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Cell Reproduction Mitosis and Cytokinesis.
The origin of all cells in all organisms
chromosome segregation and cytoplasmic division.
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MCB 143 Cell Biophysics.
History of Research on the Mitotic Spindle.
1. The Mitotic Spindle is a Protein Machine whose
moving parts are made of dynamic microtubules
and mitotic motors which generate pN scale
forces to move chromosomes over µm scale
distances with high fidelity.2. This machine
works in the noisy stochastic cellular
environment where thermal fluctuations play a
dominant role in counteracting order and
randomizing movements how does it, and other
protein machines, work so well in this
environment ? 3. The history of mitosis
research also illustrates how understanding the
molecular biology of subcellular processes
proceeds from descriptive via molecular to
physical approaches.
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The Mitotic Spindle Molecular and Quantitative
Studies of a Protein Machine
Chromosome Segregation A fundamental property of
all cells.
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The Mitotic Spindle a Protein Machine that
drives Chromosome segregation.
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History of Mitosis Research.

• Discovery of Mitosis and Chromosomal Basis of
  Heredity
• (19th Century).
• B. Classical Mechanics of Mitosis
• the spindle as a force-generating machine
  (1950-1990)
• C. Molecular Biology of Mitosis - molecular
  components
• and biochemical/biophysical mechanisms
  (1960-present).

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(A) Discovery of Mitosis.

1. The principles of Heredity Mendels Laws.
2. The principle of Evolution by Natural Selection.
3. The Cell Theory.

1805 Lorenz Oken, Professor of Anatomy at
University of Zurich all organisms made of
infusoria (cells) that multiply and
differentiate. 1840 Cell Theory formalized by
Zoologist Theodor Schwann and Botanist Matthias
Schleiden -all organisms made of cells -cells
are fundamental units of all organisms -how do
they arise?
Mitosis discovered in context of origins of cells
by cell division and chromosomal theory of
heredity.
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Discovery of Cell Division.
LeeuwenhoekTrembleySpallanzani (1700-1780)
Cell Division.
Robert Remak, (1844-53) Nuclear division.
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Chromosomes and Mitosis.
1876 Balbiani and Van Beneden saw rods
separating into nuclei. 1878-1880 Walther
Flemming (Prof. Anat. Kiel) proposed
chromosomes split longitudinally and move to
daughter nuclei named mitosis.
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Mitosis and Chromosome theory of Heredity
1.Van Beneden (1885) chromosome number and
morphology is species-specific. 2. Boveri
(1905) each chromosome is distinct and carries
specific part of genome. 3. Sutton (1905)
Mendels Laws and chromosome transmission. 4.
T.H. Morgan (1910) specific chromosomes (X)
carry specific genes (Red eye).
-Mitosis segregates genes on chromosomes. -Mechani
sm? Role of Spindle Fibers?
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(B)The Spindle as a Machine. Spindle fiber
dynamics, sliding filaments and spindle forces.
Reality of the Spindle Fibers?
Advocates 1. Flemming and others spindle fibers
are contractile elements. 2. Dujardin, 1835,
cytoplasm full of contractile fibers made of
sarcode. 3. Franz Unger, 1832, intracellular
particle transport (blocked by EtOH) observed in
pollen tubes. Driven by contractile fibers. 4.
Belar, 1929 (and later Bajer) saw vigorous
particle transport along spindle fibers. 5.
Druner, 1894 spindle fibers contract to pull
chromosomes to poles and expand to push poles
apart. 6. Ris, 1949 found that 0.5 chloral
hydrate blocks spindle elongation but not
chromatid-to-pole motility. Defined anaphase A
and anaphase B. Critics However, E.B. Wilson
and many others questioned the reality of the
spindle fibers. E.g. could a force field move
chromosomes and deform cytoplasm like a magnetic
field acting on Fe filings. Formed passively.
Fixation artifact.
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1952 Mazia and Dan mass isolation spindles SU
eggs.
Spindle fibers, chromosomes and asters coherent
physical entity (But did not contribute much to
spindle biochemistry).
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1953 Shinya Inoue uses polarizing light
microscopy
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Dynamic Equilibrium (Inoue, 1967). Poleward
Flux (Forer, 1965).
Steady state fibers polymerize at
equator depolymerize at poles.
Polymerize/push depolymerize/pull.
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E.M. analysis by McIntosh et al, 1969-80 Sliding
Filaments
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Force Generation by Spindle Fibers.
Nicklas, 1983 1 nN stall force.
Ostegren, 1951 Force balance.

• Antagonistic poleward forces.
• Force proportional to distance
• from pole.
• Forces balance at metaphase.
• Poleward forces drive anaphase A

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(C) Molecular Mechanisms of Mitosis

• Discovery of Tubulin.
• 1967, Borisy and Tayor purified tubulin as a
• H3-colchicine binding protein.
• 1972, Bob Weisenberg, tubulin assembly into
• MTs in vitro using 0.1M PIPES EGTA.
• 2. Discovery of MT-based motors proteins.
• 1963 Gibbons purifies dynein an ATPase from
  cilia.
• 1985 Vale, Reese and Sheetz purify kinesin from
• bovine brain and squid axons.

