CH - 44 - Mitosis and Cytokinesis PDF

CHAPTER 44
Mitosis and Cytokinesis
M itosis is the division of a somatic cell (a vegetative

Prophase Early prometaphase
cell in yeast) into two daughter cells. The daughters are CDK activity high
usually identical copies of the parent cell, but the process
can be asymmetrical. For example, division of some stem
cells gives rise to one stem cell and another daughter cell
that goes on to mature into a differentiated cell. See Box
41.2 for examples.
Traditionally, mitotic events are subdivided into six Late prometaphase Metaphase
phases: prophase, prometaphase, metaphase, ana-
phase, telophase, and cytokinesis (Fig. 44.1). The
dramatic reorganization of both the nucleus and cyto-
plasm during the mitotic phases is brought about by
activation of a number of protein kinases, including
Cdk1cyclin Bcks (abbreviated here as Cdk1 kinase;
Anaphase Telophase
see Chapter 40). After activation by Cdc25 phosphatase,
APC/C activity high
Cdk1 kinase accumulates in the nucleus, where it joins
Cdk1cyclin Acks, which was activated somewhat
earlier (see Fig. 43.6). These two Cdk1 kinase complexes
operate as both master controllers and workhorses that
directly phosphorylate many proteins whose functional
and structural status is altered during mitosis. Their
Early cytokinesis Late cytokinesis
progressive inactivation following the correct attach-
ment of the chromosomes to spindle microtubules drives
the orderly exit of cells from mitosis.
Mitosis is an ancient process, and a number of varia-
tions emerged during eukaryotic evolution. Many single-
celled eukaryotes, including yeast and slime molds,
undergo a closed mitosis, in which spindle formation FIGURE 44.1 OVERVIEW OF THE PHASES OF MITOSIS.
and chromosome segregation occur within an intact APC/C, anaphase-promoting complex/cyclosome.
nuclear envelope to which the spindle poles are
anchored. This chapter focuses on open mitosis, as
Prophase
used by most plants and animals, in which the nuclear
envelope disassembles before the chromosomes segre- Prophase, the transition from G2 into mitosis, begins
gate. Fig. 44.2 summarizes some of the important events with the first visible condensation of the chromosomes
during the various mitotic phases. and disassembly of the nucleolus (Fig. 44.3). In the
755
756 SECTION X n Cell Cycle
A. Interphase B. Prophase C. Prometaphase D. Metaphase

Nucleus
Centrosome
Chromosomes
NE
Microtubules
Centrosomes separate Begins with nuclear Chromosomes align

Chromosomes condense envelope (NE) break-down on spindle equator
Chromosomes attach
to spindle
E. Anaphase A F. Anaphase B G. Telophase H. Cytokinesis
NE
CS
Midbody
CF CF CS remnant
CS
Pole
Sister chromatids separate Organized central spindle (CS) Cleavage furrow (CF) constricts Chromosomes decondense
and move to poles assembles Nuclear envelope (NE) Interphase microtubule
Poles (arrows) separate reassembles network reforms
Cleavage Furrow (CF)
assembles Daughter cells separate
FIGURE 44.2 KEY EVENTS OF MITOSIS. AC, Prophaseprometaphase: Cdk1 kinase triggers condensation of replicated sister chromatids,
disassembly of the nuclear envelope and Golgi, and a dramatic reorganization of the cytoskeleton. As the barrier between the chromosomes and
cytoplasm is abolished, microtubules contact the condensed chromosomes and attach at the kinetochores (see Fig. 8.21). Interaction of kineto-
chores with dynamic microtubules culminates with the chromosomes aligned at the midplane of the bipolar spindle. DF, Metaphaseanaphase:
Once all chromosomes achieve a bipolar attachment to the spindle, an inhibitory signal is silenced, leading to activation of a proteolytic network
that destroys proteins responsible for holding sister chromatids together and also inactivates Cdk1 by destroying its cyclin B cofactor (see Fig.
40.15). Sister chromatids separate and move toward opposite spindle poles, which themselves move apart. GH, Telophasecytokinesis: Targeting
of nuclear envelope components back to the surface of the chromatids leads to the reformation of two daughter nuclei. In most cells, the two
daughter nuclei and the surrounding cytoplasm are partitioned by cytokinesis. (Micrographs courtesy William C. Earnshaw.)
cytoplasm, the interphase network of long microtubules a century ago, the biochemical mechanism remains a
centered on a single centrosome (see Fig. 34.18) is mystery. Protein kinases trigger mitotic chromosome
converted into two radial arrays of short microtubules condensation and onset of condensation is correlated
called asters. Most types of intermediate filaments disas- with phosphorylation of histones H1 by Cdk1 kinase and
semble, the Golgi apparatus and endoplasmic reticulum H3 by Aurora-B protein kinase. However, chromosomes
fragment, and both endocytosis and exocytosis are still condense when both of these phosphorylation
curtailed. events are blocked. It is possible that a combination of
histone modifications promotes mitotic chromatin con-
Nuclear Changes in Prophase densation (see Fig. 8.3).
Chromosome condensation, the landmark event at the Two pentameric protein complexes, condensin I
onset of prophase, often begins in isolated patches of and condensin II are major regulators of mitotic chromo-
chromatin at the nuclear periphery. Later, chromosome some architecture. These complexes share the SMC2 and
condense into two threads termed sister chromatids SMC4 (structural maintenance of chromosomes) ABC
that are closely paired along their entire lengths. Although adenosine triphosphatases (ATPases), but have two dif-
chromosome condensation was first observed more than ferent sets of three auxiliary proteins (see Fig. 8.18).
CHAPTER 44 n Mitosis and Cytokinesis 757
A. Prophase Chromosome B
condensation
Phosphorylated begins
condensin enters nucleus
Histone H3 Duplicated
phosphorylation centrioles begin
begins to separate
Cell surface Microtubule half-
markers life decreases
internalized and asters form
Intracellular membrane DNA
Cell begins to Microtubules
networks remodeled round up Centrosomes
FIGURE 44.3 INTRODUCTION TO PROPHASE. A, Summary of the major events of prophase. B, Distribution of DNA (blue), microtubules
(red), and -tubulin (centrosomes [green]) in a prophase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the University of
Dundees School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
Condensins are required to disassemble the topologically

TABLE 44.1 Comparison of Microtubule Dynamics
associating domains (TAD) of interphase chromatin (see
in Interphase and Mitotic Newt Lung Cells
Fig. 8.13) and promote the formation of the linear array
of loops that characterizes mitotic chromosomes (see Parameter Interphase Mitosis
Fig. 8.14). The decisive experiment was to target SMC2 Elongation rate 7m/min 14m/min
for rapid degradation, with the result that no well- Elongation time before 71s 60s
organized mitotic chromosomes formed. The chromatin catastrophe
compacted but was less organized. Slower depletion of Shortening rate 17m/min 17m/min
condensins by RNA interference (RNAi) or conditional Probability of rescue 0.046/s 0
gene disruption gave less-dramatic results. from catastrophe*
Condensin complexes can encircle DNA and also Length 100m 14m
promote its supercoiling in vitro, but how these activi- *Most cellular microtubules grow constantly by addition of subunits to
ties help them to orchestrate the changes in chromatin their free ends but they occasionally stop growing and begin shrinking
rapidly (a catastrophe). Unless shrinking is reversed (a rescue), the
architecture is not known. Condensin II is nuclear during microtubule completely disappears.
interphase, and has an important role in prophase chro- Data from Gliksman NR, Skibbens RV, Salmon ED: How the transition
mosome formation following its activation by phos- frequencies of microtubule dynamic instability regulate microtubule
dynamics in interphase and mitosis. Mol Biol Cell. 1993;4:10351050.
phorylation. Condensin I acts both in prophase and
prometaphase in further compacting the chromosomes.
DNA topoisomerase II and other scaffold proteins also properties of the microtubules (Table 44.1; also see
function during mitotic chromosome formation, but Chapter 34). Interphase microtubules have a high prob-
condensin appears to play the major role. ability of recovering from catastrophes, so they grow
quite long. Mitotic microtubules grow more rapidly
Cytoplasmic Changes in Prophase but exist only transiently, because rescues are rare fol-
Most of the cytoskeleton reorganizes during prophase. lowing a catastrophe. Thus, they usually shorten all
The microtubule array changes from an extensive the way back to the centrosome, with little chance of
network permeating the cytoplasm into two dense, rescue. These differences in dynamic instability can be
radial arrays of short, dynamic microtubules around the reproduced in vitro in mitotic and interphase cellular
duplicated centrosomes (see Fig. 34.17). Each of these extracts. They appear to arise, at least in part, from
asters eventually becomes one pole of the mitotic counterbalancing interactions between microtubule-
spindle. During prophase, the two asters usually migrate associated proteins that promote microtubule stability,
apart across the surface of the nuclear envelope, signal- and kinesin-13 (see Fig. 36.13), which promotes micro-
ing the start of spindle assembly (Fig. 44.3). tubule disassembly.
Mitotic microtubules behave like interphase microtu- Other cytoskeletal elements that disassemble during
bules in many ways (see Chapter 34). They are mostly prophase include many, but not all, classes of interme-
nucleated at their minus ends, they grow by addition of diate filaments (including the nuclear lamins) (see
tubulin subunits at their plus ends, and they undergo Fig. 35.1A) and specialized actin filament structures,
random catastrophes during which they rapidly shorten. such as stress fibers. However, the junctional complexes
To a large extent, the prophase changes in microtubule between adjoining cells are maintained in epithelial
organization can be explained by two simple biochemi- cells. As a result of the cytoskeletal reorganization, most
cal changes: (a) increased microtubule-nucleating activ- cells round up during prophase. This is particularly
ity of centrosomes and (b) altered dynamic instability evident for animal cells that are cultured on a flat
substrate, but cells in tissues also change their shape bly of new ribosomes. Phosphorylation of several nucleo-
dramatically during mitosis. lar proteins leads to disassembly of the nucleolus.
RNA transcription of the chromosomes stops during The Golgi apparatus and endoplasmic reticulum frag-
mitosis except for highly specialized transcription at ment and disperse during prophase (Fig. 44.4). Several
centromeres. Phosphorylation of components of the kinases, including Cdk1 drives Golgi apparatus disas-
transcriptional machinery by Cdk1 kinase appears to be sembly, the first step being fragmentation into smaller
responsible for this shutoff. Cdk1 kinase phosphoryla- ministacks following phosphorylation of Golgi stacking
tion of ribosomal elongation factor 2a (EF2a) also stops proteins and tethers. Later steps are still being investi-
most (but not all) ongoing protein synthesis and assem- gated. Many lines of evidence argue that Cdk1 phos-
phorylation of key components prevents the fusion of
transport vesicles back into Golgi stacks (see Chapter
21), the net result being that the Golgi buds away into
Interphase Mitosis
small vesicles that disperse throughout the mitotic cell
cytoplasm. Other evidence suggests that an imbalance of
vesicle flow between the Golgi and the endoplasmic
Golgi Ministacks reticulum results in the Golgi being absorbed into the
Golgi stacking GM130 endoplasmic reticulum during mitosis. Whatever the
proteins Cdk1
cyclin Bp9 mechanism of its disassembly, Golgi reassembly begins
p115
again during late anaphase/early telophase, following
inactivation of Cdk1 kinase.
Prometaphase
In cells that undergo an open mitosis, prometaphase
begins abruptly with disassembly of the nuclear envelope
FIGURE 44.4 GOLGI APPARATUS DYNAMICS IN INTER- (Fig. 44.5). Microtubules growing outward from the
PHASE AND MITOSIS. Disassembly in mitosis is driven by phos- spindle poles penetrate holes in the nuclear envelope,
phorylation of components blocking fusion of Golgi membranes. make contact with the chromosomes, and attach to them
A. Early prometaphase C. Late prometaphase E. Kinetochore attachments

