Lecture 15. Microfilaments, Muscles and Myosins

Question 1

What are the microvilli in the gut epithelial cells made of?

Answer 1

Actin fillaments

Question 2

Where are microfilaments found in fibroblast cells?

Answer 2

Fibroblasts need to move towards source of wound to fill in gap, microtubules found on the leading edge

Question 3

Do microfilaments have to be nucleated/emerge from the centrosome?

Answer 3

No, microvilli microfilaments emerge from the apical membrane

Question 4

What are the three different structures that are made up of actin in epithelial cells?

Answer 4

Microvilli: extends the plasma membrane area
Cell cortex: branched network around inside of cell - processes include endocytosis
Adherens belt: contractile belt that uses myosin motor proteins in order to respond to force from adjacent cells
All nucleated from different places

Question 5

What are the four different structures that are made up of actin in migrating cells?

Answer 5

Cell cortex: branched network around inside of cell - processes include endocytosis
Filopodia, lamellipodium/leading edge and stress fibres all act coordinately to move in a specific direction

Question 6

In a migrating cell, where are the stress fibres located?

Answer 6

Ends of stress fibres buried in the focal adhesion site which contracts to allow the back end of the cell to follow the forward end of the cell

Question 7

What happens to actin in cells when the cell undergoes cell division?

Answer 7

Cell focuses on the process of cytokinesis and forming a contractile, psychokinetic active myosin ring
This constricts the two cells apart after nuclear division to enable cellular division and absolution in order to create another epithelial or fibroblast cell

Question 8

What is actin?

Answer 8

A globular protein (G-actin) which is divided by a central cleft that binds ATP. Actin filament appears as two strands of subunits. One repeating unit consists of 28 subunits (14 in each strand) covering a distance of 72nm in length. The filament has a clockwise helical twist such that the filament has symmetry every 36nm. The ATP binding cleft always binds to the opposite side of the adjoining actin molecule. This gives the filament polarity with the actin binding cleft exposed at the minus end. Filamentous actin is called F-actin.

Question 9

How does actin assembly occur?

Answer 9

If given ATP, ATP-G actin will start binding to both ends, preferentially at the plus end where addition is 10 times faster. The dissociation rates from both ends however are similar.
In the filament ATP hydrolyses to ADP-Pi and Pi is released slowly giving rise to a filament containing ATP-actin, ADP-Pi-actin and ADP-actin. ATP-actin is added preferentially at the plus end while ADP-actin disassembles at the minus end, giving rise to treadmilling of subunits.

Question 10

What is the interaction between actin and cytochalasin D and latrunculin A?

Answer 10

Binds actin monomers and prevents actin polymerisation

Question 11

What is the interaction between actin and phalloidin?

Answer 11

Binds and stabilises actin filaments. This can be labeled with a fluorescent dye for staining actin filaments in cells. Stabilises F-actin

Question 12

What is the role of thymosin-β4 (monomer binding protein)?

Answer 12

In cells G-actin is 1000 times more concentrated than the critical concentration for actin filament formation. However most G-actin is bound to thymosin-β4 and this cannot be incorporated into filaments. Raising thymosin-β4 levels by micro-injection inhibits actin filament assembly by sequestering actin from dissociation filaments

Question 13

What is the role of profilin (monomer binding protein)?

Answer 13

Profilin binds G-actin more weakly than thymosin-β4, but stronger than actin + ends. Uniquely it allows ADP/ATP exchange and can promote ATP replacement and promotes actin incorporation into filaments. Profilin is mostly at the plasma membrane bound to PIP₂

Question 14

What is the role of cofilin (filament severing proteins)?

Answer 14

Cofilin (also known as Actin destabilising factor or ADF) binds to the sides of ADP-actin in the filament, inducing them to fragment. In this manner cofilin replenishes the pool of free ADP- actin which can be recharged by profilin to be used again.
Cofilin chops actin into 14 sub-filaments

Question 15

How do you end up with either straight or branched actin filaments?

Answer 15

Formin makes straight filaments of actin
Arp2/Arp3 complex makes branched actin filaments

Question 16

How does formin induce straight actin filaments?