Allowed study of spindle MT dynamics and mitotic
motors.
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Microtubule Polymer Dynamics
Teadmilling. (Margolis and Wilson, 1978).
Dynamic Instability. (Mitchison and Kirschner,
1984).
vg , vs, fres, fcat
dn/dt KonC - Koff
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The Spindle Machine Drosophila spindle MTs
display poleward flux AND dynamic instability.
t1/2 5s

1. How do microtubules and motors drive mitotic
   motility.
2. How are smooth, accurate movements produced if
   the microtubule tracks are constantly growing and
   shrinking.

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Purification of Mitotic Motors Kinesins in Sea
Urchin Embryos.
Scholey et al (1985) Nature, 318, 483 Wright,
Terasaki and Scholey (1993) J. Cell Biol., 681.
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Clarification of the roles of motor proteins in
mitosis and cell division may be complicated by
their number and variety. Wright et al, 1993
suggest that some kinesins may have essential
mitotic functions, but kinesin-1 does not.
Rappaport (1996) Cytokinesis in Animal Cells.
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Mitosis in Drosophila embryos.

• Biochemistry and inhibitor microinjection.
• Cytology of specific proteins in transgenic
  embryos.
• Mutants and sequenced genome.

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Drosophila Mitotic Motors
Protein Family Localization Function
KLP61F Kinesin 5 Central spindle Bipolar spindle maintenance and elongation
Ncd Kinesin 14 Spindle MTs and poles Pole organization, centrosome separation
KLP10A Kinesin 13 Spindle poles and kinetochores Maintenance of steady state length, poleward flux and anaphase A
KLP59C Kinesin 13 Kinetochores Congression and anaphase A
KLP67A Kinesin 8 Kinetochores, central spindle MT destabilization in prometaphase, metaphase, and early anaphase MT stabilization in late anaphase, telophase, and cytokinesis
CENP-meta Kinesin 7 Kinetochores Chromosome congression maintenance
KLP3A Kinesin 4 Chromosomes and spindle MTs Spindle structure and elongation
Nod Kinesin 10 Chromosomes Chromosome alignment
KLP38B (tiovivo) Kinesin 3 Chromosomes Cytokinesis, spindle bipolarity, polar ejection force
PAV-KLP (pavarotti) Kinesin 6 Spindle MTs, cortical actin Central spindle assembly and/or maintenance, cytokinesis
Dhc64C Cytoplasmic dhc Cell cortex, kinetochores Spindle pole separation, chromosome congression, anaphase A, checkpoint
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The bipolar kinesin-5 KLP61F drives MT-MT
sliding.
130
The kinesin-13s, KLP10A and KLP59C depolymerize
MTs.
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Homotetrameric kinesin-5 drives a sliding
filament mechanism
Rs 16.2 nm
S-value 7.6 S
MWholo 490 Kd
MWsubunit 130 Kd
Subunits 4 x 130
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Kinesin-5 localizes to spindle MTs including ipMT
bundles (Sharp et al, 1999, JCB, 144, 125
Cheerambathur et al, Unpublished)
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Antagonistic Sliding Filament Model for Kinesin-5
and -14
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Test Purified Kinesin-5 and Kinesin-14.
(Tao et al, 2006, Curr. Biol, 16, 2293)
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The kinesin-5 - kinesin-14 Force Balance.
Balance Point-Submicron Oscillations.
Model for V versus X.
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Prometaphase Force-Balance.

• Conclusions
• KLP61F and Ncd display competitive motility
• Active - Power Strokes
• Braking - Protein Friction
• Could maintain pole-pole spacing during
  prometaphase

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Mechanism of Anaphase A
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MT Flux, Sliding and Pacman
sliding KLP61F (Ncd brake turned off) flux
KLP10A
Metaphase
sliding KLP61F flux KLP10A pacman KLP59C
Dynein
Anaphase A
sliding KLP61F flux turned off
Anaphase B
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Coupled kin-I dependent flux-pacman model for
anaphase A. (Rogers et al, 2004, Nature 427,
364.)
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Mechanism of Anaphase B
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Anaphase B Spindle Elongation
1. A motor-dependent sliding filament mechanism
generates force to push the poles apart. 2.
Poleward flux acts as a regulatory switch.
Brust-Mascher, Civelekoglu-Scholey, Kwon,
Mogiler, Scholey (2004).
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Model Equations describing the dynamics of
Anaphase spindle elongation.
1. dS/dt 2 (vspeckle - vflux) 2 (vsliding -
vflux) 2. dL/dt 2 (vpolymerization -
vsliding) 3. µ(dS/dt) / 2 kNLFm(1 -
vsliding / Vm)
Unknown variables S(t) L(t) vsliding
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CONCLUSIONS.
1. The Mitotic Spindle is a Protein Machine whose
moving parts are made of dynamic microtubules
and mitotic motors which generate pN scale
forces to move chromosomes over µm scale
distances with high fidelity.2. This machine
works in the noisy stochastic cellular
environment where thermal fluctuations play a
dominant role in counteracting order and
randomizing movements how does it work so
well? 3. The history of mitosis research
illustrates how understanding the molecular
biology of subcellular processes proceeds
from descriptive to molecular and physical
approaches.