Microtubules: Kar3 Eats up the Track

Current Biology Vol 15 No 16 R622

Dispatches

Microtubules: Kar3 Eats up the Track Molecular motors are quintessential bio-machines found throughout phylogeny. A new application of in vitro assays highlights an unexpected dual functionality for the motor domain of the microtubulebased kinesin-14 type motor protein from budding yeast. Paul S. Maddox Microtubules are ubiquitous protein polymers which provide the structural basis for force production in basic cellular processes as varied as vesicle transport and cell division. There are two general mechanisms by which microtubules contribute to force production: motor protein translocation along stable polymers; and dynamic coupling of microtubule growth and shortening. In both cases, microtubule motor proteins play important roles: in the first, the motor proteins use the intrinsic A Mating Pathway

polarity of microtubules to move cargo in a directional manner; in the second, the motor proteins use their motor domain to depolymerize microtubules, and in the case of kinesin-13 family motors, at least, do not move on the microtubules in the process. As they report in a recent issue of Current Biology, Sproul et al. [1] have now shown that the kinesin14 motor protein Kar3p of the budding yeast Saccharomyces cerevisiae not only translocates along microtubules, but also catalyzes microtubule depolymerization. So, unlike the kinesin-13 depolymerases, Kar3p B Karyogamy Models

Diploid vegetative cycle

α-factor

(i) Depolymerization

a

α a-Factor

Projection formation aα

α

a

α

Ka

ry og am y

a

Nuclear fusion

(ii) Motor sliding a

α

Nuclear congression a

α

Cell fusion

Current Biology

Figure 1. Mating and karyogamy in budding yeast. (A) Mating in budding yeast begins with haploid cells releasing pheromone. Upon sensing pheromone of opposite mating type, cells arrest in G1 of the cell cycle and repolarize their cytoskeleton to form a mating projection (shmoo). Microtubules emanating from the spindle pole body on the nucleus attach to the shmoo tip, orienting the haploid nucleus to the future site of cell fusion. After cell fusion, microtubules from the two haploid nuclei facilitate karyogamy: nuclear migration and subsequent fusion to form a diploid nucleus. The new diploid cell then re-enters the vegetative cycle by producing a bud and undergoing DNA replication. (B) Models for the function of Kar3p during karyogamy. In the depolymerization model, Kar3p (red) at the former shmoo tip depolymerizes microtubule plus ends, pulling the nuclei close to one another at the site of cell fusion. In the sliding model, Kar3p walks in a minus-end direction on overlapping antiparallel microtubules emanating from each haploid nucleus pulling the nuclei together. Sproul et al. [1] have shown that Kar3p in vitro has depolymerase activity that is specific to microtubule plus ends, supporting the depolymerization model.

uses its motor domain for both modes of force production, translocation and depolymerization. This marks the first detailed characterization of such a dual activity motor protein. Kar3p was first identified in a mutant screen for genes required for karyogamy in budding yeast — hence the name ‘Kar’ [2]. Karyogamy, or nuclear fusion, is an important step in the mating cycle of budding yeast, and is equivalent to pronuclear migration after fertilization in metazoans. During mating, haploid cells of opposite mating type, a or α, respond to mating pheromone by arresting in G1 and reorganizing their cytoskeleton to create a mating projection, known as a shmoo tip, rather than a bud (Figure 1A). When two shmoo tips of opposite mating type meet, the cells fuse creating a zygote with two haploid nuclei in a common cytoplasm. The haploid nuclei then migrate toward one another and fuse to create a diploid nucleus [3]. Cells lacking Kar3p fail in the nuclear migration stage of karyogamy and thus are unable to efficiently mate [2]. The shmoo tip is an interesting model for the regulation of dynamic microtubule attachments, such as those found at kinetochores during cell division. In budding yeast, microtubule minus ends remain attached to the spindle pole body, while plus ends dynamically probe the cytoplasm. Importantly, budding yeast cytoplasmic microtubules are only dynamic at their plus ends, while the minus ends do not grow or shorten [4]. After shmoo formation, but prior to cell fusion, microtubule plus ends attach to the cell cortex at the shmoo tip, tethering the nucleus to the future site of cell fusion. Interestingly, this attachment is not static — microtubules persistently interacting with the cortex remain dynamic [5], much like microtubules attached to kinetochores during mitosis

