PDCB Module 3

Where is actin localized?

- in the microvilli on the apical end of the epithelial cell

- smooth muscle that surrounds the intestine

What is the structure and function of the epithelial cells that line the intestine?

structure: polarized morphology & junctions that form a tight single cell layer supported by the cytoskeleton

function: nutrients are imported from the intestinal lumen, provides support via bundles of actin filaments

What is the function of actin filaments for white blood cells?

- leading edge is oriented in direction of locomotion

- used at the leading edge for chemotaxis and phagocytosis to pursue, capture, and destroy infectious agents

What is the significance of the cytoskeleton?

cell shape, internal organization, polarity

What are the three types of filaments?

microfilaments (actin), microtubules (tubulin), intermediate filaments

How is the cytoskeleton regulated by cell signaling?

cell surface receptors transmit external signals from the extracellular matrix to activate specific cytosolic signaling pathways that regulate cytoskeleton organization and function

How does G-actin reversibly assemble into polarized F-actin filaments?

two protofilaments (actin subunits are oriented in the same direction) are wound around each other to form a helix with the actin nucleotide-binding site exposed on the minus end of each protofilament

What is actin?

Thin filament protein. Twisted into a double helix and appears like a double-stranded chain of pearls. Contains the myosin-binding site.

- comprises up to 10% of the total protein in muscle cells

G-actin

a globular subunit of F actin with an active site for binding a myosin head

- actin monomer structure is divided by a central cleft into two approximately equal-sized lobes and four subdomains

- ATP/ADP binds at the bottom of the cleft and contacts both lobes

F-actins

2 helical strands that direct local traffic, accumulate in axon terminals and spines

- the ATP binding cleft of each actin subunit is oriented toward the same end of the filament. The filament end with an exposed binding cleft is the (-) end; the opposite end is the (+) end.

How does F-actin have structural and functional polarity?

all actin subunits are assembled in the same orientation, making the filament ends distinguishable from each other

What is the arrowhead decoration experiment?

Myosin S1 head domains, proteolyzed from intact myosin, bind to all actin subunits in a filament at the same angle around the filament

Coating of myosin heads produces a series of arrowhead-like decorations

Polarity in decoration defines an arrowhead pointed end (the less preferred end for polymerization; [-]) and a barbed end (the preferred end for polymerization; [+] end).

What is treadmilling?

ATP-actin is added at the (+) end, ATP is hydrolyzed to ADP and P1; P1 is lost slowly, and ADP-actin dissociates from the (-) end.

What are the 3 phases of G-actin polymerization?

- Nucleation phase

- Elongation phase

- Steady state

(addition of short-actin filaments bypass the slow nucleation phase)

What is the nucleation phase?

- lag phase during which G-actin subunits oligomerize

- inefficient formation of 3 ATP-G actin "nucleus/seed) initiates formation of a filament

What is the elongation phase?

- rapid growth via the addition of actin monomers to (+) end

- actin subunits rapidly assemble onto each end of a filament

What is steady state?

- G-actin monomers exchange with subunits at plus and minus ends, but NO NET change in total length

What is Critical concentration (Cc)?

- below, filaments cannot assemle

- above, filaments assemble and G-actin addition is balanced by disassembly of G-actin from filament ends

actin polymerization is dependent upon critical concentration

Which side do actin filaments grow faster at?

the plus end

Growth rate experiment

Short actin filaments arrowhead decorated with myosin S1 heads added to G-actin nucleate (seed) actin assembly on both ends of the filament

G-actin assembles ~10 times faster at the (+) end than at the (-) end, reflecting different ratios of on-off rates at each end

Which side do ATP-actin subunits assemble faster at?

+, lower Cc, resulting in treadmilling at a steady rate

- 10x faster at positive vs negative end

- rate of disassembly is similar at both ends

What is the process of actin treadmilling?

- ATP-actin binds to plus end

- ATP is hydrolyzed to ADP and Pi; Pi is slowly released

- At steady state, ATP-G actin subunits assemble preferentially on the (+) end, while ADP-G actin disassemble from the end (-) end

How is actin treadmilling powered by ATP hydrolysis?

- When ATP-actin binds to plus end, ATP is hydrolyzed to ADP, and Pi is slowly released. This causes a conformational change, which explains the different association/dissociation rates for the plus and minus ends

- Two actin subunits (blue) assemble on the (+) end. Over time, more actins assemble onto the (+) end while actins disassemble from the (-) end, and the blue subunits treadmill to the (-) end

How do actin binding proteins regulate filament turnover?

