Biology 12 Final New Content

what does the cytoskeleton help with

the cytoskeleton helps dictate cellular structures, change in shape, and motility

3 filaments that comprise the cytoskeleton

1. microfilaments

2. microtubules

3. intermediate filmanets

actin

makes up microfilaments

g-actin

monomeric actin hanging around in space

asymmetrical due to monomeric subunit’s ability to bind to ATP (ATP binding cleft)

plus and minus ends

stacks, allows for filament assembly

f-actin

assembled/polymerized g-actin

what do actin filaments contribute to in the cell

contributes to transport, motility, shape, and force

actin polymerization

forms filaments

2 actins together is not strong enough of an interaction

can push forward the plasma membrane

actin polymerization steps

1. lag phase: 3+ actins must come together for nucleation

2. growth phase: elongation, add in more g-actin subunits

3. equilibrium phase: steady state, not constant f-actin

   1. subunit composition, subunits get replaced

   2. subunits keep coming on and falling off

   3. rate of coming off = rate of coming on

interestingly, by adding a seed/preformed nucleus, no more lag/nucleation phase

critical concentration (Cc)

concentration of g-actin above which polymerization can take place

if [g-actin] < Cc → no filament

if [g-actin] ≥ Cc → filament forms

plus end forms at a lower Cc compared to the minus end (allows polymerization at one end and no polymerization/falling off at the other end, steady state)

actin hydrolyzes ATP

monomers exhibit weak ATPase activity, weak hydrolysis, usually in ATP form

once monomers joins filament, conf change, boosts ATPase activity

ATP hydrolysis activity of actin filaments

once actin subunits are incorporated, ATP hydrolysis occurs randomly. Older filaments are more likely to have hydrolyzed their ATP (time based since longer time = higher chance of getting hydrolyzed)

hydrolysis acts as a molecular timer/indicator of how old the filament is

ADP bound actin are more weakly attached to its neighbouring filaments

ATP hydrolysis is not required for polymer formation

what does ATP hydrolysis do to actin-actin interactions

decreases the stability of actin-actin interactions, ADP bound = weaker attachment

minus end growth of actin filaments

rate of subunit addition < rate of ATP hydrolysis

more ADP-actin which is more likely to dissociate

plus end growth of actin filaments

rate of subunit addition ? rate of ATP hydrolysis

more ATP-actin → ATP cap (a buildup of ATP actin)

What does the actin ATP cap do?

Helps facilitate treadmilling, stability, and contribute to cellular functions

what do actin-binding proteins do

adding proteins that regulate intrinsic actin allows regulation of actin dynamics

main function of actin acting proteins

1. dictate concentration of ATP-actin

2. promote exchange of ADP for ATP on actin monomer

3. makes ends unavailable

4. break filaments (increases ends)

5. alter rate of reactions

proteins that bind g-actin to regulate actin polymerization

1. profilin

2. thymosin beta 4

3. cofilin

profilin

binds to ADP g-actin and promotes nucleotide exchange (ATP replace ADP)

thymosin beta 4

sequesters ATP g-actin, maintains pool of monomeric actin available for polymerization by binding to ATP g-actin and keeping it away

cofilin

stress and severs filaments to promote disassembly by binding to ADP-actin filaments

• helpful for moving filaments

• increases availability of ADP actin (run into profilin to be able to join in after NT exchange)

• ADP actin can become ATP actin on its own eventually (even without profilin)

toxins that alter actin monomer-polymer equilibrium

1. phalloidin

2. cytochalasin

3. latrunculin

these toxins can be used to study actin cytoskeleton

phalloidin

binds alongside actin filaments and stabilizes them in place, prevents dynamism

cytochalasin

binds to the plus end of actin filaments, prevents addition of subunits, causes size restriction

latrunculin

binds to g-actin and inhibits its addition to filament, similar effect to thymosin which sequesters ATP g-actin

actin-binding proteins

proteins that bind and link actin, contributes to different structures

depending on the protein and its property, different structures are formed

ex. fimbrin links actin but alpha-actinin interacts with itself, crosslinking actin filaments/creates actin bundles

