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Molecular Cell Biology > Cytoskeleton: Structure & Dynamics > Flashcards

Cytoskeleton: Structure & Dynamics Flashcards

1
Q

3 types of cytoskeletal elements:

A

Actin - 7nm Diameter

Microtubules - 25nm

Intermediate filaments - (eg Vimentin-10nm)

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2
Q

Purification of cytoskeletal elements:

A

filaments form in cell by polymerisation
can purify specific elements if their polymerisation conditions are unique:
-place under unique polymer conditions
-centrifuge - filaments + some contaminants in pellet
-then introduce unique depolymerisation conditions- makes the subunits soluble - will remain in supernatant upon centrifugation
-centrifuge to purify from contaminants into supernatant

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3
Q

conditions for the three filament types:

A

Actin- polymerised at high pysiological salt, depoly at low

MT - depolymerise from temp shift 37 -> 4 degrees

IF - polymerised under normal physiological conditions - depoly at 8M Urea.

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4
Q

benefit of using polymers

A

individual protein subunits too small

allows building of large structures the size of the cell

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5
Q

Actin filament structure-mechanical strength

A

filament forms in two strands
out of register by half a subunit
-braces the weak points where subunits join in one strand
subunits rotate by ~13degrees
-causes strands to twist around each other so when pulled they twist closer

these allow the filaments to endure diff mechanical stresses

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6
Q

Actin directionality basic info

A

Decorating filament w myosin gives it an arrowhead shape illusion
Barbed + pointed ends
G-actin addition rate is greater at the Barbed end - Barbed end is preferred end of addition

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7
Q

Microtubule filament structure basic info

A

tubule with helical shape
alpha and beta tubulin subunits form heterodimers
heterodimers make a protofilament
protofilaments form a sheet which comes around to form the tubule

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8
Q

Microtubule directionality basic

A

Minus end
Plus end
Rate of subunit addition is greater at PLUS end
Alpha subunit binds to Plus end (beta faces out)
Beta subunit binds to minus end

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9
Q

Intermediate filament Example

A

eg Lamins:
phosphorylated- dissolve nuclear lamina (eg at prophase)
dephosphorylated- nuclear lamina reforms (around chromosomes in each daughter at end of mitosis after cytokinesis)

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10
Q

Intermediate filament structure

A

share Alpha Helical Coiled Coil in common
repeat every 3-4 AA with same amino acid on inside/outside
Inside residues form a Hydrophobic Core
individual coils interdigitate - resist pulling - Forms the Apha helical coiled coil dimer

2 dimers assemble into Anti-parallel tetramer- dimers are staggered to brace weak points (kinda like actin monomers)

Tetramers come together head to tail to form Protofilament
protofilaments form Protofibril
Protofibrils form the Filament

Overall filament made of many individual protofilaments twisted together
Apes together Strong 🦧

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11
Q

Latrunculin

A

Depolymerises ACTIN
Binds to G-actin monomers
Prevents them from binding to the filament
off-rate now > on rate

OVERALL DEPOLYMERISATION

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12
Q

Finding the binding site of a cytoskeleton drug (latrunculin)

A

Use mutant yeast strains
each with ONE surface mutation in ONE type of actin surface molecules

Test each strain w latrunculin
look for escape mutants where the mutation reduced latrunculin sensitivity

can use this to narrow down latrunculin binding site
for latrunculin- Just above ATP binding site
Pulls the subdomain inward
preventing the part of one subunit from inserting into the next - can’t join filament
stabilising the Globular form

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13
Q

Phalloidin

A

Stabilises ACTIN filaments (prevents depoly)
BUT paralyses it
no more ADDITION

Paralyses motile cells
as a TURNOVER of actin between Filamentous and monomer is necessary for motility (treadmilling)
Also there is a limited pool of structures so prevents recycling of used ones for new filament formation

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14
Q

Colchicene

A

MICROTUBULE DRUG
from Colchicum autumnale
prevents heterodimer addition onto filament

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15
Q

Taxol

A

from Pacific Yew

Stabilises microtubules
prevents turnover of old structures to prevent new structure formation

the correct dose can slow cancer cells without affecting others too much (due to cancer cells dividing a lot - MTs needed in division)
can allow immuni system to catch up to them

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16
Q

Cytoskeleton monomer sequesterering drugs:

A

Actin:
Latrunculin
Cytochalasin D

MTs:
Colchicene

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17
Q

Cytoskeleton Stabilising drugs

A

Actin:
Phalloidin

MTs:
Taxol

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18
Q

Measuring actin polymerisation methods

A

Viscosity
Electron microscopy (direct)
Fluorescence

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19
Q

Actin poly- measurement: Viscosity:

A

Fill capillary w G-actin
measure polymerisation by how fast/slow ball bearing sinks
Filament formation causes them to mesh with each other dragging on the ball bearing - more F actin=slower

get G actin at low salt
introduce physiological salt back to polymerise

simple assay
good for assaying other proteins that may aid in actin gel formation

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20
Q

Actin poly- measurement: electron microscopy:

A

Direct Measurement of No. of subunits added onto either barbed or pointed end
(tedious)

use seed to start polymerisation
need to know time 0 - then measure speed of growth

have a Seed
add G actin
let it go for a bit
stop process and look at it under EM
see growth of filaments (more readily at barbed end)
Darker part shows the seed can see any additional - use that new poly to measure how much/speed

do this at diff time points w diff actin concs
v tedious but only ever need to do once

