Lecture #20 Microfilaments

Intermediate filaments, microtubules, microfilaments

3 major elements of cytoskeleton

Actin

What are microfilaments made of ?

Intermediate filaments, microtubules, microfilaments

3 major elements of cytoskeleton

Actin

What are microfilaments made of ?

G actin

Nonpolymerized actin. Usually an atpase, "free actin", atp binding cleft

F actin

actin in a microfilament, double helix with defined polarity

+, -

Actin grows from the ___ end and disassembles from the ___ end

+

Barbed end of actin

-

Pointed end of actin

critical concentration

what determines if the growth of actin occurs, rate of assembly=disassembly

grow

When [actin] is above CC we ____ filament

disassemble

When [actin] is below CC we _____ the filament

+, -

Cell free actin concentration will be below the concentration of the _ and _ ends

profilin

_____binds to ADP-G-actin opposite the nucleotide-binding cleft, opening the cleft and catalyzing the exchange of ADP for ATP. ____ 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-____ complex assembly on the (+) end dissociates ____ to interact with another ADP-G-actin.

Cofilin

fragments ADP-actin filament 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

gelsolin

•severs actin filaments by inserting itself between actin subunits of the helix - blocks the new (+) end. Some ___family members are activated by a rise in Ca2+ concentration.

CapZ

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

CapZ, gelsolin

Two + end capping proteins

Tropomodulin

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

tropomodulin

- end capping protein

formins, Arp 2/3

•Functionally different actin-based structures are nucleated by ____ and ____complexes.

Arp 2/3 dependent actin polymerization

-moves pathogenic bacteria and endocytic vesicles within cells

pushes the leading edge membrane forward in moving cells

Formin

fh2 dimer grabs 2 actins to nucleate a new microfilament. Rocks back and forth for another atp actin, binds to + end, stops capping

Rho GTPase

What activates formin to allow it to nucleate actin into long microfilament chains

Formins

assemble long actin filaments in muscle cells, stress fibers, filopodia, and the contractile ring that forms during cytokinesis.

Arp 2/3

The ____complex nucleates branched filament assembly.

coincidence detection

A ____mechanism (two input signals) activates WASp 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).

Microfilaments

participate in both endocytosis and phagocytosis.

Opsonization

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

Myosins

What is the actin based motor protein

single head

What makes the myosin I molecule unique

Myosin I

•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 associate directly with membranes through tail-lipid interactions.

Myosin II

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

•Heavy chain long helical tail homodimerizes through a coiled-coli interaction

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

Myosin V

•Two head domains and six light chains per neck.

•Heavy chain helical tail homodimerizes through a coiled-coli interaction

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

myosin VI

•Only _____ is a (-) end-directed motor.

+

All three classes of myosins except for VI move toward wha end ____

Powerstroke

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

greater

Longer neck domain of a myosin means ____velocity of a power stroke

ATP hydrolysis

What drives sliding filament sarcomere contraction

thin

-Skeletal muscle contraction - ___ filament Ca2+ regulation

thick

-Smooth/nonmuscle cell contraction - ___filament Ca2+ regulation

Myofibrils, sarcomeres

•Skeletal muscle fibers: huge multinucleated cells composed mostly of ____: 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 ____

myosin

Thick filament of a sarcomere

actin

Thin filament of a sarcomere

sarcomere, thick filaments, thin filaments

•Each ____ contains ---- of myosin II (blue ) in its center and parallel arrays of ___of actin (red) extending in from either end of the sarcomere (the Z discs, purple).

CapZ

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

Nebulin

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

Titin

•long elastic molecules

•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.

sarcoplasmic reticulum, voltage gated ion channels

•Neuronal signaling stimulates release of Ca2+ from the _______ via ____

Troponin, tropomysoin

The switch in muscle cell contraction is composed of two proteins: ____ and _____

Tropomysoin

binds the groove of actin filaments and prevents myosin heads from interacting with the filament. A protein complex that includes a Ca2+ sensitive protein associated with tropomyosin

Rho GTPase

•family proteins regulate formation of different actin filament organizations and myosin II activity to direct cell motility.

Rho family g-proteins

•Specific extracellular signals, such as growth factors and lysophosphatidic acid (LPA), activate cell-surface receptors that activate the ______ Rac, Rho, and Cdc42.

Z line

Where is actin anchored in sarcomere

M line

Where is myosin anchored in sarcomere

fh2

Formins are associated with what domain

proline

FH1 domain is _____ rich

arp 2/3

Branched microfilament assembly is regulated by