kinesin-8s: motoring and depolymerizing

NATURE CELL BIOLOGY VOLUME 8 | NUMBER 9 | SEPTEMBER 2006 903

N E W S A N D V I E W S

Kinesin-8s: motoring and depolymerizingClaire E. Walczak

Kinesins conventionally act as molecular motor proteins that translocate along microtubules. However, several kinesins also control microtubule polymerization dynamics. New work shows that the yeast kinesin-8 Kip3p has a unique combination of plus-end motor and plus-end depolymerase activities. These activities facilitate the positioning of the mitotic spindle at the cell cortex.

The kinesin superfamily consists of micro-tubule-dependent ATPases that function in a vast array of cellular processes. They have been grouped phylogenetically into 14 sub-families based on the sequence of their con-served ATPase and microtubule-binding motor domain1. In general, kinesins translo-cate cargo toward the plus- or minus-end of microtubules. In some cases, they have also been shown to regulate microtubule polym-erization dynamics. Unexpectedly, two studies on page 913 and 957 of this issue show that Kip3p, the budding yeast kinesin-8 family member does both: it undergoes plus-end directed motility and can perform plus-end microtubule depolymerization2,3.

Previous genetic studies had suggested that members of the kinesin-8 family may be micro-tubule motors and/or microtubule depolymer-ases4–7, but no one had successfully demonstrated depolymerization activity either in vitro or in vivo. Physiologically, kinesin-8 activity is needed for nuclear movement, spindle posi-tioning and chromosome segregation8–12. In principle, a motor protein exhibiting either a translocation or a microtubule depolymerase activity could control these events, but it may be that being able to do both is better. Whereas Drosophila and most vertebrate organisms con-tain one or more members of the kinesin-13 subfamily, which act solely as microtubule depolymerases, the yeast Saccharomyces cer-evisiae lacks kinesin-13 family members, suggesting that it relies on a different motor protein for microtubule depolymerization. The kinesin-8 family is closely related to the kinesin-13 family; however kinesin-8 proteins do not have several motifs of the kinesin-13 family members that are essential for their depolymerization activity13.

Using bacculovirus-expressed Kip3p, both Gupta et al.2 and Varga et al.3 demonstrated that the budding yeast kinesin-8 Kip3p is indeed a

plus-end directed microtubule depolymerase in vitro. In addition, it was also a plus-end directed motor, consistent with previous find-ings14. In an elegant demonstration of this motility, Varga et al. used total internal reflec-tion microscopy to observe the movement of individual Kip3p molecules in vitro. The in vitro velocities measured were similar to the rates of movements observed in vivo (~3.5 µm min–1)2,3. Varga et al. also found that Kip3p is highly processive (that is, it is able to stay attached to the microtubule for a long time) and exhibits run lengths of about 12.4 µm, which is approximately twenty-times longer than that of conventional kinesin. Interestingly, Varga et al. noticed that the rate of depolym-erization varied depending on the length of the

microtubule (Fig. 1). This is the first example of microtubule-length-dependent regulation of motor activity. It is important to note that the rate of movement of Kip3p is greater than the rate of microtubule polymerization (~1 µm min–1), so this robust and processive motility may provide a mechanism for Kip3p to accu-mulate rapidly at the ends of long microtu-bules, where it could then induce microtubule depolymerization. It will be of great interest to learn how this length regulation is achieved and whether other dynamic regulators are also con-trolled in this manner.

As kinesin-8 proteins are thought to be closely related to the kinesin-13 family, both groups examined whether the kinesin-8 depolymerization mechanisms were

Claire E. Walczak is in the Medical Sciences Program, Indiana University, Myers Hall 262, 915 East 3rd St., Bloomington, IN 47405, USA.e-mail: [email protected]

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Figure 1 Kip3p is a microtubule length-dependent plus-end depolymerase. Kip3 motors (green) walk processively towards the plus-end of the microtubule (large arrow), where they accumulate at the end of longer microtubules and induce microtubule depolymerization. As the microtubule depolymerizes from the plus end (small arrow), the Kip3 motors dissociate.

