katanin-60: Biological Overview | References
Gene name - Katanin-60
Cytological map position - 82F6-82F6
Function - enzyme
Symbol - Katanin-60
FlyBase ID: FBgn0040208
Genetic map position - 3R:1,053,011..1,057,940 [-]
Classification - AAA+ ATPase
Cellular location - cytoplasmic
Regulation of microtubule dynamics at the cell cortex is important for cell motility, morphogenesis and division. This study shows that the Drosophila katanin Dm-Kat60 functions to generate a dynamic cortical-microtubule interface in interphase cells. Dm-Kat60 concentrates at the cell cortex of S2 Drosophila cells during interphase, where it suppresses the polymerization of microtubule plus-ends, thereby preventing the formation of aberrantly dense cortical arrays. Dm-Kat60 also localizes at the leading edge of migratory D17 Drosophila cells and negatively regulates multiple parameters of their motility. Finally, in vitro, Dm-Kat60 severs and depolymerizes microtubules from their ends. On the basis of these data, it is proposed that Dm-Kat60 removes tubulin from microtubule lattice or microtubule ends that contact specific cortical sites to prevent stable and/or lateral attachments. The asymmetric distribution of such an activity could help generate regional variations in microtubule behaviours involved in cell migration (Zhang, 2011).
Microtubules form complex and dynamic arrays with pivotal roles in the development and function of eukaryotic cells. Although microtubules are intrinsically dynamic, their cellular behaviours are tightly regulated by a host of other factors. Thus, the microtubule cytoskeleton is responsive to a variety of cues and can locally adapt its dynamic properties accordingly. These regulatory inputs seem to be particularly relevant at the cell cortex, where localized alterations in microtubule dynamics and organization are central to cell migration, polarization, morphogenesis and division (Zhang, 2011).
Katanin is a phylogenetically conserved enzyme that uses the energy of ATP hydrolysis to generate microtubule breakage in vitro (Roll-Mecak, 2010; seeSchematic representation of the domain architecture of microtubule-severing enzymes). Katanin was originally purified from sea urchin eggs as a heterodimer of p60, a catalytic subunit of relative molecular mass 60,000 (Mr 60,K) and p80, a targeting and regulatory subunit Mr (K) 80 (McNally, 1993). Katanin p60 and p80 homologues have now been identified in evolutionarily diverse systems and many organisms contain several genes encoding distinct p60 and/or p80 proteins. Functional analyses reveal diverse roles for katanin in mitosis and meiosis (Buster, 2002; McNally, 2006; Srayko, 2006; Zhang, 2007), in neuronal morphogenesis (Karabay, 2004; Yu, 2008), and in the assembly and disassembly of cilia and flagella (Casanova, 2009; Quarmby, 2007; Rasi, 2009; Sharma, 2007). In addition, a katanin in higher plants has been shown to regulate the assembly of cortical microtubule arrays, which, in turn, determine the directional deposition of cellulose and thus impact cell morphogenesis. In this context, katanin releases new microtubules nucleated from the walls of pre-existing microtubules (Roll-Mecak, 2010; Nakamura, 2010; Zhang, 2011 and references therein).
Previous studies have found that the Drosophila katanin p60, Dm-Kat60, associates with mitotic chromosomes and stimulates the depolymerization of kinetochore-associated microtubule plus-ends during anaphase A (Zhang, 2007). The present study tested the hypothesis that Dm-Kat60 also functions to regulate microtubule dynamics during interphase -- a topic that has not been addressed in any other animal system (Zhang, 2011).
The results identify Dm-Kat60 as an important regulator of microtubule dynamics and cell migration. The human katanin KATNA1 behaves similarly. In addition to its cellular roles, in vitro analyses indicate that Dm-Kat60 has the capacity to function as both a microtubule-severing enzyme and a microtubule end depolymerase. On the basis of its cortical localization, RNAi phenotypes and catalytic activity, it is proposed that Dm-Kat60 (and the human p60-like protein KATNA1) contributes to the generation of a dynamic interface between the microtubule cytoskeleton and the interphase cortex by removing tubulin subunits from any region of the microtubule making contact with Dm-Kat60-rich cortical sites (Zhang, 2011).