- cycle 1, profilin

- cycle 2, cofilin

- cycle 3, thymosin-beta4

What does profilin do?

It promotes the addition of G-actin to filaments to the plus end in concert with formins.

- catalyzes exchange of ADP for ATP

- blocks minus end assembly while promoting plus end assembly

ATP-G actin profilin cannot initiate spontaneous F-actin polymerization

What does cofilin do?

Binds to the sides of actin filaments and changes their structure, destabilizing them and causing them to break or dissociate at the minus ends, i.e. enhancing minus end disassembly

What does thyomisin-beta4 do?

Thyomisin-beta4 buffers ATP-G actin - sequesters G-actin at high concentration; releases G-actin at low concentrations to polymerize

What do actin capping proteins do?

block assembly and disassembly at filament ends to control the rates

What are plus end capping proteins?

CapZ binds with high affinity to the plus end to inhibit the addition/subtraction of ATP-G actin

- Highly concentrated in cells and can rapidly cap most + ends, thus, other plus-end proteins can bind and inhibit CapZ while still promoting plus-end polymerization

Minus end capping proteins

Tropomodulin caps the minus end, Blocks the end where filament disassembly normally occurs, thereby stabilizing the filament. This is necessary for cells in which actin filaments must remain stable, like muscle cells.

What does Arp2/3 dependent actin polymerization do?

- moves pathogenic bacteria and endocytic vesicles within cells

- pushes the leading edge membrane forward in moving cells

What are formins?

actin-nucleating proteins that promote microfilament growth containing two adjacent FH1 and FH2 domains

What do FH2 domains do?

form a dimer from two formins

Step 1: Two FH2 domains from two formin monomers associate and form a doughnut-like complex that binds two actin subunits (bypass nucleation lag phase)

Bypasses formation of a three monomer trimer seed)

Step 2: one FH2 rocks up to permit association with additional actin monomer

Allows assembly of one actin subunit onto a protofilament (+) end

Step 3: Second FH2 rocks up to permit association with additional actin monomer

Allows assembly of one actin subunit onto the other protofilament (+) end

Step 4: The cycle repeats to elongate an unbranched filament.

What are the components of the Rho-GTPase cycle?

Rho-GDP, Rho-GTP, GDI, GEF, GAP, Pi

Rho-GDP

The inactive form of Rho GTPase bound to GDP.

Rho-GTP

The active form of Rho GTPase bound to GTP, capable of interacting with downstream effectors

GDI (GDP dissociation inhibitor)

Maintains Rho-GDP in the cytosol, preventing its activation by inhibiting the exchange of GDP for GTP.

GEF (guanine nucleotide exchange factor)

Catalyzes the exchange of GDP for GTP, activating Rho GTPase.

GAP (GTPase-Activating Protein)

Accelerates the hydrolysis of GTP to GDP, inactivating Rho GTPase.

Pi (Inorganic phosphate)

released during the hydrolysis of GTP

Steps of the Rho GTPase Cycle

1. Inactive state

Rho GTPase is in its inactive GDP-bound form (Rho-GDP) and associates with GDI, which stabilizes it in the cytosol.

Steps of the Rho GTPase cycle

2. Activation by GEF

When a signal is received, GEF promotes the exchange of GDP for GTP on Rho GTPase.

This converts Rho-GDP into its active GTP-bound form (Rho-GTP).

Steps of the Rho GTPase cycle

3. Effector Interaction

Active Rho-GTP translocates to the plasma membrane and interacts with downstream effectors to regulate cellular processes like cytoskeleton dynamics, cell migration, and adhesion.

Steps of the Rho GTPase Cycle:

4. Inactivation by GAP

GAP enhances the intrinsic GTPase activity of Rho GTPase, leading to the hydrolysis of GTP into GDP and Pi.

This returns Rho GTPase to its inactive GDP-bound state.

Steps of the Rho GTPase Cycle:

5. Reassociation with GDI

GDI binds to Rho-GDP, extracting it from the membrane and maintaining it in the cytosol until reactivation.

Formins

Formin Rho-binding domain (RBD) binds Rho-GTP, a monomeric G protein, exposing the FH2 domain

FH1 domain can bind Profilin-ATP actin and increase the local concentration of actin monomers, which are fed into the neighboring FH2 domain

FH2 domain prevents (+) end capping while still allowing actin monomer binding, permitting microfilament polymerization

Formin inactive state

folds back on itself to inhibit FH2 domain activity

Formin activation state

Membrane receptor activation of Rho to the GTP-bound form (Rho-GTP)

Formin Rho-binding domain (RBD) binds Rho-GTP, exposing FH2 to dimerize and nucleate a new actin filament

FH1 domain:

the proline-rich FH1 domain recruits profilin-ATP-G-actin complexes that can assemble on the growing filament (+) end in the adjacent FH2 domain

Arp2/3 complex

A protein complex that binds actin filaments and initiates the formation of branches composed of actin-related polypeptides Arp2 and Arp3.