to the spindle
2 4
1 Tension
3 5
(+) ends
1. Nuclear envelope disassembles 4. Chromosome slides rapidly poleward Amphitelic (correct)

along microtubule
2. Microtubules grow and shrink in aster 5. Microtubule from opposite pole is
3. Kinetochore captures microtubule captured by sister kinetochore
6. Chomosome attached to both poles
congresses to middle of spindle
B D
Syntelic (errors)
DNA
Microtubules
Centrosomes Merotelic (errors)
FIGURE 44.5 INTRODUCTION TO PROMETAPHASE. A, Summary of the key events of early prometaphase. B, Distribution of DNA (blue),
microtubules (red), and -tubulin (centrosomes [green]) in early prometaphase human cells. C, Summary of the key events of late prometaphase.
D, Distribution of DNA, actin, microtubules, and centrosomes in late prometaphase PtK1 cells. E, Terms used to describe the orientation of
kinetochore attachments to the mitotic spindle. (B and D, Images were recorded by Dr. Melpomeni Platani on the University of Dundees School
of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
at specialized structures called kinetochores (see Fig. as an extensive tubular (or flattened cisternal network
8.19). Interactions of the two opposing kinetochores of another source of discussion) throughout mitosis.
paired sister chromatids with microtubules from oppo- Further experiments are required to answer this ques-
site poles of the spindle ultimately result in alignment of tion, and both mechanisms could contribute. Lamin B
the chromosomes in a group midway between the poles. remains associated with the dispersed nuclear envelope,
An important cell-cycle checkpoint (see Chapter 40) whereas lamins A and C and many proteins of the nuclear
known as the spindle assembly checkpoint (SAC) pore complexes disperse as soluble subunits.
delays the onset of chromosome segregation until all During prophase, kinetochores transform from non-
kinetochores are attached to microtubules. descript balls of condensed chromatin into structures on
the surface of the chromosomes. By early prometaphase,
Nuclear Envelope Disassembly in Prometaphase the characteristic trilaminar disk structure (see Fig. 8.19)
Nuclear envelope disassembly involves the removal of can be seen. Each sister chromatid has a kinetochore.
two membrane bilayers coupled with disassembly of the Sister kinetochores are located on opposite faces of
nuclear pores and the fibrous nuclear lamina mesh- the mitotic chromosome.
work that underlies the inner bilayer (Fig. 44.6). Phos-
phorylation causes the nucleoporin Nup98 to dissociate Organization of the Mitotic Spindle
from nuclear pores. This removes the permeability The mature metaphase spindle is a bilaterally sym-
barrier between nucleus and cytoplasm. Phosphoryla- metrical structure with centrally located chromosomes
tion of other proteins causes the pore to disassemble to flanked by arrays of microtubules radiating from the
soluble subcomplexes. Phosphorylation of the nuclear poles (Fig. 44.7).
lamins at two sites flanking the coiled-coil causes the Three predominant classes of microtubules are
lamina network to disassemble into subunits. Interaction present in the metaphase spindle (Fig. 44.12). Kineto-
between microtubules and dynein associated with the chore microtubules have their plus ends embedded in
nuclear envelope can rip holes in the envelope, although the kinetochore and their minus ends at or near the
this is not required for nuclear envelope disassembly. spindle pole. They characteristically form bundles, called
Nuclear envelope membranes are dispersed in the kinetochore fibers, which contain anywhere from 1
cytoplasm from prometaphase until telophase (Fig. microtubule in the budding yeast to more than 200
44.6), but the mechanism is not settled. Some experi- microtubules in some higher plants. Each human kineto-
ments suggest that the nuclear membranes break up into chore binds approximately 20 microtubules. Up to
small vesicles that disperse in the cytoplasm. Other approximately 80% of the approximately 2200 spindle
experiments suggest that the nuclear envelope is microtubules in humans may be present in kinetochore
absorbed into the endoplasmic reticulum, which remains fibers, but not all microtubules in those fibers stretch all
A. Interphase B. Mitosis C D. Lamins A and C E. Lamin B

Nuclear Endoplasmic
membrane: reticulum
Outer
Inner
HO PO4 O PO4 O
Lamina
Interphase O O
disassembly C C
OCH3 O
Nuclear
lamina Cdk1 G2 / M G2 / M
cyclin Bp9
Condensing
chromosomes
PO4 PO4
O O
Eggs? Somatic C C
cells? Earliest OCH3 O
prometaphase OH O PO4 OH O PO4
Lamin B
+ or
Chromosomes Vesicles derived Lamin B

from the nuclear dispersed through the Soluble Subunits on
Metaphase Lamina Lamina
envelope endoplasmic reticulum subunits membrane vesicles
FIGURE 44.6 DISASSEMBLY OF THE NUCLEAR ENVELOPE DURING MITOSIS. AB, Two contrasting models to explain the fate of the
nuclear envelope during the transition from interphase to mitosis in a higher eukaryote. C, Micrographs showing solubilization of lamin A fused
to green fluorescent protein (GFP) (green) during mitosis. DNA is blue. Scale bar is 10 m. DE, Reversible disassembly of lamins A, C, and B
is driven by posttranslational modifications of the lamin polypeptides. (C, Courtesy William C. Earnshaw.)
A. Metaphaseforces balanced, spindle length stable B. Anaphaseforces elongating the spindle dominate
Cytoplasmic dynein moves

toward minus () end
Microtubule disassembly at kinetochores
Microtubule (+)
plus flux moves sister chromatids toward poles
(+) (+)
(+) (+)
(+) Flux
(+) (+)
(+)
Ordered central
(+) spindle assembles
() () () (+) () (+)
() () () ()
() ()
() () () () ()
(+) Kinesin-13
(+)
(+) ()
(+) ()
(+)
() (+)
() (+) NuMA
Bipolar plus-enddirected kinesin-5 dominates
Inward force from minus-enddirected kinesin-14 and microtubule to elongate spindle, pushing poles apart
disassembly at poles by kinesin-13 is balanced by outward force
from kinesin-5 plus dynein stretching poles apart
Microtubule assembly at kinetochores and disassembly at poles causes tubulin subunit flux along kinetochore microtubules
FIGURE 44.7 ROLE OF MOTOR PROTEINS IN SPINDLE DYNAMICS. Mitotic spindle structure depends on microtubule assembly/
disassembly plus balanced forces that slide microtubules relative to one another and to pull the poles together or push them apart. A, In
metaphase, the structure is at steady state. Forces that tend to elongate the spindle, including cytoplasmic dynein (which moves toward
microtubule minus ends, pulling the poles out toward the cell cortex) and bipolar kinesin-5 (which moves toward microtubule plus ends, pushing
the poles apart), are counterbalanced by cohesion between sister chromatids and kinesin-14, which moves toward microtubule minus ends (and
pulls the poles together) and microtubule disassembly at the spindle poles. Dynein and its associated protein, NuMA (nuclear mitotic apparatus),
also help to organize a focused spindle pole. B, In anaphase, sister chromatids separate, the balance of kinesin activity shifts, microtubule disas-
sembly at the poles declines, and the spindle undergoes a dramatic elongation. During anaphase, bipolar kinesin-5, chromokinesin KIF4A, and
protein regulated in cytokinesis 1 (PRC1) also have important roles in organizing the central spindle, which is essential for subsequent assembly
and function of the cleavage furrow.
the way from the kinetochores to the spindle poles. microtubules. As a result, the highly dynamic spindle
Interpolar microtubules are distributed throughout changes shape as a delicate balance of forces shifts
the body of the spindle and do not attach to kineto- between the various motors. For example, inactivating
chores. Many interpolar microtubules penetrate between one or more kinesins with drugs or switching a
and through the chromosomes and extend for some temperature-sensitive mutant to the nonpermissive
distance beyond them. Thus, the central spindle contains temperature can cause the spindle to collapse rapidly
a large number of interdigitated antiparallel microtu- on itself.
bules. Tracking these spindle microtubules by electron
microscopy revealed a tendency for the interdigitated Spindle Assembly
microtubules of opposite polarity to pack next to one In metazoans, spindle assembly starts in prophase with
another. During late anaphase, these antiparallel micro- the separation of the asters. In most cells, each aster is
tubules bundle to form a structure called the central organized around a centrosome, consisting of a centriole
spindle that plays important roles in signaling during pair and associated pericentriolar material. -Tubulin
cytokinesis. Astral microtubules project out from the ring complexes in the pericentriolar material efficiently
poles and have a role in orienting and positioning the nucleate microtubules (see Fig. 34.16), so each centro-
spindle in the cell through interactions with the cell some acts as a microtubule organizing center
cortex in somatic cells. All microtubules within each (MTOC). By the end of prophase, the spindle consists of
aster have the same polarity, with their minus ends two asters linked by a few interpolar microtubules.
proximal to the pole. Each unit of a spindle pole, with Cytoplasmic dynein at the cell cortex exerts an outward
its associated kinetochore and interpolar and astral force separating the centrosomes, whereas kinesin-14
microtubules, is referred to as a half-spindle. motors (which move toward microtubule minus ends)
Spindle structure is largely determined by a combina- on the interpolar microtubules exert a counterbalancing
tion of microtubule dynamics plus the action of at least force holding the asters together.
seven different types of kinesins and cytoplasmic dynein This balance of forces changes when the nuclear
(see Chapter 36). These motors often work in opposition envelope breaks down. Bipolar kinesin-5 motors phos-
to one another. Furthermore, forces exerted by motors phorylated by Cdk1 kinase concentrate in the central
can influence the dynamic assembly/disassembly of spindle, where they crosslink adjacent antiparallel
interpolar microtubules. Kinesin-5 moves toward the enable chromosomes to control the activity of key
plus ends of microtubules, so when attached to two spindle assembly factors. The nuclear import receptors
adjacent antiparallel microtubules, its action will cause importin and bind these factors, as though they were
them to slide and push the spindle poles apart (Fig. going to transport them into the nucleus. This blocks
44.7). However, the two half-spindles do not separate their spindle assembly activity. During mitosis, chromo-
because they are physically linked via the chromosomes bind high levels of RCC1, the guanine exchange
somes, with sister kinetochores attached to opposite factor for Ran (Ran-GEF in Fig. 9.18). This RCC1 creates
spindle poles. a gradient of the active guanosine triphosphatase
Also at this time, the asters mature into focused (GTPase) Ran-GTP (guanosine triphosphate) around the
spindle poles. The pericentriolar material efficiently chromosomes. During interphase, Ran-GTP is confined
nucleates the assembly of new microtubules with their to the nucleus, where it releases importins from their
minus ends at the pole. Microtubule assembly at two cargo. In mitosis the chromosome-associated cytoplas-
other sites also contributes to spindle morphology. At mic Ran-GTP gradient locally liberates the spindle assem-
kinetochores, a complex derived from interphase nuclear bly factors including motor proteins and NuMA from
pores recruits -tubulin. This locally nucleates microtu- sequestration by importins. They then stabilize nearby
bules that grow by inserting subunits at the kinetochores, microtubules and organize them into a bipolar spindle.
pushing the minus ends with -tubulin outwards toward If centrosomes are removed or destroyed experimen-
the spindle poles. These preformed kinetochore fibers tally in cells about to enter mitosis, somatic cells can also
are then incorporated into the spindle. In the central use motor proteins to organize microtubules into bipolar
spindle, microtubules are nucleated on the walls of other spindles that lack asters but are otherwise remarkably
microtubules by -tubulin recruited by the multi-subunit normal. Most treated cells manage to complete mitosis
augmin complex. The action of augmin creates branched successfully but normal mammalian cells then either
microtubules throughout the central spindle, contribut- arrest in the next cell cycle prior to replicating their DNA
ing to an even fir tree-like distribution of microtubules. or commit suicide. Both outcomes depend on the pres-
The daughter microtubules may be released from their ence of the important tumor suppressor protein p53 (see
nucleation site, free at both ends, and move toward the Fig. 43.11). Thus, centrosomes are not required to form
spindle poles as part of the flux of tubulin from the spindles, but they contribute to cell-cycle progression in
center of the spindle toward the centrosomes. many cells. This dependence on centrosomes is not
The microtubule array focuses at the poles partly universal; Drosophila, for example, can live without
because centrosomes tether microtubules, and partly centrosomes.
due to the concerted action of various motors and micro-
tubule crosslinking proteins such as dynein and nuclear Chromosome Attachment to the Spindle
mitotic apparatus (NuMA) protein. NuMA is released Dynamic microtubules of prometaphase asters scan the
from the nucleus when the nuclear envelope breaks cytoplasm effectively searching for binding sites that
down, and it accumulates near the poles at the minus will capture and stabilize their distal plus ends. Captured
ends of microtubules. microtubules are approximately fivefold less likely to
Large cells that lack centrosomes, such as eggs, form depolymerize catastrophically than free microtubules.
spindles by an alternative pathway that also functions in When catastrophes do occur, the microtubules depoly-
the background in cells with centrosomes (Fig. 44.8). merize back to the pole, recycling tubulin subunits for
This mechanism hijacks the nuclear trafficking system to incorporation into other, growing microtubules.
Unstable microtubule
g radient
TP Microtubule
G
stabilized by
n-
Ra
Ran-GTP
gradient
Stabilized Motors sort Other motors and

