Gene name - pavarotti
Cytological map position - 64A12
Function - cytoskeletal motor protein
Symbol - pav
FlyBase ID: FBgn0011692
Genetic map position - 3-18--20
Classification - Kinesin motor domain, P-loop containing nucleotide triphosphate hydrolases
Cellular location - nuclear
|Recent literature||Tao, L., Fasulo, B., Warecki, B. and Sullivan, W. (2016). Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motor. Nat Commun 7: 11182. PubMed ID: 27091402
Centralspindlin is essential for central spindle and cleavage furrow formation. Drosophila centralspindlin consists of a kinesin-6 motor (Pav/kinesin-6) and a GTPase-activating protein (Tum/RacGAP). Centralspindlin localization to the central spindle is mediated by Pav/kinesin-6. While Tum/RacGAP has well-documented scaffolding functions, whether it influences Pav/kinesin-6 function is less well-explored. This study demonstrates that both Pav/kinesin-6 and the centralspindlin complex (co-expressed Pav/Tum) have strong microtubule bundling activity. Centralspindlin also has robust plus-end-directed motility. In contrast, Pav/kinesin-6 alone cannot move microtubules. However, the addition of Tum/RacGAP or a 65 amino acid Tum/RacGAP fragment to Pav/kinesin-6 restores microtubule motility. Further, ATPase assays reveal that microtubule-stimulated ATPase activity of centralspindlin is seven times higher than that of Pav/kinesin-6. These findings are supported by in vivo studies demonstrating that in Tum/RacGAP-depleted S2 Drosophila cells, Pav/kinesin-6 exhibits severely reduced localization to the central spindle and an abnormal concentration at the centrosomes.
|McLaughlin, C. N., Nechipurenko, I. V., Liu, N. and Broihier, H. T. (2016). A Toll receptor-FoxO pathway represses Pavarotti/MKLP1 to promote microtubule dynamics in motoneurons. J Cell Biol 214: 459-474. PubMed ID: 27502486
FoxO proteins are evolutionarily conserved regulators of neuronal structure and function, yet the neuron-specific pathways within which they act are poorly understood. To elucidate neuronal FoxO function in Drosophila melanogaster, a screen was performed for FoxO's upstream regulators and downstream effectors. On the upstream side, genetic and molecular pathway analyses is presented indicating that the Toll-6 receptor, the Toll/interleukin-1 receptor domain adaptor dSARM, and FoxO function in a linear pathway. On the downstream side, it was found that Toll-6-FoxO signaling represses the mitotic kinesin Pavarotti/MKLP1 (Pav-KLP), which itself attenuates microtubule (MT) dynamics. In vivo functions were probed for this novel pathway, and it was found to be essential for axon transport and structural plasticity in motoneurons. Elevated expression of Pav-KLP underlies transport and plasticity phenotypes in pathway mutants, indicating that Toll-6-FoxO signaling promotes MT dynamics by limiting Pav-KLP expression. In addition to uncovering a novel molecular pathway, this work reveals an unexpected function for dynamic MTs in enabling rapid activity-dependent structural plasticity.
|Costa, M. F. A. and Ohkura, H. (2019). The molecular architecture of the meiotic spindle is remodeled during metaphase arrest in oocytes. J Cell Biol. PubMed ID: 31278080
Before fertilization, oocytes of most species undergo a long, natural arrest in metaphase. Before this, prometaphase I is also prolonged, due to late stable kinetochore-microtubule attachment. How oocytes stably maintain the dynamic spindle for hours during these periods is poorly understood. This study reports that the bipolar spindle changes its molecular architecture during the long prometaphase/metaphase I in Drosophila melanogaster oocytes. By generating transgenic flies expressing GFP-tagged spindle proteins, it was found that 14 of 25 spindle proteins change their distribution in the bipolar spindle. Among them, microtubule cross-linking kinesins, MKlp1/Pavarotti and kinesin-5/Klp61F, accumulate to the spindle equator in late metaphase. The late equator accumulation of MKlp1/Pavarotti is regulated by a mechanism distinct from that in mitosis. While MKlp1/Pavarotti contributes to the control of spindle length, kinesin-5/Klp61F is crucial for maintaining a bipolar spindle during metaphase I arrest. This study provides novel insight into how oocytes maintain a bipolar spindle during metaphase arrest.