Inactive Arp2/3 complex

Arp2 and Arp3 are in the wrong configuration to nucleate filament assembly

Arp2/3 activation and actin filament nucleation:

Step 1: two NPFs WH2 (W) domains each bind an actin monomer

Arp2/3 activation and actin filament nucleation:

Step 2: binding of two NPF-actin complexes bind to the Arp2/3 complex (through connector and acidic domains) induces a conformational change that activates the Arp2/3 complex

Arp2/3 activation and actin filament nucleation:

Step 3: the activated Arp2/3 complex binds to the side of an existing actin filament and binds the negative ends of actin subunits transferred from the NPF W domains

Arp2/3 activation and actin filament nucleation:

Step 4: additional G-actins assemble onto the (+) end of a new actin filament, which makes a characteristic 70 degree angle with the old filament

Regulation of Arp2/3 complex by WASp and PIP2

NPFs link the actin cytoskeleton to the cell membrane

WASp is a modular protein activated at the cell membrane upon interaction with both PIP2 and Cdc42 (a rho-family G protein) - this is called coincidence detection

Regulation of the Arp2/3 complex by WASp and PI(4,5)P2

Actin nucleation by the Arp2/3 complex is finely controlled by NPF regulatory processes

Inactive WASp (NPF)

Intramolecular interaction blocks WASp WCA domain activity

Activated WASp

•The W domain binds and transfers a G-actin to an activated Arp2/3 complex.

•The acidic A domain activates Arp2/3 complex binding to the side of an existing actin filament to initiate formation of a new actin filament branch.

Arp2/3 dependent actin assembly during endocytosis

Endocytosis assemblly factprs recruit NPFs that activate Arp2/3 complexes

Clathrin-mediated endocytosis

- endocytosis assembly factors recruit NPFs that activate Arp2/3 complexes

- a rapid burst of Arp2/3-dependent actin assembly drives internalized endocytic vesicles away from the plasma membrane

Arp2/3 directed actin assembly

- implicated in other steps of the endocytic pathway & secretory pathway

- not essential for several steps, but facilitates transport between different compartments of the secretory and endocytic pathways

Leukocyte phagocytosis and degradation of a bacterium

1. Opsonization

2. Fc receptor binds the Fc region

3. Fc receptor-antibody binding signals cell to activate Arp2/3 complexes

4. fusion of lysosome with the phagosome delivers enzymes that degrade the bacterium

What do Rho GTPases (Rho, Rac, Cdc42) do?

small GTPases that orchestrate cytoskeletal rearrangements essential migration; regulate actin dynamics, myosin contractility, and other processes involved in the motility and polarity of cells

ROCK (RhO assoCiated Kinase)

Increases phosphorylation of MYPT and pMLC (phosphorylated myosin light chain), enhancing actomyosin contractility. This generates contractile forces for cell migration.

What does Cdc42 activate?

MRCK: contributes to myosin II

WASP: activates the ARp2/3 complex, promoting actin branching and polymerization

LIMK: phosphorylates and inhibits cofilin

Rac activates:

PAK: influences actin filament dynamics and regulates LIMK/cofilin

WAVE: activates the Arp2/3 complex

NOX: produces reactive oxygen species

Arpin: inhibits Arp2/3

Crosstalk

These GTPases interact with each other to balance contractility, protrusion, and polarity:

Fimbrin

Bundling protein which promote the formation of tightly-packed, non-contractile actin filament bundles

A-actinin

two binding domains on opposite ends of an antiparallel dimer cross-links actin filaments into looser bundles

Spectrin:

Two binding sites on opposite ends of a flexible tetramer cross-links actin filaments much farther apart

Filamin

Two binding sites on opposite ends of a spring-like region cross-links actin filaments into gels and networks, such as that found in the leading edge of a motile cell

Dystrophin

actin binding site on N-terminus. C-terminus binds membrane protein dystroglycan

Spectrins attach to the membrane through two adapter proteins:

Ankyrins and Band 4.1 proteins stabilize the structure

What is the structure of myosin II?

a hexameric protein composed of 2 heavy chains and 2 pairs of light chains

skeletal muscle myosin II

assembles into bipolar filaments in which the tails associate to form the shaft of the filament and the heads are exposed at both ends.

