Cell Organization and Movement, Part 1: Microfilaments

Lecture 20

Actin is a

highly conserved and abundant eukaryotic cell protein

cells assemble

diverse structure of actin filaments for different functions

G-actin reversibly assembled into

polarized F-actin filaments

F-actin filaments strcutures

• compoised of two protofilaments, in which the actin subunits all oriented in the same direction

• protofilamwnts are wound around each other to form a helix with the actin nueclotide-binding site exposed on the (-) end of each protofilaent 

the cytoskeleton is responsible for

maintaining cell shape, change cell shape, cell motility, intracellular transport, location of organelles, cell division

the cytoskeleton is a network of

filamentous structures: intermediate filaments, microtubules, and microfilaments

cell reversibly and dynamically assemble each type of 

filament from specific subunits 

a single can have all

three filament systems in its cytoskeleton

each filament system has

a distint organization in cells

cell-surface receptors transmit external signals from the ECM, other cells, or soluble factors across the plasma membrane to 

activate specific cytosolic signaling pathways that regulate cytoskeleton organization and function

integration of signals from more than one type of receptor leads to a

variety of cytoskeleton organizations and activities, some of which may be localized in cells 

microfilaments (actin filaments) can be organized into a 

variety of different structures with distinctive activities within a cell/in different types of cells 

eukaryotic actin is structurally related to

bacterial MreB, which also forms filament

actins from amoebae and animal actins are identical at 80 percent of their amino acid positions despite

a billions years of evolution 

six human actin genes are expressed in

different cell types, but the proteins are 93 percent identical

actin compromises up to 10 percent of the total

protein in muscle cells and 1-5 percent in other cell types

G-actin structure

• the 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 

actin monomers polymerized into

a long, helical F-actin polymer (microfilament)

F-actin polarization

the ATP-binding cleft of every 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 

polymers are twisted threads of a

single actin monomeric protein

actin filaments assemble into a two stranded

helical structure with a rapidly growing plus end, and a minus end that does allow polymerization, but much more slowly than the minus end 

F-actin has structural and functional polarity:

all actin subunits assembled in the same orientation, and therefore, establish filament polarity making the filament ends distinguishable from each other 

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 (arrows).

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

arrowhead decoration reveals the

(+) end of actin filaments in cells associated with membranes

actin filament polymerization

• in vitro mechanism

• kinetics

• critical concentration

regulation of actin filament polymerization and stability by

G-actin and F-actin binding proteins

in vitro polymerization of G-actin monomers to from F-actin filaments can be monitored to 

determine filament formation 

G-actin polymerization into F-actin filaments - nucleation (lag) phase:

inefficient formation three ATP-G actin “nucleus/seed” (more stable than two actin associations because of the extra bonds) intiates formation of a filament 

G-actin polymerization into F-actin filaments - elongation phase:

actin subunits rapidly assemble onto each end of a filament

G-actin polymerization into F-actin filaments - steady state phase:

G-actin monomers exchange with subunits at the filament ends, but there is no net change in the total length of filaments

addition of short actin filament “nuclei/seeds” bypasses the

slow nucleation phase - elongation proceeds without any lag period

1-10% of cell protein is

actin; half polymerized; other half monomers

critical concentration (Cc) for polymerization is the concentration:

• below which filaments cannot assemble

• above which filaments assemble and G-actin from filament ends to yield a steady state mixture of a constant concentration of G-actin and filaments (mass = total actin concentration - Cc concentration)

each monomer binds a molecule of ATP that is

hydrolyzed upon polymerization

once ATP is hydrolyzed, the association between actin monomers is

reduced, therefore nucleotide hydrolysis promotes depolymerization

ATP-actin subunits assemble faster at the

(+, lower Cc) end than the (-, higher Cc) end of an actin filament, resulting in tread milling at steady state

actin filament assembly-disassembly at each end:

rate of ATP-G actin assembly is almost ten times faster at the (+) end than at the (-) end

• rate of ADP-G actin disassembly is similar at the two ends 

actin tread milling - at steady state, 

ATP-actin assembly on the (+, lower Cc) end is faster than actin ATP hydrolysis in the filament, giving rise to a filament with a short region of ATP-actin and regions of ADP-Pi-actin and ADP-actin toward the (-) end 

actin0binding proteins regulate the

rate of assembly and disassembly of actin filaments as well as the availability of G-actin for polymerization 

Profilin binds to 

to ADP–G-actin opposite the nucleotide-binding cleft, opening the cleft and catalyzing the exchange of ADP for ATP.   Profilin binding sterically blocks ATP–G-actin assembly on the filament (–) end but allows the unblocked G-actin monomer end to assemble onto the filament (+) end.  ATP–G-actin–profilin complex assembly on the (+) end dissociates profilin to interact with another ADP–G-actin

coffin fragments ADP-actin filaments regions, enhancing

overall depolymerization by making more filament (-) ends

Thymosin-B4 provides a

buffered reservoir of ATP-G-actin for polymerization; sequesters G-actin at high concentration; releases G-actin at low concentration to polymerize 

capping proteins block

assembly and disassembly at filament ends

(+) end capping proteins - CapZ

limits actin assembly and disassembly dynamics to that at the (-) end

(+) end capping proteins - Gelsolin

severs actin filaments by inserting itself between actin subunits of the helix - blocks the new (+) end

