Journal of Science 113, 2821-2827 (2000) 2821 Printed in Great Britain © The Company of Biologists Limited 2000 JCS0718

COMMENTARY Cellular Samurai: katanin and the severing of

Lynne Quarmby Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6 ([email protected])

Published on WWW 20 July 2000

SUMMARY

Recent biochemical studies of the AAA ATPase, katanin, difficult to establish the role of katanin in , new provide a foundation for understanding how microtubules genetic evidence indicates that a katanin-like protein, MEI- might be severed along their length. These in vitro studies 1, plays an essential role in in C. elegans. Finally, are complemented by a series of recent reports of direct in new proteins involved in the severing of axonemal vivo observation of breakage, which indicate microtubules have been discovered in the deflagellation that the in vitro phenomenon of catalysed microtubule system of Chlamydomonas. severing is likely to be physiological. There is also new evidence that microtubule severing by katanin is important for the production of non-centrosomal microtubules in cells Key words: Microtubule severing, Katanin, mei-1, C. elegans, such as neurons and epithelial cells. Although it has been Chlamydomonas

INTRODUCTION around the circumference of the tubule each to be dissociated from tightly bound neighbours above, below and on two sides. Microtubules are highly dynamic structures, and their dynamic It seems likely that a modest disruption of the lattice would be behaviour is essential for fundamental processes such as cell propagated spontaneously, given the large amount of energy division and differentiation (for recent reviews, see Anderson, stored in the microtubule polymer. Structural studies have 1999; Cassimeris, 1999; Walczak, 2000). Much of the dynamic revealed that the protofilaments change from a ‘straight’ nature of microtubules is attributed to regulated growth and conformation to a ‘curved’ conformation upon hydrolysis of shrinkage of the polymer plus ends (dynamic instability) or to GTP by β- (Hyman et al., 1995). If the αβ-tubulin dimer the addition of subunits at the plus end while they are is part of a microtubule lattice when the GTP is hydrolysed, simultaneously lost from the minus end (treadmilling). But then the lattice restricts the conformational change, and the there exists an additional pathway by which microtubule tubulin is held in the ‘straight’ conformation (Downing and dynamics can be affected: microtubules can be broken or Nogales, 1998). Thus, perhaps the problem of severing a severed along their length. Whether microtubule severing is a microtubule reduces to one of disrupting a section of the lattice physiological pathway, used by cells to mediate changes in while restricting (or not) total disassembly along the length of their microtubule array, is a topic of increasing interest to the the tubule. Recent biochemical studies of the microtubule- cell biology community. severing ATPase katanin suggest a mechanism by which the requisite disruption could be generated. Katanin is a heterodimer consisting of a 60-kDa HOW ARE MICROTUBULES SEVERED? microtubule-stimulated ATPase that requires ATP hydrolysis to disassemble microtubules, and an 80-kDa subunit that targets It is relatively easy to understand how a linear actin polymer the complex to and regulates the microtubule- can be severed along its length by the catalysed dissociation of severing activity of the p60 subunit (McNally and Vale, 1993; two adjacent actin subunits. In contrast, the problem of Hartman et al., 1998; McNally et al., 2000). On the basis of severing a microtubule is much more difficult to get one’s mind the presence of a conserved 230-residue module, katanin p60 around. Microtubules are made of α- and β-tubulin dimers has been shown to be a member of the AAA family of arranged head-to-tail into protofilaments that assemble, (ATPases Associated with various cellular Activities; McNally probably through the formation of a sheet, to form hollow tubes and Vale, 1993). Sequence analysis indicates that this is an that most commonly comprise 13 protofilaments (Downing and ancient class of proteins, which today are found in all five Nogales, 1998). Therefore, severing of a microtubule at a kingdoms and have a wide range of functions, including specific spot along its length requires thirteen or so subunits proteolysis, DNA replication, recombination, restriction and 2822 L. Quarmby transcription, sporulation, chelation, vesicle fusion, and