Perspective
Perspective
Cell Cycle 9:4, 715-719; February 15, 2010; © 2010 Landes Bioscience
Emerging functions of force-producing kinetochore motors
Yinghui Mao, Dileep Varma and Richard Vallee*
Department of Pathology and Cell Biology; Columbia University College of Physicians and Surgeons; New York, NY USA
M
ore than two decades of research
has resulted in the identification
of some 60 microtubule motor proteins,
several of which have been implicated in
mitosis. Although some kinesin superfamily proteins function as microtubule
depolymerases at kinetochores, such as
Kinesin-8 and -13, it is now appreciated
that there are only two force-producing
kinetochore associated motors, the plus
end-directed microtubule motor CENP-E
and the minus end-directed microtubule
motor cytoplasmic dynein. Defining their
roles at kinetochores has been hampered
by the complexity of mitosis itself, and a
multiplicity of mitotic roles, at least for
cytoplasmic dynein. Nonetheless, recent
advances have served to define the primary roles of the two kinetochore motors
in detail.
Kinetochore Motors
Key words: CENP-E, checkpoint,
dynein, microtubule, mitosis, motor
Submitted: 11/20/09
Revised: 11/23/09
Accepted: 11/30/09
Previously published online:
www.landesbioscience.com/journals/cc/
article/10763
*Correspondence to: Richard Vallee;
Email: [email protected]
www.landesbioscience.com
Cytoplasmic dynein is a 1.3 mDa multisubunit complex of heavy, intermediate, light intermediate and light chains,
involved in a very broad array of cellular
functions, from vesicular and macromolecular transport to directed cell migration.1 During mitosis dynein is found at
kinetochores, spindle poles, and the cell
cortex, and has been implicated in spindle
assembly and orientation, chromosome
behavior, and mitotic checkpoint control. Cytoplasmic dynein is a fast motor
(1–3 µm/sec), but can also serve in much
slower functions.1
CENP-E is a member of the kinesin-7
family with a molecular mass of 310 kDa,
containing an N-terminal conserved 35
kDa motor domain. It has a 230 nm-long
flexible coiled-coil tail in between its motor
and C-terminal globular kinetochore-
Cell Cycle
binding domains,2 and includes PEST
sequences possibly for regulated degradation at the end of mitosis. CENP-E
functions exclusively at the kinetochore.
Single-molecule assays have indicated
CENP-E to be very slow (∼8 nm/sec) and
highly processive.2,3
Recruitment of Motors to
Kinetochores
Cytoplasmic dynein appears at kinetochores in early prometaphase, and localizes
to the fibrous corona, a diffuse outer kinetochore-associated matrix.4 Kinetochore
dynein levels decline sharply upon microtubule end-on attachment.5,6 CENP-E also
localizes to the fibrous corona during prometaphase and persists to a lesser extent
during metaphase.7
CENP-F has emerged as a potential
master recruitment factor for cytoplasmic
dynein and CENP-E (Fig. 1). CENP-F
was pulled out in a CENP-E yeast twohybrid screen,8 appears at kinetochores
before CENP-E, and, at least in one study,
was required for CENP-E recruitment.9
Localization of CENP-E to kinetochores
may also depend on the mitotic checkpoint
protein kinases Aurora B, Mps1, Bub1 and
BubR1,10 but whether these factors control
CENP-E recruitment directly or through
a signaling cascade is unknown.
Cytoplasmic dynein recruitment to
kinetochores is dependent on a number
of protein factors (Fig. 1), each of which
lies downstream from CENP-F, including
dynactin, NudE and NudEL. CENP-F
appears at kinetochores first, and is necessary for recruitment of NudE and NudEL,
which, in turn, recruit dynein through its
intermediate and light chains.11-13 CENP-F
can also interact with the zwint-1 complex,
715
Figure 1. Disposition of kinetochore motor proteins. Kinetochore is shown with component proteins drawn roughly to scale. CENP-F is shown as a highly elongated, coiled-coil containing master
regulator, associated with the kinetochore outer plate through its C-terminus, and extending into
(and possibly in part comprising) the fibrous corona. CENP-E, itself containing a highly elongated
coiled-coil domain, interacts directly with CENP-F, whereas several complex recruitment factors
lie between CENP-F and cytoplasmic dynein.