|Lie-Jensen, A., Ivanauskiene, K., Malerod, L., Jain, A., Tan, K. W., Laerdahl, J. K., Liestol, K., Stenmark, H. and Haglund, K. (2019). Centralspindlin Recruits ALIX to the Midbody during Cytokinetic Abscission in Drosophila via a Mechanism Analogous to Virus Budding. Curr Biol. PubMed ID: 31607533
Abscission, the final step of cytokinesis, cleaves the thin intercellular bridge connecting the two daughter cells. The scaffold protein ALIX is a core component of the abscission machinery with an evolutionarily conserved role in midbody recruitment of ESCRT-III, which mediates the final cut. In mammalian cells, the centralspindlin complex recruits the major midbody organizer CEP55 that directly binds and recruits ALIX and ESCRT-I, which in turn cooperatively recruit ESCRT-III. However, CEP55 is missing in Drosophila melanogaster and other invertebrates, and it is unknown how the abscission machinery is recruited to the midbody in the absence of CEP55. This study addresses how Drosophila ALIX is recruited to the midbody. Surprisingly, ALIX localizes to the midbody via its V-domain, independently of the GPPX3Y motif in the proline-rich region that recruits human ALIX. It was elucidated that the centralspindlin component Pavarotti (H.s.MKLP1) interacts with the V-domain of ALIX to recruit it to the midbody. Specifically, the results indicate that an LxxLF motif in Pavarotti directly interacts with a conserved hydrophobic pocket in the ALIX V-domain, which in human ALIX binds (L)YPXnL/LxxLF motifs of virus proteins. Thus, this study identifies that ALIX is recruited by an analogous mechanism during abscission in Drosophila as during virus budding in mammalian cells and an ancestral role for centralspindlin in recruiting the abscission machinery to the midbody.
|Nakamura, M., Verboon, J. M., Prentiss, C. L. and Parkhurst, S. M. (2020). The kinesin-like protein Pavarotti functions noncanonically to regulate actin dynamics. J Cell Biol 219(9). PubMed ID: 32673395
Pavarotti, the Drosophila MKLP1 orthologue, is a kinesin-like protein that works with Tumbleweed (MgcRacGAP) as the centralspindlin complex. This complex is essential for cytokinesis, where it helps to organize the contractile actomyosin ring at the equator of dividing cells by activating the RhoGEF Pebble. Actomyosin rings also function as the driving force during cell wound repair. Previous work has shown that Tumbleweed and Pebble are required for the cell wound repair process. This study shows that Pavarotti also functions during wound repair and confirm that while Pavarotti, Tumbleweed, and Pebble are all used during this cellular repair, each has a unique localization pattern and knockdown phenotype, demonstrating centralspindlin-independent functions. Surprisingly, it was found that the classically microtubule-associated Pavarotti binds directly to actin in vitro and in vivo and has a noncanonical role directly regulating actin dynamics. Finally, this actin regulation by Pavarotti was shown to not be specific to cellular wound repair but is also used in normal development.
|Sechi, S., Frappaolo, A., Karimpour-Ghahnavieh, A., Fraschini, R. and Giansanti, M. G. (2020). A novel coordinated function of Myosin II with GOLPH3 controls centralspindlin localization during cytokinesis in Drosophila. J Cell Sci 133(21). PubMed ID: 33037125
In animal cell cytokinesis, interaction of non-muscle myosin II (NMII) with F-actin provides the dominant force for pinching the mother cell into two daughters. This study demonstrates that celibe (cbe) is a missense allele of zipper, which encodes the Drosophila Myosin heavy chain. Mutation of cbe impairs binding of Zipper protein to the regulatory light chain Spaghetti squash (Sqh). In dividing spermatocytes from cbe males, Sqh fails to concentrate at the equatorial cortex, resulting in thin actomyosin rings that are unable to constrict. cbe mutation impairs localization of the phosphatidylinositol 4-phosphate [PI(4)P]-binding protein Golgi phosphoprotein 3 (GOLPH3, also known as Sauron) and maintenance of centralspindlin at the cell equator of telophase cells. These results further demonstrate that GOLPH3 protein associates with Sqh and directly binds the centralspindlin subunit Pavarotti. It is proposed that during cytokinesis, the reciprocal dependence between Myosin and PI(4)P-GOLPH3 regulates centralspindlin stabilization at the invaginating plasma membrane and contractile ring assembly.
