Exam 4 Cell Biology

Front 1

Formin

Back 1

Binds two actin subunits to create nucleation site.

Rocks back and forth to enhance binding of each additional actin subunit

Remains at the growing plus end

Front 2

Elongation

Back 2

Monomers are quickly added to both ends of nucleated actin

ATP on actin monomers slowly hydrolyzes to ADP after addition of microfilament.

ADP-actin more stable

Front 3

Critical concentration

Back 3

Monomer (actin) concentration is slowly decreasing until monomeric actin and filamentous actin are at equilibrium.

[Actin-ATP]>CC growth

[Actin-ATP]

CC at (+) 120nM, CC at (-) 600nM.

Growth occus at (+) at lower concentrations of soluble G actin in the cytoplasm.

Front 4

Caps

Back 4

CapZ (+)

Tropomodulin (-)

Front 5

Profilin

Back 5

Binds G actin and promotes nucleotide exchange of ADP to ATP.

Increases the concentration of G-Actin-ATP

Front 6

Thymosin Beta4

Back 6

Transiently binds to G actin-ATP, temporarily lowering the concentration of available competent monomers.

Regulates the relationship between competent monomer concentration [G-Actin-ATP] and CC and controls elongation

Front 7

Coflin

Back 7

Binds to G-actin-ADP within the microfilament, destabilizing the minus end.

Promotes the releasse of G-Actin-ADP and becomes available for profilin.

Front 8

Fimbre and alpha-actinin

Back 8

Parallel arrays bundled together control microfilament organization

Front 9

Filamin

Back 9

Loose mesh network to control microfilament organization

Front 10

ERM or Spectrin

Back 10

Attach to plasma membrane or nuclear envelope to control microfilament organization

Front 11

Myosin Head

Back 11

Microfilament and ATP binding site

Front 12

Myosin Neck

Back 12

Myosin light chain binding site, conformation changes for motility

Light chains- Calcium binding protein that regulate myosin ATP binding activity and motility

Front 13

Myosin Tail

Back 13

organelle, membrane, or other myosin binding tail. Divergent region of heavy chain.

Heavy chain- a dimer or 2 heavy chains

Front 14

Myosin uses ATP to move along microfilaments

Back 14

1) ATP binding causes lift

2) ATP hydrolysis causes Pivot

3) Pi release causes pull

Other leg counters

3)--> 2) --> 1)

Front 15

Myosin V

Back 15

Moves organelles within cells

In budding yeast, myosin V brings organelles into new bud.

Front 16

Myosin II

Back 16

Muscle cell contraction

Cytokinesis in animal and fungal cells

Amoeboid cell movement

Front 17

Muscle Cell Contraction

Back 17

Within the muscle cell cytoplasm, myofibrils contain repeating sarcomeres. Each sarcomere contains overlapping myosin II thick filaments and actin microfilaments.

Front 18

Muscle cells: Sarcomere contraction requires Calcium and ATP

Back 18

Nerve signal reaches the muscle cell

Calcium is released from ER into the cytoplasm

Elevation of cytoplasmic Calcium

Front 19

Troponin and Tropomyosin

Back 19

In skeletal muscle they lie along actin filaments and block myosin binding sites. Calcium binds troponin, changing conformation of tropomyosin filaments, freeing myosin binding sites on actin.

Front 20

Myosin II contraction

Back 20

Uses ATP hydrolysis to contract opposing microfilament bundles. Sarcomere (+) are pulled toward center. Myosin II tails bind tails of Myosin II facing the opposite direction. Myosin II doesn't move only the opposing microfilaments!

Front 21

Returning muscle cells to relaxed state after contraction

Back 21

Calcium is pumped back into ER to end contraction.

Troponin/tropomyosin block myosin binding sites on actin. Now myosin heads cannot bind microfilaments.

Passive tension on compressed titin now expands sarcomere.

Front 22

Calcium dependent regulation of smooth cells

Back 22

No troponin and tropomyosin

Increased cytoplasmic Ca2+ activates the phosphorylation of myosin light chain.

Myosin heas then unfolds and binds the microfilament.

Contraction occurs.

Ca2+ pumped back into the ER, myosin light chains are dephosphoylated so myosin heads no longer bind microfilaments.

Passive tension on compressed titin now expands sarcomere.