which recruits ZW10, a component of the
“R22” complex, to kinetochores.14 ZW10
dynactin binds,15 which directly recruits
dynein.5,16 Curiously, p38 MAPK phosphorylation of the dynein intermediate
chains causes dynein to bypass dynactin
and bind directly to ZW10,17 suggesting
that dynein recruitment to kinetochores
may be highly regulated during mitotic
progression. Recent studies have identified
yet another protein, spindly, which functions downstream of the RZZ complex
and is also involved in dynein recruitment
to kinetochores.18
Functional Analysis of
Kinetochore Motors
CENP-E inhibition in cells19 or mutational analysis in mice20 each revealed late
prometaphase mitotic arrest associated
with unattached or monooriented kinetochores. The latter were typically found
near the spindle poles, strongly suggesting a role for CENP-E in congression to
716
the metaphase plate. This possibility has
been supported by analysis of cells subjected to a sequence of mitotic inhibitors
to allow more detailed analysis of congression movements.21 Monooriented chromosomes exhibited slow progression toward
the spindle equator via lateral attachments
with kinetochore microtubule fibers, an
effect impaired by CENP-E RNAi.
CENP-E has also been implicated
in mitotic checkpoint control. Purified
recombinant CENP-E interacted with
and stimulated the kinase activity of
recombinant BubR1.22,23 Using BubR1conditional knockout cells and BubR1
domain mutants, kinetochore-associated
BubR1 kinase activity has been demonstrated to be important for prolonged
mitotic checkpoint activation.24 CENPE-depletion from Xenopus egg extracts
caused checkpoint inactivation,25 and
reduced expression of CENP-E in mammalian cells caused premature anaphase
onset with one or a few polar chromosomes.20,23 These results support a role for
CENP-E-dependent BubR1 activation
in amplifying the checkpoint signal at
kinetochores to prevent single chromosome loss. Furthermore, the interaction
between CENP-E and BubR1 has also
been implicated in achieving metaphase
chromosome alignment.26
Kinetochore dynein was initially
implicated in a then little understood
phenomenon, the poleward movement
of chromatid pairs during early prometaphase27 (Fig. 2A). Chromosomes moved
laterally along spindle microtubules at
0.4–0.9 µm/sec, more consistent with fast
vesicular transport than chromosome segregation rates (∼1–2 µm/min). A role for
dynein was confirmed by ZW10 RNAi
and anti-dynein antibody injection.28
Poleward movement of paired chromatids
during prometa phase might seem premature, but it has been proposed to reflect the
initial “capture” of microtubules to facilitate engagement of chromosomes with the
mitotic spindle.
Inhibition of cytoplasmic dynein also
interferes with metaphase chromosome
alignment, not from defects in chromosome translocation, but, rather, in kinetochore-microtubule attachment. This event
represents a critical shift in the mode of
kinetochore-microtubule
interaction
from a lateral to an end-on association.
Chromosomes could reach the spindle
equator in dynein- or NudE- and NudELinhibited cells, but with imperfect metaphase alignment.12,29 Live analysis revealed
misorientation of kinetochore pairs relative
to the plane of the metaphase plate, and
a dramatic destabilization of kinetochore
microtubules upon cold treatment,12,29 an
effect thought to result from exposure of
kinetochore microtubule ends.
The switch to end-on microtubule
attachment correlates with the decrease
in kinetochore dynein levels.6 However,
some dynein remains at kinetochores as
judged by persistence of a dynein phosphoepitope.17 Furthermore, inhibition of
dynein by antibody injection or expression
of dynein tail constructs reversed end-on
kinetochore-microtubule attachment and
dramatically increased chromosome oscillations at the metaphase plate.29 Dynein
inhibition also reduced the rate of anaphase A poleward chromosome movement
by ∼30–40%.30,31 Whether these results,
Cell CycleVolume 9 Issue 4
Figure 2. Roles of CENP-E and cytoplasmic dynein at the kinetochore. (A) Dynein and CENP-E produce opposite directed forces laterally along
microtubule. (B) Kinetochore is shown in metastable condition, having reached plus end of microtubule through forces proposed to be generated by
CENP-E. Dynein is speculated to draw kinetochore into proper orientation to allow other factors to stabilize this model of kinetochore-microtubule
binding (From Varma et al. 2008). (C) “Dynein cycle”. With end-on microtubule attachment, dynein is thought to remove itself, CENP-E, and mitotic
checkpoint factors by transport along kinetochore microtubules. Return of dynein to kinetochores establishes a steady-state flux from prometaphase
through anaphase. (D) Speculative model suggests dynein-mediated tension within kinetochore may trigger release of dynein and associated factors
for poleward transport. (See text for details).
indeed, reflect a direct role for dynein in
poleward anaphase chromosome movement or defects in kinetochore-microtubule attachment remains to be tested more
fully.