Mutations in the Drosophila gene pavarotti result in the formation of abnormally large cells in the embryonic nervous system. In mitotic cycle 16, cells of pav mutant embryos undergo normal anaphase but then develop an abnormal telophase spindle and fail to undertake cytokinesis. The septin Peanut, actin, and the actin-associated protein Anillin do not become correctly localized in pav mutants. pav encodes a kinesin-like protein related to the mammalian MKLP-1. In cellularized embryos, the protein is localized to centrosomes early in mitosis, and to the midbody region of the spindle in late anaphase and telophase. Polo kinase associates with Pav, with which it shows an overlapping pattern of subcellular localization during the mitotic cycle and this distribution is disrupted in pav mutants. It is suggested that Pav is required both to establish the structure of the telophase spindle to provide a framework for the assembly of the contractile ring, and to mobilize mitotic regulator proteins (Adams, 1998).
Cytokinesis is the quintessential act of mitosis, whereby segregated daughter nuclei are partitioned into two separate cells. In eukaryotes, this is accomplished by an actin-myosin contractile ring that forms around the cell equator during mitosis and constricts inwards at telophase. In higher eukaryotes the correct positioning and assembly of the contractile ring requires the mitotic spindle, although the mechanism by which this occurs is a matter of controversy. Classical experiments suggest that the spindle poles are sufficient to stimulate cytokinesis. Two mechanisms have been put forward to demonstrate how spindle poles might induce cleavage furrows. In the first model, the poles signal directly to the equatorial cortex of the cell, perhaps by the action of astral microtubules. Alternatively, the astral relaxation model proposes that spindle poles induce a relaxation of the cell cortex nearest to the poles, leading to a tension differential between the poles and the equator that results in equatorial contraction. However, this model has been discredited by force measurements showing that tension increases at the equator without a concomitant decrease at the cell poles (Adams, 1998 and references therein).
There is growing evidence that the central spindle plays an essential role in the positioning and assembly of the contractile ring. The central spindle is composed of a dense network of overlapping antiparallel microtubules that forms between the separating daughter nuclei during anaphase. In cultured cells induced to develop multipolar spindles as result of treatment with low concentrations of the microtubule-destabilizing drug colcemid, the formation of a cleavage furrow absolutely depends on the presence of central spindle microtubules that are required throughout cytokinesis. Creation of a barrier between the central spindle and the cell cortex prevents cleavage, indicating that a signal for cytokinesis may emanate from the central spindle. Manipulation of grasshopper neuroblast spindles results in the formation of a cleavage furrow when the spindle is moved close to the cell cortex (Adams, 1998 and references therein).
In addition to the contribution made by the spindle, several proteins, known as the chromosomal passenger proteins, dissociate from chromosomes at the metaphase-anaphase transition to be deposited at the cell equator. The inner centromere proteins (INCENPs), for example, transfer to the central spindle and the cell cortex and are necessary for completion of cytokinesis, whereas TD-60 forms a disc structure in the position of the metaphase plate anticipating the cleavage plane. This disc is proposed to link the central spindle to the cell cortex (Adams, 1998 and references therein).