motor (a) activity (Neuwald et al., 1999). Many AAA proteins play roles in the assembly, operation, or disassembly of protein complexes. Orthologs of katanin p60 are present in humans, Chlamydomonas, Drosophila, C. elegans, and Arabidopsis, but not yeast. On the basis of data from rotary shadowing electron microscopy, and by analogy to other AAA proteins, Hartman et al. (1998) hypothesised that (b) p60 katanin functions as an oligomeric ring complex. However, in solution studies, katanin behaves as a dimer (McNally and Vale, 1993; Hartman and Vale, 1999). Hartman and Vale (1999) recently resolved this paradox, using mixtures of CFP-p60 and YFP-p60 fusion proteins, in which CFP and YFP function as donor-acceptor pairs for fluorescence resonance energy transfer (FRET) when the p60 subunits are neighbours in an oligomer (see McNally, 2000). In order to capture the Spindle pole p60 katanin as an oligomer, the authors Treadmilling used an active-site mutant of p60 katanin (E334Q) designed to block the protein in its ATP-bound state. The Fig. 1. Microtubule severing by katanin. Katanin p60 subunits (swords) are shown as a ATPase activity of wild-type katanin is hexameric complex inserted into the face of a microtubule. Tubulin dimers are blue-green; maximal at a microtubule concentration red sword handles represent ATP; blue sword handles represent ADP; protective MAPs of 2-10 µM tubulin dimer; at higher (microtubule-associated proteins) are shown in purple. The samurai swordsmen represent concentrations of microtubules the accessory/regulatory proteins, possibly including katanin p80. (a) Accessory proteins are ATPase activity decreases (Hartman et absent in the in vitro assay (top panels), in which ATP binding triggers oligomerization, and al., 1998). Hartman and Vale (1999) assembly on the wall of the microtubule, which in turn triggers ATP hydrolysis and observed the same microtubule- phosphate release, which results in conformational changes in the katanin (the swords twist); concentration dependence for katanin the p60 subunits then dissociate, and directly affected tubulin dimers are wrenched from the microtubule lattice; disassembly of the lattice propagates around the circumference of the ATPase, microtubule severing and tubule (based, in part, on the data of Hartman and Vale, 1999). (b) Three of several possible oligomerization (as measured by in vivo contexts for microtubule severing. In cells, microtubule severing is likely to be FRET). controlled by regulatory factors, some of which might hold the severing complex together Taken together, the accumulated data after ATP hydrolysis (e.g. the samurai at the spindle pole), whereas others, such as protective on katanin and related AAA ATPases MAPs, might prevent the immediate disassembly of the new plus and minus ends. At the suggest the following model for spindle pole, severing might produce free microtubule ends, allowing for the poleward flux microtubule severing by katanin (see of tubulin and poleward movement of the microtubule. At the centrosome, microtubule Hartman and Vale, 1999). Microtubules severing might release microtubules. And in the , microtubule severing might act as a scaffold upon which katanin facilitate treadmilling. Other in vivo contexts for microtubule severing are discussed in the oligomerizes after it has exchanged its text. ADP for ATP. Once a complete katanin ring is assembled on the microtubule, the ATPase activity of could be reversibly masked by regulatory factors; and other katanin is stimulated. As a consequence of ATP hydrolysis factors could stimulate or inhibit ATP hydrolysis and severing. and subsequent phosphate release, the katanin undergoes a Different types of regulation might be used in different cellular conformational change leading to destabilisation of tubulin- contexts (see below), but the ubiquitous p80 subunit might well tubulin contacts. The ADP-bound katanin has lower affinity play a fundamental role. both for other katanin molecules and for tubulin; this leads to The in vivo function of the p80 subunit remains enigmatic, the dissolution of the complex and the recycling of the katanin but it is likely to play roles both in targeting and in regulation (see Fig. 1). of the p60 subunit. The N-terminal WD40 domain of p80 can This model suggests several possible points of regulation: a target a p80WD40-GFP chimera to centrosomes nucleotide-exchange factor could regulate loading of p60 and is thus implicated in the targeting p60 to centrosomes with ATP; accessibility