Front 23

Myosin II Contractile movement causes cytokinesis in animal and yeast cells

Back 23

Nuclear envelope reassembles and assembly of contractile ring forms in telophase

Cytokinesis reformation of interphase microtubule array and contractile ring cleavage furrow.

Front 24

Myosin II Amoeboid movement driven by actin polymerization and myosin contractions

Back 24

Extension- microfilament polymerization by ARP2/3 complexes and profilin pushes cell forward.

Adhesion-Cell grabs external substrate at front

Translocation- Myosin II bundles pull the back of the cell towards the front by contracting microfilament bundles, making the cell move forward.

Front 25

ARP2/3 forms branched network of many microfilaments

Back 25

ARP2/3:

Binds nucleation promoting factor (NPF) with an Actin-ATP subunit

Complex then binds along a microfilament to create a nucleation point for a new microfilament

As more Actin-ATP subunits are added, the new microfilament (+) elongates away from ARP2/3 at 70 degree angle from the existing filament.

Front 26

Intermediate Filament

Back 26

Organize cell structure in cytoplasm and nucleoplasm.

Stabilize the plasma membrane and the nuclear envelope in animal cells.

No direct role in motility.

Ex) Keratin and lamins

Front 27

Intermediate Filament Assembly

Back 27

When subunit concentration is high enough, subunits assemble into polymers

Does not require ATP or GTP

Front 28

Intermediate Filament Disassembly

Back 28

Requires ATP or GTP

Subunits are phosphoylated to promote disassembly.

Phosphate binding unravels the filament structure.

Front 29

Microtubules

Back 29

Hollow tubes w/ walls made of tubulin protein dimers

Organize cytoplasm, position organelles.

Motors generate organelle motility during interphase

Divide chromosomes during mitosis and meiosis

Usually single tubes, but doublet and triplet too

Front 30

Microtubules assembly

Back 30

Alpha/beta tubulin dimers and GTP

Nucletion of dimers is a rate limiting step

When [dimer] exceeds CC, assembly occurs.

(-) are at the Microtubule organizing centers (MTOC) and contains Gamma tubulin Ring Complex, which creates a template from which the (+) elongates.

Front 31

Microtubule disassembly

Back 31

(+) is preferential site of BOTH assembly and dissassembly

After tubulin dimer addition, GTP on BETA-tubulin hydrolyzes. GTP + Beta --> GDP + Beta

GDP tubulin dimers curve, making them less stable and more likely to fall off.

Front 32

Microtubule dynamics governed by 2 factors

Back 32

1) Variable concentration of GTP-dimers at (+)

2) Constant rate of hydrolysis of GTP to GDP on beta tubulin after binding to microtubule

Cause rapid assembly when [dimer] is high so GTP hydrolysis does not reach the (+). Rapid disassembly when [dimer] is low and GTP hydrolysis reaches the (+).

Front 33

Kinesin 13

Back 33

Binds to individual dimers at protofilament (+) end and promotes curvature so dimers are released from end (+)

A molecular motor but ATPase activity just makes it release the dimer.

Front 34

Stathmin

Back 34

Binds two tubulin dimers at protofilament (+) end and promotes curvature

Dimers are released from end (+)

Front 35

EB1

Back 35

Binds and stabilizes actively growing (+) so the (+) continues to elongate

A reduced protein that stabilizes the (+)

Evidence that other proteins and some organelles (ER tubules) bind to EB1 and move through cell by riding the (+)

Front 36

XMAP215

Back 36

Stabilize (+) to promote elongation

Front 37

MAPs

Back 37

Stabilize microtubules by binding along sides

Front 38

Katanin

Back 38

Sever microtubules to promote instability

Front 39

TIPs

Back 39

connect to plasma membrane at (+)

Front 40

Plektin

Back 40

Connect to intermediate filaments

Front 41

MAP2 and Tau

Back 41

Cross link parallel microtbules into bundles

Front 42

Microtubule motors

Back 42

Dyneins and Kinesins

Front 43

Dynein Head (middle)

Back 43

Site of ATP hydrolysis

Front 44

Dynein Stalk (Bottom)

Back 44

Binds microtubules

Front 45

Dynein Stem (top)

Back 45

Binds Dynactin, Intermediate chains, and light chains.