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21
Q

Seed for Actin electron microscopy

A

Acrosomal Process of Horseshoe crab sperm
comes from coil of actin filaments that activates upon meeting ovum and uncoils to pierce vitellin layer

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22
Q

Actin poly- measurement: Fluorescence

A

Penultimate residue Cysteine 374
modified w Pyrene to make Pyrene-actin

upon polymerisation- fluorescence emission of F-actin is 20x than in G-actin

Illuminate at pyrene excitation wavelength of
measure excitation wavelength
as polymerisation occurs - Fluorescence increases
Can measure Fluorescence over time and figure out a polymerisation rate

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23
Q

Real time imaging of single actin molecules

A

Measure length in Microns
knowing symmetry and length of subunits can work out how many subunits/second are being added

Visualised using Total Internal Reflection Microscopy

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24
Q

Total Internal Reflection Microscopy method

A

can see single filament
use proteolytic fragment of Myosin II (binds actin) attached to slide to hold filament close

from other side of slide illuminate it from greater than its Evanescent angle
produces Evansecent wave in excitation wavelength of fluorophores on the actin

the Evanescent wave quickly falls off in intensity as it goes further from slide
So avoids Background noise from the G-actin as it is not near enough slide to be reached

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25
Q

Methods for measuring MT polymerisation:

A

Light Scattering
Electron microscopy

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26
Q

MT poly- measurement - Light scattering

A

MTs can become v long/stiff
so give light scattering interference

Light scattering (proxy for extent of polymerisation) against time:
Have long lag phase of low scattering
then rapid extension - quick increase in scattering
Then Oscillations at higher steady state (due to GTP hydrolysis)

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27
Q

MT poly- measurement - Electron microscopy

A

Use cilium from a Paramecium
Remove membrane w detergent giving premade nucleation point for MT growth

original Cilium is darker - so can see new Tubulin addition
More dimer addition at PLUS end

can measure extent of poly- at certain time points
BUT have to stop it to measure
can only do this once per
so need to do many experiments to do diff time points
repeat for multiple repeats and organisms

so can be issues w continuity between them?

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28
Q

Basic overview of MT kinetics (shudder)

A

Plot of amount of filament against time:
-Lag phase of v low filament for period of time
-Then sudden quick growth phase, amount of filament quickly increases here
-then the Equilibrium phase - steady state of higher levels of polymer

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29
Q

Reason for the Lag phase in cytoskeleton filament polymerisation:

A

Need to form a Minimally Stable Nucleus
-for actin polymerisation this is 3 subunits (2 in one strand, 1 in the other-beginnings of one strand buttressed by subunit in another)
due to the weak bonds along protofilaments needing to be strengthened by lateral bonds

and then for more subunits to be added
The more subunits attached - the less likely the filament is to disappear
and then reach growth phase

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30
Q

Equilibrium phase

A

AKA Steady state phase
where addition rate = rate of depolymerisation

Never reaches 100% filamentous
there will always be a Critical Concentration of monomers that does not polymerise

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31
Q

Critical concentration

A

– the conc of monomers that will never polymerise - means can never reach 100% filamentous

– also is the conc beneath which no new polymerisation can occur - as no new nuclei are formed

– also is a measure of affinity for the end of the filament
– greater affinity gets it closer to 100% Filamentous

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32
Q

Lag phase benefit

A

allows cell to control where actin polymerisation occurs to some degree as it need the nucleus to begin

even a small conc of nuclei added obliterates the lag phase

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33
Q

Critical Concentration and Kd:

A

Cc is Kd
Kd is given by:
Conc of F-actin*Conc of free actin
divided by conc of F actin

so Kd=conc of free actin = CC at equilibrium

addition of subunit to F-actin doesnt change no. of F-actin molecules (just No of subunits in it)

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34
Q

Measuring Cc

A

plot two lines
Fluorescence intensity against Actin conc
one line for low salt, one for high

low salt: v small increase in fluorescence with actin conc due to higher proportion of G-actin

High-salt: same actin conc gives much higher fluorescence (higher F-actin)

These two lines dont cross at 0
but in fact at 0.11 micromolar
tells us that Cc/Kd=~0.11 micromolar

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35
Q

Affinity at pointed and barbed ends

A

is the same at each
-use binding proteins-preferentially bind P or B end
-so can look at Cc of one end specifically
-Plot polymerisation state against Actin conc
-lines have diff gradients - so DIFF rates of addition
-BUT they cross at same point so have SAME Cc/Kd

makes sense as addition at P and B ends both entail making the same No. of lateral and pointed/barbed contacts
so subunit affinities are identical

so no energy here to make polymerisation go in one direction rather than other

at steady state filaments wont grow or shrink
subunits come on and off at fixed rates
therefore cannot do work here

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36
Q

How to allow actin polymerisation to do work?