Kinetochore Dynein Cycle
The marked loss of cytoplasmic dynein
from kinetochores upon end-on microtubule attachment has been proposed to
reflect dynein self-removal, along with
checkpoint proteins and other kinetochore
proteins, possibly including CENP-E.
This remarkable behavior was suggested
by a number of observations. First, Mad2
and a number of other kinetochore proteins have been observed to spread along
kinetochore-to-pole microtubules during
prometaphase and through anaphase,30,32
results supported by live imaging of poleward transport of fluorescent Mad2,30 and
GFP-tagged dynactin puncta.17 Although
movement was at very slow rates (∼2–3
µm/min), a role for dynein in this process was supported by its accumulation
and that of checkpoint proteins at kinetochores in dynein-inhibited cells.29,30
These results suggest a kinetochore dynein
www.landesbioscience.com
“cycle” involving dynein turnover through
a combination of self-removal and recruitment through a non-microtubule-based
mechanism (Fig. 2C). Depolymerization
of kinetochore microtubules also inhibits dynein loss, but, in addition causes a
gradual, dramatic expansion of the fibrous
corona to form almost circular super
kinetochore structure.5,6,30 These results
suggest an increase in a critical organizing component of the fibrous corona, but
whether it is dynein itself or an upstream
recruitment factor is unknown.
How kinetochore dynein knows to
leave the kinetochore is incompletely
understood. Dynein molecules seem likely
to experience increased load upon endon microtubule attachment (Fig. 2D), in
view of the drastic decrease in poleward
chromosome transport rates following
this event. Increased load may result from
opposite-directed tension on kinetochore
pairs, but dynein depletion occurs at
monoattached chromosomes as well, suggesting that forces within individual kinetochores are important. Despite persistent
turnover of tubulin subunits at kinetochore attached microtubule ends, poleward rates of chromosome movement are
Cell Cycle
far below those for unloaded dynein, suggesting that any remaining dynein forces
at the kinetochore encounter extreme
resistance. Under these conditions a weak
element in the dynein-kinetochore linkage
could be disrupted, or a signaling cascade
regulating release of dynein and associated proteins might be activated through
a tension-sensing mechanism.
Regulatory Pathways
SUMO-2/3 modification has recently
been reported to regulate CENP-E kinetochore localization.33 Global inhibition of SUMOylation resulted in a loss
of kinetochore associated CENP-E and
chromosome misalignment. Mutation of
the predicted SUMO-interacting motif
in CENP-E tail domain impaired its
ability to localize to kinetochores. How
SUMOylation of CENP-E may affect its
ability to interact with its known binding partners, such as BubR1, remains
uncharacterized.
A recent study has shown that the
CENP-E tail domain was able to completely block CENP-E motility in vitro34
due to a direct interaction with the motor
717
domain. Mps1 or cyclin B/Cdk1 phosphorylation of the CENP-E tail relieved the
autoinhibition and restored the motility
of CENP-E. The in vivo consequences of
such a possible control of CENP-E motility through phosphorylation remains to be
further explored.
