BIOM 3530 - Week 9

Actin Depolymerization Factors (ADF)/cofilins: can bind ADP-actin monomers with more

•affinity than ATP-actin

Actin Depolymerization Factors (ADF)/cofilins: inhibits

nucleotide exchange, not polymerization

Actin Depolymerization Factors (ADF)/cofilins: also involved in

severing filaments

ADF/cofilin binds to

ADP-actin in filaments

ADF/cofilin binds to ADP-actin in filaments and induces

twist in the filament

•ADF/cofilin binds to ADP-actin in filaments and induces

twist in the filament which leads to

severing, breaking it into smaller fragments

inhibit nucleotide exchange, not polymerization, once in ADP state, needs to be

exchanged, so it blocks ADP -> ATP

ADP containing filament is

older filament

with actin, coffin grabs and

condenses the filament

Key initiator of actin polymerization:

Actin related proteins (Arps)

Actin related proteins (Arps), large family of

actin binding proteins found in all cells

Actin related proteins (Arps), not as conserved as

actins

Actin related proteins (Arps), look like actin, but is not, regions are

highly conserved, which is also why there are no non-lethal mutations

Arp 2/3 complex consists of

•Arp2, Arp3 and five other

proteins

Arp 2/3 complex contains ____ proteins

7

Arp 2/3 complex caps

pointed end of filaments

•Arp 2/3 complex caps pointed end of filaments and can

nucleate

polymerization in the barbed direction

Arps anchors the

•capped pointed end of new filament to

the side of existing filament

Arps can form branching of the

actin filament at 70o angle

Arp 2/3 complex is a key nucleator of

actin polymerization

Actin related proteins (Arps), can bind to f-acrin filament to initiate

branching

Arps form branching of the actin filament at 70o angle due to the structure of

Arp 2/3 complex when bound

Arps nucleates 3 trimers at

- end, starting polymerization in + direction

Arp 2/3 is not

always active

Arps bind at the junction and creates

a new branch

branching allows for: 1) creation of more

f-actin chains (if branching)

branching allows for: 2) pushing mb forward through

polymerization

branching allows for: 3) over 50x more

actin to be created

Adapter proteins: used to link

signaling proteins with actin rearrangement

Wiskott-Aldrich Syndrome:

immunodeficiency and bleeding disorder, blood system is being inhibited as neutrophils contain actin

Wiskott-Aldrich Syndrome:

immunodeficiency and bleeding disorder

mutation in

WASp (adaptor protein) causes disease

WASp: involved in nucleating actin filament assembly on

the sides of existing filaments

WASp: induces ________ complex (C-terminus)

Arp 2/3, activating it

WASp: also interacts with

G-protein Cdc42, a GTPase

also interacts with G-protein Cdc42, which controls actin

filament induction

Neutrophils contain actin, which if there is a mutation, it inhibits

activity of WBC to attach to viruses

Profilin exchanges ADP for

ATP, recharging it

Rho family GTPases

WASp protein

WASp activates

Arp 2/3 complex

Some bacteria can hijack the

actin cytoskeleton for motility

•____________________is the most common food borne pathogen

in humans

Listeria Monocytogenes

Listeria Monocytogenes: is

Facultative anaerobic

Facultative anaerobic

Loves no O2 conditions, but can live in O2

Listeria Monocytogenes: is Facultative anaerobic and requires

intracellular entry into host cells

•Bacterial protein (____) is homolog of host WASp protein

ActA

Listeria cells recruit Arp2/3 complex in host to induce

actin polymerization

ActA looks like

WASp to cell

Actin polymerization propels cells through the

host cell and generates 'comet tails' of actin

Bacteria needs ActA to be a

pathogen, without it cant move, therefore not allowing it to infect neighbouring cells

How is monomeric actin recruited to the growing actin filament?

Formins

Formins are

•dimeric proteins (>15 formin genes exist)

•Formins are dimeric proteins (>15 formin genes exist)

that have binding sites for

G-actin and profilin-actin complexes

•Formins have "whiskers" which are long filaments that bind

profilin-actin

formin heterodimer goes back and forth up the filament, leaving a site for

g-actin molecule to bind, only 1 site at a time

each formin has whiskers that extend out, are binding sites for

profilin actin

while the heterodimer puts actin in, the whickers can bend, slotting actin into

the next open binding site

Cross-linking of filaments increases

stability and strength, 1 bond is not strong but 1000s of bonds are

Actin filament cross-linking proteins: proteins that contain

two actin binding sites

Actin filament cross-linking proteins: used to

stabilize and link actin filaments together

Actin filament cross-linking proteins: some promote

actin bundling, a very stable structure

a-actinin: found in

cortical actin, along stress fibers, and in

cell adhesion zones

a-actinin: found in cortical actin, along stress fibers, and in

cell adhesion zones, being zones of

membrane physically attached to ECM

Fimbrin and villin: stabilize

actin bundles in microvilli

Filamin: links cortical actin to

integrins

Filamin: links cortical actin to integrins which attach to the

extracellular matrix (focal adhesions)

Spectrin and dystrophin: bind to

integral membrane proteins

and actin filaments

integrins are cell

mb proteins

integrins are cell mb proteins, grab outside of cell to

keep in place

Spectrin is a

trimer

fimbrin is a

monomer

alpha-actinin is a

dimer

filamin is a

dimer

actin filaments and a-actinin has a

longer distance between actin filaments

actin filaments and a-actinin structure is a

contractile bundle, with loose packing allowing myosin 2 to enter bundle

actin filaments and fimbrin has a shorter

distance between actin filaments

actin filaments and fimbrin structure leads to

parallel bundle, tight packing blocking myosin 2 from entering bundle

filamin dimers hold a

web/mesh-like structure

•Periventricular heterotropia results from mutations in

filamin A gene

•mutations in filamin A gene causes Errors in

neuronal migration during development

mutations in filamin A gene causes reduced

•brain size and epilepsy

mutations in filamin A gene Causes reduced brain size and epilepsy but not

•mental impairment

Actin signaling pathways mediated by

small G protein family Rho

small G protein family Rho: three main members

Rho, Rac, Cdc42

Rho

Stress fibers

Rac

Cortical actin

(membrane ruffles)

Cdc42

Filopodia, disruptive in cell division

Actin signaling pathways: all members are active bound to

GTP

Rho family: all members are active bound to GTP and inactive with

GDP

bacteria reduces chemicals, WBCs binds them and

drives actin polymerization

Dynamic Actin Rearrangement: most cytoplasmic F-actin turns over every

few minutes

half of cellular actin is in

polymerized state

G-actin (GDP) pool concentration is 500-1000 X higher than the

concentration needed to polymerize actin

G-actin pool concentration is mostly bound to

•other proteins

(e.g. profilin/b-thymosin/CAPs)

-very little is free in cell

cell can respond rapidly to changes requiring

•actin polymerization

•cell can respond rapidly to changes requiring actin

polymerization (e.g. cell motility at the

leading edge of fibroblasts/WBCs)

Why are there so many actin binding proteins? many actin binding proteins are

•essential genes (e.g.Arp2/3,

profilin, cofilin and capping protein in yeast)

Why are there so many actin binding proteins? knock out of others causes more

•mild effects (some have redundant functions)

if there is not severe affects, another proteins is

taking over its job, reductant functions

some (dystrophin) have effects

•later in life if mutated (muscular dystrophy)

most actin binding proteins have some

•basic function in all cells