3. cytoskeleton (filament proteins)

7/21/2019 3. Cytoskeleton (Filament Proteins)

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BIO3153 – Cell Biology – Caroline Petit-Turcotte

Cytoskeleton (Filament Proteins) – Lecture Shivan Desai

T!e Cytoskeleton"

- Unique to eukaryotic cellso Tubulin and actin homologs are found in bacteria (transient

structures)o In eukaryotes they are not permanent and dynamic

o In prokaryotes they are !ed structures "ith a specic function- Dynamic #D structures that ll the cell- $oth muscle and skeleton for the cells

o %llo"s movement

o &ives shape- 'rganiation of intracellular

membraneso In * and &olgi (brings protein

to membrane+ "hich are carried

along the cytoskeleton)- ,ells have elaborate arrays of protein

bers "hich serve functions such as

establishing cell shape+ structural

support+ mechanical strength+ locomotion+ chromosome separation in

mitosis and meiosis+ and intracellular transport of organelles- # Types of ilaments.

o %ctin laments (%s or microlaments) ound in the periphery of the cell (outside+ occupies the

corte! of the cell)

• The cell corte! is a specialied layer of cytoplasm on

the inner face of the plasma membrane "hich

functions as a mechanical support of the plasma

membraneo In animal cells it is an actin-rich layer

responsible for movements of the cell surfaceo %ctin net"orks give a sti/ cell corte! "hich

provides mechanical support for the plasma

membrane !0 *ed blood cells springing back into

shape after squeeing through a capillary



They are also involved in cell migration (for pseudopodia+

"hich are stretched e!tensions of the cytoplasm) They give shape to the cell

o 1icrotubules (1Ts) 1aintains the cell structure+ providing platforms of

transport "here organelles can move They contribute in the formation of the mitotic spindle and

in other cellular processes In neurons they play the role of transport from cell bodies

to terminals T"o isoforms form a dimer

• Dimers form a long chain to form a lea2et

o Intermediate laments (Is) *einforces cells to organie them into tissues

They provide mechanical support for the plasma

membrane "hen it comes into contact "ith other cells or

the e!tracellular environment



• !0 3eratin intermediate laments are found in nails

and hair

• 4ery brous proteins (not globular)

• 1ore 2e!ible than 1Ts

Cytoskeletal Fi#ers"

- 1onomers of proteins connected end to end to form polymers- Diversity is in combination of bers+ proteins and proportions

Filament Construction"

- There are small subunits "hich form laments

- %ctin and tubulin are compact and globular (Ifs are bers)- Disassembly+ di/usion+ reassembly are the steps in formation- Under given conditions+ reverse assembly can occur "here large

polymers can disassemble into a common pool of smaller subunits

"hich is al"ays maintained- Steps of construction.

o 5) Signals such as a nutrient source signals for the disassembly

of actin laments



o 6) The subunits di/use

o #) The subunits reassemble at a ne" site into laments

- 7eak noncovalent bonds are important for speed of formationo 8ydrophobic+ ionic+ dipole-dipole bonds

o 8arder to break a microlament than a protolaments

o The ends are less stable since there are fe"er bonds present 'ne subunit is only bonded "ith # other subunits

% subunit in the middle is bonded "ith 9

other subunits

• This is "hy addition and removal

of subunits occur at the ends of

the laments- If they build every lament one by one they are

unstableo To become stable+ addition must occur as a sheet and become

stacked 1akes the lament very stable

:rotolaments in sheets is the most stable conguration

• Thermally stable Sheet structure gives stability to the center

• ;eaves the ends able to interact (dynamic)o 1Ts form in the manner in "hich protolaments (long linear

strings of subunits <oined end to end) "ill stack and align in a

circular fashiono 5# protolaments = microtubule

$ucleation"

- ;imiting step



- >ucleation is the term used for

polymeriation+ or assembly "here the

rst steps are energetically less

favourable than the continuation of

gro"th- %ctin subunits are proteins "hich get

the formation of the laments activated- The monomers "ill form a dimer+ "hich

"ill then form a protolaments+ "here

t"o protolaments "rap around each other- The concentration and availability of the species is called nucleation- In the cytoplasm there is a concentration of ions+ and salt+ "here salt

favours the formation of dimers making it more stable to assembleo 7hen conditions change+ they become less favourable (for

assembly)o Salt induces nucleationo The lag phase is the time it takes to nucleate to form the initial

oligomer ("here lagging occurs) %fter+ elongation occurs at a faster rate

- The dimers required until equilibrium is "here a stable protolaments

is formedo The equilibrium state is called treadmilling (at steady-state)

Treadmilling is the addition and deletion of subunits at the

same rate (hence at equilibrium)

Tu#ulin"

- 8eterodimer = 5 subunit- %dded head to tail gives polarity- :lus ? ends and minus @ ends of tubulin

o ? ends binds &T: on its top (Aend = ?-tubulin+ -end = @-tubulin)

o @ ends also binds it to the top (@-tubulin)

8ence+ ?-tubulin does not have &T: "hich is accessible (it

is sequestered)

• 1eans it cannot become hydrolyed- 8ydrolysis of &T: promotes addition of subunits

o $inding and hydrolysis of &T: occurs at the positive end sinceelongation of the polymer occurs

o 7hen &T: is hydrolysed it forms &D:



- 5# protolaments t in #BC- Tubulin is made of globular

proteins of t"o di/erent

subunitso Dimers attach to each

other end to end

%ctin"

