Cytoskeleton I: Filament structure and regulation

cytoskeleton is a complex and highly regulated network of interconnected - (three things) in the cytoplasm

microtubules, actin, and intermediate filaments

cytoskeleton serves as - and -

stable infrastructure, dynamic scaffold

intermediate filaments are - in diameter made of - proteins, providing major support in - and -

~10 nm, fibrous IF, nuclear lamina, axons

microtubules are long hollow tubes - in diameter made of - assembled into a - providing rigid support in a variety of cell structures

~25 nm, tubulin dimers, ringed tube

actin filaments are - assembled from many a - with a diameter of -

helical polymers, actin monomers, ~5-9 nm

microtubules are like the - of the cell

MTs emanate from - within -

bones, MTOC, centrosomes

in pre-mitotic G1 cell, all cytosolic - project from a - outward to the -

MTs, single chromosome, PM

centrosomes duplicate during S-phase and migrate to - poles of the cell during M-phase where specific types of Mts attach to - to align them and pull - apart

opposite, chromosomes, sister chromatids

actin filaments act like the - of the cell

actin filaments are found in all - cells and all throughout their -

muscles, eukaryotic, cytosol

concentrated actin gel forms underneath the plasma membrane called the -

cell cortex

actin fibers can also be bundled into - that impart physical - with -

longer stress fibers, strength, flexibility

reorganization of actin filaments mediates dramatic changes in - (three things)

cell shape, structure, and movement

intermediate filaments act like the - of the cell

nuclear lamins are the - found in all eukaryotic cells that underly the - of the nuclear envelope

ligaments, ancestral intermediate filaments, inner membrane

cytosolic IFs provide structural support to cells, especially those subjected to -

- are specialized IFs that are crucial to the formation of neuronal axons

shearing forces, neurofilaments

all three types of subunits self-assemble into -

helical filaments

intermediate filaments assemble from - proteins winding around each other

two alpha helical fibrous

actin “nucleus” is a trimer of -

MT “nucleus” is a large ringe of more than thirteen -

globular monomers, globular tubulin dimers

nuclear lamins are present in all - cells, the - proteins that line the inner membrane of the nuclear envelope

nuclear lamins are essential for -, serving as anchoring sites

eukaryotic, filamentous, nuclear integrity

cytosolic IFs are found in only certain -, including nematodes, molluscs, and vertebrates

cytosolic IFs are not in all cell types of these animals and are abundant in cells subjected to lots of -

metazoans, mechanical stress

individual IF filamentous proteins assemble into -

the multimers further assemble to create the -

rope-like multimers, intermediate filaments

actin monomer is a - subunit in the cell, a highly conserved - present in all eukaryotic cells

free soluble, ATPase

actin filaments assemble from identical monomers of actin in a - configuration with plus/minus -

head-to-tail, polarity

minus end of monomer contains opening of -, allowing for entry of - and exit of -

ATP binding domain, ATP, ADP

ATP-bound actin subunits have an - (increased/decreased) affinity with each other

filament ends with unhydrolyzed ATP-bound subunits favour - of monomers, resulting in -

filament ends with hydrolyzed ADP-bound subunits favour - of monomers, resulting in -

increased, addition, polymerization, dissociation, de-polymerization

in reality an actin filament grows by stacking two monomers -

side by side

actin filaments are - compared to microtubules; but can be - or - into stronger actin networks

flexible, bundled, cross-linked

actin monomers can associate or dissociate from - (plus/minus/either) end of the filament, if the end - (is/isn’t) capped

either, isn’t

at low concentrations of available actin monomers, there is a net - from either/both ends, resulting in -

at high concentrations of available actin monomers, there is a net - from either/both ends, resulting in -

dissociation, de-polymerization

association, polymerization

due to structural differences between actin plus and minus ends, the - end has greater -, meaning it is more sensitive to changes in -therefore de/polymerization occurs more at the - end

plus, dynamic instability, monomer concentration, plus

at steady state, there is no net - or - of the filament, resulting in treadmilling

growth, shrinkage

formins are a family of - proteins that nucleate the formation of - that can be assembled into parallel bundles

dimeric, straight, unbranched actin filaments

each formin subunit binds to an actin monomer, bringing - together in a ring-like configuration that nucleates growth of a - or joins both monomers to the - end of an -

two monomers, new filament, plus, existing actin filament

formin dimer maintains its association with one of the two actin monomers at the growing - end, allowing a new actin monomer to be added to the available -

plus, formin binding site

profilin bound to actin monomers interact with - and promotes association between - and - to grow the plus end of actin filaments

formin whiskers, actin, formin

free, soluble form of tubulin in the cell is a heterodimer of - and -

alpha, beta tubulin

alpha and beta tubulin monomers are both - and can binds one molecule of - but only beta tubulin has - in the dimer or MT filament