Dispatch R623

(reviewed in [6]). The result of the dynamic microtubule shmoo tip attachment is that, as microtubules shrink and grow, the nucleus oscillates towards and away from the shmoo tip without loss of attachment [5]. After cell fusion, the microtubules from each haploid nucleus are prepositioned to quickly interact and facilitate karyogamy. Studies using green fluorescent protein (GFP) fusions, high resolution imaging and genetic manipulation, have shown that Kar3p is required for depolymerization of microtubule plus ends at the shmoo tip [7]. Following cell fusion, Kar3p facilitates karyogamy. Previous models suggested that Kar3p functions to pull the nuclei together either by crosslinking overlapping microtubule bundles from each haploid nucleus and using its motor to walk along microtubules, or by depolymerizing microtubules from their plus ends (Figure 1B) [2,3,5]). Sproul et al. [1] have taken these in vivo models to an in vitro testing ground to further our understanding of how Kar3p functions. Published studies have presented evidence for both mechanisms of Kar3p function. Early in vitro motility assays [8] showed that the Kar3p motor domain can support minus end directed microtubule motility at a rate of ~1.3 µm min–1. This study also reported that the Kar3p motor domain depolymerized taxol-stabilized microtubules specifically at their minus ends — the first report of active depolymerization of a microtubule end. Immunofluorescence studies [9] showed that cells lacking Kar3p contain unusually long cytoplasmic microtubules. These observations, together with the fact that Kar3p localizes in part to the spindle pole body, led to speculation that Kar3p functions as a minus-end depolymerase [9,10]; this hypothesis was contradicted, however, by the observation that budding yeast minus ends are stable [4]. The in vitro assays used by Sproul et al. [1] tested the proposed roles of Kar3p in karyogamy and have

Motility (–)

(+)

Disassembly (+)

(–) ATP

Catastrophe ADP (–)

(+)

Kinesin-13

(–)

(+)

Kinesin-14 Current Biology

Figure 2. Kinesin-14 uses a mechanism distinct from that of kinesin-13 for microtubule depolymerization. While kinesin-13 is thought to cause microtubule catastrophe and can bind and depolymerize either the plus or minus end of a microtubule (left), kinesin-14 binds and depolymerizes only the plus ends of microtubules (right). Sproul et al. [1] have now shown that kinesin-14 does not bind tubulin dimers; they propose that kinesin-14 depolymerizes microtubules by removing a single heterodimer of tubulin with each step. Importantly, the motor does not dissociate from the microtubule upon liberating a heterodimer.

demonstrated a novel function for Kar3p. They found that purified Kar3p, complexed with its light chain Cik1p [11], can walk along microtubules in a minus-end directed fashion, albeit slightly faster than previously reported (2.4 µm min–1) [1]. Interestingly, they saw that the motor complex bound preferentially to microtubule ends, unlike conventional kinesin, which binds along the length of microtubules. Using both steadystate and time-lapse assays, Sproul et al. [1] went on to show that the Kar3p–Cik1p complex depolymerizes microtubules from their ends, almost exclusively the plus ends, consistent with the proposed in vivo activity of Kar3p during mating [7]. Kinesin-13 family members have been shown to have depolymerase activity both in vitro and in vivo (reviewed in [12]). These proteins are generally referred to as catastrophe factors, binding both microtubules and tubulin heterodimers and biasing microtubules toward shortening. Kar3p is a kinesin-14 family member, with a characteristic carboxy-terminal motor domain, and bears little structural resemblance to a kinesin-13, which has an internal motor domain. One might therefore expect that the molecular mechanism for the depolymerase activity of Kar3p is different from that of the kinesin-13 MCAK. Sproul et al. [1] found that

