Biol-K 324 Lec 17: Microfilaments

Front 1

cytoskeleton

Back 1

1. microfilaments (actin)

2. microtubules (ab-tubulin dimer)

3. intermediate filaments

Front 2

microfilaments

Back 2

1. 7-9 nm

2. polymers of actin proteins organized into bundles and networks

3. involved in polarized, migratory, dividing cells

3. used in myosin/motor proteins to move cargo cells

Front 3

microtubules

Back 3

25 nm

Front 4

intermediate filaments

Back 4

10 nm

Front 5

actin

Back 5

1. basic building block of microfilaments

2. isoforms, pseudogenes, etc

3. abundantly expressed in eukaryotic cells

4. highly conserved in eukaryotes

5. monomeric, G-actin, filamentous polymer, F-actin

6. polar

7. function in cells is different than polymerization of purified actin

8. binding proteins control polymerization and functions

9. reversibly assemble into filaments that consist of two helices of actin subunits

Front 6

alpha actin

Back 6

contractile structures

Front 7

beta actin

Back 7

enriched in cell cortex

Front 8

gamma actin

Back 8

stress fibers

Front 9

F-actin

Back 9

1. has polarity

2. "+" end is barbed

3. "-" end is pointed

4. polarity is recognized by actin-binding proteins and motor molecules

5. filamentous actin

6. bound to Mg++ ion and ATP or ADP

Front 10

G-actin

Back 10

1. asymmetric molecule

2. polymer has inherent polarity

3. monomeric actin

4. bound to Mg++ ion and ATP or ADP

Front 11

S1 fragments

Back 11

1. myosin proteolytic fragments

2. bind to actin filaments

3. produce a pointed-barbed arrow appearance

(pointed = minus end; barbed = plus end)

Front 12

actin polymerization

Back 12

1. nucleation

2. elongation

3. steady state

Front 13

critical concentration

Back 13

1. concentration of free G-actin in equilibrium with filaments

Front 14

G-actin concentration below Cc

Back 14

1. no polymerization occurs

2. actin will depolymerize

3. filament shrinks

Front 15

actin filament polarity

Back 15

1. filaments grow at different rates on the different ends of the polymer

2. different on and off rates at different ends --> specific assembly dynamics

3. regulated by Cdc42

Front 16

what controls actin polymerization and function?

Back 16

various actin binding proteins

Front 17

actin filament assembly mechanism

Back 17

1. profilin binds monomeric ADP actin

2. opens up the binding cleft

3. release ADP and binding more abundant ATP

4. Cofilin cycle severs ends of filaments via binding and destabilizing filament structure

5. produces new free filament ends

6. thyomosin-beta4 sequesters charged ATP-actin

Front 18

actin filament assembly

Back 18

Profilin cycle --> "+" end --> "-" end --> Cofilin cycle --> Thymosin-beta4 cycle

Front 19

capping protein

Back 19

1. CapZ

2. bind "+" end

3. stabilization

4. blocking assembly and disassembly

Front 20

tropomodulin

Back 20

1. binds "-" ends of actin filaments

2.

Front 21

formins

Back 21

1. diverse class of actin nucleating proteins

2. contain FH1 and FH2 domains

3. regulated by Rho GTPase signaling

Front 22

Arp2/3

Back 22

1. composed of a multi-subunit complex

2. assembly can power actin movements in cell

3. regulated by binding to WCA proteins

4. control endocytosis

Front 23

nucleation

Back 23

1. Arp2/3 complex binds to WCA proteins

2. induces binding to lateral surface of existing filaments

3. activating actin filament nucleation

Front 24

WASp proteins

Back 24

1. contain WCA sequences

2. activated by a Ras like GTPase switch protein (Cdc42)

3. control endocytosis

Front 25

Cdc42

Back 25

1. Ras-like GTPase switch protein

2. activate WCA sequences

Front 26

actin toxins

Back 26

1. promote depolymerization of filaments (cytochalasin D an dlatrunculin)

2. promote assembly (jasplakinolide) or stabilize filaments (phalloidin)

