Slide set 11 Flashcards
cytoskeleton
- protein polymers composed of actin subunits, tubulin subunits, and intermediate filaments
microtubules are made up of
tubulin subunits
microtubule growth
microtubules continually grow from the centrosome added to a cell extract
dynamic instability
some microtubules suddenly stop growing and then shrink back rapidly (rapid disassembly)
constant polymerization and depolymerization cycle
actin filaments aka
microfilaments
actin filaments
- helical polymers of actin
- flexible
- organize into linear bundles, 2D networks, and 3D gels
- actin filaments are dispersed in the cell but most highly concentrated in the cortex (right below the plasma membrane)
where do you find actin filaments?
microvilli, striated msucle
microtubules
- long, hollow cylinders made of tubulin
- more rigid than actin
- long, straight
- usually have one end attached to a microtubule-organizing center (MTOC) called a centrosome
where do we find microtubules and what are they made of
tubulin!
cilia!
intermediate filaments
- ropelike fibers
- made of intermediate filament proteins
intermediate filament example
forms meshwork called nuclear lamina beneath the inner nuclear membrane
extend across cytoplasm
gives mechanical strength!
in epithelial tissue, they span cytoplasm from one-cell junction to another (strengthening epithelium)
cytoskeletal polymers are….
DYNAMIC!
- cells can rapidly reorganize cytoskeletal organization
- it uses subunits of polymers to build new structures
- cytoskeletal polymers assemble from subunits AND undergo self-assembly
cytoskeletal polymers determine…..
cell polarity and internal organelle organization!
image is small intestine
actin increases SA for food absorption
actin monomers bind together to form….
a polymer/filament!
- actin monomers have ATP binding pocket (ATP can be hydrolyzed to ADP)
- actin monomers have plus and minus end
- subunits bind together head-to-tail
- subunits are added to the end of growing polymer (NOT inserted in the middle)
polarity of actin filament image
barbed = plus end
pointed = minus end
2 Ps don’t go together!
blocking actin filament assembly/disassembly has what effect on cells
toxic!
actin chemical: Latrunculin
effect: depolymerizes
mechanism: binds actin subunits
source: sponges
L in latrunculin bc larry the lobster is in spongebob
actin chemical: Cytochalasin B
effect: depolymerizes
mechanism: caps filament plus ends
source: fungi
actin chemical: Phalloidin
effect: stabilizes
mechanism: binds along filaments
source: Amanita mushroom
actin assembly graph
A. polymerization of pure actin subunits into filaments occurs after a lag phase
B. polymerization occurs more quickly in presence of preformed fragments of actin filaments (act as nuclei for filament growth)
% of free subunits after polymerization reflects critical concentration (Cc) at which there is no net change in polymer
how do we study actin polymerization?
observe change in light emission from a fluorescent probe (pyrene)
fluorescent probe covalently attaches to actin
pyrene-actin fluoresces more brightly when incorporated into actin filaments
nucleation
- a helical polymer is stabilized by multiple contacts between adjacent subunits
- in actin, 2 molecs binds weakly to each other, but a 3rd actin (forms trimer) makes the whole group more stable
- once further monomer addition occurs, this now is a nucleus for polymerization. (tubulin nucleus is larger)
- assembly of nucleus is slow (explains the lag phase seen during polymerization)
- lag phase can be reduced or abolished entirely by adding premade nuclei (EX: fragments of already polymerized microtubules or actin filaments)
how do reduce or eliminate lag phase of actin polymerization?
lag phase can be reduced or abolished entirely by adding premade nuclei (EX: fragments of already polymerized microtubules or actin filaments)
on rate vs off rate
polymerization = assembly
depolymerization = disassembly
A linear polymer of protein molecs (ex: actin filament or microtubule) assembles and disassembles by addition and removal of subunits at the ends of the polymer
rate of addition of these subunits (called monomers) is given by rate constants of kon or koff
Plus end
- fast growing end
- difference in rate of growth is bc the changes in conformation of each subunit as it enters polymer
- ratio of koff/kon is the same at both ends for simple polymerization (no ATP or GTP hydrolysis
minus end
slow growing end
nucleotide hydrolysis
- each actin carries a tightly bound ATP molecule that is hydrolyzed to a tightly bound ADP soon after its assembly into the polymer
- each tubulin has a GTP converted to a tightly bound GDP
- hydrolysis of bound nucleotide reduces the binding affinity of subunit for neighboring subunits and makes it more likely to dissociate from each end of the filament
- T form adds to filament and D form leaves
- this is steady state, not equilibrium
- bc ATP or GTP that is hydrolyzed must be replenished by a nucleotide exchange rxn of the free subunit
ATP and GTP caps
rate of addition of subunits to growing actin or microtubule can be faster than rate at which their bound nucleotide is hydrolyzed
in these conditions, end has a “cap” of subunits containing the nucleoside triphosphate
ATP cap for actin filaments
GTP cap for microtubules
treadmilling graphs
A. explains diff critical concentrations (Cc) at plus and minus ends
- subunits with bound ATP polymerize at both ends of a growing filament and then undergo nucleotide hydrolysis within the filament.