Dynactin binds cargo and connects to dynein

Front 46

Dynein (dynactin) walks to the microtuble (-)

Back 46

Head rotates during ATP hydrolysis

Rotation ratchets the stalk towards the microtubule (-)

Dynactin holds cargo and also binds to microtubule ahead of dynein. Dynein pushes dynactin along towards (-)

Front 47

Kinesin Head

Back 47

Binds microtubule and ATP

Front 48

Kinesin Linker

Back 48

pivots to swing head during ATP binding linker has a secondary docking site within the head

Front 49

Kinesin Stalk

Back 49

Connects head and linker to tail

Front 50

Kinesin Tail

Back 50

Binds light chains at cargo binding site

Light chains regulate cargo binding.

Front 51

Model for Kinesin motility Initiation

Back 51

Starts with leading head with no nucleotide tightly bound to microtubule, trailing head with ADP weakly bound to microtubule

Front 52

Model for Kinesin motility step 1)

Back 52

1) Leading head binds ATP

Front 53

Model for Kinesin motility step 2)

Back 53

2) ATP binding changes the conformation of the leading head linker. Leading linker swings forward and docks within leading head. This docking swings the trailing head with ADP forward to become the new leading head and moves the rest of the kinesin and any cargo forward.

Front 54

Model for Kinesin motility step 3)

Back 54

New leading head with ADP now loosely binds to microtubule. New trailing head continues to bind ATP along with the docked linker.

Front 55

Model for Kinesin motility Step 4)

Back 55

new leading head releases ADP and tightly binds to microtubule. This promotes ATP hydrolysis on the trailing head and the release of Pi. Trailing head with ADP now weakly bound to microtubule. The trailing linker domain on the trailing head undocks from the trailing head.

Front 56

Kinesin 1

Back 56

Moves organelles (+) directed, conventional

Front 57

Kinesin 2

Back 57

Moves organelles (+) end directed but has two different heavy chains and an organelle binding subunit. (heterotrimeric)

Front 58

Kinesin 5

Back 58

4 Heavy chains and 4 Heads. Slides opposing microtubules

Front 59

Kinesin 13

Back 59

Removes tubulin dimers from microtubule ends

Front 60

Mitosis

Back 60

Interphase

Prophase

Prometaphase

Metaphase

Anaphase A

Anaphase B

Telophase

Cytokinesis

Front 61

Interphase

Back 61

Cytoskeleton and motors transport organelles and organize cell

S phase- Centrosome duplication

Chromosomes replicate into sister chromatides held together by cohesin proteins

Front 62

Cell Cycle Control

Back 62

Checkpoints in the cell cycle are controlled by cyclin dependent protein kinases

Activated kinases phosphorylate target proteins to regulate the cell

Front 63

Mitotic CDKs (Cyclin dependent protein Kinases that control mitosis)

Back 63

Mitotic Cyclins- Accumulate through translation during S and G2. They bind to cyclin dependent protein kinases (CDK).

Mitotic-CDK is kept inactive during G2 by phosphorylation by Wee 1 kinase.

Mitotic CDK is Activated when CDC25 phosphatase removes the phosphates

Active Mitotic-CDK now phosphorylates several different target proteins to start mitosis.

Front 64

Myosin II

Back 64

Inactivated to prevent premature cytokinesis

Is a target protein phosphorylated by Cyclin B dependent protein kinase in the cytoplasm

Front 65

XMAP215

Back 65

Normally inhibits Kinesin 3 during interphase. Inactivated by CDK so elevated kinesin 13 activity makes microtubules unstable and more dynamic

Is a target protein phosphorylated by Cyclin B dependent protein kinase in the cytoplasm

Front 66

Condensin

Back 66

Activated, binding DNA to promote chromosome coiling

Is a target protein phosphorylated by Cyclin B dependent protein kinase in the cytoplasm

Front 67

Nucleoporin

Back 67

Activated, causing the nuclear pore complexes to break into subunits

Is a target protein phosphorylated by Cyclin B dependent protein kinase in the cytoplasm

Front 68

Prophase

Back 68

-Chromosome condensation

-Disruption of interphase microtubules

-Spindle formation

-Most cohesin proteins between sister chromatids are degraded by kinases

-Kinetochore cohesins remain, leaving sister chromatids attached at kinetochore

-Existing interphase microtubules are disrupted (Kinesin-13)

-Overlapping microtubules are aligned (Kinesin 14)

Front 69

Prometaphase

Back 69

-Nuclear envelope breaks apart

-Chromosome captured by kinetochore microtubules

Microtubules grow and shrink from the spindle poles. If growing (+) hits the kinetochore, phosphorylated ndc80 will lossely hold onto microtubule. Microtubules from the other spindle pole must also capture the chromosome to complete alignment.