A

centre of Actin subunit:
Nucleotide (ATP) binding site

ATP hydrolysis differences between the ends causes them to be more different

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37
Q

ATP hydrolysis in the Actin filament

A

When affinities Differ

G-actin: bound ATP is protected by hydrolysis - stays as ATP form
F-actin: subunit conformational change when G-actin joins filament, increases hydrolysis rate of the nucleoside triphosphate

ATP G-actin predominates ADP form
so most actin joining filament is ATP form
the longer the subunit stays in the filament the higher the chance of its ATP being hydrolysed

Rate of addition higher at Barbed end
so hydrolysis catches up to Pointed first

this now gives a difference between P and B ends (ATP form vs ADP form)

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38
Q

Nucleotide hydrolysis changes affinities

A

Repeat the experiment used to find the Cc/Kd of each end (P/B)
except with the B in ATP form
and P in ADP form

the lines cross the [Actin] line at diff points
the ADP form has a higher Cc/Kd - hence lower affinity

this causes growing at B end and depoly at P end - Allowing treadmilling

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39
Q

Treadmilling possible region

A

when [Actin] is:
Below Cc for pointed end (ADP-actin)
Above Cc for barbed end (ATP-actin)

cause net addition at B end
and net depoly at P end

this difference between the ends allows there to be WORK
The ENERGY INPUT comes from the ATP hydrolysis - used in the timing mechanism of ADP vs ATP forms likelihood to come off Filament

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40
Q

Work from Actin treadmilling

A

focus on Same subunits over time
subunits coming on more on one side
and subunits coming off more from the other

looks like filament is “moving forward” relative to these subunits
could push against membrane to move it forward

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41
Q

Dynamic instability basics

A

MTs growing and shrinking
similar phenomenon to actin stuff

this causes the oscillations seen around the MT steady state due to GTP hydrolysis

Cc at plus end much lower than at minus end
there is a point of [tubulin] between these where the off rate of minus end is matched by in rate of Plus end
Ahead of it is region of Growth
Region behind it (still between the Ccs) is Region of Filament Instability

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42
Q

region of filament instability

A

there will be overall depolymerisation
(catastrophe)
this is what causes MT to grow and shrink (dynamic instability)

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43
Q

Dynamic instability mechanism

A

Tubulin that adds onto filament is GTP form
GTP form has v low Cc - so high affinity for ends

the longer a subunit is in the MT - Hydrolysis will catch up
if it manages to catch up to the Plus end of the MT then affinity of Tubulin addition for that end is reduced

this gives CATASTROPHIC DEPOLYMERISATION - rapid shrinkage

but while this happens if enough GTP tubulin has managed to bind - it forms a stable GTP cap - rescuing the MT from catastrophe

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44
Q

uses for dynamic instability

A

is a good search mechanism
eg could be used by mitotic spindles to feel out and capture chromosomes
-capture chromosome = STOP
-no chromosome = retreat + try again

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45
Q

Dynamic instability structural basis

A

When the GTP is hydrolysed
causes the individual protofilaments making up the tubule to become curved
protofilaments become less stabilised by neighbours
so more likely to depoly at this end
basically unfurl in catastrophic depoly

When GTP cap present there is stronger associations between neighbour protofilaments

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46
Q

Actin binding proteins general classes

A

-Sequestering proteins

-Filament Cleavers

-Gel strengtheners

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47
Q

Actin sequestering proteins

A

bind monomeric actin
prevent it from joining filament

used to keep a reserve of G-actin for when it is necessary

Profilin: binds polyprolene in PM - brings G-actin to site of action
also binds actin at P end preventing addition there funneling it to the more productive end

Capz: binds 2 actin monomers at barbed end - prevents addition of subunit to B
Caps the B end of older filaments so G-actin is used more productively in newer filaments

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48
Q

Actin filament cleavers

A

cleave filament
break down the gel

eg Gelsolin
can study using viscosity

Cofilin preferentially destabilises Pointed end
severs them
has a greater affinity for ADP subunits so targets older pointed ends
-releases ADP actin so it can replenish ATP and join barbed end

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49
Q

Gel strengtheners

A

Filamin: antiparallel actin binding heads - one end binds one filament and the other end binds the other

Alpha-actinin is similar; but has a shorter linker between binding heads so forms actin cables

can also study w viscosity ig

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50
Q

MT binding proteins: MAPS

A

microtubule associated proteins
have MT binding domains

some proteins have larger extensions which emanate from surface and form connectiosn btwn MTs to control spacing

Tau protein can fold and form filaments associated w Alzheimer’s

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51
Q

Tau altering cell morphology

A

Tau often found in neuron axons

binds MTs and affects spacing w its spacer
transfect Tau expression plasmid into non-neuronal cell
causes development of processes that resemble axons

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52
Q

Assaying Actin filament nucleating

A

hard to do biochemically as need to fractionate cells - erasing the mechanism

Instead use LISTERIA ROCKETING
rocket through cells allowing travel between cells w/out going extracellular

they subvert actin polymerisation to themselves
Fluorescent Phalloidin tag shows this

add actin to Listeria (w Rhodamine Phalloidin stain)
then add diff fractions of the eukaryotic cell
certain fractions allow actin Poly at listeria
SO it is binding something in the cell allowing this

53
Q

actin nucleation complex

A

complex of 7 proteins (because come out in same fraction)
come together form Arp2/3 complex

can be recruited toward membrane by activated WASP or WAVE complex

54
Q

Arp2/3 complex

A

Arp= actin related proteins
Arp2 and 3 are more similar to barbed end
the other non-Arp proteins in the complex come together to hold them in place
so Arp2 and 3 can act as barbed end of stable nucleus and form a template to control where actin nucleation occurs

55
Q

Arp2/3 nucleation process

A

occurs by side binding of the complex to an existing filament at 70degree angle

causes formation of reticulated network/gel
-maximally non-commital about direction in which cell will go
-net movement is the angle which bisects the processive 70 degree angles

is signal came that stimulated movement to the left then would just need to favour the filaments going toward the left
makes for better poised machinery that can move should a signal its following change direction