- There are 6 protolaments in

a right-handed heli!- %T: is sequestered due to the

structure of the proteino $arbed on Aend and blunt on Eend confers polarity

o :olarity has nothing to do "ith charge+ it is simply based on

presence or absence of 1Tso %T: is sequestered since it is inside the protein

'nce hydrolysed+ %D: is trapped inside

Trea&milling an& 'ynamic Insta#ility"

- &ro"th and shrinking of lament proteins- %ctin and tubulin catalye hydrolysis of %T: and &T: respectively

(catalysis is faster as laments)o ,atalysed by enymes

o ;oss of phosphate reduces %T:F&T: to %D:F&D:

o %T: cap lost gives %D:+ &T: to &D:

o %T:F&T: caps are built up %T: or &T: since hydrolysis is lagged %t some point+ hydrolysis catches up

1Ts have a &T: cap since they are present on the ? unit

• asier to gro" "hen &T: is bound to ?-tubulin

• -end is easier to dissociate since &T: is hydrolysed

and sequestered- GTH or GDH form indicates if a triphosphate or diphosphate form e!ists- ,ritical concentration (,c) is "here subunit addition = subunit loss (=

rate constant lossFgain ratio = 5 at treadmilling equal)o Treadmilling occurs both "ith actin laments and microtubules

,an say treadmilling occurs "ith actin laments anddynamic instability occurs "ith microtubules

- Treadmilling.o $ased on rate of hydrolysis and ,c

o ;inear relationship of subunitJ vs0 elongation rate $oth ends are dynamic hence+ there are t"o separate lines

on a graph



o Eend gro"s less than the Aend of actin laments (di/erence is

re2ected in ,c for addition of monomers at both ends of the

lament) %ctin bound to %T: associates "ith rapidly gro"ing Aends+

"here %Tp bound to actin is then hydrolysed into %D: Since %D:-actin dissociates from laments more readily

than %T:-actin+ the ,c of actin monomers is higher for

deletion at the Eend than the Aend for actin laments Treadmilling is "here the net dissociation of monomers

(%D: bound) on Eend = net association of monomers (%T:

bound) at the Aendo Same method goes for tubulin "here &T: cap binds monomers

since they are accessible

- %ctin laments in treadmilling.o :erform an e!periment to vie" formation

5) ilaments added to %T:-actin

6) %T:-actin addition occurs at both ends

#) %T:-actin drops+ addition is greater at Aend

(aKnity) 9) Steady state (gain and loss)

L) Treadmilling (no change in length+ gain =

loss) >ucleation is not a factor since pre-formed

laments "ere added to actin solution &ro"th or deterioration is based on cytosolic

concentrations of available monomer subunits

in the given area

• If concentration of subunits is high+ additionMloss

• If concentration drops until equilibrium+ gain=loss



• If concentration of %T:-actin is limiting lossMgain

o ;oss more at Eend+ but if concentration drops

critically+ there is loss at both ends- 1icrotubules in treadmilling.

o Same systematics as in actin

o Tubulin can be labelled "ithrhodamine

;ength is constant but the

tubulin moves aroundo Dynamic instability of

microtubules implies on ho" they

may gro" steadily+ but then shrink

rapidly by the loss of tubulin dimers

at the Aend Dynamic instability only on 1Ts

• &T: cap on 1Ts are not al"ays blunt

• $ased on the gro"th and shrinkage of microtubules

dependant on the concentration present "ithin the

cytosol The rapid disassembly is referred to as a catastrophe

The tendency to gro" or shrink may be a function of

tubulin concentration %s microtubules gro"+ tubulin dimers are depleted

%s tubulin concentration decreases the rate of hydrolysis

catches up+ "here rapid shrinkage at the Aend is

attributed to the loss of the &T:

cap 8ydrolysis a/ects conformation

• If &T: tubulin dimer is

hydrolyed then it is less

stable since &T: has a more

stable shape than &D:o 'nce the cap is

hydrolyed there is

shrinkage since thereare less bonds at the

endso 'ccurs "hen the ends

are tapered since &T: is becoming che"ed upo The rigid and strong cap becomes curved and

loses its ability to become a rigid structure



o The dimers cannot stay attached and then a

catastrophe occurs

• *ecovery is possible

o ,an reattach the &T: cap and elongate as

necessary

o 3inetics dependent on tubulin concentration

The actual shape changes the kinetics

• Tapered end adds more dimers than

blunt endo Treadmilling and dynamic instability

ultimately results in spatial and temporal

2e!ibility "ith a high turnover+e!ploration for attachment sites and

remodelling+ and is the fastest "ay to

gro" laments "ithout nucleation >ecessary for cell gro"th and

division



Interme&iate Filaments"

- Di/erent from actin and tubulin since they are not

small globular proteins (they are brous)- They are organied di/erently than actin and tubulin

o Is are able to "ithstand a greater amount of

deforming force "ithout breakingo %llo"s the cell endure force+ torque+ crushing+ etc0

- They have a coiled dimer "here t"o "ill assemble- Since they are antiparallel there is no polarity

- *ope-like appearance- ormation is by spontaneous interaction (no energy required)- Disassembly likely regulated by phosphorylation- ormations.

o Dimer formation

o Tetramer formation

T"o dimers



:acked into an array of 5B dimers in cross-section

o Tetramer-tetramer association ormation of N molecules

- pithelial Is (keratins+ etc0)o 1ore diverse family

o Type I and II keratin chains

o Strength in hair+ nails+ etc0

- %!onal Is (neurolaments+ etc0)o ound in central and peripheral a!ons of vertebrate neurons

o Type ;+ 1 or 8

o &ro"th by increase in a!on diameter

3. cytoskeleton (filament proteins)

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