Answer 16

Formin only works in the cytoplasm when Rho bound to plasma membrane
Formin is a multi -domain protein containing Rho GTPase binding domain (RBD), and profilin- ATP-actin binding domain (formin homology domain 1, FH1) and a filament nucleating domain (FH2). When not bound to Rho the RBD binds and inhibits the FH2 domain. When Rho GTPase becomes activated formin is recruited to the plasmamembrane. This causes a conformational shift which releases the FH1 and FH2 domains to trigger straight actin filament formation

Question 17

How does Arp2/Arp3 complex induce branching of actin filaments?

Answer 17

The angle at which branch filaments are nucleated is fixed at 70º. The Arp2/Arp3 complex is frequently located near the cell membrane and can be activated by proteins such as WASp and WAVE

Question 18

How does WAsp trigger the release of Arp2/Arp3 complex to trigger branch actin filaments formation?

Answer 18

WASp is a multi-domain protein containing Rho GTPase binding domain (RBD), an actin binding domain and an Arp2/Arp3 complex binding domain. When not bound to Rho, the RBD prevents binding to the Arp2/Arp3 complex. When Cdc42 GTPase becomes activated (for instance in respond to a chemotactic signal) WASp is recruited to the plasma membrane. This causes a conformational shift which releases the Arp2/Arp3 binding domain to trigger branch actin filament formation.

Question 19

How can straight actin filaments be bundled?

Answer 19

Tightly-packed bundles of straight actin filaments (microvilli, filopodia) or loosely-packed (adherens belt, stress fibres, contractile ring)

Question 20

What molecule is required to form loosely-packed straight actin filaments and how does it do this?

Answer 20

α-actinin forms contractile bundle (loose packing allows myosin-II to enter bundle)
α-actinin form dimers which bundle either parallel or anti-parallel filaments that are loosely packed. This allows Myosin II to get to the actin filament in tress fibres (need for chemotaxis) and the contractile actomyosin ring (needed for cytokinesis)

Question 21

What molecule is required to form tightly-packed straight actin filaments and how does it do this?

Answer 21

Fimbrin forms parallel bundle (tight packing prevents myosin-II from entering bundle)
Fimbrin only bundles filaments of the same polarity and packs them more tightly so Myosin II is excluded. This is observed in structures such as microvilli

Question 22

In single cell organisms, what types of myosin exist?

Answer 22

Myosin I, II and V and utterly essential

Question 23

What determines what myosin binds to and therefore what it does?

Answer 23

The nature of the myosin tail

Question 24

What is the role of myosin I?

Answer 24

Myosin I binds membranes and is involved in endocytosis (step size of 10-14nm)

Question 25

What is the role of myosin II?

Answer 25

Myosin II forms dimers that associate to each other in a bidirectionally symmetrical configuration to give rise to a Myosin II bouquet (step size of 5-10nm)

Question 26

What is the role of myosin V?

Answer 26

Myosin V dimerises but does not form large complexes - but binds via adaptor molecules to vesicles to transport then to the plus ends of actin filaments (step size of 36nm)

Question 27

How are myosin 2 filaments formed?

Answer 27

Formed from bi-symmetrical bouquets of Myosin II dimers. Each dimer is tied together by a coiled-coil tail region which can mostly be removed by Chymotrypsin cleavage but the heads remain together. Further protease digestion with Papain removes the remainder of the tail (called the S2 region) leaving monomeric S1 heads which have the motor activity and both essential and regulatory light chain binding sites

Question 28

How can the polarity of actin filaments be observed?

Answer 28

The polarity of actin filaments can be shown in electron micrographs following saturation binding of the actin filaments in vitro with purified monomeric myosin S1 heads
This gives actin filaments a repeated arrowhead appearance with the arrow pointing to the minus end of the filament.

Question 29

What is the role of tropomyosin on the muscle actin?

Answer 29

The binding site of tropomyosin changes on muscle actin to inhibit myosin binding when muscle is inactive and is moved out of the way to allow myosin to bind when muscle is active

Question 30

How can myosin motility be studied?