Front 27

phalloidin

Back 27

toxin that stains F-actin

Front 28

jasplakinolide

Back 28

toxin that promotes assembly

Front 29

actin cross-linking proteins

Back 29

1. into bundles or meshworks in the cell

2. regulate actin organization and cell function

3. short, rigid, or flexible, depending on the type of structure

Front 30

spectrin

Back 30

1. assemble a meshwork that stabilize membrane domains and prevent injury

2. inherited anemias

Front 31

myosin motors

Back 31

1. cell movement

2. myosin converts ATP hydrolysis into mechanical work

3. myosin II has a head, neck and tail conformation

4. myosin II assembles into bipolar thick filaments

5. powered by ATP hydrolysis

Front 32

myosins

Back 32

1. superfamily includes motor proteins

2. membrane association, contractility and cargo movements

3. produce body movements by action of muscle cells

Front 33

Myosin I

Back 33

1. binds membranes

2. participating in endocytosis

Front 34

Myosin II

Back 34

1. forms bipolar filaments

2. participating in cell contraction

3. non-muscle cells produce contractility

ex: epithelial cells and dividing cells

Front 35

Myosin V

Back 35

1. binds vesicles

2. transports them on actin filaments

Front 36

myosin-actin crossbridge cycle

Back 36

1. myosin head binds ATP

2. head released from actin

3. hydrolysis of ATP to ADP + Pi

4. myosin head rotates into "cocked" state

5. myosin head binds actin filament

6. "power stroke"

7. ADP released, ATP bound

8. head released from actin

9.

Front 37

power stroke

Back 37

1. release ofP and elastic energy straightens myosin

2. moves actin filament left

Front 38

striated muscle cells

Back 38

1. multinucleated

2. large

Front 39

sarcomeres

Back 39

1. basic unit of the contractile apparatus in muscle cells

2. composed of thick and thin filaments

Front 40

striated muscle movement

Back 40

1. presence of ATP and Ca+2

2. myosin heads in the bipolar myosin filaments move actin filaments

3. sarcomere structural proteins hold structures together

4. calcium ions low in cytoplasm of resting muscle cell

5. calcium stored in high concentration in sarcoplasmic reticulum

Front 41

sarcomere structural proteins

Back 41

1. tropomodulin

2. CapZ

3. nebulin

4. titin

Front 42

muscle cells: Ca+2 release

Back 42

produces muscle contraction

Front 43

tropomyosin

Back 43

1. associated with actin filaments in contractile apparatus

2. bound by one troponin protein

Front 44

muscle relaxation

Back 44

myosin-binding site masked

Front 45

muscle contraction

Back 45

1. ATP dependent sliding of myosin thick filaments along actin thin filaments

2. shorten the sarco mere and myofibril

3. myosin-binding site exposed

Front 46

muscle cells: Ca+2 absence

Back 46

troponin blocks myosin binding to actin --> blocking muscle contraction

Front 47

Ca+ induced contraction regulation

Back 47

1. release of Ca+ produces muscle contraction

2. tropomyosin is associated with actin filaments in the contractile apparatus

3. bound by one troponin protein

4. Ca+2 troponin triggers tropomyosin movement and allows myosin to bind

5. allows muscle contraction

Front 48

calcium-calmodulin

Back 48

activates myosin light chain (MLC) kinase

Front 49

MLC kinase

Back 49

Myosin Light Chain kinase

1. activated by MLC

2. phosphorylates myosin light chains

3. changes myosin conformation from folded to unfolded ---> thick filament assembly and contraction

Front 50

actin orientation

Back 50

1. all actin subunits are oriented in the same direction

2. nucleotide-binding site exposed on the "-" end

Front 51

rate limiting step in actin assembly

Back 51

1. formation of short actin oligomer

2. can then be elongated into filaments

Front 52

G-actin concentration above Cc

Back 52

filament end will grow

Front 53

ATP-G actin

Back 53

1. adds much faster at (+) end than at (-) end

2. lower Cc at (+) end than at (-) end

Front 54

steady state

Back 54

actin subunits treadmill through a filament

Front 55

what end is ATP-actin added to a filament

Back 55

(+) end

Front 56

what end does ADP-actin dissociate?

Back 56

(-) end; via ATP hydrolysis

Front 57

Profilin

Back 57

enhances exchange of ADP for ATP on G-protein

Front 58

cofilin

Back 58

enhances the rate of loss of ADP-actin from filament (-) end

Front 59

thymosin-b4

Back 59

binds G-actin to provide reverse actin when needed

Front 60

proteins involved in nucleation

Back 60

1. formins

2. Arp2/3

Front 61

latrunculin

Back 61

1. toxin

2. bind and sequester actin monomers

Front 62

how are actin filaments organized?

Back 62

cross-linking proteins that have two F-actin binding sites

Front 63

what end attaches to membranes

Back 63

(+) ends

Front 64

thick filaments

Back 64

myosin

Front 65

thin filaments

Back 65

actin

Front 66

low levels of free calcium

Back 66

1. muscle is relaxed

2. tropomyosin blocks interaction of myosin and F-actin

Front 67

elevated levels of free calcium

Back 67

1. troponin complex associated with tropomyosin binds calcium

2. moves tropomyosin to uncover myosin-binding site on actin

Front 68

contractile bundles

Back 68

thich filament regulation

Front 69

Back 69

intermediate filaments

Front 70

Back 70

microtubules (tubulin)

Front 71

Back 71

microfilaments (actin)