Among the more unexpected outcomes of this study is the observation that Dm-Kat60 induces microtubule end depolymerization in vitro. However, given present models of the interaction of katanin with the microtubule, such a finding is not entirely surprising. Biophysical and biochemical studies have indicated that severing by katanin is mediated by the transient hexamerization of p60 proteins at the C terminus of a single tubulin within the microtubule (Roll-Mecak, 2010; Hartman, 1999). ATP hydrolysis and/or the subsequent disassembly of the hexamer is believed to generate a mechanical force, which, through multiple iterations, induces the removal of the tubulin from the lattice. If katanin works by 'tugging' on a single tubulin heterodimer, then the exposed tubulins at the microtubule end are likely to be the easiest to remove because they lack a longitudinal contact. However, the possibility cannot bevrule out that Dm-Kat60-mediated end depolymerization is a manifestation of multiple severing events occurring very near the tip (Zhang, 2011).
Within the cell, the severing and depolymerase activities of Dm-Kat60 probably remain under very tight spatial constraints. In this regard, the recruitment of Dm-Kat60 to the cell cortex seems to be central to its interphase functions. Although the data indicate that this process is reliant on the presence of actin, but not microtubules, the specific mechanisms that deliver Dm-Kat60 to the cortex remain a mystery. One appealing hypothesis is that Dm-Kat60 is directly or indirectly linked to the cortical actin array through Drosophila p80. The p80 subunit contains repeated WD40 motifs known to mediate protein-protein interactions (Smith, 1999). Similar motifs have been identified in some actin-binding proteins. The WD40 repeats of p80 are essential for the centrosomal targeting of katanin in other organisms (Hartman, 1998; McNally, 2000). It has also been suggested that p60 acts independently of p80 in some circumstances (Yu, 2005). The identification of the binding partners of Dm-Kat60 represents an important next step in understanding its cellular activities (Zhang, 2011).
At the cortex, Dm-Kat60 suppresses microtubule growth primarily by inducing plus-end catastrophes and transitions from growth to pause. Although other classes of proteins are known to induce microtubule depolymerization, in vitro, the presumptive ability of Dm-Kat60 to remove tubulins from any region of the microtubule -- end or lattice -- may be particularly useful in the more complex cellular environment. For example, such an activity could enable Dm-Kat60 to prevent sustained microtubule growth along the cortex regardless of whether the microtubule contacts the cortex end-on or side-on. The newly created plus-end at the cortical-microtubule interface would then initiate catastrophe or enter the pause state depending on its association with other microtubule-binding proteins. Moreover, the ends of polymerizing microtubules in cells are often 'capped' by plus-end-binding proteins such as EB1. Dm-Kat60 could remove these by severing the microtubule at the base of the EB1 'cap' and/or directly removing EB1-bound tubulins from the plus-end. The acidic tail of EB1 could mimic the C terminus of tubulin, thereby providing a substrate for Dm-Kat60 (Mishima, 2007; Zhang, 2011 and references therein).
Of course, Dm-Kat60 is not alone in its ability to stimulate the catastrophe of microtubule plus-ends near the cortex of interphase S2 cells, as the Drosophila kinesin-13 KLP10A also shows this activity (Mennella, 2005). However, aside from the ability of both proteins to promote catastrophes, the activities of Dm-Kat60 and KLP10A are quite distinct. The most notable difference is that KLP10A does not concentrate on the cortex, but instead binds to the ends of polymerizing microtubules to which it is recruited by EB1. Intriguingly, recent work indicates that EB1 can inhibit the depolymerase activity of kinesin-13 proteins by shielding the plus-end (Montenegro, 2010). If Dm-Kat60 were to generate plus-ends lacking EB1, then it could relieve this inhibition (Zhang, 2011).
