Molecular motor proteins and cytoskeletal organization

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Molecular motor proteins and cytoskeletal
organization - Microtubules and myosin V -
Yin, et al., 2000 - Dynein and the cell cortex
Yamamoto, et al., 2001 - Kinesins with
exotubulase activity Desai, et al., 1999
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Myosin V orientates the mitotic spindle in
yeast Yin, Pruyne, Huffaker, Bretscher Nature,
2000
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DeZwaan, et al., 1997
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• Spindle orientation early in mitosis requires
  actin and MTs.
• MTs from the bud-proximal SPB are recruited into
  the bud.
• Many genes have been identified that are
  required for this
• process, but the link between MTs and actin
  remains unknown .

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• Actin localized to polarized arrays of cables
  and polar patches.
• Myo2p is a member of the Myosin V family, with
  N-terminal
• motor domain and C-terminal cargo-binding
  domain.
• Myo2p is localized to the tips of emerging buds.
• Myo2p moves along actin cables, delivering
  post-Golgi secretory
• vesicles.

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Figure 1. Spindle orientation depends on Myo2.
Yin, et al., 2000
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Figure 2. Kar9p polarization is affected by only
those myo2 alleles That disrupt spindle
orientation.
Yin, et al., 2000
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• Results
• MYO2 mutants are defective in spindle
  orientation.
• Phenotype does not require proper vesicle
  transport.
• kar9D has a similar phenotype and Kar9p
  localization as Myo2.
• Kar9p localization is dependent on Myo2p.
• Myo2p and Kar9p physically interact.

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• Conclusions
• Myo2p translocates along cortical actin to bud
  tip.
• Kar9p interacts with Myo2p to become localized to
  bud tip.
• Bim1p interacts with MT-ends and Kar9p, but
  creating the connection between the MTs and
  cortical actin required
• for proper spindle orientation requires
  Kar9p to bind Myo2p

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Figure 4. Working model for the establishment of
spindle orientation by Myo2.
Myo2
Kar9
Bim1
Yin, et al., 2000
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• Questions
• Kar9p binding competitive with other Myo2p cargo?
• Myo2p tail required?
• Myo2p motility required?
• Regulation of interactions at SPBs

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• Whats been learned since 1999
• 1.   Kar9p localization to the bud-pole (and not
  the mother-pole) is dependent on its
  phosphorylation by CLB4/CDC28.
• 2.   Kar9p-Clb4/Cdc28p accumulate at the SPB,
  then move to the
  ends of MTs by the kinesin Kip2.
• 3.   Fusion chimera of Bim1p Myo2p removes the
  need for Kar9p, further indicating that Kar9p is
  the link between Bim1p Myo2p.
• 4.   Rate of movement of MT to bud tip depends on
  the rate of movement of Myo2p along actin
  filaments.

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MT-actin interactions in spindle orientation in
yeast
Gundersen Bretscher, 2003
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Dynamic behavior of microtubules during
dynein- dependent nuclear migrations of meiotic
prophase in fission yeast Yamamoto, Tsutsumi,
Kojima, Oiwa, and Hiraoka Molecular Biology of
the Cell, 2001
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Dynein-Dynactin complex
Hirokawa, 1998
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Figure 1. Microtubule organization during
nuclear oscillation.
Yamamoto, et al., 2001
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Figure 2. Two phases of SPB movement during
nuclear oscillation
Yamamoto, et al., 2001
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Figure 3. Interaction of directing MTs with the
cell cortex.
Yamamoto, et al., 2001
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Figure 10. Model for meiotic nuclear oscillation
in fission yeast.
Yamamoto, et al., 2001
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• Results
• Nuclear migration is two phases, fast movement
  pauses.
• Migration follows vector of directing MTs to
  the cortex.
• Directing MTs have lateral association with the
  cortex curl around the cell end.
• Shortening of leading MTs at their distal end
  correlates with nuclear migration.
• MT dynamics are greatly altered in dynein mutant.
• Dynein accumulates at the site of MT-cortex
  interaction.

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• Conclusions
• SPB migration follows forward-extending MTs.
• SPB migrations start when the directing MTs
  interact with the cell cortex.
• Dynein is anchored at the cortex of the tip, and
  pulls on directing MTs to move nucleus.
• Dynein interacts in something (?) with a MT
  depolymerase at the cortex to further promote
  nuclear movement to the tip.

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• Questions
• What anchors dynein to the cortex?
• What depolymerizes the MTs at the cortex?
• How is the SPB released when in arrives at the
  cortex?
• What regulates dyneins interaction with the
  cortex MTs?

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• Whats been learned since 1999
• CLIP-170 LIS1 are required for Dyneins
  accumulation at MT Plus ends probably through
  interactions with dynactin.
• Num1 ApsA are required for dynein-cortex
  interactions.
• Dynein-cortex interactions also important for
  cell morphology in vertebrates.

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Dynein-cortex interactions
Lee, et al., 2003
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Kin I kinesins are microtubule-destabilizing
enzymes Desai, Verma, Mitchison, Walczak. Cell,
1999
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Kin I kinesins are microtubule-destabilizing
enzymes Desai, Verma, Mitchison, Walczak. Cell,
1999
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• Isolated from Xenopus extracts (XKCM1 Walczak,
  1996) and
• CHO cells (MCAK- Wordeman, 1995) as
  MT-destabilizing
• activity.
• Changes the catastrophe rate of MT dynamic
  instability.
• KinI (internal motor domain).

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Figure 1. Purified XKCM1 inhibits MT assembly
and induces catastrophe.
Desai, et al., 1999
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Figure 2. XKCM1 and XKIF2 depolymerize
stabilized MT substrates.
Desai, et al., 1999
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Figure 5. Targetting and accumulation of XKCM1
at GMPCPP MT ends.
Desai, et al., 1999
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Figure 6. XKIF2 forms a nucleotide-sensitive
complex with tubulin dimer
Desai, et al., 1999
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• Results
• XKCM1 inhibits MT assembly and induces
  catastrophes.
• XKCM1 catalytically destabilizes MTs.
• Depolymerization at MT ends, and not internal
  severing.
• XKCM1 induces a structural change at the MT ends.
• ATP hydrolysis is not required for XKCM1 binding
  to ends.
• XKIF2 forms ATP-sensitive complex with tubulin
  dimer.

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• Conclusions
• Direct targeting of KinI to MT ends.
• Induce structural change to MT ends.
• ATP-dependent release of Tubulin-KinI complex.

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• Questions
• Require dimer of protein?
• Require domains outside motor domain?
• Linear or lateral progression along MT lattice?
• Structural distinction between motile and
  exotubulase kinesins?
• Regulation of activity.
• Cellular functions in mitosis and other times?

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• Whats been learned since 1999
• Dimer not required.
• Only the motor domain required (neck?)
• Follows single protofilaments (linear, not
  lateral)
• ADP-KinI binds MTs, releases ADP at MT-end,
  ATP-binding distorts MT structure, ATP hydrolysis
  releases
• tubulin-KinI.
• ICIS (inner centromere KinI stimulator)
  cofactor stimulates activity.
• Broad regulator of MT dynamics, and antagonized
  by
• the TOG1/XMAP215 proteins.

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KinI bound to microtubules
Moores, et al 2002
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