microtubules microtubules, () end-binding proteins
accumulate on which bind to organize spindle poles
chromosomes kinetochores (note absence of asters)
FIGURE 44.8 ASSEMBLY OF A BIPOLAR SPINDLE IN THE ABSENCE OF CENTROSOMES. A gradient of Ran-guanosine triphosphate
(GTP) stabilizes microtubules around chromosomes, which contain high concentrations of bound Ran GEF RCC1. This releases spindle assembly
factors from importin and . Microtubules that accumulate around the chromosomes are sorted, organized, and focused to make poles by
motors and () end-binding proteins such as NuMA. These spindle poles lack prominent astral microtubules.
Breakdown of the nuclear envelope makes the con- chromosome toward the middle of the spindle. These
densed chromosomes accessible to the microtubules. movements are accompanied by coordinated shrinkage
Chance encounters allow kinetochores to capture of the microtubules at the leading kinetochore and
microtubule plus ends. Capture probably involves the growth of microtubules at the trailing kinetochore.
nine-component KMN network, which includes the rod- More recent studies revealed that chromosomes
shaped Ndc80 complex (see Fig. 8.21) that binds along attached to only one spindle pole can move away from
the sides of microtubules near their plus ends. Another that pole if the unattached kinetochore associates with
member of the complex, the scaffolding protein Knl1 the kinetochore fiber of a chromosome already aligned
(its name in vertebratesthe K of KMN; see later), at the spindle equator. In this case, the kinetochore of
anchors Ncd80 in the kinetochore. the mono-oriented chromosome glides toward the
Historically, it was thought that forces generated by equator, where it is more likely to capture microtubules
bipolar attachment of the kinetochores of sister chro- emanating from the opposite pole. This motion of one
matids center chromosomes midway between the two chromosome along the kinetochore fiber of another
spindle poles. This hypothesis was based on the observa- chromosome requires the kinesin-7 motor centromere
tion that when a kinetochore first attaches to a microtu- protein E (CENP-E) (see Fig. 36.13) associated with the
bule, the chromosome moves along the side of that kinetochore of the moving chromosome. Recognition of
microtubule toward the spindle pole (Fig. 44.9). Subse- a tubulin posttranslational modification leads CENP-E to
quent capture of a microtubule emanating from the move the chromosome toward the spindle equator,
opposite spindle pole by the sister kinetochore would rather than out into the aster.
provide a counterforce pulling the chromosome in the The attachment of microtubules to kinetochores can
opposite direction. Chromokinesin family motor proteins be reconstituted in vitro from mixtures of chromosomes,
distributed along the chromosome arms were also isolated centrosomes, and tubulin subunits. The plus
thought to contribute to the gradual movement of the ends of microtubules grow out from centrosomes and
A E
Spindle
pole
Spindle pole
4 Microtubule
Kinetochore
Distance traveled toward
3
spindle pole (m)
B 1
1 Start of
movement
2
0 20 40 60 80
Time (seconds)
C D
Kinetochore Microtubule
10 m
FIGURE 44.9 INITIAL CHROMOSOMAL MOVEMENTS DURING PROMETAPHASE. AB, Capture of a microtubule by the kinetochore
results first in movement along the side of the microtubule toward the pole from which that microtubule originated. These images come from a
study in which living cells, observed by differential interference microscopy, were subjected to rapid chemical fixation just after a chromosome
had attached to the spindle (arrow). C, Attachment of the chromosome to the spindle was confirmed by indirect immunofluorescence staining
for tubulin and thin-section electron microscopy (D). E, The graph shows the movements of the chromosome before and after attachment. (From
Rieder CL, Alexander SP, Rupp G. Kinetochores are transported poleward along a single astral microtubule during chromosome attachment to
the spindle in newt lung cells. J Cell Biol. 1990;110:8195, copyright The Rockefeller University Press.)
attach to the chromosomes. Surprisingly, chromosome- Chromosome Attachment Errors: The Spindle Assembly
bound microtubules can either lengthen or shorten at Checkpoint below) delays mitotic progression to allow
the attached end without detaching from the chromo- the correction process to occur.
some. Similar experiments with kinetochores isolated When syntelic attachments occur, one or both kineto-
from budding yeast cells showed that kinetochores can chores must detach for the chromosome to achieve a
remain attached to a shortening microtubule plus end bipolar orientation. Chromosome attachment to oppo-
even against an applied force of 9 pN (piconewtons). site spindle poles is more stable than attachment to a
Physiological levels of tension actually stabilize the single pole, because the tension generated by bipolar
attachments of kinetochores to microtubules in vitro, as attachment (where forces pull a chromosome simultane-
in vivo. This tethering of kinetochores to disassembling ously toward opposite spindle poles) preferentially sta-
microtubules is essential for chromosome movements bilizes microtubule connections to both kinetochores.
during mitosis. Merotelic attachments are more dangerous, as the kineto-
chore is under tension and the attachments are therefore
Correcting Errors in Chromosome Attachment to stable. Merotelic attachments are the most common
the Spindle cause of chromosome segregation errors in cultured
The goal of mitosis is to partition the replicated chromo- mammalian cells.
somes accurately between two daughter cells. Therefore, Syntelic and merotelic chromosome attachments are
all chromosomes must attach correctly to both spindle corrected through the action of Aurora B protein kinase,
poles (known as amphitelic attachment; Fig. 44.5) which forms the chromosomal passenger complex
before being segregated. Three other sorts of attachment (CPC), along with inner centromere protein (INCENP),
are seen: (a) chromosomes with one kinetochore lacking survivin, and borealin (Fig. 44.10). The other subunits
attached microtubules (known as monotelic attach- target Aurora B to its various sites of action during mitosis
ment; this is a normal intermediate), (b) chromosomes and regulate the kinase activity. The complex concen-
with both sister kinetochores attached to the same trates at inner centromeres (the heterochromatin beneath
spindle pole (known as syntelic attachment), and (c) and between the two sister kinetochores) during pro-
chromosomes with a single kinetochore attached simul- metaphase and metaphase. At anaphase onset, the CPC
taneously to both spindle poles (known as merotelic moves to the overlapping interpolar microtubules of the
attachment). Correcting monotelic and syntelic errors central spindle and to the cell cortex, where the cleav-
takes time, and the SAC (see Finding Time to Fix age furrow will form, ultimately winding up in the
A Borealin
B C
DNA
Survivin
Microtubules
D E. CPC Aurora-B F
INCENP
Borealin
Survivin
Histone H3 phosphorylation
Kinetochore targets
Central spindle targets
Cleavage furrow targets
FIGURE 44.10 CHROMOSOMAL PASSENGER COMPLEX (CPC) REGULATES MITOTIC EVENTS. The CPC (green) is present at
centromeres in prometaphase and metaphase (B), but transfers to the spindle midzone at anaphase (C) and midbody at anaphase (D).
E, Diagram of Aurora B protein kinase complexed with INCENP (inner centromere protein), survivin, and borealin with some key targets of the
CPC. F, If CPC function is inhibited (in this case by RNA interference [RNAi] depletion of borealin), chromosome attachment errors are common
and many chromosomes fail to segregate properly in anaphase. Distribution of DNA (blue), microtubules (red), and survivingreen fluorescent
protein (GFP) (green) in human mitotic cells. Inset in A, Distribution of kinetochores (red), and borealin (green) in a prometaphase cell.
(AD, Micrographs by Sally Wheatley and William C. Earnshaw. F and Inset in A, Micrographs by Ana Carvalho, Reto Gassmann, and William
C. Earnshaw. Insets in A and F, From Gassmann R, Carvalho A, Henzing AJ, etal. Borealin: a novel chromosomal passenger required for stability
of the bipolar mitotic spindle. J Cell Biol. 2004;166:179191, copyright The Rockefeller University Press. AC, From Wheatley SP, McNeish IA.
Survivin: a protein with dual roles in mitosis and apoptosis. Int Rev Cytol. 2005;247:3588.)
intercellular bridge during cytokinesis. The CPC regu- lose a chromosome only once in 100,000 cell divisions.
lates mitotic events from prophase through cytokinesis. The frequency of chromosome loss may be 20-fold to
Along the way it contributes to the correction of 400-fold higher for human cells grown in culture. To
chromosome attachment errors and to the operation of achieve even this level of accuracy, most cells delay
the checkpoint that delays the cell cycle in response to entry into anaphase until all chromosomes have achieved
those errors. amphitelic attachment to the spindle. This delay is
Aurora B corrects chromosome attachment errors by caused by the spindle assembly checkpoint (SAC), which
phosphorylating Ndc80 in the microtubule-binding KMN senses the completion of microtubule binding to kineto-
complex (see Fig. 8.21). Aurora B phosphorylation chores at metaphase (Fig. 44.11). The spindle checkpoint
strongly inhibits Ndc80 binding to microtubules, causing differs from DNA damage checkpoints in that its default
the kinetochore to release attached microtubules. When setting is on as cells enter mitosis. It is silenced only
a chromosome is correctly attached to both spindle when every chromosome is properly attached to the
poles, tension stretches the kinetochore away from the spindle.
CPC buried in the chromatin beneath. This can stabilize The SAC involves the products of the mitotic arrest
chromosomemicrotubule interactions by preventing defective (MAD) genes, the budding-uninhibited-by-
the kinase from phosphorylating Ndc80. benzimidazole (BUB) genes, the monopolar spindle
(Mps1) kinase and the CPC. The MAD and BUB genes
Finding Time to Fix Chromosome Attachment Errors: were identified in yeast genetic screens for cells that
The Spindle Assembly Checkpoint continued to divide (and die) when the spindle was
Segregation of replicated chromosomes into daughter disassembled by drugs. These genes are conserved
cells is extremely accurate. For example, budding yeasts from yeast to humans. SAC proteins accumulate at
A D
C
23 minutes
Time
B
Seat
belt
N
Time Mad1 Mad2