It is proposed that Dm-Kat60 and KLP10A work together as follows. (1) Dm-Kat60 removes tubulins (EB1 bound or otherwise) from regions of the microtubule that come in close proximity to the cortex, thereby creating a free plus-end at the cortical interface. Many of these newly created plus-ends immediately enter a paused state (depletion of EB1 has been shown to strongly promote pause). (2) Next, KLP10A, which has already been accumulated near the end by EB1, promotes the transition of this end from pause to shrinkage -- this transition can occur rapidly and may often appear as a catastrophe. The present study also indicates that KLP10A increases the rate of plus-end depolymerization and thus a small, difficult-to-detect, portion of the protein may remain associated with the microtubule end as it depolymerizes. Why such an effect was not noted in the initial analysis of KLP10A is unknown, but may be due to the more limited region of the cortex analysed in that study (Mennella, 2005; Zhang, 2011 and references therein).
The finding that Dm-Kat60 targets the leading edge of motile D17 cells and alters their migration provides a broader biological context through which the current findings can be viewed and interpreted. Although the depletion of Dm-Kat60 had no obvious influence on the establishment of cell polarity, it did increase both the frequency and displacement of membrane protrusions, at least in S2 cells. The localized suppression of protrusions at the leading edge of motile D17 cells could exert negative control over the rate and persistence of cell movement (Zhang, 2011).
The observation that Dm-Kat60 RNAi results in faster and more persistent migration seems consistent with other studies demonstrating that growing microtubules stimulate the GTPase Rac at the leading edge, which may promote adhesion-complex remodelling, needed to drive and sustain protrusions. In addition, because Dm-Kat60 depletion at the leading edge of migratory cells should decrease catastrophes, microtubules could become unusually persistent and abundant in the extending protrusion, which may intensify other processes that favour protrusion, such as increased kinesin-mediated delivery of vesicles to the protrusion zone (Zhang, 2011).
A recent study examining haemocyte migration in developing Drosophila embryos, for which D17 cells may be a model, showed that haemocytes migrate less efficiently in response to guidance cues following the disruption of microtubule dynamics (Stramer, 2010). Under these conditions, an increase in cell velocities and a decrease in directional persistence were observed, similar to D17 cells depleted of Dm-Kat60 by RNAi. Thus, Dm-Kat60 may modulate microtubule dynamics at the leading edge to 'fine-tune' cell migration by suppressing protrusions (Zhang, 2011).
The findings of this study uncover unexpected roles for the Drosophila katanin p60 Dm-Kat60 in the regulation of cortical microtubule dynamics and provide insights into how the microtubule cytoskeleton affects cell migration. The ability of cells to move and change shape is central to organismal development. Defects in these processes have been linked to human diseases such as cancer. Thus, the finding that human KATNA1 has many of the same functions as Dm-Kat60 suggests the former as a potentially useful therapeutic target (Zhang, 2011).
Chromosomes move toward mitotic spindle poles by a Pacman-flux mechanism linked to microtubule depolymerization: chromosomes actively depolymerize attached microtubule plus ends (Pacman) while being reeled in to spindle poles by the continual poleward flow of tubulin subunits driven by minus-end depolymerization (flux). Pacman-flux in Drosophila melanogaster incorporates the activities of three different microtubule severing enzymes, Spastin, Fidgetin, and Katanin. Spastin and Fidgetin are utilized to stimulate microtubule minus-end depolymerization and flux. Both proteins concentrate at centrosomes, where they catalyze the turnover of γ-tubulin, consistent with the hypothesis that they exert their influence by releasing stabilizing γ-tubulin ring complexes from minus ends. In contrast, Katanin appears to function primarily on anaphase chromosomes, where it stimulates microtubule plus-end depolymerization and Pacman-based chromatid motility. Collectively, these findings reveal novel and significant roles for microtubule severing within the spindle and broaden the understanding of the molecular machinery used to move chromosomes (Zhang, 2007).
The results of this study show that three closely related MT severing enzymes, Dm-Kat60, Spastin, and Fidgetin, are important for mitosis in D. melanogaster S2 cells. Interestingly, the activity of these proteins is segregated both spatially and temporally, allowing them to perform complementary functions throughout the spindle. This is most apparent during anaphase A, when all three are integrated into the Pacman-flux machinery used to move chromosomes (Zhang, 2007).