In Drosophila, mutational analysis has identified several genes encoding structural elements of the contractile ring essential for cytokinesis, such as myosin light chain, cofilin (Twinstar in Drosophila), profilin (Chickadee in Drosophila), and Peanut, a homolog of the yeast septins. Kinesin-like proteins (KLPs) are microtubule motor proteins responsible for many of the dynamic aspects of mitosis such as centrosome separation, spindle assembly, and anaphase movement of chromosomes (Adams, 1998).
pavarotti encodes a fly homolog of the mammalian mitotic kinesin-like protein-1 (MKLP-1). In pav mutants, anaphase B occurs normally but cytokinesis is defective, due to a disruption of the central spindle structure and subsequent failure to assemble a contractile ring. In mitosis, the Pav first associates with the centrosomes and then becomes localized at the spindle equator at late anaphase. Pav associates with Polo kinase and is required for its localization to the centrosome and central spindle. The Polo-like kinases are known to be required for centrosome function: Mutations in the Drosophila gene polo and its fission yeast homolog plo1 lead to spindle defects including the formation of monopolar structures. Injection of antibodies against human plk also result in the formation of monopolar structures in HeLa cells. An additional role for plks in cytokinesis is suggested by the late mitotic phenotype of fission yeast plo1 and its budding yeast counterpart cdc5. Multiple septation has been shown to result from plo1+ overexpression in fission yeast, or the expression of mammalian Plk in budding yeast. In light of the association of Polo kinase with PAV-KLP, the plks might also have a role in cytokinesis in metazoan cells that has been obscured by the earlier requirement for mitosis (Adams, 1998).
Pav mutant embryos show cytokinesis defects in virtually all dividing cells at cycle 16. As with other genes required for cell cycle progression in Drosophila, the lethal phase depends on the role and stability of the wild-type protein, the abundance of its maternal provision to the embryo, and the strength of the mutant allele. The majority of cell cycle proteins are supplied maternally, and the rate at which this supply is exhausted depends on the particular protein. The maternal supply of Pav appears to be depleted early in development, either by degradation or its expulsion from newly divided cells in the remnants of the midbody. Consequently, the lethal phase occurs in embryonic development leading to the defects in the PNS, embryonic tissues in which cells continue dividing beyond a sixteenth cell cycle. Lethality at this stage is a consequence of the inability of each mutant allele to produce functional gene product, since all alleles of pav show the same lethal phase, whether homozygous or hemizygous against a chromosome deficiency for the region (Adams, 1998).
As the cell progresses through its division cycle, the distribution of Pav follows a highly dynamic pattern that is very similar to that of MKLP-1 (Nislow, 1990). Both the mammalian and fly proteins show a punctate distribution in the region of the nucleus at interphase. They are present at the centrosomes from metaphase onwards and localize to the spindle midzone at late anaphase and telophase. Many proteins with roles in cytokinesis, including Pav and Peanut, become concentrated in midbodies, the remnants of which are expelled from the two daughter cells as they separate, and are seen as discrete spots of staining between interphase cells in cellularized embryos (Adams, 1998).
The pattern of distribution of Pav in syncytial embryos differs in some respects from that seen in individual cells, presumably reflecting the absence of cytokinesis. The protein still shows a pronounced association with the central region of the spindle at late telophase but then follows a pattern that suggests association with actin or actin-associated molecules. Although cytokinesis does not take place at this stage of development, the organization of the actin cytoskeleton is important in maintaining the distribution of and spacing between the rapidly increasing numbers of syncytial nuclei. Thus, an actin-containing structure analogous to the contractile ring forms at the position of the former metaphase plate, but in interphase this is redistributed to the cortex where it will form a 'cap' that sits between the nucleus and plasma membrane. Upon entry into mitosis the cortical actin is reorganized into the 'pseudocleavage furrows' that form a network capturing individual metaphase figures. In many respects these are analagous to cleavage furrows except that they are formed earlier in the mitotic cycle. Pav appears to concentrate in this network but is then liberated to associate with the spindle and move to the central region at late anaphase. It has been proposed that actin is continually binding to microtubules at this stage and moving toward their plus ends. One might speculate that Pav could provide the requisite motile force (Adams, 1998).
