s mediate the attachment of chromosome arms with the MTs.

Front 286

Lecture 12. What are the two tasks of kinesin-14s?

Back 286

1) Focus spindles.
2) Counteract the forces of kinesin-5.

Front 287

Lecture 12. Can the kinesin motors function even with the presence of a centrosome?

Back 287

Yes.

Front 288

Lecture 12. What are dyneins, what do they do, where do they localize? which end of the MTs to they orient towards? What happend to this protein when there are no centrosomes? which kinesin does a similar job as this protein?

Back 288

The dyneins are localized at the cellular cortex. They are minus end oriented (towards the centrosomes). They exert a pulling force on the aster and pulls the spindles apart. They cannot function without centrosomes, because there would no asters to pull on. They can also be used to focus spindles. Kinesin-14.

Front 289

Lecture 12. Minus end MTs generally stay associated with the ____ end of a MT. The kinesin-5 can fall off the _____ end and re-bind another MT.

Back 289

Minus. Plus.

Front 290

Lecture 12. What would be the effect of inhibiting kinesin-5?

Back 290

You would get a mono-astral spindle.

Front 291

Lecture 12. What is the mechanism with which chromosomes are aligned at the metaphase plate in mitosis? What happens afterwards? All this is in what phase of the cell cycle? What kinesins are crucial in this step in aligning the chromosomes?

Back 291

Search and capture. Afterwards, with one kinetochore attached to the MT spindle of one pole, the chromosome oscillates back and forth, then moves closer to the metaphase plate, where it is most likely to encounter another MT from opposite pole.
-The chromosome will still oscillate after the other kinetochore has attached to the MT from the opposite pole. This is done by having the plus end of the MT of one pole polymerize and the other depolymerize rapidly. The MTs are still depolymerizing SLOWLY at BOTH plus ends. The alignment is finished when the length of the MTs of both spindles are about the same. Prometaphase. Kinesin-10, 13, 7.

Front 292

Lecture 12. What kinds of forces are seen in metaphase in the MT of the spindle? What does this process depend on? How can it be visualized?

Back 292

1) Poleward MT flux: even after the chromosomes have stabilized at the equator, the polar and kinetochore MTs continue to depolymerize and polymerize. There is still slow depolymerization at BOTH plus ends of the spindle, but there is also slow polymerization at BOTH minus ends. This leads to the treadmilling of subunits, where they come on the MT at the + end and come off at the - end.

-This process depends on polymerization/depolymerization and also a variety of motors.

-This process can be viewed in photoactivatble fluorescence microscopy.
-A small amount of tbulin is attached to fluorescent probe, whic fluoresces only when shined with UV light. When UV light is shone, the tubulin is seen to move towards the POLES of the spindle.

2) Projection force: kinesins 10s, N-type kinesins, located on arms of chromosomes, move chromosomes toward the + end of the MTs, towards the spindle. It is this force along with the MT poleward flux that gives the chromosome oscillations at the spindle equator.

Front 293

Lecture 4. What are the two functions that profilin performs in terms of actin polymerization.

Back 293

It serves as a sequestering agent and also promotes elongation at the plus end.

Front 294

Lecture 4. Are the orientations of actin fibers polarized or not? What about in filapodia and lamellipodia?

Back 294

In stress fibers, there is no net polarity of actin filaments. In filapodia and lamellipodia, there is polarity with the plus end of the actin filaments oriented towards the leading edge.

Front 295

Lecture 4. What are the two main actin bundling proteins? Which one has a longer spacer domain? Whtat about compared to filamen?

Back 295

Fimbrin and alpha actinin. Fimbrin has a slightly longer space domain. Both have shorter spacer domains compared to filamen.

Front 296

Llecture 4. In cell movement, what is membrane protrusion driven by in terms of actin dynamics, what about elongation?

Back 296

Protrusion is driven by nucleation of actin filaments. Eloingation is driven by retrograde flow as actin filaments are depolymerized at the rear and polymerizing at the front in a treadmilling fashion to push the membrane forward.

Front 297

Lecture 4. What two mechanisms does profilin use to promote plus end elongation?

Back 297

1) Binding to the end opposite to the ATP cleft.
2) Promoting exchange of ADP for ATP in the actin monomer that it's bound to.

Front 298

Lecture 4. In speckle microscopy, how exactly do you make the individual actin monomers fluorescent? How do you know that the lit up monomers are in filaments, since you can't see any actin under the microscope?

Back 298

Using GFP-actin. These actin monomers only light up when incirporated into actin filaments.