Spastin and Fidgetin emerge from this study as new regulators of poleward MT flux. Specifically, inhibition of either protein results in a significant reduction in flux velocity. In addition, it was found that both proteins similarly promote the turnover of γ-tubulin at spindle poles and γ-tubulin at centrosomes. In sum, these data are consistent with a general model for flux and chromosome motility, in which Spastin and Fidgetin function to release MT minus ends from their nucleating γ-TuRCs, which are believed to cap and stabilize MT ends. In turn, severing exposes minus ends to depolymerization by spindle pole-associated Kinesin-13 (KLP10A in D. melanogaster), which has been shown to also contribute to flux. During anaphase A, the MT minus-end depolymerization of flux 'reels in' chromosomes to the poles (Zhang, 2007).
Based on the proposal that Spastin and Fidgetin work in concert with KLP10A to promote flux, one would expect many similarities in the phenotypes resulting from the inhibition of these proteins. Indeed, as in Spastin or Fidgetin RNAi-treated cells, depletion of KLP10A also inhibits flux and slows anaphase A. A notable difference, however, is that KLP10A RNAi induces spindle elongation, whereas Spastin or Fidgetin RNAi does not. One possible explanation for this apparent inconsistency stems from the fact that spindles probably elongate as a result of continued plus-end polymerization and MT sliding when minus-end depolymerization (i.e., flux) is decreased after KLP10A RNAi. Thus, the puzzling absence of spindle elongation after Spastin or Fidgetin RNAi might be explained by the observation that plus-end polymerization is also significantly decreased in these cells (Zhang, 2007).
Although these data demonstrate roles for Spastin and Fidgetin in regulating flux and MT-centrosome interaction (i.e., catalyzing the turnover of γ-tubulin and regulating abnormal spindle-mediated attachments with MT minus ends), it is currently unclear whether centrosomes are the sole or even primary site of action of these proteins in the spindle. Indeed, the presence of centrosomes is not required for spindle assembly, flux, and chromosome segregation in some D. melanogaster cell types and other systems such as oocyte spindles. Additionally, even in centrosome-containing cells, the majority of MT minus ends are often positioned at a distance from centrosomes, and many spindle MTs are thought to arise from noncentrosomal sources (e.g., chromosomes/kinetochores). These MTs may still be capped by cytoplasmic γ-TuRCs, and it is conceivable that severing within the spindle (i.e., away from centrosomes) is required for their normal dynamics and flux. Thus, although this model depicts Spastin and Fidgetin as functioning only at centrosomes (where these proteins concentrate), this may be an oversimplification (Zhang, 2007).
Why both Spastin and Fidgetin would be used for the same task is unclear. At present, there is no clear evidence for a functional or physical interaction between these proteins. Each protein might sever a distinct subset of centrosomal MTs, but this would be unprecedented, and it is therefore considered unlikely. Unfortunately, coinhibition of these proteins by RNAi causes a high degree of cell death, making it difficult to assess this possibility. Alternatively, a degree of functional redundancy may explain why a small portion of D. melanogaster carrying null mutations in the spastin gene survive to adulthood. Genetic analysis of the relationship between these proteins should be revealing and may help answer this question (Zhang, 2007).
It is notable that Dm-Kat60 also localizes to centrosomes but performs no obvious function there, at least based on the assays used in this study. However, Dm-Kat60 RNAi does impact the mitotic index, which is likely indicative of subtle preanaphase Dm-Kat60 activities, which are beyond the sensitivity of current visualization techniques (Zhang, 2007).
Although Dm-Kat60 does not appear to function at centrosomes, the data indicate that this protein plays an important role in moving anaphase chromosomes (McNally, 1993). Dm-Kat60, which localizes to both chromosome arms and kinetochores, functions during anaphase to stimulate the depolymerization of MT plus ends, thereby moving chromosomes by a Pacman mechanism. It is proposed that Dm-Katanin functions in this regard by uncapping MT plus ends -- much the same as Spastin and Fidgetin do at minus ends -- and exposing them to depolymerization by centromere/kinetochore-associated Kinesin-13, which is also required for Pacman. Although Pacman-inhibiting plus-end caps have not been identified, several MT-stabilizing microtubule-associated proteins (such as the plus-end tracking proteins CLASP, EB1, and CLIP-190) associate with kinetochore-associated MT plus ends. Whether the association of these proteins with plus ends inhibits depolymerization by Kinesin-13s is unknown (Zhang, 2007).