to microtubules could be regulated by (Hartman et al., 1998). Recent work from McNally et al. (2000) removal of protective MAPs; oligomerization sites on p60 demonstrates that both p60 and the C-terminal domain of p80 Cellular Samurai: katanin and the severing of microtubules 2823 bind to microtubules in vitro and that the association of the two end factor is a severing protein that releases short fragments of subunits increases their affinity for microtubules and also microtubules near the minus end. Whatever the mechanism, microtubule-severing activity. In addition, indirect evidence minus-end shortening in cells that use centrosome-associated implicates the WD40 domain in microtubule-dependent microtubules could keep the cytoplasm ordered, because, in targeting of katanin to the spindle pole as well as inhibition of general, in vivo breakage tends to promote microtubule microtubule-severing activity (McNally et al., 2000). disassembly (Waterman-Storer and Salmon, 1997; Odde et al., 1999). In contrast, in neurons, myocytes and epithelial cells, the minus end might be capped, which would allow the IS MICROTUBULE SEVERING AN IMPORTANT persistence of centrosome-free microtubules (Rodionov et al., SOURCE OF NON-CENTROSOMAL 1999). MICROTUBULES? In neurons, a large number of non-centrosomal microtubules are required for the growth and maintenance of neuronal In many cell types, such as fibroblasts, microtubules of the processes (Ahmad et al., 1999). Injection of an anti-katanin interphase array have their minus ends anchored near the into neurons leads to an accumulation of centrosomal centrosome, whereas their plus ends are oriented towards the microtubules and a loss of neuronal processes, which indicates cell periphery. In other cells, such as myocytes, epithelial cells that centrosomal katanin is important for the production of and neurons, non-centrosomal microtubules are critical for the non-centrosomal microtubules primarily through severing of activity of the differentiated cell. For example, the axonal and the microtubules near the centrosome. In addition, Dent et al. dendritic processes of neurons require a robust supply of non- (1999) have observed that microtubules fragment in pausing centrosomal microtubules both for structural support and for neuronal growth cones and at sites of branching. These data transport of materials. There are three possible sources of non- suggest that the sites of microtubule breakage/severing are centrosomal microtubules: (1) de novo nucleation and growth locally regulated, and Dent and co-workers (1999) suggest that in the cytoplasm; (2) release from the centrosome; or (3) a protein such as katanin is likely to be the principal means for severing of microtubules at sites remote from the centrosome. generating the short microtubules observed in growth cones With respect to this last mechanism, note that in reports of in and interstitial branch points. Waterman-Storer and Salmon vivo studies, the terms ‘breakage’ or ‘fragmentation’ are used (1997) similarly concluded that microtubule breakage is the to refer to the phenomenon of microtubules breaking along most likely source of the abundant non-centrosomal their length. I think that this is because the term ‘severing’ is microtubules of migrating newt lung cells. more value laden in that it has been used primarily in in vitro studies in which the breakage of microtubules is mediated by the activity of severing proteins. Although ‘breakage’ does not IS MICROTUBULE SEVERING IMPORTANT IN imply a mechanism, I think that the observed in vivo breakage, MITOSIS? such as that seen in the studies reviewed below, will ultimately be shown to be regulated protein-dependent events. A role for microtubule severing in mitosis has been suggested A recent study supports the idea that breakage is an since the first in vitro assays of microtubule severing, in which important source of non-centrosomal microtubules. Odde et al. Vale (1991) observed that a severing activity is specifically (1999) performed a quantitative study of the relationship activated in extracts of M-phase Xenopus eggs. McNally and between curvature and breakage of microtubules in living cells Vale (1993) used the in vitro assay to purify the severing (fibroblasts). They observed that microtubules that have greater protein, katanin. McNally and Thomas (1998) subsequently curvature are more likely to break than straight microtubules, demonstrated that katanin is responsible for the M-phase but that, importantly, straight