Mutations leading to cytokinesis defects have been described in genes encoding two other KLPs in Drosophila: KLP3A, and KLP38B. It is difficult to make comparisons between the mutant phenotypes, as in the case of KLP38B, a cytokinesis phenotype is inferred in larval neuroblasts that attain low levels of polyploidy, and from the observation of binucleate follicle cells in the ovary. KLP3A mutants also show a central spindle defect leading to a failure of cytokinesis, but this is seen in male meiosis. The differing lethal phases of the pav and KLP3A mutants suggest a specialized role for the two proteins for cytokinesis in different cell types, but an overlapping function of Pav in meiosis cannot be ruled out at the present time. Both Pav and KLP3A accumulate in the midbody at telophase, although Pav has the distinctive attribute of being associated with the centrosome (Adams, 1998 and references therein).
pav embryos initially show dramatic defects in the morphology of the central spindle at telophase. The simplest model for Pav function is that it participates in reorganizing the central spindle region after anaphase B has occurred, in a manner that is vital for assembly of the contractile ring. How the central spindle assists formation of the contractile ring is poorly understood. The septin Peanut, itself essential for cytokinesis in Drosophila, fails to localize to a ring-like structure in the pav mutants. This may be a direct consequence of the disrupted structure of the telophase spindle, since recent work with Xenopus shows that septins are microtubule-associated proteins, and that the injection of anti-septin antibodies inhibits contractile ring function. The septins may link the central spindle to the cell cortex, and consequently the delocalization of Peanut in pav mutants would disrupt the formation of the contractile ring. Anillin and actin also fail to associate with the equatorial region of the cell during anaphase and telophase, and consequently cells fail to divide and become tetraploid (Adams, 1998).
The role of the spindle poles in cytokinesis is less clear. Do they signal the onset of cytokinesis, or, rather, direct the positioning of cell cleavage? In Drosophila male meiosis, study of the male sterile mutation asterless reveals that in the absence of astral microtubules, cell cleavage occurs to completion but in the wrong place. A normal central spindle is seen in these mutants, indicating that in this system the central spindle may play some role in the recruitment of contractile ring components, whereas astral microtubules dictate the position of the cleavage furrow (Adams, 1998 and references therein).
The finding that pav is required for cytokinesis seems at odds with previous work suggesting that the mammalian homolog might function during either metaphase or anaphase. In vitro assays demonstrate an ability to cross-link and slide apart antiparallel microtubules, leading to the suggestion of a role in spindle pole separation during anaphase B, whereas the injection of anti-MKLP-1 antibodies into mitotic HeLa cells results in a metaphase arrest. The in vitro assay may not be sufficiently specific to dissect the exact role of MKLP-1, since the system lacks centrosomes and the ancillary mechanisms necessary to generate a contractile ring, both of which influence microtubule behavior during cytokinesis. Thus, the ability to cross-link and slide along microtubules in vitro may be a property required of the KLP when associated with contractile ring structures. In any event, the pav mutant phenotype shows no indication of a defect in anaphase B, and spindles extend to the same length as in wild-type embryos at late anaphase and telophase. Moreover, the rare binucleate cells that have lost pav function in the previous mitosis can perform anaphase perfectly well. Thus, if Pav does play a role in anaphase spindle elongation or spindle assembly, it would seem that this role is redundant and can be fulfilled by other KLPs. Although MKLP-1 is the most closely related klp to Pav, the sequence homology outside the conserved motor domain is not particularly high (20%), so there could be nonidentical functions. The idea that KLPs in the same subfamily can have distinct functions in different organisms is not without precedent. For example, the chromokinesin subfamily of DNA-binding KLPs involved in chromosome congression includes Drosophila KLP3A, which does not appear to bind DNA and is also required for cytokinesis. Nevertheless, because the size, domain structure, ability to associate with plks, and dynamic localization of pav-KP is so similar to MKLP-1, it would be surprising if it were not a functional homolog (Adams, 1998).