Additionally, severing by Katanin could uncap plus ends associated with chromosome arms. A vertebrate kinesin, XKLP1, which targets to chromosome arms, has been shown to bind and stabilize MT plus ends and would probably resist Pacman motility. The D. melanogaster genome encodes several potential XKLP1 homologues, and it will be interesting to see whether Katanin has an antagonistic relationship with any of these (Zhang, 2007).
The possibility cannot be ruled out that D. melanogaster Katanin directly stimulates the depolymerization of kinetochore-associated MT plus ends. Indeed, it could conceivably supplant chromosome-associated Kinesin-13s in some systems, potentially explaining why the Kinesin-13 KLP59C does not appear to play a direct role in chromosome motility in S2 cells even though it drives Pacman in D. melanogaster embryos. However, another Kinesin-13 that is needed for Pacman in S2 cells has been identified (unpublished data of Zhang, 2007), making it unlikely that Dm-Katanin directly depolymerizes plus ends (Zhang, 2007).
It is notable that Spastin and Fidgetin also target to chromosomes before anaphase, where they may function similarly to Dm-Katanin. FRAP analysis indicates that both proteins normally enhance the turnover of chromosome-associated plus ends on preanaphase spindles. Why the chromosome activity of these proteins is down-regulated at the onset of anaphase while Katanin, which associates with chromosome throughout mitosis, is up-regulated is unclear. The loss of Spastin and Fidgetin from chromosomes may result from the underlying dependence of this targeting on MTs. Both are released from chromosomes in the presence of colchicine, and alterations in MT dynamics that accompany the onset of anaphase may have the same effect. Alternatively, Katanin's activity may be up- or down-regulated by phosphorylation. Indeed, the primary sequence of Dm-Kat60 contains several putative CDK1 phosphorylation motifs. Finally, Katanin's severing activity may be negatively regulated by MT-coating microtubule-associated proteins (Zhang, 2007).
Interestingly, Katanin does not appear to target to chromosomes or kinetochores in many cell types. In fact, the only system besides D. melanogaster in which a Katanin homologue has been reported to associate with chromosomes is C. elegans, which does not use Katanin for mitosis. This raises the question of whether the mitotic functions of MT severing proteins, particularly Katanin, are conserved throughout phylogeny. In this regard, it is noted that several additional Katanin p60 homologues whose functions have not yet been analyzed have been identified within vertebrate and invertebrate genomes. Any of these could target to chromosomes and stimulate Pacman-based anaphase A. Moreover, a recent yeast two-hybrid study has shown that Fidgetin associates with the protein kinase A anchoring protein, AKAP95, which targets to chromosomes throughout mitosis. D. melanogaster contain no obvious AKAP95 homologue, perhaps explaining why Fidgetin does not impact Pacman in this system. Future studies to examine the possible mitotic functions of vertebrate Fidgetin and Katanin homologues would address this question (Zhang, 2007).
In closing, this study suggests a general mechanism in which appropriately positioned and tightly regulated MT severing proteins provide a means to rapidly create free MT ends, which are then exposed to the actions of other regulatory proteins. During anaphase, such an activity works in close coordination with Kinesin-13s, stimulating poleward chromatid motility by a combined Pacman-flux mechanism. In other instances, the creation of free ends could have a very different impact on MT behaviors. Future analyses examining the interactions between severing proteins and Kinesin-13s, as well as other regulators of MT dynamics, will help test this proposal (Zhang, 2007).