microtubules do break, and regulated severing activity of Xenopus eggs. curved microtubules do not always break. This suggests that A microtubule-independent focus of katanin is found at the the in vivo breakage is mediated by severing proteins and that centrosome in several cell types (McNally et al., 1996; tubulin, protective MAPs and/or the severing proteins have McNally and Thomas, 1998). A possible role for katanin intrinsic curvature sensitivity. during the cell cycle would be to release centrosomal The consequence of microtubule severing strongly depends microtubules and thereby facilitate the rapid disassembly of the upon what happens to the new polymer ends. Borisy and interphase array that occurs as cells enter mitosis. Although a colleagues (e.g. Rodionov et al., 1999) observe treadmilling of dramatic increase in catastrophe frequency is clearly important microtubules in cytoplasts prepared from melanocytes or in this process, severing and minus-end disassembly might fibroblasts at rates 2-3 orders of magnitude higher than those nevertheless also be important. In fact, both might be indirectly reported for in vitro treadmilling. By contrast, Rodionov et al. activated by the removal of protective MAPs. In Xenopus egg did not observe treadmilling in cytoplasts obtained from extracts, the -related protein, XKCM1, promotes epithelial cells. The rapid treadmilling observed in fibroblasts catastrophe (Belmont and Mitchison, 1996). Importantly, this and melanocytes might be due to plus-end factors that activity is constrained during interphase because the accelerate polymerisation at the growing ends or to minus-end microtubules are stabilised by MAPs (Tournebize et al., 2000; factors that increase the rate of loss of subunits from the minus Walczak, 2000). The cell-cycle-regulated release of stabilising ends. By analogy to the action of ADF/cofilin on actin MAPs from microtubules probably also results in increased filaments, Borisy hypothesises that a minus-end dissociation rates of microtubule severing. factor exists for microtubules. Such a minus-end factor could In addition to being found at centrosomes, katanin is also be a motor, because motors have been linked with increased concentrated in a microtubule-dependent manner at mitotic depolymerisation, but it seems equally possible that the minus- spindle poles (McNally and Thomas, 1998; McNally et al., 2824 L. Quarmby

2000). Katanin is thought to play a critical role during the The most exciting result, from the perspective of those of us depolymerization of spindle microtubules at their minus ends, interested in microtubule severing, is that coexpression of a process important for chromosome movements MEI-1 and MEI-2 in HeLa cells results in microtubule (Waters et al., 1996; Desai et al., 1998; McNally et al., 2000). disassembly in vivo, as observed by a reduction in overall anti- Given that the microtubule-severing activity of katanin is well tubulin immunofluorescence (Srayko et al., 2000). These data established, and that katanin is appropriately positioned to play provide support for the idea that MEI-1–MEI-2 functions as a a role in the poleward flux of spindle microtubules, the idea is microtubule-severing complex during meiosis. The authors attractive. Severing need only provide free minus ends for speculate that short spindle microtubules and the absence of disassembly; other mechanisms, such as an actin- astral microtubules are important for keeping the meiotic system or microtubule-based motors, could provide the force spindle near the cortex (for efficient extrusion of the polar necessary for poleward movement (Silverman-Gavrila and bodies) and to allow efficient focusing of the acentrosomal Forer, 2000; Compton, 1998). Nevertheless, there is as yet no meiotic spindle of C. elegans. definitive evidence to establish whether microtubule severing There are perplexing aspects to the story of MEI-1/MEI-2 by katanin plays an essential role in mitosis, either by as it is unfolding. If katanin plays an important role in stimulating disassembly of the interphase array or by mediating vertebrate mitosis (as suggested by the data reviewed above), the poleward flux of spindle microtubules. then are there additional microtubule-severing proteins in C. elegans that mediate the essential functions, if any, of severing during mitosis? Similarly, if microtubule severing is important IS THERE A SPECIFIC ROLE FOR MICROTUBULE for meiosis in worms, might it not also be important for meiosis SEVERING