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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
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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
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• !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
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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
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- >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:
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- 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
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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
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• 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
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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
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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
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: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