56
Q

viewing treadmilling in cell - how does cell move forwards basic

A

fluorescent label actin
photobleach spot on it
looks like the mark is moving backwards as new subunits are added at barbed / removed at pointed

relative to this point it the cell is moving forwards
getting productive movement requires the actin filaments to stay stationary relative to substrate

done using FOCAL CONTACTS
since the filament is stationary relative to substrate new polymerisation at the membrane pushes the membrane FORWARDS

57
Q

Focal contacts basic

A

using interference microscopy
look from beneath at an attached cell thorugh interference phenomena

places where membrane is close to slide show as black destructive interface

then look at same cell through fluorescence of actin
see actin cables all end at places where membrane is close to substratum
at FOCAL CONTACTS
actin attaches non covalently via receptors

58
Q

Proteins in the focal contact

A

actin filaments held together by alpha-actinin

Vinculin - an adapter between Talin + the inside of the integrins AND the actin filament

then Talin
Then integrins which connect to Fibronectin in the basement membrane

means that actin is connected to the outside of the cell
any polymerisation at the ends of the filaments that are cross-linked and connected to these stress fibres causes the membrane to be pushed forwards
-because filament is connected to larger network attached to substrate
allows actin polymeriasation work to push the membrane

59
Q

Myosins

A

produce contractile forces
contract to cause hydrostatic pressure within cell and push cell contents forward

Have ATPase domain
attached to :
-Tail which can bind cell membranse (Myosin I)
-or to longer alpha helical coiled coil tails
the coiled coil allows formation of Bipolar Filaments

60
Q

Myosin II in resting cell state

A

occur in folded soluble complexes
if the light chains are phosphorylated: there is a folding back of the coiled coil connecting to the light chains
making the folded diffusable molecule

61
Q

Myosin II activation

A

myosin light chain kinase

-activated by Rho G-protein
phosphorylates the light chains interrupting the interaction
unfolds the molecule into bipolar filaments
1/2 of molecules face one way, other 1/2 the other way

-attached at each end to different actin filaments in a gel

-by each head walking over the filaments it can squeeze the gel

-causing it to contract - creating hydrostatic pressure on the more fluid cytoplasm in the centre of the cell

-pushing cell contents forwards as the cell geos forwards

62
Q

Myosin II localisation in fish keratinocytes

A

mostly in back of cell
contraction
hydrostatic pressure in back
pushes against the lamellipodium
pushed cell forward

63
Q

myosin I localisation

A

rich in the leading edge of cell connected to membrane by its C-terminus
walks the membrane towards the barbed end of the filaments
i.e. walking the filament forward

64
Q

Myosin experimental evidence

A

Dictystelium KO of Myosins
(not yeast as they chitinous cell wall and not normal eukaryotic cell motility)
though in Dictystelium mutations are not as stable

KO myosins - cells just move uncontrollabnle in all directions at once w no direction/polarity of movement

suggests WT myosin is necessary for proper cell motility

65
Q

Myoll Speckles model

A

speckles of Rhodamine Phalloidin fluorescence in Fish keratinocyte
watch movement w respect to cell

appears as if the cells stay in one place while membrane is pushed forward
but if look relative to cell boundary:
-there is a backward FLUX of actin
-due to new poly ahead of that pushing the membrane
-the actin speckles are attached to actin, indirectly attached to substratum
-speckles move back and then disappear in the regions where there is myosin II at the back (some evidence that tension on actin filaments by myosin II facilitates Actin interaction w other proteins that cause depoly)

backward flux is FASTER at front of lamellipodium
slows down as go back

Blebbistatin inhibits Mysoin II
adding it stops cell movement AND the backward actin flux
presumably due to the actin not being disassembled at back of cell to provide new monomers for the backward flux and push membrane forwards

66
Q

Integrated model for motility

A

-Gel like actin cortex just beneath PM is most developed in Lamellipodium
-less actin near the more fluid centre
-connections connect actin cytoskeleton to underlying substratum through focal contacts
-Lamellipoium moves forward through treadmilling and preferential nucleation of actin at leading edge of cell
-at same time Myosin II contracts actin cortex at back placing hydrostatic pressure on more fluid central region of cytoplasm pushing it forward
-and MIGHT also be involved in removing Focal adhesions at the back of the cell allowing new ones to form at the front of cell (specific proteases involved)

by the two mechanisms of:
CORTICAL TENSION created by myosin
and NEW POLYMERISATION at lamellipodium
the cell moved forward

67
Q

why is 50% of actin G-actin

A

even though Cc is 0.11 micromolar
certain ABP (profilin, Thymosin B) sequester it
keep it in reserve

68
Q

Actin and ABP activity at leading edge

A

profilin sequesters G-actin for when needed
binds the barbed end of the monomer so it directs addition at barbed end of filament

Nucleation of new filaments at 70degrees from last filament by Arp2/3 complex
localises nucleation toward receptor sites following external stimulus
also makes sure polymerisation is localised near the PM

Filament barbed end binding proteins (CapZ eg) prevent additions of Profilin:Actin to older filaments
so instead more likely go new filament

cross linkers (Filamin eg) connect actin filaments to each other
so that they are all at indirectly associated w focal contacts

then as filaments become older
ATP hydrolysed to ADP
Cofilin/gelsolin (prefer bind ADP actin) sti,ulate depoly
actin released from filament as ADP-G-actin
promotes exchange for ATP
ATP has larger affinity and greater ON rate for Barbed ends

69
Q

cytoskeleton cell signalling

A

integrating chemokine gradient signals
affecting the polarity and growth of the CS

through Small GTP binding proteins
eg RHO
normally binds myosin light chain kinase
Lysophosphatidic acid (LPA) binds the TM receptor, indirectly leads to Rho activation
leading to Myosin II activation