In addition to its role in dynein recruitment to kinetochores, dynactin has been
reported to increase cytoplasmic dynein
processivity in in vitro assays,35 a role presumably mediated by the microtubulebinding domains present in the p150Glued
subunit. The large numbers of dynein
molecules likely to be associated with individual kinetochores would seem to obviate
the need for a processivity factor. Dynactin
could, therefore, serve an alternative role
in prometaphase microtubule capture, a
possibility supported by the striking association of dynactin with growing microtubule plus ends in interphase cells.36
LIS1, NudE, NudEL and NudC, are
additional regulatory factors in the cytoplasmic dynein pathway with roles in
kinetochore function.40 LIS1, the causative gene for the brain developmental disease lissencephaly, interacts with dynein,
NudE and NudEL, which are phosphorylated by cdk1, cdk5, Aurora kinase
and Erk1, suggesting a role for NudE
and NudEL as intermediaries of mitotic
dynein
regulatory
mechanisms.11-13
NudC contains a binding site for the
mitotic kinase Plk1, and is a substrate.37
Localization of NudC to kinetochores has
only been detected with a NudC phosphoepitope antibody, and a direct role in
regulation of kinetochore dynein has not
been established. Phosphomutant forms
of NudC fail to rescue the mitotic NudC
phenotype, suggesting that Plk1 phosphorylation is functionally important.38
Models for Coordinate Function of
Kinetochore Motors
Both cytoplasmic dynein and CENP-E
are associated with the outermost region
of the mitotic kinetochore, and each has
been implicated in the initial stages of
microtubule interaction. We suspect,
therefore, that they play complementary
roles in this process. The participation
of cytoplasmic dynein in prometaphase
poleward chromosome movement is
718
surprising, as it counteracts the ultimate
purpose of mitosis, the separation of sister
chromatids. A role for CENP-E in opposite-directed movement would counterbalance the effects of dynein during the early
stages of mitosis, a potentially important
function for CENP-E (Fig. 2A). Thus, the
oppositely-directed motor activities might
contribute to an assisted random-walk
mechanism, which could help ensure that
chromosomes explore the full extent of
spindle microtubules in a search for their
plus ends. The complementary behaviors
of the two motors are already supported
by phenotypic data: chromosomes are left
at the spindle poles in CENP-E inhibited cells, consistent with the persistence
of minus end-direct cytoplasmic dynein
activity. Conversely, chromosomes find
the spindle equator in dynein-inhibited
cells without stable kinetochore fibers,
consistent with the persistence of plus
end-directed motor activity.
The contribution of cytoplasmic
dynein to end-on kinetochore-microtubule
attachment29 is another unexpected role
for a minus end motor protein. However,
it can be explained by opposing forces
between motors or between kinetochores
of sister chromatids. As CENP-E pushes
paired chromatids toward, and perhaps
off, the plus ends of kinetochore microtubules, cytoplasmic dynein may provide
a restraining force, with end-on attachment as a metastable compromise (Fig.
2B). Alternatively, this outcome could
result from pushing and pulling forces
on biattached kinetochore pairs. Motormediated end-on microtubule binding
could precede and facilitate interaction of
kinetochores with other microtubule associated proteins, such as the Hec1/Ndc80
complex.
CENP-E and cytoplasmic dynein each
play important roles in mitotic checkpoint silencing, but through very different
mechanisms. Prior to microtubule capture, CENP-E binds to BubR1 to promote
checkpoint activation. Upon microtubule
binding to CENP-E, this effect should
be reversed.39 Presumably, this early stage
in checkpoint inactivation would remain
to be completed by cytoplasmic dynein
as it removes itself and BubR1, Mad2,
and other checkpoint proteins from the
kinetochore.
The potential complementarity of
CENP-E and dynein functions remains
largely hypothetical, but should represent an important area for future
investigation.
Acknowledgements
We thank Sarah Weil and Tania Nayak for
critical reading on the paper. Supported
by ACS RSG-09-027-01-CCG and NIH
GM47434.
References
1. Vallee RB, Seale GE, Tsai JW. Emerging roles for
myosin II and cytoplasmic dynein in migrating
neurons and growth cones. Trends Cell Biol 2009;
19:347-55.
2. Kim Y, Heuser JE, Waterman CM, Cleveland DW.
CENP-E combines a slow, processive motor and a
flexible coiled coil to produce an essential motile
kinetochore tether. J Cell Biol 2008; 181:411-9.
3. Yardimci H, van Duffelen M, Mao Y, Rosenfeld SS,
Selvin PR. The mitotic kinesin CENP-E is a processive transport motor. Proc Natl Acad Sci USA 2008;
105:6016-21.
4. Wordeman L, Steuer ER, Sheetz MP, Mitchison
T. Chemical subdomains within the kinetochore
domain of isolated CHO mitotic chromosomes. J
Cell Biol 1991; 114:285-94.