- in this plus end, terminal subunits are in T form bc elongation is faster than hydrolysis
- at minus end, hydrolysis is faster than elongation so terminal subunits are in D form
B. treadmilling at diff concentrations
- critical concentration is T form is lower than for D form
- if conc is between these two, plus end grows while minus end shrinks (this is treadmilling!)
Cc of minus vs plus end
Cc(minus) > Cc(plus)
treadmilling
- nucleotide hydrolysis that comes along with polymer formation is to change the critical concentration at the 2 ends of the polymer
- if both ends of polymer are exposed, polymerization proceeds until concentration of free monomer reaches a value that is above Cc for the plus end but below Cc for the minus end
- at this steady state, subunits undergo net assembly at the plus end and disassembly at the minus end at an identical rate
- polymer maintains a constant length even though a net flux of subunits through the polymer
actin binding proteins
actin binding proteins regulate where and when actin polymerizes or depolymerizes
- different cells have diff collections of these proteins
- these proteins can respond to form different arrays of actin filaments
actin monomer binding proteins
- when thymosin is bound to actin monomers, they cannot be added to the plus end of an actin filament
- profilin can bind actin monomers and rapidly add the monomer to the filament
- profilin competes with thymosin for binding to actin monomers (they can’t both bind!)
- promotes assembly!
- profilin is faster
formin
nucleates assembly and remains associated with the growing plus end
thymosin
binds actin subunits, prevents assembly
Arp 2/3 complex
nucleates assembly to form a web and remains associated with the minus end (actin subunits)
profilin
binds actin subunits, speeds elongation
tropomodulin
prevents actin filament assembly and disassembly at minus end
cofilin
binds ADP-actin filaments, accelerates dissassembly
gelsolin
severs filaments and binds to plus end
gel = not glueing together
capping protein
prevents assembly and disassembly at plus end
tropomyosin
stabilizes actin filament
actin nucleating proteins
determine where polymerization occurs
actin, arp2, arp3
when an activating factor binds the complex, Arp2 and Arp3 are brought together into a new configuration that resembles the plus end of an actin filament
actin subunits can assemble onto this structure, bypassing the rate-limiting step of filament nucleation
what does Arp2/3 bind
existing filaments
result is a branching array of filaments
Arp2/3 nucleates actin filaments
growth at 70 degree angle to original filament
results in treelike web of actin filaments
Formins
formins ride along on growing plus ends and promotes assembly by binding actin monomers
- formins promote growth of linear filaments, not branched networks
- formins can interact with profilin and actin
- formins form a dimeric complex that nucleates formation of a new actin filament and stays stuck to plus end as it elongates
other actin binding proteins
- some proteins bind the sides of filaments to stabilize them
- proteins can bind the ends of filaments, which can stabilize the filament by preventing assembly or disassembly
- proteins can destabilize filaments
cofilin
binds actin filaments and twists them to make them less stable and prone to rapid disassembly
cofilin binds best to ADP actin
shape and dynamics of actin filaments is determined by….
which regulators are active and when they are active
stress fibers
contractile, exert tension
actin cortex
underlies the plasma membrane and consists of gel-like networks or dendritic actin networks that enable membrane protrusion at lamellopodia
filiopodia
spike-like projections of the plasma membrane that allow a cell to explore its environment
myosin
mechanochemical ATPases
couple ATP hydrolysis to force generation
force can be generated by….
coupling ATP hydrolysis and shape changes
myosin II cycle
the head remains bound to the actin filament for only about 5% of the entire cycle time, allowing many myosins to work together to move a single actin filament
how cells move
- the actin-polymerization-dependent protrusion and firm attachment of a lamellipodium at the leading edge of the cell move the edge forward and stretch the actin cortex
- contraction at the rear of the cell propels the body of the cell forward to relax some of the tension
- new focal contracts are made at the front, and old ones are disassembled at the back as the cell crawls forward
- this can occur quickly because all steps can be tightly coordinated
studying locomotion
fish keratinocytes
moving the plasma membrane
the whole branched array of actin treadmills and pushes the plasma membrane forward
advancement of lamellipodium
nucleation by Arp 2/3 complex at front
newly nucleated actin filaments are attached to sides of preexisting filaments
filaments elongate, pushing plasma membrane forward
at a steady rate, actin filament plus ends become capped
after newly polymerized actin subunits hydrolyze their bound ATP in the filament lattice, the filaments become susceptible to depolymerization by cofilin
this causes spatial separation between net filament assembly at the front and disassembly at the back so the actin filament network as a whole can move forward (even though individual filaments within it remain stationary)