Rac G-protein affects lamellipodium formation
activates Arp2/3 and cofilin

Cdc42 g-protein causes filipodia formation, Formin localisation

70
Q

G-protein mechanism

A

are prenylated at C-terminal cysteine
(for Cdc42 its a Geranylgeranyl modification)
is a lipophilic molecule
Interacts with membrane so they only diffuse in plane of membrane

In GDP form when receptor inactive (default)

receptor activation:
leads to GTP exchange factor localisation close to site of activation at the membrane
leads to exchange of G protein of GDP for GTP

GTP form G protein has diff conformation allowing it to bind an effector
leads to effector response near the membrane (due to membrane localisation of G-protein)

This allows local effects on cytoskeleton in response to chemokines/other signals

71
Q

G-protein Effectors

A

N-WASP

Wave complex

72
Q

N-WASP

A

is an intermediary
necessary for listeria movement?
not present in platelets

is autoinhibited in inactive form
has an acidic and basic region that bind to each other causing protein to fold back on itself

CRIB domain is adjacent to Basic domain
is the binding site for activated G-protein

Acidic domain binds/activates Arp2/3 complex
but is cryptic in inactive form

active G-protein at membrane near active receptor
CRIB domains binds
breaks interaction btwn acidic/basic regions

maked acidic Arp2/3 binding domain accessible

Arp2/3 binding to A region changes its conformation
causing the two actin like proteins to be in Filament conformation available for G-actin addition to form filaments

73
Q

WAVE complex

A

WASP family Verprolin-homologous protein

complex of many proteins
has acidic binding region
sequestered in similar way to N-WASP

Binding to Rac1 g-protein displaces the:
-V-helix
-and C-helix
so that they are available in cytoplasm (Acidic region follows C-helix) where they can bind and activate Arp2/3

WAVE complex is the most important on
found from sequence homology to a more minor actor N-WASP

74
Q

Actin nucleator that doesnt act via an intermediary

A

intermediaries:
N-WASP
WAVE complex
are activated by g-protein
then activate Arp2/3
that then nucleates

Formins are directly activated by G-protein
and also direclt nucleate Actin

75
Q

Actin in S. cerevisiae fusion

A

dont have an adapter protein
are directly activated by G-protein

in Budding yeast (S. cerevisiae)
budding is indicative of Haploid form
have A and Alpha mating types
also diploid form after A/Alpha cell fusion
fusion occurs after repolarisation of actin cytoskeleton causing cells to grow towards each other
after which nuclei fuse

controlled by a/alpha mating factor secretion
cause opposite type cell to repolarise in that direction
forming a Shmoo
-patches of actin form at shmoo tip
-pointed ends towards nucleus
-MTs also orient w minus end towards nucleus, provide track for them to move towards each other
-this allows trafficking of vesicles of membrane components for building the extending shmoo

76
Q

Formins genetic screen S. cerevisiae

A

look for cells that grow as haploid but cannot form shmoo
(or for temperature sensitive mutant that cannot do either at restrictive temp)

identified all the Smoo formation actors this way
named the mutants Sterile mutants

77
Q

Ste2 - Shmoo formation

A

is a G-protein coupled receptor
a TM spanning receptor

when inactive binds to heterotrimeric G protein complex of Alpha, Beta, and Gamma

Beta and gamma are lipidated so stay by membrane

activation causes Alpha subunit to sever its connection to beta and gamma

Beta and gamma can only diffuse by the membrane and only close to where they are activated

cdc42-GDP also prenylated- stays membrane localised
Released beta/gamma subunits localise the GEF cdc24 to cdc42 converting it to its GTP form

Active cdc42-GTP binds Formins - which nucleate actin

78
Q

Formin domain structure

A

Crib domain - G-protein binding domain
Acidic domain next to it
Basic domain at other end looping back to bind Acidic domain

Between A and B domains:
Formin homology regions 1 and 2 (FH1, FH2)
when acidic and basic domains bind each other FH1/2 are sterically inhibited

cdc42-GTP (or other active G-protein) binds the Crib domain (only happens close to membrane, close to active receptor eg Ste2)
-Acidic and basic domains’ interaction interrupted - FH1/2 exposed

FH1 is proline rich - which profilin binds to - this localises Profilin/G-actin complexes to the active receptor to create a pool for nucleation

FH2 domain forms a dimer which is site of nucleation

79
Q

Formin homology region 2- Nucleation from the Barbed End of the filament problem

A

strange way to do it as when subunits are added - need to then follow the new end which has just been created by subunit addition
(is not an issue for Arp2/3 as nucleates from other end?)

How does FH2 begin nucleation at Barbed end and continue it even though filament barbed end is growing away

80
Q

FH2 nucleation/continuation process:

A

due to the helical twist between subunits
they are at 167degrees (13 short of 180 deg two fold axis between them)

FH2 dimer forms ring
is a symmetrical dimer with 180 degrees between them
if subunit no. 1 is the furthest to barbed end
then Subunit 1, 2, and 3 are forced by the dimer into 180deg conformation (strained from normal 167)

No. 3 then goes into 167deg conformation to better bind into filament
so no longer interacts well w the formin
so one of the FH2 domains in the dimer ring so no longer binds the actin

This frees up space for new subunit to bind to the freed up formin domain previouslt bound to 2/3
Subunit 0 can bind

filaments formed like this are STRAIGHT not BRANCHED

81
Q

examples of intracellular transport on cytoskeleton

A

axons
melanocytes
Golgi
ER

82
Q

Axonal transport

A

done over cytoskeleton as regular diffusion takes too long - need motor proteins
Transcription/translation in cell body

