methods for studying the cell cycle cell fusion live and...
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Methods for Studying the Cell Cyclecell fusion
live and fixed imaging
geneticsbiochemistry
in vitro systems
inhibitors of cellular processes(transcription, replication, microtubules)
DISCUSSION SECTIONS BY STUDENT NUMBERENDING IN ODD NUMBERS 2-3, EVEN 3-4
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Genetic Screens: Yeast ‘Cell Division Cycle’ (CDC) Mutants
‘Dominos’sequential, dependent
events‘Oscillator’
central controller
can individual protein mutations block steps or whole process ?
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Cdc Genetic Screens• Lee Hartwell (cerevisiae); Paul Nurse (pombe)• Goal: find mutants unable to transit the cell cycle• Why yeast?
– Cell shape --> cell cycle stage– Grow as haploids (easier to find mutants), or diploids (can
do genetics)• Problem:
– the screen is for cells that can’t grow• Solution:
– temperature sensitive mutants– Replica plating
Genetic Screens: Yeast ‘Cell Division Cycle’ (CDC) Mutants
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In vitro Dissection of the Cell Cycle -Xenopus Egg Extracts
use to isolate proteins present at particular stagesmanipulate proteins-deplete and observe changes to cell cycle
DNA Tubulin
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Lecture 2Introduction to the Cytoskeleton
Outline:Composition of the cytoskeleton
Polymer Dynamics in theory
Polymer Dynamics in cells
Paper: Identification of pathways regulating cell size and cell-cycle progression by RNAi
Paul Nurse “Controlling the Cell Cycle” !!! Thu 4 PM, 100 GPBB
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Structural scaffold - cell shape, spatial organization
Roles of the Cytoskeleton
Dynamic assemblies - movement and force production:
cell migration cell divisionintracellular traffic contraction
cytoskeletal functions often involve motor proteins
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3 major elements of the cytoskeleton
microtubulesα/β tubulin dimers 25 nm diameter relatively stiff –
hollow, 13 protofilaments
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3 major elements of the cytoskeleton
microfilaments = actin filaments actin monomers7 nm diametermore flexible – 2 helicies
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3 major elements of the cytoskeleton
intermediate filaments – 10 nm diameterfibrous – resistant to shear forces structural – prominent in cells
subject to mechanical stress vimentin, nuclear lamins
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Introduction to polymer dynamics: 3 cases
•1) simple equilibrium polymers
•2) polar polymers: asymmetric subunitsundergo conformational change during assembly
•3) complex polymers: non-equilibrium ☞ subunit nucleotide hydrolysis (energy input)
actin andmicrotubules
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assembles & disassembles by addition & loss of subunits at ends
rates = Kon and Koff
Kon depends on concentration of subunit, units of M-1sec-1
Koff does not (unimolecular), units of sec-1
Simple Equilibrium Polymer
KoffKon
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1) lag due to kinetic barrier to nucleation 2) growth 3) equilibrium
[polymer]
lag growth
equilibrium
time
Polymer assembly timecourse
rate of subunit addition = rate of loss
polymer grows as time proceedssubunit concentration drops until Kon[C] = Koff [C] = critical concentration Cc (M-1sec-1[M] = sec -1)
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Kon[C] = Koff
Kon[C] > Koff
Kon[C] < Koff
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Equilibrium constant Keq determined by change in free energy between free subunits and polymer
Keq= Kon/Koff = 1/Cc
Critical Concentration
Concentration of free subunits at which rate of subunit addition = rate of loss
Above Cc net growth, below net shrinkage
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Polar Polymer
subunit in polymerfree subunit
plus endminus end
fastslow
Two ends polymerize and depolymerize at different ratesBECAUSE
subunit conformation changes as it incorporates into the polymer
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> the same interactions are broken when a subunit dissociates from either end
> the final state of the subunit is identical
If the plus end grows 3 times faster it must also shrink 3 times faster.