Destroy free kinetochore 23 minutes
with blast of laser light
APC/C
substrate
C
APC/C APC/C
it
Go
Wa
MCC Cdc20APC/C
Kinetochore
Mad1 Mad2 Mad2
APC/C
Microtubule Cdc20APC/C
Checkpoint proteins
not on kinetochore BubR1
Cdc20MCC U Ub Ub
Go! Wait! Ub b
Mad2 MCC
Proteins not to scale relative to kinetochore Substrate
FIGURE 44.11 SPINDLE CHECKPOINT. Signaling by unattached kinetochores stops the cell from entering anaphase until all chromosomes
have made a proper bipolar spindle attachment. A, As long as there is a chromosome that is not properly attached to the spindle (beige cells),
the cell does not enter anaphase. The cell enters anaphase approximately 20 minutes after chromosome attachment is complete (green cells).
B, In a cell with a persistently maloriented chromosome, anaphase entry is delayed (beige cells). If the unattached kinetochore is destroyed with
a high-powered laser, the cell enters anaphase about 20 minutes later. This proves that the unattached kinetochore sends an inhibitory signal.
C, Overview of the spindle assembly checkpoint (see text for details). D, Structure of the Mad1/Mad2 complex.
kinetochores early in mitosis, when the checkpoint is Silencing of the checkpoint involves several pathways.
on (ie, during prophase or prometaphase), and are Protein phosphatase 2A (PP2A) and protein phosphatase
gradually displaced as microtubules bind and the kineto- 1 (PP1) are recruited to the kinetochore in feedback
chores come under tension. loops involving the CPC and other checkpoint compo-
The target of the SAC is the APC/CCdc20, the anaphase- nents. They dephosphorylate Knl1 so that it releases
promoting complex/cyclosome (APC/C) ubiquitin- Mad1. When microtubules bind, cytoplasmic dynein
protein ligase (an E3 enzyme; see Figs. 23.3 and 40.16) motors actively strip checkpoint components from the
with its substrate recognition factor Cdc20 bound, APC/ kinetochore, dragging them away toward the centro-
CCdc20 ubiquitylates target proteins to mark them for somes. In yeast, access of Mps1 to its target sites on
destruction by proteasomes. Key APC/CCdc20 substrates Knl1 is physically blocked when microtubules bind.
are proteins that must be degraded for the cell to move Exactly how this interaction is regulated in metazoans is
from metaphase to anaphase, including cyclin B and still actively studied. In addition, the SAC appears to
securin, an inhibitor of the enzyme that triggers separa- crosstalk with the DNA damage response (see Chapter
tion of sister chromatids at anaphase (Fig. 44.16). 43), since DNA damage response components activate
During mitosis, Cdk1 activates the APC/C by phos- SAC components and vice versa. However, the network
phorylating an auto-inhibitory loop, allowing Cdc20 to of interactions is very complex and details are still being
bind. The SAC is an additional regulatory circuit that worked out.
inactivates APC/CCdc20 until all kinetochores attach to Experimental inactivation of the spindle checkpoint
spindle microtubules. Kinetochores without microtubules causes a catastrophic, premature entry into anaphase,
attract proteins that assemble the mitotic checkpoint regardless of the status of chromosome alignment. This
complex (MCC), the inhibitor that inactivates APC/CCdc20. leads to an unequal distribution of sister chromatids and
Checkpoint activation starts when Aurora B in the genetic imbalance between daughter cells known as
CPC activates Mps1 kinase, allowing it to phosphorylate aneuploidy. Yeasts can live without the checkpoint
Knl1 in the kinetochore at several sites. Mps1 phos- genes, but their loss is lethal for mice, which die early
phorylation of Knl1 creates a binding site that results in during embryogenesis. Mice heterozygous for various
Mad1 recruitment to the kinetochore. Mad1 then recruits checkpoint components show increased aneuploidy.
Mad2 to form a stable complex (Fig. 44.11). A loop on Humans with mutations in BubR1 have mosaic variegated
Mad2 wraps around Mad1 like a safety belt making aneuploidy syndrome (extra copies or loss of various
the complex particularly stable. This form of Mad 2 is chromosomes in a variety of tissues), which is associated
known as closed Mad2. Mad1/Mad2 can transiently with microcephaly (decreased brain size) and an
bind soluble Mad2 molecules (known as open Mad2), increased cancer risk.
load them onto Cdc20 in the closed safety belt conforma-
tion (this loading probably occurs at kinetochores), then
release them to form the soluble MCC of Mad2/Cdc20/
Metaphase
BubR1/Bub3. The MCC associates with the APC/CCdc20, When all the chromosomes have attained amphitelic
interfering with binding of cyclin B and other key sub- orientations and moved to positions roughly midway
strates. As each chromosome becomes attached to both between the two spindle poles, the cell is said to be in
poles of the spindle it stops producing MCC. When the metaphase (Fig. 44.12). The compact grouping of chro-
last chromosome has achieved a proper attachment, the mosomes at the middle of the spindle is referred to as
last source of MCC is extinguished, and entry into ana- the metaphase plate. In many cells, even though chro-
phase can proceed. mosomes remain, on average, balanced at the middle of
A. Metaphase B
Kinetochore
microtubules
Astral
microtubules
Interpolar
microtubules DNA
Cyclin B and Microtubules
securin degraded Chromosomes oscillate Centrosomes
FIGURE 44.12 INTRODUCTION TO METAPHASE. A, Summary of the major events of metaphase. B, Distribution of DNA (blue), microtu-
bules (red), and gamma tubulin (centrosomes [green]) in a metaphase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the
University of Dundees School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
A B C D
Spindle pole
E F G
P AP
AP P
2 m
2 min
Spindle pole
FIGURE 44.13 KINETOCHORE OSCILLATIONS BETWEEN P (POLEWARD) AND AP (AWAY FROM THE POLE) MOVEMENT DURING
LATE PROMETAPHASE AND ANAPHASE IN PTK1 (RAT KANGAROO) CELLS. AD, Images showing the movements of several pairs of sister
kinetochores, labeled with green fluorescent protein (GFP)-Cdc20 (green), combined with phase-contrast images of the cell (red). E and G, Higher-
magnification views of sister kinetochores (marked with dashed lines) in prometaphase and anaphase, respectively. F, Kymograph (collage of images
of a vertical strip showing the same two kinetochores at various time points during the movie) showing the movements of these two kinetochores.
Movements toward (P) and away from (AP) spindle poles are indicated. P movement involves microtubule shrinkage at the leading kinetochore and
microtubule growth at the trailing kinetochore (which is undergoing AP movement away from its associated kinetochore). Spindle poles are near
the top and bottom of panels E to G. (Micrographs courtesy E.D. Salmon, University of North Carolina, Chapel Hill.)
the spindle, they jostle one another and undergo numer-