The presence of Pav and its mammalian counterparts at the centrosomes and the central region of the spindle raises the possibility that it may transport a signaling molecule required for either centrosome function, cytokinesis, or both. The serine-threonine protein kinase encoded by polo is an ideal candidate for such a molecule. polo mutants show abnormal centrosome behavior, including the formation of monopolar spindles, a phenotype that is also seen following the microinjection of anti-plk antibodies into mammalian cells. In addition to the formation of monopolar spindles, the additional consequence of disruption of the fission yeast homolog is to prevent cytokinesis (Ohkura, 1995). It is not clear whether the polo mutation results in cytokinesis defects in Drosophila because the mitotic cycle is blocked at an earlier stage. Nevertheless, the localization of polo kinase throughout the mitotic cycle would be consistent with a dual role for the enzyme similar to that demonstrated in fission yeast. The association demonstrated between Polo kinase and Pav leads to a speculation that in addition to a role in assembling the central region of the spindle, Pav may also be responsible for transporting Polo kinase from one set of centrosome-associated substrates to a second set of substrates in the midzone of the spindle as mitosis progresses (Adams, 1998).
Kinesin-1 can slide microtubules against each other, providing the mechanical force required for initial neurite extension in Drosophila neurons. This sliding is only observed in young neurons actively forming neurites and is dramatically downregulated in older neurons. The downregulation is not caused by the global shutdown of kinesin-1, as the ability of kinesin-1 to transport membrane organelles is not diminished in mature neurons, suggesting that microtubule sliding is regulated by a dedicated mechanism. This study has identified the "mitotic" kinesin-6 Pavarotti (Pav-KLP) as an inhibitor of kinesin-1-driven microtubule sliding. Depletion of Pav-KLP in neurons strongly stimulated the sliding of long microtubules and neurite outgrowth, while its ectopic overexpression in the cytoplasm blocked both of these processes. Furthermore, postmitotic depletion of Pav-KLP in Drosophila neurons in vivo reduced embryonic and larval viability, with only a few animals surviving to the third instar larval stage. A detailed examination of motor neurons in the surviving larvae revealed the overextension of axons and mistargeting of neuromuscular junctions, resulting in uncoordinated locomotion. Taken together, these results identify a new role for Pav-KLP as a negative regulator of kinesin-1-driven neurite formation. These data suggest an important parallel between long microtubule-microtubule sliding in anaphase B and sliding of interphase microtubules during neurite formation (Del Castillo, 2014).
Previous work showed that microtubule sliding by kinesin-1 drives initial neurite outgrowth in Drosophila neurons and that sliding is downregulated as neurons mature. This paper, has demonstrated that the 'mitotic' kinesin Pav-KLP functions as a negative regulator of interphase microtubule sliding both in Drosophila S2 cells and in Drosophila neurons. Knockdown of Pav-KLP stimulated microtubule sliding, producing longer axons, while overexpression of Pav-KLP inhibited both sliding and neurite outgrowth. Increased length of axons after Pav-KLP depletion was also observed in vivo in Drosophila. Therefore, Pav-KLP attenuates neurite outgrowth by downregulation of kinesin-1-powered microtubule-microtubule sliding (Del Castillo, 2014).
Pav-KLP and its orthologs (members of the kinesin-6 family) were originally identified as essential components for central spindle assembly and cleavage furrow formation. Pav-KLP depletion induces defects in morphology of the mitotic spindle at telophase and failure to recruit contractile ring components. However, it has been demonstrated that CHO1/MKLP1, the mammalian ortholog of Pav-KLP, has an additional function in neurodevelopment. CHO1/MKLP1 plays a role in establishing dendrite identity in differentiated neurons. Depletion of CHO1/MKLP1 induces progressive loss of dendrites. It has concluded that CHO1/MKLP1 organizes microtubules in dendrites by transporting short minus-end-distal microtubule fragments into the dendrites. More recent work has revisited the role of CHO1/MKLP1 in developing neurons and suggested that CHO1/MKLP1 can regulate neurite outgrowth. Depletion of CHO1/MKLP1 increased transport of short microtubule fragments. The current data are in agreement with the idea that Pav-KLP regulates formation of neurites. However, the mechanisms reported in this study are clearly different from the results obtained by the mammalian studies in two significant aspects. First, this study has shown that the reorganization of microtubules required for neurite formation is driven by kinesin-1. Second, the current visualization technique clearly demonstrates that microtubules in developing Drosophila neurons are moved as long polymers. It is possible that the differences between the results and the mammalian study can be explained by different models (Drosophila versus mammalian neurons). A more attractive idea is that similar mechanisms work in both systems, but further work is required to understand the role of kinesin-1 in neurite outgrowth in mammalian neurons (Del Castillo, 2014).