DURING MEIOSIS? in other systems, including vertebrates? If so, what are the proteins involved? The first suggestion that microtubule severing might play a role in meiosis came from the discovery that MEI-1, an oocyte- specific meiotic spindle component from C. elegans, has high WHAT PROTEINS MEDIATE THE SEVERING OF sequence similarity to the C-terminal AAA ATPase domain of AXONEMAL MICROTUBULES DURING katanin (Clark-Maguire and Mains, 1994). The N-terminal DEFLAGELLATION/DECILIATION? domain of other presumptive katanin p60 orthologs is missing in MEI-1. Although this N-terminal domain is highly divergent Ciliated or flagellated cells, including sea urchin embryos, among the cloned katanin p60 genes, it does include a scallop gills, vertebrate respiratory epithelial cells, rat cerebral conserved region that is implicated in binding to microtubules ependymal cells, and protists shed their cilia or flagella in and to the p80 subunit (Lohret et al., 1999; McNally et al., response to stress (Auclair and Siegel, 1966; Anderson, 1974; 2000). Consequently it was unclear whether MEI-1 functions Stephens, 1975; Hastie et al., 1986; Mohammed et al., 1999). as a katanin-like microtubule-severing ATPase. As first noted by Blum (1971), a key event in deciliation is the Loss-of-function alleles of mei-1 result in disorganised breakage of the outer doublet microtubules at a specific site in meiotic spindle microtubules and meiotic failure, but mitosis the distal region of the flagellar transition zone (Fig. 2). This in these mutants is normal (Mains et al., 1990; Clandinin and process has been most extensively studied in the unicellular Mains, 1993; Clark-Maguire and Mains, 1994). A gain-of- alga Chlamydomonas, in which analysis of mutations that are function mutation in mei-1 causes persistence of the MEI-1 non-essential for growth indicated that at least three genes, protein, which is otherwise degraded after meiosis, into ADF1, FA1, and FA2, are necessary for pH-shock-induced mitosis. In these worms, meiosis is normal, but mitotic spindles deflagellation (Finst et al., 1998). ADF1 is essential for signal- are smaller than normal (Clark-Maguire and Mains, 1994). induced Ca2+ influx (an early event in the signalling pathway), Given that, in C. elegans, the meiotic spindle is much smaller whereas FA1 and FA2 are required for severing of the outer than the first mitotic spindle, the mei-1 phenotypes led to the doublet microtubules of the flagellum in response to the Ca2+ idea that the normal function of MEI-1 is to keep microtubules signal (Quarmby, 1996; Quarmby and Lohret, 1999). Katanin short during meiosis (Srayko et al., 2000). mutants were not isolated in this screen, possibly because mei-2 was originally identified as a suppressor of mei-1 katanin plays other roles that are essential for growth, and the (Mains et al., 1990). Genetic analysis suggested that MEI-2 p60 subunit of katanin is encoded by a single-copy gene in functions as an activator of MEI-1 (Mains et al., 1990; Chlamydomonas (Lohret et al., 1999). Clandinin and Mains, 1993; Clark-Maguire and Mains, 1994). Three lines of evidence suggest that katanin mediates the It now turns out that the 32-kDa MEI-2 protein contains a severing of axonemal microtubules during deflagellation. domain that has high sequence similarity to the C-terminal First, purified sea urchin katanin can sever microtubules of domain of katanin p80 (Srayko et al., 2000). This is the domain the axoneme (Lohret et al., 1998). Second, immunogold EM of katanin p80 that binds to katanin p60 (Hartman et al., 1998). studies reveal p60 katanin on several basal body and flagellar Both MEI-1 and MEI-2 are found at the poles of meiotic transition-zone structures, including the distal end of the spindles, and on meiotic chromatin, and both proteins appear flagellar transition zone, the site at which the doublets are to be dependent upon each other for this localisation (Clark- severed during deflagellation (Lohret et al., 1999). Third, Maguire and Mains, 1994; Srayko et al., 2000). Further support against the human p60 katanin inhibit axonemal for the idea that MEI-2 and MEI-2 form a heterodimer severing in response to Ca2+ (Lohret et al., 1998). (analogous to the p60/p80 katanin heterodimer) came from the Nevertheless, definitive evidence demonstrating that katanin co-immunoprecipitation of MEI-1 and MEI-2 coexpressed in is the microtubule-severing protein of deflagellation is still HeLa cells (Srayko et al., 2000). lacking. Moreover, katanin at this site might play some other Cellular Samurai: katanin and the severing of microtubules 2825

