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89 Cards in this Set
- Front
- Back
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What is special about actin? |
actin filament - microfilaments it has cortical function -very responsive to extracellular cues (unlike microtubules) |
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What is the main interaction pathway of Actin? |
1. signals from soluble factors from other cells, the extracellular matrix to plasma membrane with receptors 2. signal transduction pathways 3. cytoskeleton 4. organization and movement of organelles 5. cell shape, movement, and contraction |
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Where can we find Actin? |
on the cortex, on the outside, usually on the plasma membrane or right next to it actin forms a shell on the outside can form branches, netowrks and bundles => supports the plasma membrane |
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What are actin based structures? |
Epithelial cell Migrating cell Muscle and non-muscle functions |
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What are actin based structures in epithelial cells? |
Microvili Cell cortex
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What are actin based structures in migrating cell? |
Filopodia Lamellipodium, leading edge Stress fibers |
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What are actin based structures in (muscle) and non-muscle functions? |
Phagocytosis Moving endocytic vesicles Contractile ring |
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What is the structure of Actin? |
in vertebrates: isoform 4 alpha(muscle) beta and gamma -generally all the same depending on tissue |
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How is it actin produced? |
-produced as a monomer (G-actin) globular. -has ATP-binding cleft -has polarity the monomer polymerizes into F-actin (filamentous) microfilaments -double helix structure, turn is 36 nm |
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What does the polarity of actin tell us? |
(-) end is the pointed end (+) end is the barbed end |
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How do we know the polarity of actin? |
-use myosin S1 as a nucleating factor -when things bind to actin they bind with a certain orientation -take myosin S1 and add it to actin filament it forms these "arrowhead" which show the orientation of actin.. point to (-) end -when you add myosin it stabilizes it, no polymerization or depolymerizaation |
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Where does polymerizaiton of actin filaments occur? |
occurs preferentially at the (+) end |
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How does actin assembly happen? |
1. G-actin monomers 2. Nucleation 3. Elongation 4. Steady State |
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What role does the presence or absence of a nucleus (nucleating factor) play in actin assembly? |
-if you have a nucleus at t = 0, you can start elongation right away. -if not you will spend some time (lag) building one |
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What is the steady state in actin assembly? |
at or above critical concentration you get polymerization -equlibrium between adding and taking monomers.. doesnt exist in real life -what happens is that when you stop polymerizing your monomers are going to hydrolizing ATP and be in ADP form so they will not come back on, more likely that depolymerization happen in steady state rather than nothing. |
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What is the role of critical concentration in actin polymerization? |
-usually the (+) end has a lower Cc threshold -to polymerize at one end you need G-actin in ATP form concentration to be above 0.12 micromolar |
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What are the critical concentrations at of actin polymerization at both ends? |
(-) end = Cc = 0.60 micromolar (+) end = Cc= 0.12 micromolar |
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What if you are in between the two critical concentrations for (-) and (+) ends, during actin polymerization? |
get treadmilling =>> add one remove one -> get relative position movement |
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How is actin polymerization regulated? since cellular concentration of G-actin is 400 micromolnar but Cc is 0.12minormolanar |
with those concentrations we'd expect there to always be polymerization BUT! there are some proteins that regulate it |
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What are the proteins that regulate actin polymerization? |
1. Thymosin 2. Profilin 3. Cofilin |
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What is the role of Thymosin in the regulation of actin polymerization? |
-sequesters actin and provides a reservoir -binds G-actin and removes it from the pool of free monomers is such a way that G-actin is not longer in Cc -cell can regulate how much Thymosin is aroud, most of the G-actin is bound to thymosin |
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What is the role of Profilin in regulation of actin polymerization? |
Profilin promotes actin polymerization by charging G-ADP into G-ATP actin -the more profilin you have the more G-actin in the ATP form available - regulated by cell |
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What is the role of Cofilin in regulation of actin polymerization? |
in enhances depolymerization -takes actin off either end. -if a cell doesnt want polymerization it can express a lot of cofilin, doesnt matter if above or below Cc. |
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What does actin capping do? |
actin capping proteins - block assembly and disassembly |
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What actin capping protein can we find at the (+) end? |
CapZ binds to the (+) end -when bound only the (-) is active |
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What actin capping protein can we find at the (-) end? |
Tropomodulin binds at the (-) end -when bound only the (+) end is active |
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What kind of drugs allow us to study what role actin plays in cells? |
Actin-disrupting drugs Cytochalasin - depolymerizes actin filaments Phalloidin - stabilizes actin filaments, by covering it - we can make it fluorescent and see where actin is |
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How can we control the speed of polymerization and depolymerization of actin? |
Formins - assembles unbranched filaments -it's a dimer of Formin FH2 domain -stays at the + end -is regulated by Rho GTPase -> activates formin |