A B C
ous small excursions toward one pole or the other
throughout metaphase (Fig. 44.13). 0 sec
Metaphase can also be defined biochemically as the
time of destruction of cyclin B and securin (Fig. 44.16),
Fluorescent tubulin
because this begins as soon as the last chromosome 10 sec speckles move
achieves amphitelic orientation. Degradation of cyclin A toward poles
begins earlier, at the entry into prometaphase, and is
largely complete before metaphase. Loss of securin initi- 20 sec
P P
ates a process leading to the separation of sister chroma-
tids and the onset of anaphase. P P P P
30 sec
Microtubule Flux Within the Metaphase Spindle
FIGURE 44.14 MICROTUBULE FLUX IN METAPHASE. A, Cells
Although the average length of the kinetochore micro- entering mitosis were injected with tubulin subunits modified chemi-
tubules is roughly constant during metaphase, the cally by attachment of a caged fluorescent dye. This dye becomes
microtubules change continuously in three ways. First, fluorescent after being irradiated with UV light. When cells entered
there is constant net addition of new tubulin subunits metaphase, the spindle was illuminated with a narrow stripe of UV
light, activating a narrow band of fluorescent tubulin subunits. With
(approximately 10 subunits per second) to the plus end
time, these subunits approach the spindle poles (P). Because the
of the microtubules, where they are attached to the length of kinetochore microtubules is constant during this time, the
kinetochore. Second, a comparable number of tubulin labeled tubulin molecules must migrate along the microtubules toward
subunits is continuously lost from the minus end of the pole (arrows). This can occur if new subunits are added to the
the kinetochore tubules at the spindle poles. There microtubule at the kinetochore and old subunits are removed at the
pole. B, Microtubule flux at metaphase in a Drosophila embryo visual-
fore, tubulin subunits slowly migrate through kineto-
ized by fluorescence speckle microscopy. Embryos were injected with
chore microtubules from the kinetochore to the pole very low levels of fluorescent tubulin, which appears as speckles dis-
(Fig. 44.14). This subunit flux or treadmilling in kineto- tributed along the microtubules. If a very sensitive camera is used,
chore microtubules is caused by microtubule depolymer- these speckles can be seen to move toward the poles, reflecting the
ization at the poles driven by kinesin-13 family members. flux in the underlying microtubules. Scale bar is 5m. C, Movement
of labeled tubulin speckles toward the spindle poles. (A, Courtesy
In addition, tubulin moves toward the poles as by micro-
Arshad Desai and the MBL Cell Division Group, Marine Biology Labo-
tubules are transported towards the pole (many nucle- ratory, Woods Hole, MA; from Mitchison TJ, Salmon ED. Mitosis: a
ated by the augmin complex) within the kinetochore history of division. Nat Cell Biol. 2001;3:E17E21. B, Courtesy Paul
fiber. All microtubules attached to each kinetochore Maddox and Arshad Desai, University of California, San Diego.)
change coordinately in length during chromosomal omitted here for simplicity) and Scc3. Additional proteins
oscillations. are required to stabilize the loading of this complex onto
DNA. Cells with mutations in cohesin components sepa-
rate sister chromatids prematurely in mitosis, resulting
Anaphase in chaotic chromosome missegregation. This system is
The separation of sister chromatids at anaphase is one of very ancient, as bacteria depend on an SMC-related
the most dramatic events of the entire cell cycle (Fig. protein for orderly chromosome segregation.
44.15). Sister chromatids move to opposite spindle poles A variety of evidence suggests that cohesin forms a
(anaphase A), and the poles move apart (anaphase B). ring with a diameter of 35nm, large enough to encircle
Anaphase is also the time when the mitotic spindle two sister chromatids like a lasso. In yeast, the complex
activates the cell cortex in preparation for cytokinesis. functions only if it binds chromosomes during DNA
Two forms of the APC/C (see Fig. 40.15) trigger the replication. Cohesin accumulates at preferred sites on
transition from metaphase to anaphase by degrading key the chromosomes, often near centromeres in budding
proteins. APC/CCdc20 targets cyclin B for degradation, yeast or in regions of heterochromatin in fission yeast.
causing Cdk activity to fall (see Fig. 40.17). This decline In vertebrates, most cohesin dissociates from the chro-
in Cdk activity allows for activation of APC/CCdh1, because mosome arms by late metaphase, owing to the action of
Cdh1 phosphorylated by Cdk1 kinase cannot bind to the the protein kinases Plk1 and Aurora B. Importantly, a
APC/C. APC/CCdh1 targets polypeptides whose destruc- critical fraction remains associated with heterochromatin
tion by the proteasome is required for the cell to exit flanking centromeres where it is protected from cleav-
from mitosis and return to interphase. APC/CCdh1 remains age by shugoshin until the onset of anaphase (see follow-
active during G1 phase, where it is essential for the ing paragraphs and Chapter 45).
licensing of DNA replication origins (see Fig. 42.28). Sequential cleavage of two key proteins triggers sister
chromatid separation at anaphase. This proteolysis makes
Biochemical Mechanism of anaphase onset an irreversible transition. The first target,
Sister Chromatid Separation securin, inhibits the separase protease. After the last
Separation of sister chromatids is regulated by the chro- chromosome forms an amphitelic attachment to the
mosomes themselves, not by the mitotic spindle. Under spindle, the spindle checkpoint is silenced. This allows
certain circumstances, sister chromatids can separate in APC/CCdc20 to tag securin with ubiquitin, leading to its
the absence of microtubules, ruling out a requirement destruction by proteasomes throughout metaphase. When
for forces from the spindle in the process. securin levels fall below a critical threshold, separase is
Three factors regulate sister chromatid separation: a unleashed to cleave the Scc1 subunit of cohesin. Cleavage
protein complex known as cohesin, a protease known of Scc1 breaks the cohesin ring, allowing the sister
as separase, and an inhibitor of separase known chromatids to separate triggering the onset of anaphase
as securin (Fig. 44.16). This system is conserved from (Fig. 44.16B).
yeast to human. Chapter 8 discusses the functions of Efficient Scc1 cleavage requires that the protein be
cohesin in interphase. phosphorylated near its cleavage site. This allows a
Cohesin is a complex of four proteins that resembles mode of regulation where shugoshin (Japanese for
the condensin complex (see Fig. 8.18). Like condensin, guardian spirit) recruits PP2A to centromeres. PP2A
cohesin has two large subunits from the SMC ATPase keeps Scc1 dephosphorylated. This inhibits its cleavage
family. These proteins, SMC1 and SMC3, are complexed and protects cohesin until shugoshin is released follow-
with proteins called Scc1 (which has other names ing amphitelic attachment of the chromosome. This
A. Anaphase B
Sister chromatids separate
Cohesin Anaphase A: Chromatids approach
degrades poles (APC/CCdc20 active)
Interdigitated interpolar
microtubules bundled by PRC1
and stem body material to
form central spindle
DNA
Anaphase B: Spindle poles Microtubules
migrate apart (APC/C Cdh1active) Centrosomes
FIGURE 44.15 INTRODUCTION TO ANAPHASE. A, Summary of the major events of anaphase. B, Distribution of DNA (blue), microtubules
(red), and gamma tubulin (centrosomes [green]) in a mid-anaphase human cell. APC/C, anaphase-promoting complex/cyclosome; PRC1, protein
regulated in cytokinesis 1. (B, Images were recorded by Dr. Melpomeni Platani on the University of Dundees School of Life Sciences Imaging
Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
Hinge
A. S phase
Replication
fork
Cohesin
complex
Smc3
Smc1
Scc1
SA1
B C D E
Separase
Chromatin
loops Securin
APC/C
Ubiquitin Sister
Cohesin chromatids
Proteasome separate
Sister
chromatids
Active
Mitotic separase
chromosome cleaves Scc1
Metaphase/
anaphase
S phase Early mitosis transition Anaphase
FIGURE 44.16 REGULATION OF SISTER CHROMATID PAIRING BY THE COHESIN COMPLEX. AB, The cohesin complex forms a
35-nm diameter ring that links sister chromatids during DNA replication. At anaphase onset, degradation of its securin inhibitor liberates active
separase enzyme. CE, Separase then cleaves cohesin subunit Scc1, and the two sister chromatids are freed to separate from one another and
move toward opposite spindle poles.
mechanism is absolutely essential during meiosis, as Microtubule disassembly on its own can move chro-
without it, it would not be able to segregate homologous mosomes (see Fig. 37.8). Energy for this movement
chromosomes from each other (see Fig. 45.12). comes from hydrolysis of GTP bound to assembled
Securin can act as an oncogene in cultured cells tubulin, which is stored in the conformation of the
and is overexpressed in some human pituitary tumors. lattice of tubulin subunits. Microtubule protofilaments
Overexpression of securin may disrupt the timing of are straight when growing, but after GTP hydrolysis
chromosome segregation, leading to chromosome loss protofilaments are curved, so they peel back from the
and, ultimately, contributing to cancer progression. ends of shrinking microtubules (see Fig. 34.6). Several
kinesin motors influence the dynamic instability of the
Mitotic Spindle Dynamics and Chromosome spindle microtubules. Members of the kinesin-13 class,
Movement During Anaphase which encircle microtubules near kinetochores and at
Anaphase is dominated by the orderly movement of spindle poles, use adenosine triphosphate (ATP) hydro-
sister chromatids to opposite spindle poles brought lysis to remove tubulin dimers and promote microtubule
about by the combined action of motor proteins and disassembly rather than movement.
changes in microtubule length. There are two compo- Kinetochores are remarkable in their ability to hold
nents to anaphase chromosome movements (Fig. 44.15). onto disassembling microtubules. In straight (growing)
Anaphase A, the movement of the sister chromatids to microtubules, the Ndc80 complex is mostly responsible
the spindle poles, requires a shortening of the kineto- for microtubule binding. It binds to the interface between
chore fibers. During anaphase B, the spindle elongates, and tubulin subunits. This interface bends in curved
pushing the spindle poles apart. The poles separate (shrinking) microtubules, so Ndc80 cannot bind. This
partially because of interactions between the antiparallel could allow it to redistribute onto straight sections of
interpolar microtubules of the central spindle and par- the lattice and thereby move away from the curved
tially because of intrinsic motility of the asters. Most cells protofilaments at the disassembling end. In metazoans
use both components of anaphase, but one component the Ska complex in the outer kinetochore binds and
may predominate in relation to the other. tubulin subunits away from the interface, so it can bind
to curved (disassembling) protofilaments. At yeast kineto- kinetochore microtubules toward the pole. In these
chores the Dam1 ring (green in Fig. 8.21) couples the cells, subunit flux accounts for only 20% to 30% of
kinetochore to disassembling microtubules. chromosome movement during anaphase A, and this flux
Anaphase A chromosome movement involves a com- is dispensable for chromosome movement. In Drosophila
bination of microtubule shortening and translocation of embryos, in which subunit flux accounts for approxi-
the microtubule lattice that result from flux of tubulin mately 90% of anaphase A chromosome movement, the
subunits (Fig. 44.14). The contributions of the two chromosomes catch up with a marked region of the
mechanisms vary among different cell types. When living kinetochore fiber slowly, if at all.
vertebrate cells are injected with fluorescently labeled Anaphase B appears to be triggered at least in part by
tubulin subunits, the spindle becomes fluorescent (Fig. the inactivation of the minus-enddirected kinesin-14
44.17). If a laser is used to bleach a narrow zone in the motors, so that all the net motor force favors spindle
fluorescent tubulin across the spindle between the elongation. Four factors contribute to overall lengthen-
chromosomes and the pole early in anaphase, the chro- ing of the spindle: release of sister chromatid cohesion,
mosomes approach the bleached zone much faster than sliding apart of the interdigitated half-spindles, microtu-
the bleached zone approaches the spindle pole. This bule growth, and intrinsic motility of the poles them-
shows that the chromosomes eat their way along the selves (Fig. 44.7). During the latter stages of anaphase B,
the spindle poles, with their attached kinetochore micro-
tubules, appear to move away from the interpolar
microtubules as the spindle lengthens. This movement
A of the poles involves interaction of the astral microtu-
Photobleached
zone bules with cytoplasmic dynein molecules anchored at
the cell cortex.
Time Anaphase B spindle elongation is accompanied by
reorganization of the interpolar microtubules into a
Microtubule
disassembly
highly organized central spindle between the separat-
ing chromatids (Fig. 44.15). Within the central spindle,
an amorphous dense material called stem body matrix
stabilizes bundles of antiparallel microtubules and holds
B 42 70 212 422 together the two interdigitated half-spindles. Proteins
concentrated in the central spindle help regulate cytoki-
nesis. One key factor, PRC1 (protein regulated in cytoki-
nesis 1), is inactive when phosphorylated by Cdk kinase
and functions only during anaphase when Cdk activity
declines and phosphatases remove the phosphate groups
5 m
placed on target proteins by Cdks and other mitotic
kinases. PRC1 directs the binding of several kinesins to
C 40 92 210 436
the central spindle. The kinesin KIF4A targets Aurora B
kinase to a particular domain of the central spindle,
where phosphorylation of key substrates then regulates
spindle elongation and cytokinesis.
How can protein kinases such as Aurora B continue
to function during anaphase while protein phosphatases
are removing phosphate groups placed there by Cdks
and, indeed, Aurora B during early mitosis? One answer
FIGURE 44.17 CHROMOSOMES MOVE ON SHRINKING is that the phosphatase activity is highly localized, con-
MICROTUBULES DURING ANAPHASE. A, Mitotic cells were trolled by specific targeting subunits. Cdk phosphoryla-
injected with a fluorescently labeled tubulin that was incorporated into tion can inhibit targeting subunits such as the exotically
the spindle. Just after anaphase onset, a laser was used to photo-
named Repo-Man (recruits PP1 onto mitotic chromatin
bleach a stripe (white) across the spindle near the upper pole. The live
cell was monitored over time by fluorescence (B) and phase-contrast at anaphase) from binding protein phosphatase 1 or
(C) microscopy. In this mammalian cell, the chromosomes approach localizing to targets, such as chromatin in early mitosis.
the bleached stripe much faster than the stripe approaches the spindle When Cdk activity drops, Repo-Man (and other similar
pole. In other organisms with higher rates of microtubule flux in their targeting subunits) is dephosphorylated, and now targets
spindles, the bleached zone would also move appreciably toward the
PP1 to chromatin, where it removes phosphates placed
pole. The numbers are time in seconds. (BC, From Gorbsky GJ,
Sammak PJ, Borisy GG. Microtubule dynamics and chromosome there by Aurora B in the CPC. As long as phosphatases
motion visualized in living anaphase cells. J Cell Biol. 1988;106:1185 are not specifically targeted to the cleavage furrow,
1192, copyright The Rockefeller University Press.) Aurora B can continue to control events there during
mitotic exit by phosphorylating key target proteins scaffold protein ELYS to chromatin. ELYS can recognize
required for cytokinesis. DNA regions rich in A:T base pairs, so it is likely to
bind directly to the DNA. ELYS then recruits other
components of the nuclear pore scaffold and nuclear
Telophase pore trans-membrane proteins. The pore subsequently
During telophase, the nuclear envelope reforms on the matures as various peripheral components and elements
surface of the separated sister chromatids, which typi- of the permeability barrier are added.
cally cluster in a dense mass near the spindle poles (Fig. The mechanism of nuclear membrane reassembly is
44.18). Some further anaphase B movement may still debated. In cells where nuclear membranes fragments
occur, but the most dramatic change in cellular structure into vesicles during mitosis, a Ran-GTPdependent
at this time is the constriction of the cleavage furrow and pathway directs at least two discrete populations of
subsequent cytokinesis. vesicles to chromatin where they fuse to reform the
nuclear envelope. In cells where the nuclear membrane
Reassembly of the Nuclear Envelope is absorbed into the endoplasmic reticulum during
Nuclear envelope reassembly begins during anaphase mitosis, reassembly involves lateral movements of mem-
and is completed during telophase (Fig. 44.19). As in brane components within the membrane network and
spindle assembly, Ran-GTP promotes early steps of their stabilization at preferred binding sites at the periph-
nuclear envelope assembly at the surface of the chromo- ery of the chromosomes.
somes by releasing key components sequestered by Lamin subunits disassembled in prophase are recycled
importin . These include several nuclear pore com to reassemble at the end of mitosis. Lamina reassembly
ponents, and one of the earliest events in nuclear enve- is triggered by removal of mitosis-specific phosphate
lope reassembly involves binding of the nuclear pore groups and methyl-esterification of several COOH side
A. Telophase B
Cleavage plane
specified
Nuclear envelope
reassembles around Organized
chromosomes central spindle
assembles
Poles DNA
continue Microtubules
to separate Centrosomes
FIGURE 44.18 INTRODUCTION TO TELOPHASE. A, Summary of the major events of telophase. B, Distribution of DNA (blue), microtubules
(red), and -tubulin (centrosomes [green]) in a telophase human cell. (B, Images were recorded by Dr. Melpomeni Platani on the University of
Dundees School of Life Sciences Imaging Facility OMX 3DSIM Microscope and stored and processed in OMERO.)
A B C
0 min 810 min 333 nm 25 min
FIGURE 44.19 SCANNING ELECTRON MICROSCOPY OF THE STAGES OF ASSEMBLY OF MEMBRANE VESICLES ON THE
SURFACE OF CHROMOSOMES IN A XENOPUS EGG CYTOSOLIC EXTRACT. A cell lysate containing membrane vesicles was added to
isolated chromatin from Xenopus sperm, fixed, and then imaged by scanning electron microscopy. Each panel shows the time of incubation prior
to fixation. (Micrographs courtesy of K.L. Wilson, Johns Hopkins Medical School, Baltimore, MD. A and C, From Wiese C, Goldberg MW, Allen
TD, etal. Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent envelope smoothing event.
J Cell Sci. 1997;110:14891502.)
chains on lamin B (Fig. 44.6). Together with ELYS, B-type