Front 299

Lecture 4. How does speckle microscopy work?

Back 299

You incorporate low levels of fluorescent actin monomers by fusion to GFP. You then allow these monomers to be incorporated into actin filaments, which you know has happened it you see it light up. Over time, you would see speckles move backwards RELATIVE TO THE FRONT of the cell. Eventually you start to see less speckles (actin filament depolymerization) and eventually when the depolymerized speckles are re-incorporated into new actin filaments, you see a new pattern of speckles.

Front 300

Lecture 4. What can one use to prove that retrograde flow of actin monomers in cell movement occurs?

Back 300

Speckle microscopy, photoactivatable actin microscopy, treatment with drugs-use latrunculin and see that the cell stops moving.

Front 301

Lecture 4. Why can't you see GFP actin in speckle microscopy?

Back 301

Because they are diffusing too fast in the solution.

Front 302

Lecture 4. What are the three steps you would observe in a speckle microscopy during retrograde flow?

Back 302

1) Nucleation-you being to see the speckles.
2) Elongation/actin polymerization.
3) De-polymerization.

Front 303

Lecture 4. In listeria and cell movement, how can one tell where the actin monomers are first incorporated in the actin filament?

Back 303

Usea small amount of actin monomers that are fluorescent and see where they are incorporated first into the filament.

Front 304

Lecture 4. Outline the steps researchers use to reconstitute Listeria motility in cells.

Back 304

They developed a cell free system so that they can see what's SUFFICIENT for Listeria movement. The cells are homogenized and centrifuged to remove the large insoluble proteins from the cytoplasmic extracts. Then, Listeria are introduced. Cytoplasmic extracts are open systems and can be more easily manipulated and proteins necesary for movement can be purifed.

Front 305

Lecture 4. Using the reconstitution experiment with Listeria, what are the necessary proteins needed for cell movement?

Back 305

1) Acta-WASP equivalent.
2) CapZ.
3) ADF/Cofilin.
4) Profilin.
5) Arp2/3 complex.
6) Actin, ATP, salts.

Front 306

Lecture 4. In terms of actin polymerization dynamics, how are the effector actin binding proteins which control actin polymerization dynamics regulated ?

Back 306

By GTPases (G protein switches) called Ran. They are activated (GTP form) by GEFs and inactivated (GDPform) by GAPs. The GTP bound form of Ran can activate these effector proteins downstream.

Front 307

Lecture 4. Are there only one kind of Rhos, Racs, and Cdc42s in mammalian cells? What do GDIs do for the GTPases?

Back 307

No, there are multiple forms of each. GDIs stabilize the inactive GDP bound Ran.

Front 308

Lecture 4. How were the functions of Rac, Rho, and Cdc42 determined? What does each mutatnt form in excess in the cell?

Back 308

Using mutatants that can't hydrolyze their GTP into GDP.

-Rho-GTP forms excess stress fibers.
-Rac-GTP forms excess lamellipodia and ruffles .
-Cdc42-GTP causes the formation of excess filapodia.

Front 309

Lecture 4. What does Cdc42 activate, and how does it do it?

Back 309

Cdc42 activate WASP and it does so by Binding to the GBD region of WASP, which exposes its WCA region (previously bound to GBD region, thus WASP is autoinhibited in inactivated state). The WCA region is now able to bind to the Arp2/3 complex and nucleate new actin filaments.

Front 310

Lecture 5. Describe how photo-activatable actin microscopy works, and observations made when it used on a moving cell.

Back 310

You micrinject a population of actin that is photo-activatable, and shine UV light on just one region to activate the actin in that region. You later watch that part of fluorescent actin with fluorescent microscopy over time and see that the stripe of activated actin moves backwards with relation to the cell's leading edge. The strip also becomes fainter as actin monomers that are activated by the UV strip of light depolymerizes.

Front 311

Lecture 5. The strip of UV activated actin seems to move backwards with respect to the leading edge of the cell but is ______ with respect to the substratum with which the actin monomers sit on. Would this be true if the cell did not have polarized actin and the leading edge did move outwards?

Back 311

Stationary. No. If the leading edge doesn't move even with retrograde motion going on, the actin monomers that are activated by the UV light would be pushed back and the the strip of fluorescence would never become more faint.

Front 312

Lecture 5. In actin-based motility, it's either the activated actin region that moves from the original substatum area or _______.

Back 312

The cell doesn't move from the originals substratum but the leading edge moves.

Front 313

Lecture 7. What are two different ways with which stres fiber formation would be brought about in a cell?