5. Echeverri CJ, Paschal BM, Vaughan KT, Vallee RB.
Molecular characterization of the 50-kD subunit of
dynactin reveals function for the complex in chromosome alignment and spindle organization during
mitosis. J Cell Biol 1996; 132:617-33.
6. King JM, Hays TS, Nicklas RB. Dynein is a transient
kinetochore component whose binding is regulated
by microtubule attachment, not tension. J Cell Biol
2000; 151:739-48.
7. Yao X, Anderson KL, Cleveland DW. The microtubule-dependent motor centromere-associated protein
E (CENP-E) is an integral component of kinetochore
corona fibers that link centromeres to spindle microtubules. J Cell Biol 1997; 139:435-47.
8. Chan GK, Schaar BT, Yen TJ. Characterization of
the kinetochore binding domain of CENP-E reveals
interactions with the kinetochore proteins CENP-F
and hBUBR1. J Cell Biol 1998; 143:49-63.
9. Yang Z, Guo J, Chen Q, Ding C, Du J, Zhu X.
Silencing mitosin induces misaligned chromosomes,
premature chromosome decondensation before anaphase onset, and mitotic cell death. Mol Cell Biol
2005; 25:4062-74.
10. Vigneron S, Prieto S, Bernis C, Labbe JC, Castro A,
Lorca T. Kinetochore localization of spindle checkpoint proteins: who controls whom? Mol Biol Cell
2004; 15:4584-96.
11. Liang Y, Yu W, Li Y, Yu L, Zhang Q, Wang F, et al.
Nudel modulates kinetochore association and function of cytoplasmic dynein in M phase. Mol Biol Cell
2007; 18:2656-66.
12. Stehman SA, Chen Y, McKenney RJ, Vallee RB.
NudE and NudEL are required for mitotic progression and are involved in dynein recruitment to kinetochores. J Cell Biol 2007; 178:583-94.
13. Vergnolle MA, Taylor SS. Cenp-F links kinetochores
to Ndel1/Nde1/Lis1/dynein microtubule motor complexes. Curr Biol 2007; 17:1173-9.
14. Starr DA, Saffery R, Li Z, Simpson AE, Choo KH,
Yen TJ, Goldberg ML. HZwint-1, a novel human
kinetochore component that interacts with HZW10.
J Cell Sci 2000; 113:1939-50.
Cell CycleVolume 9 Issue 4
15. Starr DA, Williams BC, Hays TS, Goldberg ML.
ZW10 helps recruit dynactin and dynein to the kinetochore. J Cell Biol 1998; 142:763-74.
16. Vallee RB, Varma D, Dujardin DL. ZW10 function
in mitotic checkpoint control, dynein targeting and
membrane trafficking: is dynein the unifying theme?
Cell Cycle 2006; 5:2447-51.
17. Whyte J, Bader JR, Tauhata SB, Raycroft M, Hornick
J, Pfister KK, et al. Phosphorylation regulates targeting of cytoplasmic dynein to kinetochores during
mitosis. J Cell Biol 2008; 183:819-34.
18. Griffis ER, Stuurman N, Vale RD. Spindly, a novel
protein essential for silencing the spindle assembly
checkpoint, recruits dynein to the kinetochore. J Cell
Biol 2007; 177:1005-15.
19. McEwen BF, Chan GK, Zubrowski B, Savoian MS,
Sauer MT, Yen TJ. CENP-E is essential for reliable bioriented spindle attachment, but chromosome
alignment can be achieved via redundant mechanisms in mammalian cells. Mol Biol Cell 2001;
12:2776-89.
20. Putkey FR, Cramer T, Morphew MK, Silk AD,
Johnson RS, McIntosh JR, Cleveland DW. Unstable
kinetochore-microtubule capture and chromosomal
instability following deletion of CENP-E. Dev Cell
2002; 3:351-65.
21. Kapoor TM, Lampson MA, Hergert P, Cameron
L, Cimini D, Salmon ED, et al. Chromosomes can
congress to the metaphase plate before biorientation.
Science 2006; 311:388-91.
22. Mao Y, Abrieu A, Cleveland DW. Activating and
silencing the mitotic checkpoint through CENP-Edependent activation/inactivation of BubR1. Cell
2003; 114:87-98.