lamins are among the earliest components of the nuclear
Cytokinesis
envelope to target to the surface of the chromosomes Cytokinesis divides a mitotic cell into two daughter cells
during mid-anaphase. Either at this time or shortly there- (Fig. 44.20). Cytokinesis depends on signals to specify
after, other proteins associated with the inner nuclear the cleavage plane (Fig. 44.21), assembly and constric-
membrane, including BAF, LAP2, and lamin B receptor tion of the contractile apparatus, specific alterations of
(see Fig. 9.10), join the forming envelope. Later during the cell membrane, and the final separation (abscission)
telophase when nuclear import is reestablished, lamin of the two daughter cells.
A enters the reforming nucleus and slowly assembles In animals, protozoa, and most fungi, a contractile
into the peripheral lamina over several hours in the ring of actin filaments and myosin-II guides the separa-
G1 phase. If lamin transport through nuclear pores is tion of daughter cells at the end of mitosis (Fig. 44.2).
prevented, chromosomes remain highly condensed fol- Myosin-II pulls on the ring of actin filaments, applying
lowing cytokinesis, and the cells fail to reenter the next tension to the plasma membrane, much like contraction
S phase. of smooth muscle (see Figs. 39.23 and 39.24). Because
A. Early cytokinesis B. Late cytokinesis C

Chromatin decondenses
New membrane Actomycin
inserted Nuclear substructures
contractile reform
Actomycin ring forms
Interphase microtubule
Midbody array reassembles
begins
to form Midbody
ESCRT III action leads to

separation (abscission) of the two cells
FIGURE 44.20 INTRODUCTION TO CYTOKINESIS. AB, Summary of the major events of cytokinesis. C, Distribution of DNA (blue),
microtubules (red), and -tubulin (centrosomes [green]) in a human cell undergoing cytokinesis. ESCRT, endosomal sorting complexes required
for transport. (C, Images were recorded by Dr. Melpomeni Platani on the University of Dundees School of Life Sciences Imaging Facility OMX
3DSIM Microscope and stored and processed in OMERO.)
A. Evidence that the cleavage furrow is positioned B. An organized central spindle is required for
midway between asters in eggs cleavage furrow formation and/or function
TOP VIEW
Glass rod pushed
down into egg
90
Sand dollar egg Microtubules

Chromosomes
Ectopic furrow Actin

ring
Profilin
Metaphase 1 Cytokinesis 1 Metaphase 2 Cytokinesis 2 Wild type mutant
FIGURE 44.21 IN EGGS, A CLEAVAGE FURROW FORMS MIDWAY BETWEEN SPINDLE ASTERS. IN ANIMAL CELLS, THE CENTRAL
SPINDLE IS IMPORTANT. A, A classic experiment in which a sand-dollar egg is caused to adopt a toroid shape. At cytokinesis 2, the egg
cleaves into four cells, and a furrow forms between the back sides of the two spindles. (For a description of this and other classic experiments
in cytokinesis, see the book by Rappaport in the Selected Readings list.) B, Left, A wild-type Drosophila spermatocyte undergoing cytokinesis,
with the contractile ring stained in yellow. Right, In a profilin mutant, no central spindle forms, and the cell fails to form a contractile ring.
(Micrographs courtesy Professor Maurizio Gatti, University of Rome, Italy. B, From Giansanti MG, Bonaccorsi S, Williams B, etal. Cooperative
interactions between the central spindle and the contractile ring during Drosophila cytokinesis. Genes Dev. 1998;12:396410.)
the contractile ring is confined to a narrow band of central spindle appeared to modulate the behavior of the
cortex around the equator, it forms a cleavage furrow, furrow signaled by the poles. We now know that the
constricting the plasma membrane locally like a purse central spindle does emit a positive signal directing a
string (Fig. 44.20). Signals from the mitotic spindle and cleavage furrow to form above it, while the poles con-
cell cycle machinery control the position of this ring (ie, tribute by focusing that furrow at a point on the cortex
the relative sizes of the two daughter cells) and the midway between them.
timing of its constriction. The molecular nature of the cleavage stimulus is now
Protozoa, animals, fungi, and plants use an evolution- beginning to be understood in animals. The following is
arily conserved set of components to implement differ- a simplified scenario:
ent strategies to separate daughter cells. For example, 1. During anaphase, overlapping microtubules between
both fission yeast and metazoan cells use signals from the separating chromatids establish an ordered array
polo kinase and a Rho-GTPase to direct the assembly of known as the central spindle. A key protein compo-
a contractile ring of actin, myosin-II, and other conserved nent of this array is a protein heterodimer known as
components, even though the yeast has a closed mitosis centralspindlin. Centralspindlin is normally seques-
and the metazoans have an open mitosis. In animal tered in the cytoplasm, but phosphorylation by the
cells, contractile ring constriction provides the force that CPC enables it to target to the central spindle. Dro-
remodels the cortex to generate the two daughter cells. sophila mutants that fail to form a central spindle
In contrast, in yeasts, which have a cell wall, contractile cannot initiate cytokinesis (Fig. 44.21B). In contrast,
ring constriction is thought to guide the orderly centrip- C. elegans embryos that lack a central spindle can
etal growth of the cell wall septum, which contributes initiate but not complete the process.
force to overcome turgor pressure and invaginate the 2. One of the components of centralspindlin recruits a
plasma membrane. Plants lack myosin-II, so they divide GEF (guanine exchange factor; see Figs. 4.6 and 4.7)
by targeted fusion of membrane vesicles to build a new for the small GTPase RhoA. This Rho-GEF, Ect2, also
cell wall rather than constricting a cleavage furrow (Box has a motif for targeting to the inside surface of the
44.1 and Fig. 44.26). These differences reflect the fact equatorial plasma membrane.
that widely divergent eukaryotes use variations of similar 3. Membrane associated Ect2 locally activates RhoA,
themes for cytokinesis. Cytokinesis in prokaryotes is which then stimulates localized actin filament assem-
genuinely different, since completely different proteins bly and activation of myosin-II to begin assembly of
are involved (Box 44.2 and Fig. 44.27). the contractile ring.
Although cytokinesis has been studied for more Signals from the poles of the mitotic spindle contribute,
than 100 years, it has posed a number of challenges due particularly in large invertebrate embryos, by confining
to its complexity at the molecular level. For example the zone of active RhoA to a narrow equatorial band
genetic analysis of fission yeast revealed more than between the separating sister chromatids.
150 genes that contribute to cytokinesis. RNAi-based
protein knockdown and molecular replacement analy Assembly and Regulation of the Contractile Ring
sis indicates that similar proteins participate in cytoki- Exposure of the cell cortex to the cleavage stimulus
nesis of Caenorhabditis elegans, Drosophila, and culminates in the assembly of a contractile ring consist-
vertebrate tissue culture cells. Cytokinesis research typi- ing of a very thin (0.1 to 0.2 m) array of actin filaments
cally employs living cells, although progress is being attached to the plasma membrane at many sites around
made toward reconstituting some aspects of the process the equator (Fig. 44.22). Polymerization of the actin fila-
in cell-free systems. ments depends on formins (see Fig. 33.14). Small, bipolar
filaments of myosin-II are interdigitated with actin fila-
Signals Regulating the Position of ments. The plasma membrane adjacent to this actin-
the Cleavage Furrow myosin ring undergoes alterations in its lipid composition
Elegant experimental data from classic studies on fertil- that may help recruit proteins important for the function
ized echinoderm eggs suggest that a cleavage stimulus, of the contractile ring.
emitted by the mitotic spindle, specifies the position of Membrane furrowing requires actin and the motor
the cleavage furrow midway between the poles and activity of myosin-II (see Fig. 36.7). In animals, the small
perpendicular to the long axis of the spindle, thereby GTPase RhoA regulates actin polymerization by formins
ensuring that the cleavage process separates the daugh- as well as constriction of the ring. Many other proteins
ter nuclei (Fig. 44.21). In fertilized eggs, the poles, with are required for cytokinesis to go to completion. In their
their large astral arrays of microtubules (see Fig. 6.4B), absence, furrowing begins, but the cleavage furrows
were regarded as the source of the cleavage stimulus, as ultimately regress, producing binucleated cells. These
furrows can be induced to form midway between two supporting proteins include anillin, actin filament cross-
poles, even when no chromosomes are present. In addi- linking proteins, the CPC (Fig. 44.10) and the central-
tion, a signal emitted by the bundled microtubules of the spindlin complex, among many others. Anillin helps
A. Early anaphase B. Late anaphase C
INCENP
Myosin II
Actin pointed ends
INCENP
Myosin II
Actin barbed ends