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similar to that of the kinesin-13 family13. Whereas kinesin-13 family members depo-lymerize microtubules from both ends, Kip3p was plus-end specific, similar to Kar3p (a kinesin-14 protein that couples minus-end motility with plus-end depolymerization)15. In addition, although the motility of Kip3p was ATP-dependent, some depolymerization activity still occurred in the absence of ATP. This suggests that Kip3p uses ATPase activ-ity to power plus-end movement, but uses a mechanism distinct from kinesin-13 proteins to depolymerize microtubules. Given this important difference, it is surprising that many other aspects of the kinesin-8 mecha-nism seem to be conserved with kinesin-13 family members. For example, the ATPase activity of both families is stimulated by the tubulin dimer, and even more strongly by microtubules. Kip3p also exhibits a nucle-otide-sensitive interaction with tubulin, which is thought to be important for the cat-alytic recycling of kinesin-13 proteins. One interesting aspect of kinesin-13s is their abil-ity to induce protofilament peeling of micro-tubule ends, which is thought to be critical for induction of a microtubule catastrophe — the transition from growth to shrinkage. Do kinesin-8 proteins also induce a similar conformational change at the end of the micro-tubule, and is their action catalytic? These are just two of the open questions regarding the detailed catalytic cycle of Kip3p and other kinesin-8 family members.

Any biochemical mechanism is of interest in its own right, but it is important to understand how this mechanism relates to physiological function. Kip3p is a plus-end directed motor in vivo and associates with the plus end of growing, but not shrinking, microtubules (most notably near the cell cortex). It is likely that as Kip3p motor activity is faster than the microtubule-polymerization rate, Kip3p will accumulate and linger on microtubule ends2,3. This microtubule growing-tip-track-ing activity and localization of Kip3p are put in perspective when the analysis of cellular microtubule dynamics in the presence and absence of KIP3 performed by Gupta et al.2 is considered. Within the yeast cell, a subset of cytoplasmic microtubules occasionally interact with the cortex and are then called cortical microtubules. The cortical micro-tubules exhibit slightly different dynamics than free cytoplasmic microtubules. Gupta et al. found that in kip3∆ cells, the amount of time cytoplasmic free microtubules spend in a growing state increases by 41%, resulting in longer microtubules than in wild-type cells. Another remarkable finding was that in these cells, any microtubules that did depolymerize often depolymerized faster, suggesting that Kip3p may also be involved in governing the normal rate of depolymerization. Whether this is a direct action of Kip3p, or a change in the number and configuration of proteins that track the plus-ends of the growing microtu-bules, has yet to be determined.

The effects of loss of Kip3p on cortical microtubules were even greater. Normally the microtubule catastrophe frequency is fourfold higher at the cortex, but it was 60% reduced in the absence of Kip3p. This caused microtu-bules to remain for longer periods of time at the cell cortex, and eventually become stabi-lized there. How might this affect the cell? In yeast, the cortical microtubules are connected directly to the spindle-pole body, such that any change in cortical microtubule dynam-ics can disrupt spindle positioning. Indeed, the prolonged time spent by a microtubule at the cortex in kip3∆ cells often forced the attached spindle-pole body to move to the opposite side of the mother cell. This suggests that Kip3p contributes to spindle position-ing by preventing spindles from being pushed away from the polarized region of the bud tip (Fig. 2). It likely that it does this by motoring to the end of the long cortical microtubule using its plus-end motility and then depolym-erizing the microtubule at the bud tip. This raises the question whether these properties are also characteristic of kinesin-8 family members from other systems.

In all systems examined to date, the loss of kinesin-8 family members is associated with excess microtubule polymerization, but it is still unclear what subclasses of microtubules are regulated by the kinesin-8 family. Gupta et al. clearly demonstrate that Kip3p regulates cortical microtubules, but does its activity also affect spindle microtubules? Because yeast undergo a closed mitosis in which the nuclear envelope does not break down, this may limit the interconversion of the free tubulin pool between spindle microtubules and nuclear and cytoplasmic microtubules. In contrast, Drosophila and vertebrate cells undergo an open mitosis, so defects on spindle and astral microtubules induced by loss of kinesin-8 pro-teins may be more obvious. Although three members of the kinesin-8 family are expressed in most vertebrate cells, only the loss of one of them, Kif18A, has been correlated with any microtubule polymer defects16, so it is unclear how the other kinesin-8 proteins function. One possibility is that vertebrate cells will use different members of the kinesin-8 fam-ily to control distinct subsets of microtubule dynamics, similar to what seems to be true for the kinesin-13 family. Scientists still need to unravel the intricate regulation of microtubule dynamics performed by different members of the kinesin families.

