Filament depolymerization by motor proteins

1
Filament depolymerization by motor proteins
Meredith D. Betterton Physics Department Universit
y of Colorado at Boulder Collaborators Dick
McIntosh (MCDB) Loren Hough (MCDB) Matt Glaser
(Physics) Anne Schwabe (Biochem)
2
Intracellular transport in neurons

• Proteins manufactured in cell center move to cell
  edge
• Spine to fingertip distance 1 m
• Typical protein size 3 nm

3
Kinesin and microtubules

• First discovered function intracellular
  transport

Highways microtubules 20-nm filaments
Transporter kinesin Walks on microtubules
13 protofilaments
8-nm dimer
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Conventional kinesin
Kinesin walks on microtubules with 8-nm
steps Burns chemical fuel (ATP hydrolysis) Transpo
rts proteins, wastes within cell Takes 3 weeks to
move 1 meter with v 30 µm/min
Movie Gelles lab, Brandeis
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Kinesins that promote MT depolymerization

• Kinesin-8 and kinesin-13 proteins
• Can promote disassembly of microtubules
• Help regulate MT length in the mitotic spindle

http//www.ornl.gov/sci/ismv/images/factsheets/fs-
bio/spindle1.gif
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Kinesin family

• Conventional kinesin kinesin 1
• Kinesin-13 family includes kinesin 13 and
  kinesin 8

Dagenbach and Endow, http//www.proweb.org/kinesin
/KinesinTree.html
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Kinesin 13 proteins and the mitotic spindle

• Example MCAK
• Mitotic Centromere-Associated Kinesin
• When depleted in Xenopus extracts, MTs become
  very long
• Red MTs
• Blue DNA
• Scale 20 microns

Walczak, Mitchison and Desai, Cell 84, 37 (1996)
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MCAK in vitro

• Localizes to microtubule ends
• Promotes MT disassembly
• Hydrolyzes ATP preferentially at ends
• Reaches MT ends by 1D diffusion

Hunter et al., Mol Cell 11, 445 (2003)
http//www.mpi-cbg.de/research/groups/howard/proje
cts.html
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Relationship to Soft Active Matter?

• Active gel theory typically assumes
• Fixed length filaments
• Motors are active crosslinks
• Extensions to include filament length
  fluctuations?

http//www.nat.vu.nl/fcm/news1.htm
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New features of the problem

• Key differences from traditional problems in
  motor protein motion
• Many motors move on filament and interact
• Collective effects important
• Filament dynamics important
• Coupled dynamics of motors and track
• Transient dynamics

Parmeggiani, Franosch, and Frey, Phys Rev E 70,
046101 (2004) Klein et al., Phys Rev Lett 94,
108102 (2005) Nowak, Fok, and Chou, Phys Rev E
76, 031135 (2007) Govindan, Gopalakrishnan, and
Chowdhury, EPL 83, 40006 (2008).
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Experimental results
In cells deleting kinesin-8 genes leads to long
MTs and defects in mitosis West et al., Mol Biol
Cell 12, 3919 (2001) West, Malmstrom, and
McIntosh, J Cell Sci 115, 931 (2002). Mayr et
al., Curr Biol 17, 488 (2007) Garcia, Koonrugsa,
and Toda, Curr Biol 12, 610 (2002) Garcia,
Koonrugsa, and Toda, EMBO J 21, 6015 (2002)
Buster, Zhang, and Sharp, Mol Biol Cell 18, 3094
(2007). In purified systems kinesin-8 proteins
move on MTs and promote MT depolymerization Gupta
et al., Nat Cell Biol 8, 913 (2006) Varga et
al., Nat Cell Biol 8, 957 (2006). Kinesin-8s
involved in chromosome oscillations and MT length
fluctuations Stumpff et al., Devel Cell 14, 252
(2008) Unsworth et al., Mol Biol Cell published
online (2008)
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Kinesin 8 protein Kip3p

• Kinesin 8 from budding yeast
• Promotes MT depolymerization from plus ends
• Motor activity toward MT plus end
• Length-dependent MT depolymerization

Varga et al., Nat Cell Biol 8, 957 (2006)
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Kinesin 8 protein Kip3p

• Depolymerization rate decreases as MTs become
  shorter
• Slower rates for lower motor concentration
• Potential new mechanism for MT length regulation

Varga et al., Nat Cell Biol 8, 957 (2006)
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Theory of kinesin-8 motion and MT depolymerization

• What controls the length-dependent
  depolymerization?
• Over what lengths does LDP occur?
• What parameters control the length?

Hough, Schwabe, Glaser, McIntosh, and Betterton,
Biophys J 96, 3050 (2009)
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Kinesin-8 motors on MTs

• Features
• Binding to/unbinding from tubulin dimers
  throughout MT
• Biased motion toward MT plus end
• Binding to/unbinding from MT plus end
• MT dynamics without motors

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Motor at MT end

• Motor at end promotes MT depolymerization
• Can be processive or nonprocessive
• Motor crowding reduces processivity

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Protofilament interactions

• Typical MT has 13 protofilaments
• Depolymerization rate could depend on number of
  lateral neighbors

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Tools

• Monte Carlo simulations of full model
• Mean-field theory
• Analysis along length of MT
• Analysis at MT plus end only

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Steady-state motor distribution

• Fixed-length MT
• Exponential near minus end
• Constant in bulk
• Clump at plus end

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Depolymerization rate versus length

• For short enough MTs the depolymerization rate
  must drop
• Crossover length increases with decreasing motor
  concentration
• Note motors pre-equilibrated

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Define crossover length d

• Determine MT length when depolymerization rate
  drops by 20 from steady-state value
• Mean-field model agrees
• Phase diagram to compare to experiments

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Define crossover length d

• Determine MT length when depolymerization rate
  drops by 20 from steady-state value
• Mean-field model agrees
• Phase diagram to compare to experiments
• May explain differences between experiments?

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Conclusions

• Minimal model of MT depolymerization by kinesin-8
  motors
• Length-dependent depolymerization strongly
  dependent on motor concentration and initial
  conditions

Hough, Schwabe, Glaser, McIntosh, and Betterton,
Biophys J 96, 3050 (2009)
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Speculation on recent in vivo data

• Puzzling experimental results
• Depleting kinesin-8 increases amplitude of
  chromosome oscillations
• Deleting kinesin-8 decreases MT length
  fluctuations
• Our fluctuation results could explain this
• Motors promote MT length fluctuations
• Lower (but nonzero) concentration of motors leads
  to larger fluctuations

Stumpff et al., Devel Cell 14, 252 (2008)
Unsworth et al., Mol Biol Cell published online
(2008)