Inject large conc radiolabelled/hot amino acids
then after inject large conc non-labelled/cold to dilute out signal

leaves pulse of radiolabel (pulse chase)
cut up squid axon after certain time points
can see how far diff proteins moved

diff cargos that go diff speeds
slow and fast done by same motors, just more or less continuously

83
Q

Melanocytes

A

Colour change in african Chiclid
males fight
start black
change to white when submit

melanocytes contain melanin vesicles
dispersed vesicles = Black
aggregated to cell interior = white

Kinesins used to move them out
Dyenins create white colour by collecting the vesicles in by the MT organisation centre

manipulated via cAMP levels
decrease - outward movement
increase - inward movement

84
Q

Golgi during mitosis

A

in cell about to divide: two foci around the two MT organisation centres and spindle poles as that have duplicated before prophase

prophase: Golgi evenly distributed between the two poles

interphase: after division golgi is only on one side close to MT organising centre

suggests dyenin moving them to minus end of MTs
ensures even segregation of golgi into daughters

depolymerise MTs - golgi end up all over cell
active process with ATP hydrolysis to keep them there

85
Q

ER and MTs

A

stain for both
ER tracks along the shape of the MTs - is coincident with them

consisten w kinesin motors moving the tubular reticular ER network over MTs

86
Q

MT motor proteins

A

Kinesin - from minus to plus end

Dyenin - from plus to minus

87
Q

Kinesin structure

A

Alpha helical coiled coil
globular domain at N terminus:
-Has ATPase and MT binding
-is the motor domain

Adapter proteins at C terminus that bind vesicles

other sorts of kinesins with slightly diff organisations
motor at C terminus/ middle too
allows diff sorts of cargo binding and diff movement

88
Q

kinesin movement

A

work in hand over hand direction
move over protofilaments in the tubule

is important to have 13 protofilaments in the tubule ring
so that they are parallel to MT axis
means kinesin doesnt have to go around helical path

89
Q

Kinesin discovery

A

from giant squid axon
big sample of axoplasm

axon is process, extended out of cell so is already somewhat purified in contents

fractionate squid axoplasm and add to purified MTs
see which direction they go in

90
Q

Kinesin purification

A

Spin down MTs - brings down Motor proteins
and also MAPs w no motor activity

can Stall Motor proteins if add AMP-PNO
non hydrolysable
can restart if add ATP - lets them be removed
so:

-spin down MTs - remove axoplasm contaminants
-take pellet - resuspend in ATP presence
-then spin again to separate Kinesins and non-motor MAPs that didnt separate from ATP addition
-preferentially purifies kinesins

-Then take Pure MTs + AMP-PNO and add to this supernatant
-only kinesins will bind in AMP-PNO presence
-throw away supernatant - further purify
-then add ATP to release Kinesins
repeat cycle to further purify

91
Q

Dyenin structure

A

many heavy and light chains
2 motor heads - AAA+ repeat protein hexamer ATPase:
-hydrolyses in cyclic fashion (domain looks like donut)
-in doing so affects an insert into one of the AAA+ domain proteins
-connection to MT is localised to small domain at the top - antiparallel alpha helical coiled coil which undergoes conformational changes in the ATPse cycle and circles around the Hexameric AAA+ domain
-end of the AH coiled coil contacts the MT
-Cargo attached at other end

92
Q

Purifying cytoplasmic dyenin

A

Can use taxol to stabilise it (expensive)
OR
use high GTP in buffer (ends of MTs now capped so no catastrophe)

complex doesnt come off in pure MT in presence of GTP - remains bound
so use impure MT
because in impure purification can get enzymatic activity that takes gamma phosphate of GTP and transfers it to ADP -> ATP
converts GTP to ATP
which REMOVES dyenin
GTP alone will not (but it will remove kinesin)

so have MT w kinesin and Dyenin bound
add GTP
-removes kinesin and leaves dyenin
spin down to leave kinesin in supernatant
then resuspend pellet
add ATP to release dyenin into the supernatant when spun

93
Q
A
94
Q

Myosin V discovery

A

discovered from mouse breeding

mutation in myosin V affecting pigment distribution giving dilute coat
moves along actin network transports melanin vesicle into hair shaft
gene found because breeders keeping good records

95
Q

motor protein Adaptor proteins

A

Kinectin - vesicles to kinesin

Dynactin - connects dyenin to vesicles

Rab27a and melanophilin connect mysoin V to melanin vesicles

96
Q

Kinectin

A

binds kinesins

also evidence that may bind dyenin too
could be that vesicles have more than one motor protein
and when there is movement to plus end dyenins inactivate
and then same vesicles can be moved back by dyenin by reciprocal dyenin activation/kinesin inactivation

discovered by:
detergent treat membrane - makes microsomes
purify
then column with Ab against kinesin
pulled down with 160kd protein kinectin

take kinectin Ab
another microsomal fraction gradient
Ab staining for western blot for kinesin and kinectin
show up in same fraction
suggesting presence in same vesicle/association

97
Q

Dynactin

A

Dyenin adaptor
has 7 actin related proteins forming a mini helix - Arp1

purifying dyenin as seen before
its moving activity is lost

so sucrose gradient fractionate cell contents
see which fractions restore dyenin movement - going longer and longer before falling off
-identified dynactin

has:
-short Arp filament capped at both end by proteins
-p150 protein associated too