• some galsolin family members are activated by a rise in Ca2+ concentration

(-) end capping protein - tropomodulin

blocks the end where filament disassembly normally occurs, thereby stabilizing the filament 

functionally different actin-based structure are nucleated by

formins and Arp2/3 complexes

Arp2/3-dependent actin polymerization

• moves pathogenic bacteria and endocytic vesicles within cells 

• pushes the leading edge membrane forward in moving cells 

toxins affect the 

dynamics of actin polymerization 

two major classes of actin-nucleating proteins regulated by

signaling pathway nucleate actin assembly

FH2 domains from two forming form

a dimer

the Fh2 domain protects the

(+) end from being immediately capped by end capping proteins

regulation of forming activity - inactive state

formin folds back to itself to inhibit FH2 domain activity

regulation of forming activity - activation 

• membrane receptors 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 

regulation of forming activity - FH1 domain

the proline-rich Fh1 domain recruitments profiling-ATP-F-actin complexes that can assemble on the growing filament (+) end in the adjacent FH2 domain

formons assemble long actin filaments in

muscle cells, stress fibers, filopodia, and the control ring that forms during cytokinesis

actin nucleation - the Arp2/3 complex

nucleates the branched filament assembly

actin nucleation - inactive Arp2/3 complex

Arp2 and Arp3 are in the wrong conformation to nuclete filament assembly

inactive WASp[ (NPF)

intramolecular interaction block WASp WCA domain activity

A coincidence detect mechanism (two input signals) activated WCA NPF activity:

•The Basic domain (B) binds the regulatory phospholipid PI(4,5)P₂.

•The Rho-binding domain (RBD) binds an active (GTP form) membrane-bound G protein Cdc42-GTP (a member of the Rho family).

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.

microfilament participate in both

endocytosis and phagocytosis

clathrin-mediate endocytosis:

•Endocytosis assembly factors recruit NPFs that activate Arp2/3 complexes.

•A burst (seconds) of Arp2/3-dependent actin assembly drives internalized endocytic vesiclesaway from the plasma membrane.

opsonization

bacterium is coated by specific antibodies to a cell-surface protein

proteins of different lengths and flexibilities and F-actin-binding sites organize

different actin filament structures with specific functions

actin filaments are attached

laterally and end-on to membranes

defects in actin filaments organization and membrane attachments cause

human diseases

actin cross-linking proteins form

diverse F-actin structure

actin cross-linking proteins - fimbrin

two closely spaced binding domains cross-link actin filaments with the same polarity into a tight bundle that supports a microvillus 

actin cross-linking proteins - alpha-actinin

two binding domains on opposite ends of an anti-parallel dimer cross-link actin filaments in looser bundles 

actin cross-linking proteins - spectrin

two binding sites on opposite ends of a flexible tetramer cross-links actin filaments much farther apart in networks underlying the plasma membrane of red blood cells and other cells 

actin cross-linking proteins - 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

actin cross-linking proteins - dystrophin

one actin-binding site on its N-temrinal cross-links actin filaments to the membrane protein dystroglycan which stabilizes membrane structure 

a microfilament-based network underlying the plasma membrane provides

erythrocytes the tensile strength and flexibility necessary to survive fluid dynamic forces of blood flow and squeezing through narrow capillaries 

Ezrin, a member of the ERM family, activated by 

phosphorlyation links actin filaments laterally to the microvillar plasma membrane 

myosin superfamily protein structure -

common head and specific tail domains

cross bridge cycles converts ATP hydrolysis energy to

mechanical work on actin filaments

myosin class-specific step sizes and processivity spport

different functions

myosins:

• superfamily of motor protein - 20 different myosin types in eukaryotes; 40 human myosin genes

• all move along actin filaments by converting energy released by ATP hydrolysis into mechanical work 

myosin I molecules:

• one heavy chain with a head domain and a neck domain - only single-headed myosin 

• variable number of light chains associated with the neck domain 

• some associated directly with membranes through tail-lipid interactions 

myosin II molecules: 

• two heavy chains - each with a head and a neck domain that binds tow different light chains 

• heavy chains long helical tail homodimerizes through coiled-coli interaction 

• only class that can assemble into bipolar filaments through tail interactions

myosin V molecules:

• two head domains and six light chains per neck

• heavy chain helical tail homodimerizes through coiled-coli interaction

• end of tails interact with specific receptors on organelles, which they transport along actin filament tracks 

all three classes of myosin move toward the

(+) end of actin filaments - (_) end-directed motors

only myosin VI is a

(-) end-directed motor

myosin-II subfamily

dimers, two globular ATPase heads and a single coiled-coil tail domain

dimers bind one another through

tails forming bipolar myosin filaments with the heads at either end

because head domains are plus-end directed, when activated they

pull these actin filament in opposite directions, causing them to slide past one another

• contractile force of muscles

the sliding-filament assay is used to

detect myosin-powered movement

assay:

• absorb myosin molecules onto a glass coverslip in a chamber 

• add a solution of actin filaments stained with rhodamine (fluorescent)-labeled phalloidin and ATP

• myosin heads “walking” toward the (+) ends of the actin filaments cause filament motility toward their (-) ends

powerstroke mechanism predicts

myosin step size should be proportional to the length of the neck domain

myosin’s with different neck length, which bind different numbers of light chains, were made using

recombinant DNA techniques

the rates of actin filament movement by the myosin were determine with

the sliding filament assay

the longer the level arm, the

faster the myosin moved (assuming different in neck length do not change cross bridge cycle kinetics)

myosin II contraction:

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

–ATP hydrolysis drives sliding filament sarcomere contraction

–Skeletal muscle contraction – thin filament Ca²⁺ regulation

–Smooth/nonmuscle cell contraction – thick filament Ca²⁺regulation

skeletal muscle fibersL

huge multinucleate cells composed mostly of myofibrils: highly ordered arrays of myosin-II, actin and accessory proteins found in discrete contractile units called sarcomeres 

each myofibril consists of

a repeating array of sacromeres

each sarcomere has

a banding app terns that gives muscle fiber a striated appearance

sarcomere banding pattern thin filaments

actin (I and A bands)

sarcomere banding pattern thick filaments 

myosin (H and A bands)