Actin Microfilaments

Actin Microfilaments play a role in

-cell structure

-motillity and migration

-muscle contraction

-cell division

-endo and exocytosis

-transport

-cytoplasmic streaming

-depolarization

most abundant protein that is in a nonphotosynthetic eukaryote

actin

most abundant protein on the entire planet

RuBisCo

Actin structure

A chain of globular G-Actins called F-actin

-the F stands for filamentous meaning they have been polymerized and put in a chain

-polar molecules with + and - ends

-subunits on either end are different thats what makes them + and -

the + end

the barbed end

-the fast growing end

the - end

the pointed end

-monomers are not added to this end usually

polymerization and depolymerization of actin

1.ATP is polymerized at the barbed end and depolymerized at the pointed end

2. Therefore, added on the + side, removed on the - side

G actin monomers

The site for ATP hydrolysis to ADP on actin surfaces

How does actin polymerization resemble that of microtubules?

they also occur in three phases:

1. nucleation:

-formation of a stable starting point

2. elongation

-growth of the chain

3. Steady state

-constant addition and removal to the chain but it remains constant length

at steady state, G actin concentration is

equal to the critical concentration

-the slope is linear

if G actin concentration is less than the CC

depolymerization occurs untill cc is reached

if G actin concentration is more than the CC

Polymerization occurs until the CC is reached

the rate of addition of ATP-G-Actin is

12 micromolar per second at the plus end and 1.3 micromolar at the - end

the + end is 10 times higher than the - end

if 1 micromolar of free ATP G actin is added

12 subunits will be added to the plus end, and 1.3 will be added to the minus end

critical concentration formula

rate of dissociation/rate of association

the rate of dissociation for the + end is ____ and for the - end is

1.4 per second, 0.8 per second

toxins

can bind to actin and alter its polymerization

phalloidins

-mushrooms

lock adjacent actin subunits together

cytochalasins

-fungus

-bind to the barbed end and prevent further assembly or disassembly

Latrunculin

-sea sponge

-bind and inhibits polymerization only, increasing the change of break down

-also inhibits nucleotide exchange

jasplakinolides

a marine sponge

-stabilizes actin monomers, enhancing filament nucleation and polymerization

though Phalloidin is highly lethal, how can we use it for good?

-you can use it as a stain

-when added to cells, it freezes them in time so we can study them in vitro

how can you tell if a filament has been treated with cytochalasin?

if you image it and notice that the filaments are contracting on their own

non muscle dynamic actin binding proteins for treadmilling and polymerization

positive regulator- Profilin

Negative regulator- Thymosin-B4

Actin binding protein for nucleation and branching

Arp2/3 complex and formin

actin protein for crosslinking

filamin, fimbrin, fascin

actin protein for capping

CapZ, cofilin, and gelsolin

actin protein for upstream regulation

phosphatases

treadmilling of actin filaments can be altred by _____ and ADP to _______

Profilin, change the size of the filaments

New filaments are nucleated by ____ which binds g actins and the side of the actin filaments to create new branches

ARP2/3

____Nuckeate filaments by binding G actin and using profilin

Formins

actin crosslinking proteins like ______________ influence packing and organization

filamin and fascin

capping proteins like CapZ

promote disassembly of actin filaments

what regulates the speed of treadmilling and filament formation in cells?

profilin and cofilin (and also thymosin B4)

profilin

binds to ADP-G actin opposite the nucleotide-binding cleft, opening the cleft and catalyzing the exchange of ADP for ATP, allowing the addition of the monomer to the + end of the chain

-also blocks interaction at the - end

cofilin

binds to regions where there is elevated ADP concentration, leading to large chuncks of the - end breaking off, causing depolymerization

thymosin- B4

binds to ATP G actin, preventing it from adding to the + end (a negative regulator of growth)

motor proteins

proteins that use ATP to move along filaments

myosin/ motor protein structure

two heavy chains alpha coiled around each other forming a rod/ tail region

-two heads used for ATP and actin binding activity

-light chains wrapped around the heads for regulation

myosin 1

binds membrane lipids

myosin 2

slide along the actin to drive actomyosin contractibility

Myosin V

transports cargo by walking towards the + end

Myosin VI

the only myosin that can walk towards the - end

myosin neck length is proportional to

Step size (velocity)

-distanced traveled in one step

the longer the neck

the larger the step size, the faster the myosin moves

how do the dual head myosin motors move?

we made two different hypothesis models

1. Hand over hand

2. Permanent leading head

Hand over Hand movement

the myosin heads alternate like a person walking

-it travels 72nm per cycle

permanent leading head movement

the inchworm model

-the leading head takes a step and both come back together before moving to the next position