Fig. 2. Transmission electron micrograph of longitudinal sections through Chlamydomonas flagella during deflagellation (adapted by R. Finst from Lewin and Lee, 1985). In B, note the severing of the outer-doublet microtubules at the distal end of the flagellar transition zone, indicated by arrows. This is the postulated location of the transition-zone microtubule-severing complex. Abbreviations: M, membrane; A, axoneme; H, H-piece; TZ, transition-zone; BB, basal body; bm, blebbed membrane. role, such as proximal disassembly of axonemes (Stephens, which the first EF hand of the centrin gene carries a missense 1999). mutation, the vfl-2 strain, led Sanders and Salisbury (1989, The phenotypes of fa1 and fa2 mutants, along with the 1994) to the conclusion that Ca2+-induced contraction of the sequences of the recently cloned genes, provide hints on how stellate fibres provides both torsional and shear stresses on this system might be put together (Finst et al., 2000; L. Zhao, the outer doublet microtubules, which causes them to break. R. Finst and L. Quarmby, unpublished observations). The However, in spite of grossly disorganised stellate fibres, the axonemal microtubules of fa mutants are not severed in deflagellation phenotype of the vfl-2 strain is subtle and response to a Ca2+ signal, whether the signal is generated in observed only under special laboratory conditions (cf. Taillon vivo or in vitro. Genetic evidence supports the idea that both et al., 1992; Lohret et al., 1998; Quarmby and Lohret, 1999). gene products mediate their effects through a stable flagellar Unfortunately, deletion of the centrin gene is lethal, and transition-zone microtubule-severing complex. Fa1p is a novel therefore the role of centrin in deflagellation remains uncertain 171-kDa protein that has a predicted coiled-coil domain and (S. Dutcher, Washington University, personal communication). three consensus Ca2+/CaM-binding domains (Finst et al., In light of the recent data from Odde and co-workers (1999), 2000). Our ability to purify severing-competent flagellar basal it seems likely that the mechanical stress supplied by the body complexes (FBBCs; Lohret et al., 1998) indicates that the contraction of the stellate fibres stimulates or facilitates proteins involved in Ca2+-induced axonemal microtubule severing of the axonemal microtubules by a severing complex. severing are tightly associated with axonemal microtubules at In this context, the observation that wild-type cells can be the transition zone. Western analysis indicates that Fa1p is induced to deflagellate by exposure to severe mechanical shear associated with the basal-body/transition zone region (Finst et (i.e. treatment with a Virtis homogenizer) is interesting al., 2000). The presence of a coiled-coil domain in Fa1p (Rosenbaum et al., 1969). The axonemes recovered from cells suggests that this protein is a component of a stable protein treated in this manner are all full-length, which indicates that complex, possibly the severing complex. The putative they were ‘broken’ at the same place as axonemes that are Ca2+/CaM-binding domains are particularly intriguing in light severed in response to stress. Furthermore, when fa1 mutant of the fact that this pathway is activated by a Ca2+ signal. cells are treated with mechanical shear, the axonemes break at Perhaps the Ca2+ is detected by a calmodulin or calmodulin- random points, and the cells do not survive the treatment. This like protein, which in turn binds and activates Fa1p, eventually suggests that the mechanical stress activates the same leading to activation of axonemal microtubule severing. This microtubule-severing process that occurs in response to stress, raises the issue of the role of centrin, a calmodulin-family perhaps via activation of the ADF1 Ca2+-influx pathway protein that has been previously implicated in deflagellation (Quarmby, 1996). Therefore, either there is a weak point in (Sanders and Salisbury, 1989, 1994). wild-type axonemes (which is strengthened in the fa1 mutant Centrin is a centrosomal protein that in Chlamydomonas is strains) or there is a severing complex specifically associated associated with several structures, including the stellate fibres with the flagellar transition zone. Given the discovery that of the flagellar transition zone (Sanders and Salisbury, 1989). katanin p60 is concentrated at this site (Lohret et al., 1999), I Ca2+-induced contraction of the centrin fibres is associated favour the idea that there is a transition-zone microtubule- with a dramatic (approx. 