23. Weaver BA, Bonday ZQ, Putkey FR, Kops GJ, Silk
AD, Cleveland DW. Centromere-associated proteinE is essential for the mammalian mitotic checkpoint
to prevent aneuploidy due to single chromosome loss.
J Cell Biol 2003; 162:551-63.
www.landesbioscience.com
24. Malureanu LA, Jeganathan KB, Hamada M,
Wasilewski L, Davenport J, van Deursen JM. BubR1
N terminus acts as a soluble inhibitor of cyclin B
degradation by APC/C(Cdc20) in interphase. Dev
Cell 2009; 16:118-31.
25. Abrieu A, Kahana JA, Wood KW, Cleveland DW.
CENP-E as an essential component of the mitotic
checkpoint in vitro. Cell 2000; 102:817-26.
26. Maia AF, Lopes CS, Sunkel CE. BubR1 and CENP-E
have antagonistic effects upon the stability of microtubule-kinetochore attachments in Drosophila S2 cell
mitosis. Cell Cycle 2007; 6:1367-78.
27. Rieder CL, Alexander SP. Kinetochores are transported poleward along a single astral microtubule
during chromosome attachment to the spindle in
newt lung cells. J Cell Biol 1990; 110:81-95.
28. Yang Z, Tulu US, Wadsworth P, Rieder CL.
Kinetochore dynein is required for chromosome
motion and congression independent of the spindle
checkpoint. Curr Biol 2007; 17:973-80.
29. Varma D, Monzo P, Stehman SA, Vallee RB. Direct
role of dynein motor in stable kinetochore-microtubule attachment, orientation and alignment. J Cell
Biol 2008; 182:1045-54.
30. Howell BJ, McEwen BF, Canman JC, Hoffman DB,
Farrar EM, Rieder CL, Salmon ED. Cytoplasmic
dynein/dynactin drives kinetochore protein transport
to the spindle poles and has a role in mitotic spindle
checkpoint inactivation. J Cell Biol 2001; 155:115972.
31. Savoian MS, Goldberg ML, Rieder CL. The rate
of poleward chromosome motion is attenuated in
Drosophila zw10 and rod mutants. Nat Cell Biol
2000; 2:948-52.
Cell Cycle
32. Basto R, Scaerou F, Mische S, Wojcik E, Lefebvre C,
Gomes R, et al. In vivo dynamics of the rough deal
checkpoint protein during Drosophila mitosis. Curr
Biol 2004; 14:56-61.
33. Zhang XD, Goeres J, Zhang H, Yen TJ, Porter AC,
Matunis MJ. SUMO-2/3 modification and binding
regulate the association of CENP-E with kinetochores and progression through mitosis. Mol Cell
2008; 29:729-41.
34. Espeut J, Gaussen A, Bieling P, Morin V, Prieto S,
Fesquet D, et al. Phosphorylation relieves autoinhibition of the kinetochore motor Cenp-E. Mol Cell
2008; 29:637-43.
35. Ross JL, Wallace K, Shuman H, Goldman YE,
Holzbaur EL. Processive bidirectional motion of
dynein-dynactin complexes in vitro. Nat Cell Biol
2006; 8:562-70.
36. Vaughan PS, Miura P, Henderson M, Byrne B,
Vaughan KT. A role for regulated binding of
p150(Glued) to microtubule plus ends in organelle
transport. J Cell Biol 2002; 158:305-19.
37. Nishino M, Kurasawa Y, Evans R, Lin SH, Brinkley
BR, Yu-Lee LY. NudC is required for Plk1 targeting
to the kinetochore and chromosome congression.
Curr Biol 2006; 16:1414-21.
38. Zhou T, Zimmerman W, Liu X, Erikson RL. A
mammalian NudC-like protein essential for dynein
stability and cell viability. Proc Natl Acad Sci USA
2006; 103:9039-44.
39. Mao Y, Desai A, Cleveland DW. Microtubule capture by CENP-E silences BubR1-dependent mitotic
checkpoint signaling. J Cell Biol 2005; 170:873-80.
40. Faulkner, N. E., Dujardin, D. L., Tai, C. Y., Vaughan,
K. T., O'Connell, C. B., Wang, Y., and Vallee, R. B.
(2000). A role for the lissencephaly gene LIS1 in
mitosis and cytoplasmic dynein function. Nat Cell
Biol 2, 784-791.
719