98
Q

Myo Va adapter complex

A

dilute mouse is deficient in Exon F of this which locates the adapter for melanin vesicles
-melanophilin

complex between melanophilin and Rab27a (small G-protein)

melanophilin mutant
Myosin V diffused throughout cell
Myosin and melanophilin no longer coincident

order:
F-actin
Myosin Va bound to it by motor domain
Myosin Va exon F bound to melanophilin
melanophilin binds Rab27a-GTP
which binds vesicle

99
Q

Vesicle movement via Kinesin and Myosin Va

A

move down axon via kinesin on MTs
carry myosin along too

use myosin to span actin cytoskeleton at end to dock w synapse

100
Q

centrioles/cytoskeleton during mitosis basic

A
  • centrosomes have pair of centrioles arranged perpendicular
  • in prophase centriole pairs separate to opposite poles
  • in prometaphase spindles from them attach to proteinaceous plaques on centromeres on chromatids
  • in metaphase once all aligned, sister chromatids disconnected from each other

in anaphase sisters move apart
Purse string action of actin and myosin at midpoint draws in creating two pouches
which separate into daughters in telophase

101
Q

Cell cycle drivers

A

these cell cycle events in mitosis are deiven by cyclin/Cdks

Cyclin E important in early phases
Cyclin B important in MITOSIS

102
Q

Centrosome duplication basic

A

Centrosomes go on to become Foci for spindles
the spindle poles
duplicate to form these

Cyclin E important

103
Q

Centrosome structure

A

9-3 config of MTs
but these in the centriole are NOT CONTINUOUS with the MTs
dont form direct template

9 fold symmetry due to protein SAS-6
forms homdimers that form the 9 fold symmetric complex star
-around this form template for cartwheels of triplet MTs that then form the centriole

end up with 2 perpendicular centrioles
amorphous material around
minus ends in the material
plus ends emanate out

104
Q

MT Nucleators in centrosome

A

MTs emanating out of pericentriolar material

within this is complexes made partly from ring isoform of tubulin: Gamma Tubulin
binds the Beta tubulin (odd as minus end ends with Alpha T)

Gamma-tubulin complex forms MT nucleation template
preferentially nucleates MTs at centrosome

3D starburst of MTs radiating out

105
Q

Cyclin E and centrosome duplication

A

CDK2-Cyclin E complex initiates duplication

106
Q

initial organisation of spindles

A

nuclear envelope breakdown btwn Pro/Metaphase (lamin phosphorylation-> depoly)

Centrosomes then start to form half spindles from each pole which interdigitate

some capture sister chromatids (catastrophe search mechanism)

need large reorganisation of cells MT network
so Microtubule stability Decreases before M-phase

107
Q

MT dynamicity changes at mitosis

A

introduce fluorescent tubulin
photobleach MT w laser
see recovery time

half lives:
-interphase: ~5mins
-metaphase: ~15secs
-so much more dynamic in meta than inter

a MAP that stabilises MTs: unphosphorylated XMAP 125
CDK2-CyclinB phosphorylates it
so no longer binds MTs
so they become more dynamic as are stabilised less in tubule conformation by XMAP 125

108
Q

setup of half spindles star formation

A

more dynamic MT cytoskeleton in metaphase than inter

MINUS directed kinesins (weird) go toward MT organising centre
These kinesins are connected in a homotetramer, but are bound to diff MTs
so as approach organising centre
cause MTs to splay out
affect whole network

Kinesin C (aka BimC)

109
Q

Half spindle overlap to form full spindle

A

Plus end multimeric motors (kinesins)
bind to opposite oriented MTs from opposing half spindles
walk to plus end on both
pushes them out

ALSO
Membrane bound dyenins walk Astral MTs toward minus end
pulling the centrosomes toward opposite sides of cell toward membrane

multimeric-kinesins and membrane bound dyenins working together to setup chromosome separation axis

110
Q

Capturing Sister chromatids to opposite side spindles

A

Dynamic instability
search mechanism

If MT captured by proteinaceous plaque - gets stabilised preventing catastrophe

not captured: catastrophe, then rescue, can grow out and search again

MTs that capture kinetochore on sister chromatid become are kinetochore MTs
some never find one

111
Q

Astral MTs

A

MTs emanating from centosomes that are not connected to kinetochores

not spindles

112
Q

Alignment on metaphase plate - Kinetochore MTs/Spindles

A

prometaphase -> metaphase

chromosmes are randomly attached by MTs

due to the higher dynamicity of MTs in metaphase (XMAP 125 phosphorylation eg)
spindles are dynamic

the MTs shift POLEWARDS to centrosomes
(stain/bleach parts - can see them move back)
-poleward force occurring through MT attached to chromosome

113
Q

Alignment on metaphase plate - Astral MTs

A

parts of chromosome without kinetochore
eg the arms
are pushed away from the poles

ASTRAL EJECTION FORCE
from plus end directed motor proteins ound to these parts of chromosome + the MT

so there is a pull from kinetochore MTs to the poles
and a push away from the astral MTs
these 2 forces align the chromosomes along the metaphase plate

114
Q

nuclear import control by Ran-GTP gradient

A

-factors in cell need to be imported into nucleus before pole is set up
-are transported through Nuclear pore complex by Importin
-released by small g-protein action
-compete for binding w importin in active GTP form

-no release of cargo outside nucleus as no GEFs in cytoplasm, most Ran is GDP form
-GEFs in nucleus so cargo offload in there