this is indeed the case (Figure 2): the depolymerase kinetics of Kar3p follow a profile distinct from that of MCAK, and Kar3p appears not to cause microtubule catastrophe as MCAK does. Furthermore, in contrast to MCAK, Kar3p–Cik1p does not bind tubulin dimers, and its ATPase activity is only stimulated by intact microtubules [1,13–15]. These results imply that Kar3p–Cik1p depolymerizes microtubule plus ends one step at a time, with each motor ATPase cycle being coupled to release of a single tubulin heterodimer. Importantly, the Drosophila kinesin-14 NCD [16,17] also shows minus-end directed motility, and Sproul et al. [1] show that NCD depolymerizes microtubule plus ends with a similar kinetic profile to Kar3p. These data indicate that kinesin-14 family proteins are plus end specific microtubule depolymerizing enzymes that function in a distinct mechanistic manner to that of kinesin-13s. The Sproul et al. [1] study elegantly tests a hypothesis from in vivo observations: that Kar3p is required for plus-end disassembly at the shmoo tip during mating. Their new in vitro results support the hypothesis that Kar3p, together with its light chain Cik1p, facilitates karyogamy at least in part by depolymerizing microtubule plus ends attached to the shmoo tip [1]. Is this a common function of all

Current Biology Vol 15 No 16 R624

kinesin-14 family members? In the case of NCD, in vivo observations have not revealed a role for depolymerase activity. NCD is required for spindle assembly and maintenance of bipolarity in the Drosophila early embryo, and chromosome segregation in meiosis [16,18]. It is not clear at this time how these activities might be linked to plus end depolymerase activity, however minus end directed motility has been implemented in these functions. Testing the role of NCD depolymerase in vivo will likely prove challenging due to the complex nature of the mitotic spindle. Nevertheless, similar studies on other kinesin-14 proteins will help shed light on the biological mechanism of this interesting protein family. Given our extensive knowledge of Kar3p function during mating, what can we say about its mechanistic role during mitosis? Recently, Tanaka et al. [19] showed that, in mitosis, the minus end directed motor activity of Kar3p contributes to bi-orientation of chromosomes on the spindle. This study did not, however, present specific observations that are consistent with plus end depolymerase activity for Kar3p in the mitotic spindle. Interestingly, Kar3p in the nucleus is thought to interact with a different light chain (Vik1p) than that in the cytoplasm (Cik1p [20]). It is possible that differential light chain binding can bias Kar3p toward either motility (Vik1p) or depolymerase activity (Cik1). Future studies on the depolymerase activity of Kar3p/Vik1p complex should yield interesting results and help to better understand this important motor molecule. In any case, this fascinating bi-functional motor protein will continue to provide insight into the ubiquitous problem of microtubule based force production throughout biology.

1029–1041. 3. Marsh, L., and Rose, M. The Pathway of Cell and Nuclear Fusion during Mating in S. cerevisiae. In The Molecular and Cellular Biology of the Yeast Saccharomyces, ed. J.R. Pringle, J.R. Broach, and E.W. Jones. Vol. 3. 1997, Cold Spring Harbor Laboratory Press: Cold Spring Harbor. 827-888. 4. Maddox, P.S., Bloom, K.S., and Salmon, E.D. (2000). The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae. Nat. Cell Biol. 2, 36–41. 5. Maddox, P., Chin, E., Mallavarapu, A., Yeh, E., Salmon, E.D., and Bloom, K. (1999). Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae. J. Cell Biol. 144, 977–987. 6. Cleveland, D.W., Mao, Y., and Sullivan, K.F. (2003). Centromeres and kinetochores: from epigenetics to mitotic checkpoint signaling. Cell 112, 407–421. 7. Maddox, P.S., Stemple, J.K., Satterwhite, L., Salmon, E.D., and Bloom, K. (2003). The minus end-directed motor Kar3 is required for coupling dynamic microtubule plus ends to the cortical shmoo tip in budding yeast. Curr. Biol. 13, 1423–1428. 8. Endow, S.A., Kang, S.J., Satterwhite, L.L., Rose, M.D., Skeen, V.P., and Salmon, E.D. (1994). Yeast Kar3 is a minus-end microtubule motor protein that destabilizes microtubules preferentially at the minus ends. EMBO J. 13, 2708–2713. 9. Saunders, W., Hornack, D., Lengyel, V., and Deng, C. (1997). The Saccharomyces cerevisiae Kinesinrelated motor Kar3p acts at preanaphase spindle poles to limit the number and length of cytoplasmic microtubules. J. Cell Biol. 137, 417–432. 10. Huyett, A., Kahana, J., Silver, P., Zeng, X., and Saunders, W.S. (1998). The Kar3p and Kip2p motors function antagonistically at the spindle poles to influence cytoplasmic microtubule numbers. J. Cell Sci. 111, 295–301. 11. Page, B.D., Satterwhite, L.L., Rose, M.D., and Snyder, M. (1994). Localization of the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting protein. J. Cell Biol. 124, 507–519. 12. Walczak, C.E. (2003). The Kin I kinesins