Search PubMed for articles about Drosophila Katanin-60
Buster, D., McNally, K. and McNally, F. J. (2002). Katanin inhibition prevents the redistribution of gamma-tubulin at mitosis. J. Cell Sci. 115: 1083-1092. PubMed ID: 11870226
Casanova, M. et al. (2009). Microtubule-severing proteins are involved in flagellar length control and mitosis in Trypanosomatids. Mol. Microbiol. 71: 1353-1370. PubMed ID: 19183280
Hartman, J. J. et al. (1998). Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit. Cell 93: 277-287. PubMed ID: 9568719
Hartman, J. J. and Vale, R. D. (1999). Microtubule disassembly by ATP-dependent oligomerization of the AAA enzyme katanin. Science 286: 782-785. PubMed ID: 10531065
Karabay, A., Yu, W., Solowska, J. M., Baird, D. H. and Baas, P. W. (2004). Axonal growth is sensitive to the levels of katanin, a protein that severs microtubules. J. Neurosci. 24: 5778-5788. PubMed ID: 15215300
McNally, F. J. and Vale, R. D. (1993). Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75: 419-429. PubMed ID: 8221885
McNally, K. P., Bazirgan, O. A. and McNally, F. J. (2000). Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin. J. Cell Sci. 113: 1623-1633. PubMed ID: 10751153
McNally, K., Audhya, A., Oegema, K. and McNally, F. J. (2006). Katanin controls mitotic and meiotic spindle length. J. Cell Biol. 175: 881-891. PubMed ID: 17178907
Mennella, V. et al. (2005). Functionally distinct kinesin-13 family members cooperate to regulate microtubule dynamics during interphase. Nat. Cell Biol. 7: U235-U239. PubMed ID: 15723056
Mishima, M. et al. (2007). Structural basis for tubulin recognition by cytoplasmic linker protein 170 and its autoinhibition. Proc. Natl Acad. Sci. 104: 10346-10351. PubMed ID: 17563362
Montenegro Gouveia, S. et al., (2010). In vitro reconstitution of the functional interplay between MCAK and EB3 at microtubule plus ends. Curr. Biol. 20: 1717-1722. PubMed ID: 20850319
Nakamura, M., Ehrhardt, D. W. and Hashimoto, T. (2010). Microtubule and katanin-dependent dynamics of microtubule nucleation complexes in the acentrosomal Arabidopsis cortical array. Nat. Cell Biol. 12: 1064-1070. PubMed ID: 20935636
Quarmby, L. (2000). Cellular Samurai: katanin and the severing of microtubules. J. Cell Sci. 113: 2821-2827. PubMed ID: 10910766
Rasi, M. Q., Parker, J. D., Feldman, J. L., Marshall, W. F. and Quarmby, L. M. (2009). Katanin knockdown supports a role for microtubule severing in release of basal bodies before mitosis in Chlamydomonas. Mol. Biol. Cell 20: 379-388. PubMed ID: 19005222
Roll-Mecak, A. and McNally, F. J. (2010). Microtubule-severing enzymes. Curr. Opin. Cell Biol. 22: 96-103. PubMed ID: 19963362
Sharma, N. et al. (2007). Katanin regulates dynamics of microtubules and biogenesis of motile cilia. J. Cell Biol. 178: 1065-1079. PubMed ID:
Smith, T. F., Gaitatzes, C., Saxena, K. and Neer, E. J. (1999). The WD repeat: a common architecture for diverse functions. Trends Biochem. Sci. 24: 181-185. PubMed ID:
Srayko, M., O'Toole, E. T., Hyman, A. A. and Muller-Reichert, T. (2006). Katanin disrupts the microtubule lattice and increases polymer number in C. elegans meiosis. Curr. Biol. 16: 1944-1949. PubMed ID:
Stramer, B. et al. (2010). Clasp-mediated microtubule bundling regulates persistent motility and contact repulsion in Drosophila macrophages in vivo. J. Cell Biol. 189: 681-689. PubMed ID: 20457764
Yu, W. et al. (2008). The microtubule-severing proteins spastin and katanin participate differently in the formation of axonal branches. Mol. Biol. Cell 19: 1485-1498. PubMed ID:
Yu, W. et al. (2005). Regulation of microtubule severing by katanin subunits during neuronal development. J. Neurosci. 25: 5573-5583. PubMed ID:
Zhang, D., Rogers, G. C., Buster, D. W. and Sharp, D. J. (2007). Three microtubule severing enzymes contribute to the 'Pacman-flux' machinery that moves chromosomes. J. Cell Biol. 177: 231-242. PubMed ID: 17452528
Zhang, D., et al. (2011). Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration. Nat. Cell Biol. 13(4): 361-70. PubMed ID: 21378981
date revised: 25 May 2011
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