-it travels 36nm per step

which model of movement is more likely?

hand over hand

structure of skeletal muscle

a single cell is a long multinucleated thing made up of many myofibrils containing F actin, motors, and actin binding proteins

contents of a myofibril

thousands of sarcomeres extending from one Z disk to another

a single contractile unit of a muscle cell

sarcomere

structural components of a sarcomere

-Z disks

-CapZ

-Tropomodulin

actin filaments are stabilized on their + ends by ____ and on their - ends by

CapZ, tropomodulin

Nebulin

extends from Z disk to tropomodulin

-determines the length of the filaments

titin

stretches from X fisk to the middle of the filament, and another will stretch from here to the opposite disk

-elastic, prevents stretching of the sarcomere

how do tropomyosin and troponin regulate myosin binding

by controlling the interaction between actin and myosin filaments

-tropomyosin can block myosin binding, preventing muscle contraction

calcium

binds to troponin, altering its shape so that tropomyosin cannot block myosin binding sites on actin

-the muscle will contract

cross bridge cycle

the act of myosin heads binding to actin, pulling the thin filament, detaching and rebinding

-powered by ATP

steps of the cross bridge cycle

a. Calcium binding starts the whole process by changing tropomyosin conformation and exposing myosin binding sites on the actin

1. binding of myosin to actin: the head contains a ADP and a phosphate group

2. Power stroke: the phosphate is released, conformational change causes the myosin head and attached actin to the center of the sarcomere

3.ADP is released strengthening the bond between myosin and actin called rigor. Once all the ATP is depleted, rigor mortis occurs

4.A new ATP binds to the head, causing detachment from the actin

5. ATP becomes ADP, releasing the head and restarting the process

the cycle of cross bridge can repeat as long as

ATP and Ca2+ is available

the sliding filament model of muscle contraction

in relaxed state, a sarcomere consists of thin and thick filaments and Z disks

-when a signal is heard, ATP is hydrolyzed and Ca2+ is released, the head moves forward causing it to detach and step forward

-as this occur, Z disks move closer together, causing contraction

Cellular locomotion

Movement driven by actin microfilament dynamics.

the act of forming the protrusion at the leading edge requires

actin polymerization directly under the plasma membrane

nucleation of new actin filaments at the leading edge is mediated by

Arp2/3 complexes that bind to a preexisting filament and create waves called lamellipodium

the leading edge will be pushed forward by ___ and the back side of it is pulled forward by ____ to contract the cell body

lamellipodia, actomyosin

steps of locomotion by actin

1. rear adhesion: the trailing edge is adhered

2. Extension: leading edge stretches out to form the lamellipodium

3. Adhesion: the lamellipodium makes contact with the ECM and sticks

4. Translocation: the cell moves forward

5. De-adhesion: the lagging edge detaches from the matrix and actin is polymerized and tread milled

what protein is important when the filament needs to change direction?

Cap Z or capping proteins

-stopping polymerization in one direction can promote other filaments to polymerize, changing the direction the filament is moving

actin comet tails

caused by the dynamic polymerization of actin at the surface of one pole which can use the force of actin polymerization to propel them

paracytophagy

when a bacteria with an actin comet tail propels into a neighboring cell and get lysed, enabling the spread of infection

role of myosin 2 in nonmuscle cells

1. interaction with the contractile ring during cell division

- if missing, cells will not divide and instead form one large multinucleated cell

2. interacts with the adhesion belt of epithelial cells between tight junctions

3. contributes to the mechanical stability of fibroblast stress fibers

occluding (tight) junctions

between epithelial cells

-leaky pathways for small ions and water only

transmembrane proteins associated with tight junctions

occludin, claudin, and JAM

Anchoring Junctions

if actin based, adherans

if filimentous based, desmosomes

Adhesion belts

cell to cell adheren junctions in epithelial cells made up of cadherin proteins

-placed right below tight junctions, creating an adhesion zone

focal adhesions

cell-matrix junctions where mechanical force and signals are transmitted between the cell and ECM allowing for regulatory signaling events

examples of molecules small enought to pass tight junctions

salt and CFTR

structure of adhesion belts

bands of actin and myosin 2 filaments creating a thick belt beneath tight junctions

-use of E cadherin as the linker protein

the transmembrane linker protein is _____ while the intracellular attachment protein can include _______

cadherin, catenin and vinculin

functions for adhesion belts

-anchoring and stucture to epithelial tissue

-vertebrate development

-dependent on myosin 2

-what makes up brain and spinal cord