-Motor activity and microtubule dynamics complete alignment at the midzone

Front 70

Metaphase

Back 70

Chromosomes held at midzone

Anaphase promoting complex activated through securin and separase

Front 71

Anaphase A

Back 71

Chromosome separation

Starts after chromosome capture and alignment

Anaphase promoting complex adds ubiquitin to specific proteins, targeting them for destruction by proteasomes

Sister chromatids separate and move to spindle poles

Front 72

Anaphase B

Back 72

Spindle pole separation (similar to the mechanism for spindle pole separation in prophase)

Anaphase promoting complex activity

(cyclin B and cdc4)

Front 73

Telophase and Cytokinesis

Back 73

Nuclear reformation and cell division

Front 74

Motor proteins and microtubule dynamics power mitosis

Back 74

Kinesin 13 (Prophase)

Kinesin 14 (Prophase)

Kinesin 5 (Prophase)

Cytoplasmic dynein/dynactin (prophase)

Kinesin 7 (Prometaphase)

Kinesin 4 (Prometaphase)

Kinetochore dynein/dynactin (Prometaphase)

Myosin II (on microfilaments)

Front 75

Kinesin 13

Back 75

Rapidly disrupts interphase microtubules, creating a pool of tubulin dimers for rapid polymerization

Disassembles the dimers on (+)

The smaller the diameter of the microtubule the less stable

Front 76

Kinesin 14

Back 76

Non-motile tail is attached to microtubule from one centrosome as cargo

Motor head walks toward (-) of opposing microtubule from second centrosome. Makes opposing microtubules parallel and overlapping. These will be the polar microtubules in the spindle.

Motility also pulls spindle poles closer and increases overlap zone.

Front 77

Three functional classes for microtubules

Back 77

Defined by what the plus ends are doing

All minus ends are in the spindle pole

Specific motors walk on specific microtubule classes

Polar: Grows towards midzone and overlap

Astral: grow towards the cell cortex

Kinetochore: grow towards midzone but bind to chromosomes

Front 78

Kinesin 5

Back 78

A Bipolar kinesin with 4 heavy chains in the overlap zone.

Four motile heads, (+) directed, that bind two microtubules coming from opposite poles.

Remains stationary and pushes polar microtubules and the two centrosomes apart.

Overlapping polar microtubules

Front 79

Cytoplasmic Dynein/Dynactin

Back 79

Anchored near plasma membrane, it pulls on astral microtubules and centrosomes.

Front 80

Kinesin 4

Back 80

Attached to the ends of the chromosome arms. Walk chromosomes towards the (+) of polar microtubules

Prometaphase

Front 81

Kinesin 7

Back 81

Anchored in the kinetochore. Walks the chromosome away from the spindle pole towards the microtubule (+)

Prometaphase

Front 82

Kinetochore Dynein/dynactin

Back 82

Anchored to the kinetochore. Walks chromosome towards the microtubule (-) in the spindle pole

Front 83

Kinesin 13

Back 83

Anchored in the kinetochore. Removes dimers from the microtubule (+)

Front 84

Securin and Separase in Anaphase A

Back 84

Securin normally prevents Separase from attacking the Cohesin protein complex which is holding the sister chromatids togetherWhen the anaphase promoting complex targets Securin, it is destroyed and separase is now active. Separase then breaks the cohesin complex. Now there is nothing holding the sister chromatids together and they are pulled towards the spindle poles in Anaphase A

Front 85

Kinesin 13

Back 85

In Anaphase A it is active at both ends of the kinetochore microtubules, causing catastrophic shortening

In Kinetochore removes dimers from the microtubule (+)

In the spindle poles removes dimers from the microtubule (-)

Front 86

Kinesin 5

Back 86

Anaphase B:

Pushes polar microtubule and the two centrosomes apart.

Overlapping polar microtubules must elongate as it pushes them apart

Front 87

Cytoplasmic Dynein/Dynactin

Back 87

Pulls on astral microtubules and centrosomes