E. Myosin confocal
Central optical section

H. Contractile mechanism I. Equatorial section
G Actin
Myosin II
J. Grazing saggital section
FIGURE 44.22 ORGANIZATION OF THE CONTRACTILE RING. A, Organization of actin at the cell cortex prior to cytokinesis. B, Distribution
of actin and myosin at the start of ring contraction. C, INCENP (inner centromere protein) (red) concentrates at the site where the cleavage furrow
will form just before myosin (green). D, INCENP and myosin concentrate in the contractile ring during contraction. E, Confocal micrograph shows
the distribution of myosin in an optical cross-section contracting contractile ring. FG, Dividing invertebrate egg with DNA (blue) and actin (red)
in the contractile ring. H, Organization of actin and myosin filaments during cytokinesis. IJ, Electron micrographs showing actin filaments in
the contractile ring. Note the thick filaments that are thought to be myosin-II filaments (red arrowheads) and the thinner actin filaments (yellow
arrows). (CD, Courtesy William C. Earnshaw. E, I, and J, Courtesy P. Maupin, Johns Hopkins Medical School, Baltimore, MD. FG, Courtesy
Professor Issei Mabuchi, University of Tokyo, Japan. For reference, see Maupin P, Pollard TD. Arrangement of actin filaments and myosin-like fila-
ments in the contractile ring and actin-like filaments in the mitotic spindle of dividing HeLa cells. J Ultrastruct Res. 1986;94:92103; Maupin P,
Phillips CL, Adelstein RS, etal: Differential localization of myosin-II isozymes in human cultured cells and blood cells. J Cell Sci. 1994;107:30773090;
and Eckley DM, Ainsztein AM, MacKay AM, etal. Chromosomal proteins and cytokinesis. J Cell Biol. 1997;136:11691183.)
keep active myosin-II focused into an organized contrac- required for transport) complex, which has a key role in
tile ring throughout cytokinesis. the final separation of daughter cells (see later).
The CPC and the centralspindlin complex are both In fission yeast, with closed mitosis, the nucleus
required for animal cells to assemble the central spindle. determines the position of cleavage. Fission yeast assem
Consequently, if either of the two complexes is elimi- ble a contractile ring along a well-defined pathway by
nated in C. elegans, a contractile ring fails to form. Thus, recruiting proteins from cytoplasmic pools (Fig. 44.23).
they appear to contribute to the cleavage stimulus, and During interphase, assemblies of proteins called nodes
indeed, both require microtubules to localize to the site form on the inside of the plasma membrane around the
of cleavage furrow formation as originally shown for the middle of cell. Prior to mitosis, an anillin-like protein
cleavage stimulus. In addition, the CPC regulates the leaves the nucleus and joins these nodes. During pro-
timely completion of cytokinesis by blocking premature phase, myosin-II, a formin and other contractile ring
activation of the ESCRT (endosomal sorting complexes proteins join the nodes. When the formin polymerizes
Interphase
-60 Mid1p (anillin-like actin patches
protein) exits nucleus
Anillin-like
Cell wall
Formin Myosin-II
-10 Nodes containing
anillin, myosin-II and formin Profilin
assemble around equator
0 SPBs separate Nodes condense into

a contractile ring of
+5 Anaphase A actin filaments and myosin-II
Time (min)
+10 Anaphase B
elongates mitotic Contractile ring
spindle matures by addition of
actin binding proteins
+30 End Anaphase B
Signal from cell cycle via SIN

pathway triggers constriction of
+40 Constriction begins
contractile ring and deposition of
cell wall material to form a septum
Plasma membrane fusion

completes cytokinesis
+70 Constriction ends
FIGURE 44.23 CYTOKINESIS IN FISSION YEAST SCHIZOSACCHAROMYCES POMBE. During interphase, microtubules (red) position
the nucleus in the middle of the cell. Actin filaments concentrate in small patches (yellow) in the cortex at the two growing ends of the cell (see
Fig. 33.1). The mitotic spindle is inside the nucleus, as the nuclear membrane does not break down during mitosis. As the cell enters mitosis,
an anillin-like protein moves from the nucleus to the equatorial cortex, where it sets up nodes of proteins, including myosin-II and a formin. The
formin grows actin filaments (yellow), and myosin-II pulls the nodes together into a continuous contractile ring. At the end of anaphase a signaling
system consisting of a GTPase and three protein kinases (the septation initiation network [SIN]) triggers constriction of the contractile ring and
associated synthesis of new cell wall to form a septum. The septum is a three-layered structure, with the primary septum flanked by two secondary
septae. Digestion of the primary septum separates the daughter cells. (For reference, see Wu J-Q, Kuhn JR, Kovar DR, etal. Spatial and temporal
pathway for assembly and constriction of the contractile ring in fission yeast cytokinesis. Dev Cell. 2004;5:723734.)
actin filaments, myosin-II pulls the nodes together into a

ring around the equator of the cell (Fig. 44.23). Constriction of the Cleavage Furrow
Contractile ring assembly in animal cells shares many Contractile rings of echinoderm eggs produce enough
properties with fission yeast, but is less completely force to invaginate the plasma membrane and form the
understood. The decline in the activity of cell cycle cleavage furrow, although many details are still being
kinases at the onset of anaphase is part of the trigger, studied. Constriction of the ring probably involves a
since they inhibit centralspindlin components through sliding filament mechanism similar to muscle (see Figs.
metaphase. INCENP and anillin move from the inter- 39.9 and 39.23), but little is known about how the
phase nucleus to the cortex around the cell equator in contractile ring is attached to the plasma membrane.
early anaphase (Fig. 44.22). Formins and profilin polym- During the early stages of furrowing, contractile rings
erize some new actin filaments, but preexisting actin fila- maintain a constant volume, but then disassemble as
ments are recruited into the contractile ring from they constrict further.
adjacent areas of the cortex. Myosin-II is dispersed The role of myosin-II as the motor for cytokinesis was
throughout the cytoplasm until anaphase, when it con- established by microinjection of inhibitory antibodies
centrates in the cortex, especially around the equator into echinoderm embryos and confirmed by genetic
where the furrow forms. The myosin-II is derived from inactivation in the slime mold Dictyostelium. Slime mold
various interphase structures including stress fibers (see amoebas lacking the myosin-II heavy chain round up
Fig. 33.1) that break down during prophase. during mitosis and complete nuclear division but cannot
form a normal cleavage furrow. Mutant cells accumulate

A
many nuclei, because the mitotic cycle continues. Mutant
cells can divide on a substratum using pseudopods to
pull themselves apart into smaller cells.
Constriction of the contractile ring is regulated so that
it does not begin until after the onset of anaphase B,
when sister chromatids are well separated. In fission Midbody ring
yeast, a signaling pathway called the septation initiation B. Abscission (centralspindlin, anillin, cep55)
ESCRT I and ESCRT II
network (SIN) initiates constriction. Much less is known ESCRT III
Midbody
in other cells. Vps4
Microtubules
Abscission
As the contractile ring pulls the cell membrane inward, ESCRT I
the single cell that entered mitosis is gradually trans-