Back 313

GTP-Rho can activate two mechanisms:

1) It activates myosin-based contractility which leads to integrin clustering and stress fiber assembly.
2) Rho also activates formins, which also mediates stress fiber assembly by nucleating actin filaments.
This activation mechanism of formins is similar to how cdc42 activates WASP.

Front 314

Other than simply connecting the actin in the intracellular domains of adherin junctions, what else do beta and alpha catenins do?

Back 314

They serve to transmit signals to the inside of the cell.

Front 315

Lecture 7. How does actin stres fibers form in the pathway of activated Rho activating myosin?

Back 315

The activated myosins can form bipolar myosin bundles which can interact and form S.F.s.

Front 316

Lecture 7. Adherin junctions are where the ________ connects to the cadherins.

Back 316

Actin cytoskeleton.

Front 317

Lecture 7. Blebbistatin blocks the activity of what?

Back 317

Myosin II.

Front 318

Lecture 8. IF monomer proteins have what regions that are conserved and what regions that are not conserved?

Back 318

The coiled coil region is highly conserved among IFs, and the non-helical heads and tails are not conserved.

Front 319

Lecture 8. What does an IF tetramer look like? Protofilament? Protofibril? IFs final organization?

Back 319

The IF tetramer, which is the basic monomer, is composed of 2 dimers arranged in antiparalle fashion, forming no overall polarity. The protofilament is made of multiple tetramers formed from end to end. The protofibril is made of 2 protofilaments stuck together laterally, and the IFs are made of 2 protofibrils stuck laterally.

Front 320

Lecture 8. Are IF monomers abundant in the cell compared to actin monomers?

Back 320

IF tetramer populations are considered very small relative to the polymer form.

Front 321

Lecture 8. What were later confirmed to be a major structural component of the bud that exists between the mother and daughter yeast cells at the time of division?

Back 321

Septins.

Front 322

Lecture 8. What do the individual septin genes: cdc1, cdc3, cdc10, and cdc12 code for?

Back 322

Individual septin subunits which are needed for polymerization to form septin polymers.

Front 323

Lecture 8. What are the structures that mediate the connections between the IF and the ECM in hemidesmosomes, what about in desmosomes?

Back 323

In hemidesmosomes, you have integrins that bind to the ECM and connects with placque proteins, which connects with the IFs.

In desmosomes, you have cadherins, which bind to placque proteins, which bind to IFs.

Front 324

Lecture 8. Are desmins IFs?

Back 324

Yes.

Front 325

Lecture 8. What is one time that IFsmust all depolymerize?

Back 325

Mitosis.

Front 326

Lecture 8. Septin subunits exist as monomers or dimers and what are the four categories that make up the cytoskeleton?

Back 326

They are dimers.

1) MTs.
2) Microfilaments.
3) IFs.
4) Septins.

Front 327

Lecture 9. What are the two events that must occur for MTs polymers to form from scratch?

Back 327

1) Monomer concentration has to be above Cc.
2) Has to be at 37 degrees C.

Front 328

Lecture 9. Can speckle microscopy be done in MTs? Can you see dynamic instability of MTs in vitro AND in vivo?

Back 328

Yes. Yes.

Front 329

Lecture 10. What are the three types of MT binding proteins?

Back 329

1) Proteins that affects dynamics.
2) Proteins that organize it.
3) Motor proteins that can move MTs.

Front 330

Lecture 4. What are two examples of actin based motility discussed in class?

Back 330

1) Listeria movement.
2) Cell movement via retrograde motion.

Front 331

Lecture 10. The classification of different types of kinesins are based on what area on the different types of kinesin?

Back 331

The positioning of the motor domain within the kinesins.

Front 332

Lecture 10. What rae the two types of dyneins?

Back 332

1) Type that function in flagella.
2) Cytoplasmic dynein.

Front 333

Lecture 10. How can one test the affects of MT motor proteins for its functions in organizing the structure and location of organelles such as the ER and Golgi?

Back 333

1) Inhibit dynein.
2) Add drugs to get rid of MTs.
3) Interfere with MT polymerization.

Front 334

Lecture 10. What are kinesine-1s used for? Would kinesin motility assays be the same for ALL kinesins?

Back 334

Kinesin-1s are used for the antergrade transport of cargoes in neurons from the cell body towards the axon's end. No, fluorescent MTs would move in different directions depending on what kinesin was used.

Front 335

Lecture 10. How are the Golgi and ER positioned on a cell's MTs?

Back 335

The ER is formed on a network of tubules that are organized along MTs and branch throughout the cytoplasm.