Sliding Filament Assay

allows one to measure the speed at which different myosins can move actin filaments

Myosin I

Simplest type of myosin, present in all cells; consists of a single actin-binding head and a tail that can attach to other molecules or organelles. Only single headed myosin can directly associate with cell membrane through tail-lipid interactions.

Myosin II

Type of myosin that exists as a dimer with two actin-binding heads and a coiled-coil tail; can associate to form long myosin filaments. Two heavy chains bind to two different light chains. Only class that can assemble into bipolar filaments.

Myosin V

Two head domains and six light chains per neck

Heavy chain helical tail homodimerizes through coiled-coil interaction.

End of tails interact with specific receptors (brown box) on organelles, which they transport along actin filament tracks

Cross bridge cycle

sequence of events between binding of a cross-bridge to actin, its release, and reattachment during muscle contraction

Cross bridge cycle step 1

ATP binding causes conformational change in the head actin-binding domain and head release from athe ctin filament.

Cross bridge cycle step 2

The head hydrolyzes the ATP to ADP and P, which induces a rotation in the head with respect to the neck. This "cocked state" stores the energy released by ATP hydrolysis as elastic energy, like in a stretched spring

Cross bridge cycle step 3

Myosin remains in the "cocked state" until it binds to an actin filament.

Cross bridge cycle step 4

binding to actin causes myosin to release Pi, which releases the elastic energy to drive the "power stroke". The power stroke involves a conformational change in the neck region, which moves the actin filament with respect to the end of the myosin neck domain.

Cross bridge cycle step 5

the head remains tightly bound to the actin filament until ADP is released, fresh ATP is bound by the head, and the cycle initiates.

Hand-over-hand processive model

Visualizes myosin V as a highly processive motor protein.

Why does myosin V ATPase cycle have a higher duty ratio than myosin II?

- slower rate of ADP release

Myosin II skeletal muscle contraction

Skeletal sarcomere contractile unit - actin thin filament - myosin II thick filament structure stabilized by thin and thick filament associated proteins

The sliding filament model of contraction in skeletal muscle

Calcium concentration increase stimulates ATP dependent contraction

- relaxed sarcomere = low calcium concentrauon

- contracted sarcomere - myosin heads on both heads of the myosin bipolar filaments walking toward the (+) ends of the oppositely oriented thin filaments in each half sarcomere pull the actin filaments and attached Z disks toward the center of the sarcomere, shortening the I bands and sarcomere length

How is sarcomere structure stabilized?

by capping and scaffolding proteins

actin filaments

CapZ caps the (+) end of the thin filaments at the Z disk

Tropomodulin caps the (-) end.

Nebulin binds actin subunits and determines the length of the thin filament.

Titin

One end attaches to the Z disk; the other end attaches to the M band.

Interactions with each pole of myosin bipolar filaments centers the thick filaments.

Molecular elasticity prevents catastrophic overstrength of the sarcomere.

Titin mutations cause cardiomyopathies.

resting skeletal muscle

low cytosolic Ca2+ levels. Maintained by a Ca2+ ATPase that pumps Ca2+ from the cytosol to the sarcoplasmic reticulum (Ca2+ reservoir)

contraction

Nerve impulses stimulation of a muscle cell initiates an action potential, which is transmitted throughout plasma membrane (sarcolemma) and down transverse tubules

tropomyosin

rope-like protein that binds actin and forms a continuous chain along the microfilament; coiled-coil dimer lies along the muscle thin filament in one of two positions. Each TM covers 7 actins and associates end-to-end along the entire actin filament.

Troponin

Troponin is a three-peptide protein complex that is bound to each TM. The Ca2+-binding peptide TN-C controls the TM position through TN-I and TN-T.

What do tropomyosin and troponin do in the absence of Ca2+?

block myosin binding interactions with actin

Prior to binding cargo vesicles, Myosin V exists in an inactive conformation. What happens?

Transports organelles and secretory vesicles along actin filaments nucleated by formins into the bud before cell division.

Binds the end of cytoplasmic microtubules to orient the nucleus in preparation for mitosis.

Tail binds to amino acids on motor domain, inhibiting the function

Interaction with cargo receptor proteins relieves inhibition, activating the motor protein. Receptor protein is degraded to allow cargo release.

myosin V inactive folded state

the tails of myosin V bind and inactivate the motor head domains