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bWild type kip3∆

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Figure 2 (a) Kip3p (green) walks along the microtubules (red) towards the plus end of the bud tip.(b) In wild-type cells, Kip3p then accumulates at the cortex in the bud tip where it depolymerizes these cortical microtubules, which are attached to the spindle-pole body. As they depolymerize, they can help position the nucleus at the bud–neck junction. In cells lacking Kip3, the cortical microtubules grow longer and the nucleus is pushed away from the mother–bud junction.



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Src transforms in a Cool wayJeffrey R. Peterson and Jonathan Chernoff

Cool-1 was previously identified as an effector of activated Cdc42 and as a regulator of epidermal growth factor receptor (EGFR) trafficking. Cool-1 has now been shown to be a phosphorylation-dependent activator of Cdc42 that contributes to transformation by Src, thus proving to be an unusually versatile signalling protein.

v-src is among the best-studied oncogenes, so it is rare for new elements of its signalling mechanism to be identified. There have been a number of hints that Src signalling intersects with that of the Rho family GTPase Cdc42 (refs 1, 2), but the functional significance of this relationship, particularly in the context of trans-formation, has not been established. On page 945 of this issue, Feng et al.3 show that Cool-1 (cloned out of library 1; also known as β-Pix), a regulator and effector of Cdc42, is important for v-src-dependent transformation.

The Cool proteins p90 Cool-2 (α-Pix), p85 Cool-1 (β-Pix) and a smaller p50 splice variant of Cool-1, were first identified as binding partners of the protein kinase Pak1 (refs 4, 5). Their role as guanine nucleotide-exchange factors (GEFs) was predicted from the presence of conserved Dbl homology (DH) and pleckstrin homology (PH) domains that occur in tandem in most Rho protein GEFs. GEFs stabilize nucleotide-free forms of the Rho GTPases Rho, Rac and Cdc42, and facilitate the exchange of bound GDP for GTP, rendering them active and competent to bind downstream effectors. Interestingly, only Cool-2 was shown to exhibit GEF activity in puri-fied protein assays, whereas Cool-1 contained a negative regulatory element that inhibited its GEF activity6. Regardless, expression of Cool-1 in cells produced phenotypes consistent with Rho protein activation5, suggesting that some factor may relieve the autoinhibition of Cool-1.

Cerione and colleagues had previously observed that Cool-1 binds to the active form of its GTPase target, that is, GTP-bound Cdc42 (ref. 7), strongly suggesting that Cool-1 serves as an effector of Cdc42. This notion was strengthened by their finding that, when bound to Cdc42–GTP, Cool-1 also asso-ciates with the ubiquitin ligase Cbl and prevents

Cbl from ubiquitinating EGFR, which inhibits receptor downregulation and ultimately leads to prolonged receptor signalling7. Thus, a neat but incomplete model existed in which active Cdc42 was linked to promoting EGFR signalling by binding to Cool-1, which then sequestered Cbl. However, an important ques-tion remained: what activated Cdc42 to begin

Jeffrey R. Peterson and Jonathan Chernoff are in the Fox Chase Cancer Center, 333 Cottman Ave., Philadelphia, PA 19111, USA.e-mail: [email protected]; [email protected]

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Figure 1 GEFs are upstream regulators and downstream effectors of GTPases. (a) Activated Ras recruits the GEF Tiam1, which then activates Rac. This linkage helps to ensure that Ras and Rac activation are temporally and spatially coordinated. (b) Sos catalyses the exchange of GDP for GTP on Ras. This exchange activates Ras and promotes binding of Ras effectors. Activated Ras also binds to an allosteric site on Sos, further activating Ras in a feedback stimulatory loop. (c) Similarly to Tiam1, Cool-2 also uses allosteric regulation by one active GTPase, in this case Cdc42–GTP, to promote GEF activity toward another, Rac. Interestingly, Rac–GTP inhibits the GEF activity of Cool-2. (d) On phosphorylation by Src or Fak, Cool-1 catalyses nucleotide exchange on Cdc42. Activated Cdc42 then binds to and signals through its effectors. Activated Cdc42 also promotes the formation of a Cdc42–Cool-1–Cbl complex, sequestering Cbl away from its targets such as the EGFR, thus preventing their ubiquitination and subsequent internalization. It has not yet been established whether Cdc42–GTP also regulates Cool-1 GEF activity through an allosteric mechanism.



kinesin-8s: motoring and depolymerizing

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