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What is the role of RhoGTPase in the regulation of speed and assembly of of unbranched filaments of actin? |
(+) end shrinkage and growth is regulated by RhoGTPase b/c it activates formin and formin regulates the speed of polymerization or depolymerization |
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How do you get branching of actin filaments? |
1. actin subunit 2. Arp 2/3 complex activated -is the branch point |
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How is Arp 2/3 regulated? (in branching of actin? |
- by GTPase Cdc42 -WASp (nucleation promoting factor) can also be activated by Cdc42 and that can in turn can activate arp2/3 |
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What pathogens take advantage of the arp2/3 - GTPase cdc42 and formins GTPase Rho ? |
Listeria, it has on its cell surface the ability to trigger stuff to work like RhoGTPase and Cdc42, push itself into the cell and another cell due to actin polymerization and branching |
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How does Arp 2/3 dependent actin assembly happen during endocytosis? |
- actin polymerization by itself can be used by the cell to do stuff Time (seconds) 1. endocytic site initiation - endocytosis assembly factors (t= 0) 2. invagination (t= 20-25 s) 3. release and movement into cell ( t = 30 sec) |
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In Arp 2/3 dependent actin assembly during endocytosis, what happens during endocytic site initiation? |
-endocytosis assembly factors -nucleation promoting factors (WASp) |
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In Arp 2/3 dependent actin assembly during endocytosis, what happens during invagination? |
-arp 2/3 dependent assembly of actin filaments -endocytic cargo on vesicle -actin pulls down on the membrane -polymerization is used, polymerizing at one end, can move the vesicle |
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How does actin influence Phoagocytosis? |
actin can push on a membrane (polymerization) and this movement is aimed at encompassing something |
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What are the structural functions of actin? |
actin-binding proteins & cellular structures bundles, networks and support |
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How does actin form bundles? |
with the help of Fimbrin alpha (actinin) ===> both bind actin filaments in bundles |
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Where can we find bundles made of actin + fimbrin? |
- microvilli - filopodia - focal adhesions |
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Where can we find bundles made of actin + alpha actinin ? |
-stress fibers -filopodia -muscle Z line |
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How does actin form networks? |
with the help of Spectrin Filamin |
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Where can we find networks made up of actin + spectrin? |
cell cortex |
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where can we find networks made up of actin + filamin? |
leading edge, stress fibers = like alpha(actinin)bundles filopodia = like alpha(actinin) bundles |
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what actin binding protein links membrane proteins to actin cortex in muscle? |
Dystrophin |
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What can you find dystrophin (actin-biding protein) doing? |
linking membrane proteins to actin cortex in muscle |
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Why are blood cells so strong? |
they depend on actin binding proteins to support the cell membrane so do microvili and muscles |
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How are blood cells supported? |
the actin cytoskeleton supports the plasma membrane with the help of ankryin and spectrin networks |
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How are microvili supported? |
microvili = bundled actin structures found in intestines the actin cytoskeleton supports the plasma membrane of microvili with the help of Ezrin |
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How does the actin cytoskeleton support the muscle plasma membrane? |
Dystrophin (actin+myosin cytoskeleton found in sacromere) supports the cytoskeleton. in muscle, dystrophin links actin+ myosin to the ECM. |
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What will happen if dystrophin is mutated? |
-when actin-myosin pulls (contraction of muscle), it doesnt pull on the tendon very well, so the muscle keeps damaging itself. e.g. muscular dystrophy you are disrupting the linkage to the extracellular matrix |
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What is myosin? |
-similar to kinesin -actin's motor protein |
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What is the structure of myosin II? |
-most abundant -heavy and light chains -head is an ATPase -neck binds light chain -tail binds "cargo" |
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what is the head of myosin II? |
an ATPase |
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Which part of myosin II binds cargo? |
tail |
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Where can we find the actin-biding sites on myosin? |
on the head |
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What does the head of myosin II do? |
is an ATPase binds to actin moves along actin using ATP to the + end |
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What do the light chains on myosin regulate? |
the movement at the neck involved in speed of movement |
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How do the heavy chains on myosin work? |
work in dimers |
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What are the three functions of myosin? |
membrane association, endocytosis - Myosin I contraction - Myosin II organelle transport - Myosin V |
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Is myosin a - or + ended motor protein? |
like kinesin, it is + end directed |
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What is myosin I? |
not a dimer step size 10-14nm has one heavy chain and binds to plasma membrane during endocytosis if you have actin polymerization, myosin 1 links the cytoskeleton to the plasma membrane, polymerize the + end and myosin will pull on the plasma membrane |
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What is myosin 5? |
dimer of two heavy chains step size 36 nm -tails that get cargo, and can bind to different vesicles and moves to the + end of the actin microfilament |
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What is myosin 2?