Interestingly, work by several groups has shown that proteins that function together with kinesin-6 in the cytokinesis pathway could also regulate neuronal morphogenesis. For example, Tumbleweed or Ect2/Pebble/RhoGEF depletion increases the extent of neurite outgrowth, suggesting that Tumbleweed and RhoGEF control neurite outgrowth through actin reorganization. However, the current results demonstrate that the primary regulator of neurite outgrowth is kinesin-6 family member Pav-KLP, the essential partner of Pebble and Tumbleweed. Furthermore, the effect of Pav-KLP on process formation is independent of actin or small GTPases (although more subtle effects of Tumbleweed or Ect2 on the actin cytoskeleton in developing neurons cannot be completely excluded). Indeed, a recent work concluded that an actin-signaling pathway regulated by the Centralspindlin complex controls protrusive activity required for directional neuronal migration (Del Castillo, 2014).
The original idea that mitotic motors regulate cytoplasmic microtubules in neurons suggested that microtubule arrays in neurons are established by mechanisms that are analogous to those that organize the mitotic spindle. Supporting this idea, it was demonstrated that inhibition of other mitotic motors, e.g., kinesin-5, affected the axon length Advancing this concept, this paper proposes that Pav-KLP/kinesin-6 directly regulates cytoplasmic microtubule arrangement by crosslinking them. It has been shown that loss-of-function mutations on ZEN-4/MKLP1, the C. elegans form of Pav-KLP, produce longer spindles, suggesting that kinesin-6 motors inhibit sliding of microtubules against each other during anaphase B. If this is indeed the case, the current results suggest an important functional similarity between the molecular mechanisms of cell division and process formation in neurons. While anaphase B is driven in part by microtubule-microtubule sliding powered by bipolar kinesin-5 and negatively regulated by kinesin-6 (mammalian MKLP1/C. elegans Zen-4/Drosophila Pav-KLP), the initial formation of neurites requires microtubule-microtubule sliding by kinesin-1 and, similar to anaphase B, is negatively regulated by kinesin-6. Thus, kinesin-6 motors together with other components of the Centralspindlin complex can function as general brakes of microtubule-microtubule sliding during both cell division and postmitotic neurite formation (Del Castillo, 2014).
The fact that microtubule sliding is inhibited by Pav-KLP in mature, but not young, neurons suggests that Pav-KLP itself is temporally regulated. One potential mechanism that could affect the ability of Pav-KLP (MKLP-1) to regulate microtubule sliding is Pav-KLP phosphorylation. Phosphorylation of Ser710 in MKLP-1 (Ser743 in Drosophila Pav-KLP) has been shown to promote its binding to protein 14-3-3, preventing MKLP-1 from clustering on microtubules. Future studies using phosphomimetic variants of Pav-KLP may help to test this mechanism (Del Castillo, 2014).
The sequence of a 3.2-kb pav cDNA reveals an ORF of 2658 bases coding for an 886-amino-acid protein of 100 kD predicted molecular mass with homology to the KLP family of microtubule motor proteins. It contains a predicted microtubule motor domain of ~350 amino acids that includes a nuclear localization signal, an ATP binding site, and several motifs common to plus end-directed KLPs. A predicted coiled-coil region lies between residues 500 and 700, whereas residues 700-886 show homology only with the MKLP-1 subfamily of KLPs. Pav is most similar to this subfamily throughout its entire sequence, showing 34% identity with MKLP-1 as compared to 13% identity to other mitotic KLPs. The highest degree of identity (48%) lies in the motor and hinge domains (residues 1-450), although patches of identity extend throughout the entire carboxy-terminal region of the protein (residues 700-886), a segment of KLPs known to be highly diverged. The MKLP-1 subfamily of KLPs comprises human MKLP-1 and its Chinese hamster counterpart CHO-1, first identified as a mitotic spindle antigen (Adams, 1998).
date revised: 15 February 2002
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