Answer 30

By binding myosin to a coverslip, then watching fluorescent actin filaments moving over it. This assay and structural studies have been useful in delineating the molecular mechanism of myosin action

Question 31

How do myosin motors move?

Answer 31

Each head uses one molecule of ATP to move
Actin filament pushed forward by the dissociation of Pi
The dissociation of Pi also induces the “power stroke” for myosin action. The release of Pi causes small conformational changes in the head (motor) domain

Question 32

What are sarcomeres?

Answer 32

The base unit of muscle action that makes up the regular repeating array in myofibrils
Each sarcomere is defined as the region between the Z lines and has one dark band and one half of a light band on either side

Question 33

How are the actin molecules in the sarcomere fixed (not growing or shrinking)?

Answer 33

The plus ends of the actin filaments are capped by CapZ which allows them to be buried in the Z disk
The minus ends of the actin filaments are capped by tropomodulin
Nebulin is a large molecule that has repeated actin binding sites and is thought to determine the length of the actin filaments

Question 34

How are muscle cells formed?

Answer 34

Muscle cells are syncytial (multi- nucleated) up to ~50cm in length formed by the fusion of myoblasts. Muscle cells contain a number of myofibrils each of which consists of a regular repeating array of sarcomeres

Question 35

What makes up the dark and light bands of the sarcomere?

Answer 35

The dark band are composed of myosin II filaments
The light band is composed of actin filaments
The darkest region is the region of overlap containing both myosin filaments and actin filaments arranged in a regular pattern

Question 36

What happens to the light bands of the sarcomere when the muscle contracts?

Answer 36

The light band disappears as the region of overlap between the myosin and actin filaments increases

Question 37

How do nerve impulses trigger muscle contraction?

Answer 37

By causing depolarisation of the plasma membrane (sarcolemma)
Key to this are Transverse tubules (T-tubules) which are invaginations of the plasma membrane which lie adjacent to the outer face of the sarcoplasmic reticulum (SR) around each myofibril

Question 38

How does a nerve impulse’s action potential cause myosin to bind to actin?

Answer 38

An action potential causes opening of a voltage gated Ca²⁺ channel which release a small burst of Ca²⁺ into the cytosol. This Ca²⁺ binds to a channel in the sarcoplasmic reticulum (SR) which triggers massive and explosive release of Ca²⁺ into the cytosol. The increase in calcium concentration causes myosin to bind to actin

Question 39

What does the elevated Ca²⁺ concentration caused by the action potential result in?

Answer 39

A conformational change in two accessory proteins, tropomyosin (TM) and the troponin (TN) complex
TM normally covers up the binding site of myosin on the actin filament, but exposes it in the presence of Ca²⁺.
TP complex is made up of TH-T, TH-I and TN-C. TN-C is the calcium binding subunit of the complex. Association of Ca²⁺ to TN-C causes a conformational change which ultimately results in a conformational change in TM

Question 40

How can the step size, processivity and force generation by myosin motors be measured?

Answer 40

By laser based optical tweezers (also known as optical trap)
Myosin is bound at low density to an immobilised bead. An actin filament, which is held by two laser based light sources, is lowered towards the bead. When myosin touches the actin filament and ATP is added the ATPase cycle is stimulated and myosin moves the actin filament. The distance and force by which it does this can be calculated via the computer running the microscope

Question 41

What is the difference between the duty ratios of myosin II and V?

Answer 41

Myosin II only spends 10% of its time attached to an actin filament (Duty ratio = 10%) so one or more of the myosin heads has to be bound otherwise muscles wouldn’t be able to work
The duty ratio of Myosin V is 70% so one of its two heads are always bound to the actin filament

Question 42

How dies myosin V move?

Answer 42

Myosin V moves by taking hand-over- hand 72nm steps
Myosin V walks down only one side of the actin filament

Question 43

Why do some vesicles bind to myosin V?

Answer 43

For short range transport on actin filaments towards the plus ends of actin filaments at the plasma membrane