References

Animals possess traits that convey a selective advantage but which are often costly in terms of energy and resources, resulting in a tradeoff between costs and benefits [1]. Changes in the ecology or behaviour of the animal may

1. Sproul, L.R., Anderson, D.J., Mackey, A.T., Saunders, W.S., and Gilbert. S.P. (2005). Cik1 targets the minus-end kinesin depolymerase Kar3 to microtubule plus-ends. Curr. Biol. 15, 1420–1427. 2. Meluh, P.B., and Rose, M.D. (1990). KAR3, a kinesin-related gene required for yeast nuclear fusion. Cell 60,

13.

14.

15.

16.

17.

18.

19.

20.

are microtubule end-stimulated ATPases. Mol. Cell 11, 286–288. Hunter, A.W., Caplow, M., Coy, D.L., Hancock, W.O., Diez, S., Wordeman, L., and Howard, J. (2003). The kinesinrelated protein MCAK is a microtubule depolymerase that forms an ATPhydrolyzing complex at microtubule ends. Mol. Cell 11, 445–457. Moores, C.A., Yu, M., Guo, J., Beraud, C., Sakowicz, R., and Milligan, R.A. (2002). A mechanism for microtubule depolymerization by KinI kinesins. Mol. Cell 9, 903–909. Niederstrasser, H., Salehi-Had, H., Gan, E.C., Walczak, C., and Nogales, E. (2002). XKCM1 acts on a single protofilament and requires the C terminus of tubulin. J. Mol. Biol. 316, 817–828. Endow, S.A., Henikoff, S., and SolerNiedziela, L. (1990). Mediation of meiotic and early mitotic chromosome segregation in Drosophila by a protein related to kinesin. Nature 345, 81–83. Walker, R.A., Salmon, E.D., and Endow, S.A. (1990). The Drosophila claret segregation protein is a minus-end directed motor molecule. Nature 347, 780–782. Sharp, D.J., Brown, H.M., Kwon, M., Rogers, G.C., Holland, G., and Scholey, J.M. (2000). Functional coordination of three mitotic motors in Drosophila embryos. Mol. Biol. Cell 11, 241–253. Tanaka, K., Mukae, N., Dewar, H., van Breugel, M., James, E.K., Prescott, A.R., Antony, C., and Tanaka, T.U. (2005). Molecular mechanisms of kinetochore capture by spindle microtubules. Nature 434, 987–994. Manning, B.D., Barrett, J.G., Wallace, J.A., Granok, H., and Snyder, M. (1999). Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p. J. Cell Biol. 144, 1219–1233.

Ludwig Institute for Cancer Research, Department of Cell and Molecular Medicine, CMM East, Room 3071, G9500 Gilman Drive, La Jolla, California 92093-0653, USA. DOI: 10.1016/j.cub.2005.08.008

Brain Evolution: Getting Better All the Time? Recent studies on bats, goats and hominids suggest that some mammalian brains may have undergone dramatic evolutionary reductions in size. These studies emphasise the importance of selective pressures upon mammalian brain evolution and the need to integrate studies of neuroanatomy, neurophysiology and behaviour. Jeremy E. Niven

reduce the selective advantage of a particular trait, potentially leading to its reduction or loss. Vision, for example, is vital for predator and prey detection, conspecific recognition and navigation, but in low light environments such as caves it has frequently been lost. The brain is subject to the same