The Golgi is positioned near the centrosome

Front 336

Lecture 10. What part of MT motors allows the spindles to form in mitosis?

Back 336

The fact that MT can transport themselves and allows for cross linking and sliding motions that allows for the creation of axons,and spindles.

Front 337

Lecture 11. Is it possible to transport cargo on both MTs and actin? How?

Back 337

In actin, you have the different classes of myosins that bind and move along actin to move cargoes.

In MTs, you have kinesins that can bind and move towards both ends of an MT and transport cargo.

Front 338

Lecture 11. If a chromosome lacks a centrosome, what does it automatically not have? What kind of protein is the Cenp-E on the kinetochore? What is this protein balanced by? Why is this necesary?

Back 338

Kinetochores. The Cenp-E is a plus end driven motor protein that makes up partially rhe kinetochore. It is AKA kinesin 7. This protein is balanced by dyneins, which are minus end oriented. This allows the chromosome to move back and forth after attaching to MTs from the spindle.

Front 339

Lecture 12. What kind of MTs in the spindle do kinesin 10s attach to?

Back 339

The polar MTs.

Front 340

Lecture 12. Is the same type of treadmilling behavior seen in polar MTs during metaphase? What is the strict definition of poleward flux in MT dynamics seen mitosis?

Back 340

Yes, they exhibit this property along with kinetochore MTs. The treadmilling behavior in which MTs are not getting longer or shorter during metaphase is called polward flux

Front 341

Lecture 11. After chromosomes are attached to spindle MTs, at around prometaphase, how do they move back and forth to align themselves?

Back 341

You need Kinesin 10 that pull on the chromosome arms.

-You have kinesin 13s to help depolymerize the MTs at the plus end.

-You also have kinesin-7s to help the chromosome move towards the plus end of the MT and dyneins to balance because they help chromosomes move to the minus end of MTs.
Polymerization at the plus end happens on its own.

Front 342

Lecture 12. What places to you most commonly find dynein?

Back 342

1) Flagella.
2) Cytoplasm.
3) Cell cortex.
4) Chromosomes.

Front 343

Lecture 12. How do you get MTs attached to kinetochores and still be able to polymerize and/or depolymerize at the plus end?

Back 343

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Front 344

Lecture 5. What happens to cadherin junctions when one deprives them of Ca++?

Back 344

The cadherin/adherin junctions can no long work.

Front 345

Lecture 5. In cell to cell junctions involving IFs, you have placque proteins that link to IFs (desmosomes). What about in adherin junctions? What about in focal adhesions?

Back 345

In adherin junctions, you have cadherins, which bind to beta catenin, which binds to alpha catenin, which binds to the actin cytoskeleton.

-In focal adhesions, you have integrins, which are bound to the substratum and accessory proteins in the intracellular emnvironment, which are bound to the actin stress fibers.

Front 346

Lecture 11. Kinesin-10s are plus or minus oriented motors that pushe the chromosomes to where?

Back 346

Kinesin-10s are plus end directed motors that push the chromosomes away from the poles.

Front 347

Lecture 12. Can Xenopus eggs have dyneins pull the spindles apart?

Back 347

No, because these cell have to asters (because they have no centrosomes) for the dyneins to act on.

Front 348

Lecture 12. What is thought to move the centrosome apart during mitosis?

Back 348

The Dyneins at the plus ends of astral MTs mostly, along with other motor proteins. Their movement to the minus ends pulls the centrosomes apart. Actin-myosin bundles in the cell cortex also contirbute in pulling the centrosomes apart later on in mitosis.

Front 349

Lecture 12. Evem in cell that have centrosomes, can the chromosomes have a role in spindle assembly and organization?

Back 349

Yes.

Front 350

Lecture 12. What is meant by kinesin-5's ability to sort MTs?

Back 350

They arrange them in an antiparallel manner.

Front 351

Lecture 13. How do the kinetochores sense that the MTs attachments from the spindle are correct?

Back 351

They sense for equal amounts of tension from the MTs that are attached at both kinetochores. Detachment occurs if it is unstable and attachment, with other MTs attaching at same site occurs it stable.

Front 352

Lecture 4. Describe how photoactivatable fluorescent miscroscopy works.

Back 352

Photoactivatable fluorescent monomers are added and after polymers are formed, a flash of UV light (a line) is used to activate and light up a segment of the polymers. You later observe that line of photoactivated part of the polymer.

Front 353

Lecture 12. Dyneins typically need what (binds to what) in addition to itself to move cargo?

Back 353

Dynactin complexes.