actin bundles

if actins are sticks, actin bundles are a group of sticks joined together by alpha actinin proteins

arp 2/3

special actin binding protein that makes filaments that join another filament at a classic 70º angle

arp 2/3 activation is triggered by nucleation promoting factor (NPF) bringing a 3rd actin subunit

has a structure that resembles 2 actin monomers so NPF bringing in a 3rd actin subunit causes nucleation

nucleation promoting factor (NPF)

triggers arp 2/3 activation when NPF is activated by bringing an actin subunit to Arp 2/3

tripartite system

actin subunit, arp 2/3, and activated NPF find groove on existing actin (mother) filament

how arp 2/3 leads to branched filament

1. NPF is activated

2. active NPF brings 3rd actin subunit to arp 2/3, activating arp 2/3 by causing a nucleation

3. nucleation creates a tripartite system consisting of actin subunit, arp 2/3, and activated NPF

4. tripartite system finds groove on an existing actin (mother) filament

5. assembly localizes and NPF finishes its job. Arp 2/3 polymerizes a daughter filament at a characteristic 70º angle against the mother filament → creates branched filament structure

where does arp 2/3 go afterwards?

some arp 2/3 complexes have the potential to hang around on the mother filament

after some time, daughter filament can detach, becoming its own separate filament

recent research reveals that even after the daughter filament falls off, arp 2/3 can stay on mother filament and form another daughter branch (many possibilities for arp 2/3)

leading edge

side of the cell leading the cell to where it is trying to go

lagging edge

ensures dynamism of the cell in the back so that back of the cell can follow front of the cell

contains formation of stress fibers, particularly contractile bundles

qualities of the leading edge

plays role in more directional process, pushes membrane out in an exploratory direction

pushes the membrane in the direction the cell wants to go

has more actin branch filaments with arp 2/3 which enables the cell to send out membrane protrusions in a dynamic and directed way

leading edge feedback

positive feedback from signalling loop encourages branching in the right direction

negative feedback upon loss of membrane pushing signal, prevents further branching in undesired direction

role of lagging edge

contractile bundles contracts at the back of the cell. Tension propels back of the cell forward (almost like a pump that provides momentum)

this occurs simultaneous to the positive/negative feedback happening at the leading edge

contractile bundles

organized assemblies of actin filaments and motor proteins

formins

rho g-protein that directs formation of unbranched actin bundles (predominantly but not restricted to the lagging edge)

how formins work

1. rho g-protein binds to rho binding domain of formin

   1. activated mostly by extracellular signal, sometimes internal

2. formin switches from self-inhibitory to active conformation

3. FH1 domain of formin recruits ATP-actin via profilin (actin binding protein)

   1. deliver actin to growing actin regulated by FH2 domain, acts as a delivery agent), accelerates plus end polymerization

rac g-protein

activates another form of nucleation promoting factor called WASP

controlled polymerization and rearrangement of actin filaments

1. Rac g-protein activates another form of NPF called WASP

2. Rac GTP, NPF like WASP and Arp 2/3 activity generates branched networks

at the leading edge → signalling pathways activate rac g-protein

at the lagging edge → signalling pathways activate rho g-protein

signals and actin binding proteins

signals trigger actin binding proteins in various ways, ex. activate Arp 2/3 for branch formation or other actin binding proteins that sever branches → all of this contributes to dynamism

cell crawling

involves controlled polymerization and rearrangement of actin

during cell crawling

1. protrusion of leading edge (lamellipodium)

2. adhesion of lamellipodium to lower surface of substratum

   1. mediated by integrins in resident plasma membrane

3. movement of the bulk of the cell forward over the site of stationary attachment

   1. accomplished by contractile force exerted against the substratum

4. cell after the attachment with the substratum have been severed and rear end has been pulled forward

lamellipodium

protrusion/extension at the leading edge of a migrating cell

myosin

motor protein that associates with actin filaments

many kinds of myosins, most walk to the plus end of actin filaments

myosin domains

1. head

2. neck

3. tail