115
Q

Cyclin control of Spindle assembly factor import

A

-in mitosis: Cyclin B/CDK2 phosphorylates near kinetochore
-leads to localisation of RCC1 (a GEF) next to kinetochore
-catalyses exchange of GDP to GTP -> Ran-GTP in nucleus localised by the kinetichore

-so SAF is released from importin close to needed site
-Free un-occluded SAF leads to setup of half spindle in this region near chromosomes

116
Q

Evidence for the Ran-GTP gradient

A

FRET
reporter constructs w Ran binding domain
fused to FRET pair:
-CFP- as long as no Ran bound - its emission wavelength excites YFP next to it

Ran-GTP binds
interferes w association of the FPs in the FRET pair
no longer any yellow fluorescence (too far apart for FRET to occur)

lowest Yellow intensity nearest where the chromosomes are
where Ran-GTP is highest

also other way round
similar reporter but w an importin binding domain
no yellow where no Ran-GTP
higher Ran-GTP
=more reporter outcompeted for importin binding
=more yellow

117
Q

MTs at anaphase

A

MTs depolymerise at anaphase
no poleward flux now - can be seen with photobleaching strips

instead the region between the bleached stripe and kinetochore reduces in size
-suggests depolymerisation from plus end (the one at the kinetochore)

118
Q

How do chromosomes track the depolymerising MT

A

kinetochore has CENP-E
a plus end directed kinesin
forms fibrous corona around the kinetochore MT
allows kinetichore to keep binding to the dynamic end of the depoly plus end

MCAK protein on the kinetochore displays MT depolymerisation activity

119
Q

Dam1 complex in yeast - Kinetochore Tracking

A

may not be general to other organisms

-forms ring complex binding around + end of MT
-GTP hydrolysis reaches plus end
-Now GDP tubulin at end
-causes end to depoly and SPLAY OUT
-creates BIAS where ring cannot diffuse off backwards toward the splaying
-BUT under thermal motion can only move other way to the poles
-biases kinetochore movement to poles and allows tracking

120
Q

Anaphase A

A

when the chormosomes are tracking MTs to the spindle poles

spindle poles not moving w respect to each other at this stage

121
Q

Anaphase B

A

MT based
Gap between spindle poles increases
so moving chormosomes pulled even further apart

in overlap zone between ASTRAL MTs (not connected to chormosomes):
-proteins like BimC act as homotetramers to work on opposite orientated MTs from opposing spindle poles
-Tubulin added to the ends of these to increase astral MT overlap extension even more - push even further apart

membrane bound dyenins also pull poles apart like before - indirectly pulling chromos apart

The overlap then decreases over Anaphase B

122
Q

Cytokinesis basic

A

sets up midpoint - perpendicular bisector
formation of Anulus around this bisection
pinch to form two pockets

forms daughters

123
Q

3 hypotheses for setup of anulus ring in cytokinesis

A

Astral stimulation

Polar Relaxation

Central Spindle

124
Q

Astral stimulation

A

only at midpoint do you get overlapping Astral MTs
somehow this delivers some factor toward the cell actin cortex causing the setting up of the Anulus ring of straight F-actin and bipolar myosin

125
Q

Polar relaxation

A

opposite of Astral Stim

around cell actin is under tension
tension only released at poles

something moves out along astral MTs to inhibit this tension
leading to the drawing in of the actin in the middle

126
Q

Central spindle

A

signalling from central spindle
signals to mid zone to set up actinomyosin anulus structure

central spindle starts as astral MTs

then becomes separate array of opposite oriented MTs foming the central spindle
some proteins left behind from chromatids after separation:
-CHROMOSOMAL PASSENGER PROTEINS
-eg kinase Aurora B

could set up central tension

127
Q

Rappaports experiment - Evidence for astral stimulation

A

overlap of astral MTs in midpoint sets up the cleavage furrow

Take urchin oocytes
glass needle - push through centre of cell exclude cytoplasm into anulus shape
-got cleabage furrow forming, because of needle could not go through entire cell - reaches one side of midpoint - but blocked by the “donut hole”

end up w one cell, 2 nuclei (no full division, just the cleavage on bottom half)
-these nuclei then duplicate
-2 pairs on each side
-got cleavage furrows between them (expected)
-BUT also got a third one where the astral MTs from 1/2 of each pair overlapped at top of anulus shaped cell

-EVIDENCE for astral MT overlap causing cytokinetic cleavage furrows

128
Q

Evidence for central spindle hypothesis

A

signal from central spindle leads to cleavage furrow forming

Plus end directed kinesin ZEN-4 can form complex w CYK-4 (a row gap? idk what that means)
form a tetramer
Cyk-4 organises kinesin into a tetrametic kinesin complex
bind oppositely oriented MTs
and so locates itself at midpoint of central spindle
mechanism for cell being able to tell where this is

129
Q

Down stream effectors of central spindle hypothesis stuff

A

Aurora B kinase - chromosomal passenger left behind on central spindle at mid point where sisters separate
-phosphorylates protein bound to the CYK-4:Zen4 and releases them into cytosol at the central spindle (ONLY here)
-then later in cell cycle Polo kinase activates Cyk4 to bind and activate ECT2 (a Rho GEF)
-causes Rho activation to GTP form
-Rho activates Formins
-Activated formins located at midpoint (as everyting began here)
-Forms the Anulus of Straight actin filaments at the mid point

-Rho G-protein also activates myosin Light chain kinases - causes unfolding of Myosin II - bipolar myosin II filaments - causes contraction of actin ring

Molecular Cell Biology (5 decks)

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