above Cc both ends grow, below Cc, both shrink
• different Kon and Koff
• BUT !Koff / Kon ratio or Cc must be the same for both ends:
Plus and minus ends
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Complex Polymer (non-equilibrium):microtubules, actin filaments
due to nucleotide hydrolysis upon assembly of subunit into polymer
nucleotide hydrolysis reduces binding affinity
TD= nucleotide
diphosphate
T= nucleotidetriphosphate
D
D
D
D
D D
D
D
T
D
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internal subunits have different dynamic properties than the ends
T
D
D
D
D
D D
D
D
T
D
Complex Polymer properties
Css = “steady state” concentrationKTon[C] = KDoff
Css = KDoff / KTon
KTon>>KDon KDoff>>KToff
T form binds, D form dissociates
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no longer true equilibrium, rather steady statebecause
ATP or GTP subunits must be replenished
D=diphosphate
T=triphosphate
T
D
D
D
D
D D
D
D
T
D
energy
Steady State Dynamics
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Consequences for polymer dynamics
treadmilling (actin and microtubules)
• critical concentration different• Cc(- end) > Cc(+ end)
_ T
D D
D
D
D D
D
D
T
D
+ T
D
• two different reactions at each end of the polymer
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both ends exposed Steady state occurs at concentration between
Cc(- end) and Cc(+ end)
net assembly at the plus end net disassembly at the minus end
subunits “flux” through the polymer
Treadmilling
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D
D
D
D
D
T
T
D
D
D
D
D
T
D
D
D
D
D
T
D
D
D
T
T
Treadmilling
+-
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Dynamic instability (microtubules)
• subunit addition is faster than nucleotide hydrolysis
• CAP of GTP-tubulin on polymer ends KDoff >> KToff : GTP CAP favors growth
GTP CAP present: growthGTP CAP lost: rapid disassembly
• stochastic (unpredictable) transitions
• frequency correlates with tubulin concentration
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5 10 15 20 25 30
Classic experiments by Mitchison and Kirschner 1984
Tubulin concentration (µM)
[PolymerizedMicrotubules]
1) determine steady state concentration (Css ) = 14 µM
microtubules nucleated from seeds - no lag
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2) dilution experiment:grow microtubule seedsdilute into tubulin solution above or below Csswait 10 minutesmeasure Mt number-concentration, Mt length(spun onto EM grids)
before dilution 15 uM 7.5 uM#concentration: x108/ml
average length: (µM)32
18
32
40
15
22
size is dependent on the concentration of tubulin
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Dynamic instability in vitro
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Microtubules are really....tubes, not simple polymers
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non-equilibrium:
exchange only at the ends turnover only with dramatic changes in subunit concentration
Summary
Dynamic instabilityTreadmilling complete and rapid polymer turnover at steady stateenergy required
simple equilibrium:
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Polymer properties regulated in cells
1) nucleation2) polarity3) dynamics
1) Nucleation: kinetic barrier - slow step
monomer dimer trimer = nucleation site
trimer for actin
more complexfor microtobules
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Actin: protein complexes (Arp2/3)
Nucleating factors in cells
Microtubules: centrosomes
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2) Polarity: due to asymmetry of subunits
structural polarity of polymer lattice
visualized by decoration of actin filaments and microtubules
allows cell to generate asymmetric structures and shapesbasis of motility
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Actin decorated with myosin subfragment 1
Actin lattice polarity revealed
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actin structural and dynamic polarity revealed
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Microtubules decorated and viewed in cross section
Microtubule lattice polarity revealed by hook direction
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microtubule dynamic polarity revealed
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Motor proteins recognize polymer asymmetry
mechanochemical enzymes hydrolyze ATP to move along filament produce force or carry cargo
polarity generates directionality
more on motors in later lectures ...
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3) dynamics
regulated by many cellular factors
end-cappingsubunit sequestering
polymer binding proteins
regulators also regulated
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MT dynamics in pombe
role in nuclear positioning and deformation
movement of kinesin motors on growing and shrinking MTs
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MT dynamics in vertebrate cells
impact of destabilization of actin
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D
D
D
D
D
T
D D
D
D
D
T
D D
D
D
D
T
T
T
growing
Dynamic Instability
catastropherescue
D
D
shrinking
D
D
D
D
D
D D
D
D
D
D D
D
D
D