formed into two daughters joined by a thin intercellular
bridge (Fig. 44.20). This process requires a significant
net increase in the surface area of the cell. New plasma
FIGURE 44.24 ABSCISSION IN ANIMAL CELLS. Proteins
membrane is inserted adjacent to the leading edge of associated with overlapping bundles of microtubules form the midbody.
the furrow. The source of the new membrane appears The midbody ring links the midbody to the membrane, and nucleates
to be recycling endosomes (see Chapter 22), so addition the formation of ESCRT (endosomal sorting complexes required for
of membrane to the cleavage furrow is a specialized form transport) III filaments that constrict the membrane to divide the
daughter cells.
of exocytosis. Fusion of vesicles providing the new
membrane depends on specific syntaxins, t-SNAREs
(soluble N-ethylmaleimide-sensitive factor attachment
protein receptors) (see Fig. 21.15) that promote vesicle
fusion along the secretory pathway.
The plasma membrane in the cleavage furrow has a
discrete composition. In budding yeast, this compart-
ment is delineated by rings made from polymers of
septins, a family of GTP-binding proteins recruited by
anillin. Septins are essential for cytokinesis in Saccharo-
myces cerevisiae but not fission yeast.
In most animal cells, constriction of the cleavage
furrow ultimately reduces the cytoplasm to a thin inter-
cellular bridge between the two daughter cells. The
FIGURE 44.25 INCOMPLETE CYTOKINESIS IN A DRO-
intercellular bridge contains a highly ordered, antiparal-
SOPHILA EGG CHAMBER LEAVES CELLS JOINED BY RING
lel array of microtubules derived from the spindle with CANALS. Colocalization of actin (red) and the ring canal protein
a dense knob, the midbody, at its center (Fig. 44.20). HtsRC (green) in the ring canals makes them appear yellow. In the
Isolated midbodies contain more than 160 proteins, with Drosophila egg chamber, ring canals connect nurse cells to each other
approximately one-third involved in various aspects of and to the oocyte. Late in oocyte development, nurse cell contraction
forces their cytoplasmic contents through the ring canals and into the
membrane trafficking.
oocyte. This helps the oocyte gain the stockpile of components that
The midbody is encircled by a dense ring of proteins is needed for early development of the fly embryo. (Courtesy Andrew
that includes centralspindlin, anillin, and a centrosomal Hudson and Lynn Cooley, Yale University, New Haven, CT.)
protein known as Cep55. Interactions between anillin
and membrane-associated septin filaments tether the
membrane to the ring. Cep55 recruits the ESCRT (endo- Ultimately, disassembly of the ESCRT filaments leads to
somal sorting complexes required for transport) III separation of the two daughter cellsabscission.
complex, proteins with important roles in vesicle In some tissues, intercellular bridges remain open as
budding events such as formation of multivesicular ring canals. After several rounds of nuclear division
bodies (see Chapters 22 and 23). In the intercellular with incomplete cytokinesis, the network of cells main-
bridge, ESCRT III forms a helical filament that spirals tains cytoplasmic continuity as each former contractile
around the inner surface of the membrane, becoming ring matures into a larger ring canal. During Drosophila
more and more constricted as it grows away from the oogenesis, four rounds of nuclear division with persis-
midbody (Fig. 44.24). ESCRT III also recruits factors that tent ring canals creates 15 nurse cells, all in continuity
disassemble the bundled microtubules in the intercellu- with the oocytes (Fig. 44.25). The cytoplasmic continu-
lar bridge, allowing the membrane to constrict further. ity through ring canals allows nurse cells to transfer
BOX 44.1 Cytokinesis in Plants
Chromosome segregation is similar in plants and animals, but materials (see Fig. 32.13), move along phragmoplast micro-
cytokinesis is very different because plants lack myosin-II tubules to the equator, where they fuse due to the action of
and do not form a conventional contractile ring (Fig. 44.26). cytokinesis-specific soluble N-ethylmaleimide-sensitive factor
Myosin-II appeared during evolution in the common ances- (NSF) attachment protein receptor (SNARE) proteins (see
tor of amoebas, fungi and animals, after branching from Fig. 21.15), forming a membrane network that becomes the
plants (see Fig. 2.4B). Plants also lack dynein, so microtubule new plasma membrane and laying down the material that
dynamics in mitosis are regulated by some of the more than will become the new cell wall. Dynamin-related proteins also
20 different plus-end and minus-enddirected kinesins that participate in shaping the newly forming plasma membrane.
are expressed in mitotic cells. In a further difference from Thus, the membrane fusion machinery used for cytokinesis
animals, plants also lack centrosomes, and during interphase, by eukaryotes likely came from the last eukaryotic common
microtubules radiate out from the surface of the cell nucleus ancestor. Actin filaments polymerized by formins and
in all directions. In mitosis, the spindle does not focus to myosin-VIII help position the phragmoplast in the cell. As
sharp poles at metaphase; instead, it assumes a barrel shape the zone of newly deposited membrane expands radially, the
with broad, flat poles. Early in mitosis, a band of microtu- ring of microtubules surrounding it similarly expands. Even-
bules and actin filaments forms around the equator of the tually, the new membrane reaches the lateral cell periphery,
cell adjacent to the nucleus. This so-called preprophase band and fusion with the plasma membrane separates the two
disassembles as cells enter prometaphase. Because the entire daughter cells. The cortical division site, not the spindle,
cell cortex is covered by a meshwork of actin filaments, determines the site of cleavage. This was shown by centri-
disassembly of the preprophase band actually leaves an actin- fuging mitotic cells to displace the spindle from the central
poor zone in a ring where cytokinesis will ultimately occur. location where it initially formed. Late in mitosis, the phrag-
This is called the cortical division site, and it is marked by moplast formed at the midzone of the displaced spindle, but
the tethering of specific kinesin motors. In late anaphase, this phragmoplast then migrated to the plane of the prepro-
two nonoverlapping, antiparallel arrays of microtubules phase band, where cytokinesis occurred. Since plant cells
form over the central spindle. This structure, the phragmo- have cell walls and do not move, the orientation of cleavage
plast, gradually expands laterally until it makes a mirror- planes critically determines the morphology of the organism.
symmetric double disk of short microtubules oriented The hormone auxin can influence cleavage, giving rise to
parallel to the spindle axis with their plus ends abutting the asymmetric division of daughter cells, but the underlying
plane of cell cleavage. Golgi vesicles, containing cell wall mechanism is not yet known.
Chromosomes Cytokinesis in higher plants Early phragmoplast Late phragmoplast
Cortical
actin
Preprophase Cortical
band actin- Golgi
(microtubules) depleted vesicles
zone
Prophase Metaphase Early cytokinesis Late cytokinesis Daughter cells
FIGURE 44.26 CYTOKINESIS IN HIGHER PLANTS. See the text for details.
their cytoplasm into the developing egg, thus greatly Yeast cells use a signaling pathway to terminate
increasing its stockpile of proteins and messenger RNAs mitosis, promote contraction of the contractile ring, and
available for use in early development. In mammals, initiate septation. These pathways, called the mitotic exit
incomplete cytokinesis in the testis results in ring canals network (MEN) in budding yeast and the SIN in fission
connecting several hundred developing sperm cells. yeast, involve a small GTPase and protein kinases. Cdk
kinase activity suppresses the pathway until anaphase,
when Cdk activity drops sharply. The MEN GTPase is
Exit From Mitosis associated with one spindle pole body (the yeast version
To exit from mitosis, cells must inactivate the Cdk1 of the centrosome), while its key regulator, a GTP
kinase. This reverses the biochemical and structural exchange factor, is located in the bud. Elongation of the
changes that are characteristic of mitosis and prepares mitotic spindle during anaphase B moves the GTPase
the cell for proliferation in the next cell cycle. into the bud, where it is activated.
BOX 44.2 Cytokinesis in Bacteria
The strategy for cytokinesis in bacteria is similar to that in to the cell cortex, where it inhibits Z-ring formation. MinE
animal cells (Fig. 44.27), but the molecules are completely is an antagonist of MinC/MinD action. This system works in
different. Cleavage of most bacterial cells depends on a truly remarkable way. MinE forms a ring at the cell equator
a ring of the FtsZ protein (filamentous temperature-sensitive; that migrates along the inner surface of the cell membrane
mutants in fts genes cannot divide and make long filaments until it reaches the end of the cell, at which point it disas-
on cells). This is called the Z ring. FtsZ is the prokaryotic sembles. The ring then reforms in the center of the cell and
homolog of eukaryotic tubulins, but it assembles into fila- sweeps toward the other end of the cell. As it moves, MinE
ments rather than tubules. As for tubulins (see Fig. 34.4), inactivates the MinC/MinD inhibitory complex on the cell
FtsZ polymerization requires bound GTP and hydrolysis of cortex. The inhibitory complex rapidly reestablishes itself on
this GTP destabilizes the polymers. Although purified FtsZ the cell cortex behind the moving MinE ring. It takes
forms rings that use energy from GTP hydrolysis to deform approximately 2 minutes for each sweep of the MinE ring
lipid vesicles, the main function of the Z ring seems to be to along half of the cell, and this cycle is repeated continuously
coordinate the assembly of a complex of proteins (divisome) until the FtsZ ring assembles at the cell center. Bacillus
including an actin homolog FtsA and number of transmem- subtilis uses an alternative mechanism to position the Z ring
brane proteins. The transmembrane proteins synthesize cell for cytokinesis.
wall materials to form the cleavage furrow. Chloroplasts use a homolog of FtsZ for their division, and
The Z ring is positioned at the cell equator of Escherichia FtsZ has been detected in mitochondria of certain primitive
coli by the action of three gene products: MinC, MinD, and eukaryotes. Mitochondria of higher eukaryotes appear to use
MinE (minicell mutants divide at inappropriate locations and another GTPase, dynamin, to coordinate their fission (see
give birth to tiny cells). MinD is an enzyme that recruits MinC Biogenesis of Mitochondria in Chapter 19).
Min C/D Min E Cytokinesis in E. coli FtsZ ring

inhibitor Nucleoid
2 minutes
Zone of minimal
Min C/D
FIGURE 44.27 CYTOKINESIS IN THE BACTERIUM ESCHERICHIA COLI. See the text for details.
The MEN kinases downstream of the GTPase activate throughout cells by their specificity, determining sub-
the phosphatase Cdc14p by releasing it from sequestra- units PP2A and PP1 remove many of the phosphates
tion in the nucleolus. Cdc14p inhibits Cdk kinase activity placed on target proteins by the mitotic kinases. Targets
in two ways: (a) it inhibits the degradation of a Cdk include chromatin, where phosphorylation during
inhibitor protein, and (b) it dephosphorylates Cdh1, mitosis had displaced factors involved in both gene
which binds the APC/C and triggers the degradation of activation and repression. Removal of those phosphates
B-type cyclins and other proteins. Cdc14p also triggers allows the interphase regulation of gene expression to
other events during anaphase, including the transfer of resume. Dephosphorylation of other targets allows
chromosomal passenger proteins to the central spindle. intermediate filaments to reform, nuclear envelope reas-
In metazoans mitotic exit is triggered by the inactiva- sembly plus the resumption of RNA transcription, protein
tion of Cdk1 and other mitotic kinases. This transition translation, and membrane trafficking.
is irreversible, in part because cyclins and Aurora
(and other kinases) are degraded. PP2A and its inhibitory
ACKNOWLEDGMENTS
kinase Greatwall (see Chapter 40) replace Cdc14
in mitotic regulation in metazoans. Greatwall activity We thank David Burgess, Iain Cheeseman, Per Paolo
requires Cdks, so when Cdk activity declines, PP2A DAvino, Arshad Desai, Tatsuo Fukagawa, Gary Gorbsky,
is released from inhibition. When directed to targets Karen Oegema, Jonathon Pines, and Graham Warren for
their suggestions on revisions to this chapter. We thank Jrgens G. Plant cytokinesis: Fission by fusion. Trends Cell Biol. 2005;
the Dundee Imaging Facility for access to the OMX and 15:277-283.
McIntosh JR. Mitosis. Cold Spring Harb Perspect Biol. 2016;(in press).
help with microscopy. Mller S, Jrgens G. Plant cytokinesisno ring, no constriction but
centrifugal construction of the partitioning membrane. Semin Cell
SELECTED READINGS Dev Biol. 2016;53:10-18.
Nasmyth K, Haering CH. Cohesin: its roles and mechanisms. Annu Rev
Carmena M, Wheelock M, Funabiki H, etal. The chromosomal pas- Genet. 2009;43:525-558.
senger complex (CPC): from easy rider to the godfather of mitosis. Qian J, Winkler C, Bollen M. 4D-networking by mitotic phosphatases.
Nat Rev Mol Cell Biol. 2012;13:789-803. Curr Opin Cell Biol. 2013;25:697-703.
Collas P, Courvalin J-C. Sorting nuclear membrane proteins at mitosis. Rappaport R. Cytokinesis in Animal Cells: Developmental and Cell
Trends Cell Biol. 2000;10:5-8. Biology Series. Cambridge, England: Cambridge University Press; 1996.
Glotzer M. Cytokinesis in metazoa and fungi. Cold Spring Harb Per- Snchez-Huertas C, Lders J. The augmin connection in the geometry
spect Biol. 2016;(in press). of microtubule networks. Curr Biol. 2015;25:R294-R299.
Green RA, Paluch E, Oegema K. Cytokinesis in animal cells. Annu Rev Sharp DJ, Rogers GC, Scholey JM. Microtubule motors in mitosis.
Cell Dev Biol. 2012;28:29-58. Nature. 2000;407:41-47.
Haeusser DP, Margolin W. Splitsville: structural and functional insights Stukenberg PT, Burke DJ. Connecting the microtubule attachment
into the dynamic bacterial Z ring. Nat Rev Microbiol. 2016;14: status of each kinetochore to cell cycle arrest through the spindle
305-319. assembly checkpoint. Chromosoma. 2015;124:463-480.

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CHAPTER 44

Mitosis and Cytokinesis

M itosis is the division of a somatic cell (a vegetative

A. Interphase B. Prophase C. Prometaphase D. Metaphase

Centrosomes separate Begins with nuclear Chromosomes align

E. Anaphase A F. Anaphase B G. Telophase H. Cytokinesis

Condensins are required to disassemble the topologically

A. Early prometaphase C. Late prometaphase E. Kinetochore attachments

1. Nuclear envelope disassembles 4. Chromosome slides rapidly poleward Amphitelic (correct)

A. Interphase B. Mitosis C D. Lamins A and C E. Lamin B

Chromosomes Vesicles derived Lamin B

Cytoplasmic dynein moves

Stabilized Motors sort Other motors and

Time Mad1 Mad2

the spindle, they jostle one another and undergo numer-

0 min 810 min 333 nm 25 min

chains on lamin B (Fig. 44.6). Together with ELYS, B-type

A. Early cytokinesis B. Late cytokinesis C

ESCRT III action leads to

Sand dollar egg Microtubules

Ectopic furrow Actin

A. Early anaphase B. Late anaphase C

Actin pointed ends

Actin barbed ends

Central optical section

J. Grazing saggital section

0 SPBs separate Nodes condense into

+30 End Anaphase B

Signal from cell cycle via SIN

Plasma membrane fusion

actin filaments, myosin-II pulls the nodes together into a

form a normal cleavage furrow. Mutant cells accumulate

the single cell that entered mitosis is gradually trans-

BOX 44.1 Cytokinesis in Plants

Chromosomes Cytokinesis in higher plants Early phragmoplast Late phragmoplast

Prophase Metaphase Early cytokinesis Late cytokinesis Daughter cells

BOX 44.2 Cytokinesis in Bacteria

Min C/D Min E Cytokinesis in E. coli FtsZ ring

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