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tails of the other heavy chains will form a thick filament with other myosin IIs |
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How can we detect myosin-powered movement? |
sliding filament assay |
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what determines the rate of movement of myosin II? |
length of myosin II neck longer the neck the higher the velocity neck movements (bending) is regulated by the binding of light chains |
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What is the movement of myosin V? |
"hand over hand" put label on neck and trace it myosin V steps are 72 nm |
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what are the conformational changes in myosin movement? |
1. start in rigor state - means myosin is bound to actin and there is no ATP present 2. Binds ATP, head released from actin 3. Hydrolysis of ATP to ADP+Pi, myosin head rotates into "cocked" state, Pi is still present in the myosin head. Conformational change in the neck and head 4. myosin head binds actin filament 5. phosphate is released, head and neck conformational change so that actin molecule is pulled "Power stroke" - elastic energy, moves actin filament left. 6. myosin moves to the + end |
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What can we find in the skeletal muscle sacromere? |
myosin II filaments are the A band (doesnt change size) Z disk come closer together during contraction (actin filaments) |
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What does contraction require? |
ATP, ca2+ |
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what end does formin act on? |
it regulates polymerization and depolymerization speed at + end only |
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How is formin regulated? |
GTPase, RhoGTPase, in the same family with RAS -it activates formin |
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What ultimately regulats the growths of the + end of actin? |
activation of formin by GTPase |
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What is the molecule that causes branching of actin? |
arp 2/3 |
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what regulates Arp 2/3? |
GTPase Cdc42 activates arp 2/3 and also -Cdc42 activates WASP and that activates arp2/3 |
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why is listeria so bad?
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it causes polymerization when there shouldnt be... actin polymerizes so fast it pushes the listeria through the cell, works like Rho and cdc42 |
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What kind of work can actin do in the cell? |
endocytosis! by causing invagination it's bending b/c actin is pulling down on part of the membrane. actin polymerization is used to pulling the membrane away from the cell surface. - membrane cell shape |
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How can you move the endocytic vesicle? |
actin polymerization at one end can move it around |
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What other kind of work can actin do in the cell? |
phagocytosis, it pushes the membrane so the membrane engulfs the bacterium. actin pushing on the membrane encompasses something. |
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What are the two roles of actin polymerization in normal cellular function? |
endocytosis - actin pulling on membrane phagocytosis - actin pushing on membrane |
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What do fimbrin and alpha actinin do? |
fimbrin and and allpha actin bind microfilaments together into bundles -found in microvili projections -push on the membrane to increase surface area |
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How can actin be made in complex networks? |
spectrin and filamin can take branched actin networks and make even more complex actin systems |
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What do we use filamin for? |
actin networks |
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what do we use spectrin for? |
actin networks |
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How can actin bind to the plasma membrane? |
dystrophin, in the muscle |
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How does actin binding proteins help to support the cell membrane in red blood cells? |
-underside of the plasma membrane of the red blood cell is a network of actin networks -involves actin + spectrin (cross links actin) -actin + spectrin form the cytoskeleton, they are bound to the plasma membrane by ankryin |
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How is the actin + spectrin cytoskeleton found under the plasma membrane of red blood cells linked to the plasma membrane? |
through ankryin - it is anchoring the spectrin and actin to the plasma membrane of the red blood cell. ankryn links it to membrane proteins!!! |
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How are is the actin cytoskeleton in microvili linked to the plasma membrane (to membrane proteins specifically)? |
by using ezrin (protein) |
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Another protein linking the plasma membrane to the actin+ myosin (sacromere) cytoskeleton in muscle? |
dystrophin links cytoskeleton to tendon (ECM) (mediates the pulling that happens in contraction). -actin cytoskeleton to transmembrane protein (cell membrane) and ECM (tendon) by dystrophin |
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in muscle dystrohy what kind of problems do we see? |
mutation is dystrophin, pulling on tendon is messed up. |