40%) reduction in the diameter of the severing complex. axoneme in the flagellar transition zone. In addition, the angle The outer-doublet microtubules of isolated flagellar basal of the outer doublets relative to the circumference of the body complexes (FBBCs) sever at the transition zone in axoneme twists by approx. 12° (Sanders and Salisbury, 1989). response to Ca2+ (Lohret et al., 1998). Exogenous ATP is not These data, and experiments on a Chlamydomonas strain in required in this in vitro system; therefore, if katanin is the 2826 L. Quarmby severing protein of the deflagellation pathway, these data Desai, A., Maddox, P. S., Mitchison, T. J. and Salmon, E. D. (1998). suggest that an ATP-loaded katanin oligomer is docked on the Anaphase A chromosome movement and poleward spindle microtubule flux microtubule. In this case the regulated step might be a ‘twist’ occur at similar rates in Xenopus extract spindles. J. Cell Biol. 141, 703- 2+ 713. provided by Ca binding, possibly involving Fa1p, centrin and Downing, K. H. and Nogales, E. (1998). Tubulin and microtubule structure. p80 katanin. Curr. Opin. Cell Biol. 10, 16-22. The role of Fa2p might be quite different from that of Fa1p. Finst, R. J., Kim, P. J. and Quarmby, L. M. (1998). Genetics of the Preliminary sequence analysis suggests that Fa2p is a protein deflagellation pathway in Chlamydomonas reinhardtii. Genetics 149, 927- 936. kinase (L. Zhao, R. Finst and L. Quarmby, unpublished Finst, R. J., Kim, P. J., Griffis, E. R. and Quarmby, L. M. (2000). Fa1p is observations). If Fa2p does prove to be a kinase, perhaps it a 171 kDa protein essential for axonemal microtubule severing in regulates the assembly and/or targeting of a transition-zone- Chlamydomonas. J. Cell Sci. (in press). destined microtubule-severing complex. Hartman, J. J., Mahr, J., McNally, K., Okawa, K., Iwamatsu, A., Thomas, S., Cheesman, S., Heuser, J., Vale, R. D. and McNally, F. J. (1998). Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40 domain. Cell 93, 277-287. CONCLUSIONS/PERSPECTIVES Hartman, J. J. and Vale, R. D. (1999). Microtubule disassembly by ATP- Dependent oligomerization of the AAA enzyme katanin. Science 286, 782- It is now well established that katanin p60 hydrolyses ATP and 784. Hastie, A. T., Dicker, D. T., Hingley, S. T., Kueppers, F., Higgins, M. L. severs microtubules in vitro, and we are beginning to and Weinbaum, G. (1986). Isolation of cilia from porcine tracheal understand the mechanism by which this feat might be epithelium and extraction of dynein arms. Cell Motil. Cytoskel. 6, 25-34. accomplished. But what does katanin do in vivo? There is now Hyman, A. A., Chretien, D., Arnal, I. and Wade, R. H. (1995). Structural abundant evidence for the idea that microtubules break in vivo changes accompanying GTP hydrolysis of microtubules: information from α β and that this breakage is likely to serve important roles in the a slowly hydrolyzable analog guanylyl- ( , )-methylene-diphosphonate. J. Cell Biol. 128, 117-125. production of non-centrosomal microtubules and possibly in Jarvik and Suhan (1991). the function of meiotic and mitotic spindles. But is in vivo Lewin, R. A. and Lee, K. W. (1985). Autotomy of algal flagella: electron microtubule breakage mediated by severing proteins such as microscope studies of Chlamydomonsa (Chlorophyceae) and Tetraselmis katanin? These are still early days, but the tentative answer is (Prasinophycee). Phycologia 24, 311-316. Lohret, T. A., McNally, F. J. and Quarmby, L. M. (1998). A role for katanin- ‘yes.’ The continued application of complementary mediated axonemal severing during Chlamydomonas deflagellation. Mol. biochemical, molecular genetic and classical genetic Biol. Cell 9, 421-437. approaches is likely to yield an understanding of the basic Lohret, T. A., Zhao, L. and Quarmby, L. M. (1999). Cloning of physiological significance, mechanisms and regulation of Chlamydomonas p60 katanin and localization to the site of outer doublet microtubule severing within the next decade. severing during deflagellation. Cell Motil. Cytoskel. 43, 221-231. Mains, P. E., Kemphues, K. J., Sprunger, S. A., Sulston, I. A. and Wood, W. B. (1990). Mutations affecting the meiotic and mitotic divisions of the I warmly thank Frank McNally (U.C. Davis) and Paul Mains (U. early embryo. Genetics 126, 593-605. 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