GTP in beta-tubulin is exposed on the - end and can dissociate or be hydrolyzed to GDP by its GTPase activity

GTPases, GTPase activity, GTP

plus

GTP-bound tubulin dimers have - (higher/lower) affinity for each other and for MT ends still containing -

higher, GTP

tubulin dimers self-assemble as helical rings, forming - and - lateral contacts between tubulin subunits in -

result is a stiff microtubule containing 13 parallel protofilaments with a defined polarity: - : -

alpha-alpha, beta-beta, adjacent protofilaments

alpha→beta, minus→plus

a microtubule polymerizes - to - end

because minus end of microtubule is usually -, polymerization and de-polymerization tend to occur at the - end

minus, plus

stabilized, plus

multiple ATP (-) or GTP (-)-bound subunits can form a - at the growing end of a filament

actin, microtubule, T cap

presence of T cap favours - due to the - affinity of incoming ATP or GTP-bound subunits for ATP or GTP-bound ends

polymerization, higher

loss of T cap occurs when hydrolysis of ATP/GTP is - (slower/faster) than subunit addition

faster

kinetics of - and - at one or both filament ends are highly regulated and can result in net filament -

ATP/GTP hydrolysis, subunit addition/loss, de/polymerization

treadmilling results in steady-state balance between addition of - and dissociation of - from filament ends

new ATP-bound subunits, ADP-bound subunits

changes in - of soluble subunits affect the kinetics of subunit association and shift steady-state towards filament polymerization or depolymerization at - or - subunits concentrations, respectively

available concentration, high, low

rate of association and dissociation gives koff/kon = Kd = -

critical subunit concentration

critical subunit concentration (Cc) is the subunit concentration at which - is achieved

equilibrium

Kd for hydrolyzed --bound subunits is higher than for --bound subunits

ADP, ATP

de/polymerization of actin is regulated by -

accessory proteins

what is regulation of actin polymerization is mediated by?

two actin monomer binding proteins: thymosin and profilin

thymosin binds and - actin monomers from associating with either end of the filament, preventing -

inhibits, polymerization

profilin competes with - for binding to actin monomers, promoting - of monomers with filament ends and -

thymosin, association, polymerization

stathmin performs similar inhibitory function in microtubule polymerization by sequestering - and - (decreasing/increasing) the effective concentration of free tubulin in the cell

tubulin subunits, decreasing

phosphorylation of stathmin inhibits its binding to - and favours microtubule -

free tubulin, polymerization

what happens after?

GTP hydrolysis catches up and microtubule shrinks

tropomyosin reduces - with each other

actin filament interactions

cofilin increases -

actin filament turn-over

cofilin is a small protein able to bind to - and -

free actin subunits, filaments

cofilin binds all along the length of actin filament and twists it into a tighter coiled-coil, applying -, weakening the -, resulting in - of actin filaments

mechanical stress, subunit interactions, depolymerization

cofilin preferentially binds - to facilitate filament turn-over for re-organization

older ADP bound actin filaments

MAPs are -

microtubule-associated proteins

MAPs have - or - MT binding domains that laterally link microtubules together into more -

two, three, stable parallel bundles

- of MAP domain that projects and contacts the adjacent MT dictates the - between filaments and thickness of bundle

length, packing distance

short domain of MAP results in - packing (-)

long domain of MAP results in - packing (-)

tighter, tau

looser, MAP2

MAPs are particularly abundant in - (three things)

axons, dendrites and neurites

MAPs are regulated by - in response to signalling

phosphorylation

capping proteins bind to the - of the filaments and have dramatic effects on net - or - of filaments

ends, growth, shrinkage

CapZ binds to the - end of actin filaments and makes it -

plus, inactive

gamma-tubulin nucleates and caps MT - ends and organizes them into a growth center ( - and -)

minus, MTOC, centrosome

MAPs can also bind to MT - and stabilize the -

ends, length

catastrophe factors take microtubule protofilament ends -, resulting in - of MT length

apart, destabilization

severing filaments creates new ends, facilitating - and/or - of filaments

rapid turn-over, reorganization

specialized - (GTPase/ATPase) called - is required to break the 13-protofilament bonds in a microtubule

crucial to - during mitosis

ATPase, katanin

spindle fiber reorganization

severing of actin filaments requires breaking of only - bonds and does not require -

two, ATP

actin severing proteins belong to - superfamily which all respond to high levels of -

gelsolin, Ca2+

gelsolin can also cap -, preventing -

severed ends, depolymerization

cofilin severs both filaments but also promotes their -

depolymerization