InteractiveFly: GeneBrief

no distributive disjunction: Biological Overview | References

Chromosome congression, the process of positioning chromosomes in the midspindle, promotes the stable transmission of the genome to daughter cells during cell division. Congression is typically facilitated by DNA-associated, microtubule (MT) plus end-directed motors called chromokinesins. The Drosophila melanogaster chromokinesin No distributive disjunction (NOD) contributes to congression, but the means by which it does so are unknown in large part because NOD has been classified as a nonmotile, orphan kinesin. It has been postulated that NOD promotes congression, not by conventional plus end-directed motility, but by harnessing polymerization forces by end-tracking on growing MT plus ends via a mechanism that is also uncertain. This study demonstrates that NOD possesses MT plus end-directed motility. Furthermore, NOD directly binds EB1 through unconventional EB1-interaction motifs that are similar to a newly characterized MT tip localization sequence. It is proposed that NOD produces congression forces by MT plus end-directed motility and tip-tracking on polymerizing MT plus ends via association with EB1 (Ye, 2018).

Equal distribution of duplicated DNA is required to maintain genomic stability through cell division. The microtubule (MT) cytoskeleton is reorganized to form a bipolar spindle as cells enter mitosis or meiosis. Chromosomes are positioned at the spindle equator during a process known as congression. Chromosome movements within the spindle are predominantly mediated by motor proteins that walk directionally on spindle MTs. An important class of congression motors is the chromokinesins, which include the kinesin families, kinesin-4, kinesin-10, and kinesin-12. Chromokinesins work cooperatively to promote chromosome alignment during cell division. Kinesin-10 and kinesin-4 are chromosome-associated, plus end-directed motors, although KIF4A suppresses plus end MT dynamics and may dampen polar ejection forces (PEFs) that push chromosome arms away from spindle poles, whereas kinesin-10's role is more intuitive and likely the predominant PEF-producing motor. The function of vertebrate kinesin-10 (Kid) was first described in Xenopus laevis egg extracts in which Xkid was required to establish and maintain chromosome arm congression. Although data from human cells have consistently shown that hKid contributes to congression, the alignment defects observed in tissue culture cells have not been as severe as in egg extracts. Vertebrate kinesin-10s have been shown to possess plus end-directed motility and to generate force when bound to chromatin (Ye, 2018).

The Drosophila melanogaster chromokinesin NOD shares sequence homology in its N-terminal motor with both kinesin-10 and kinesin-4 motor domains and in its C terminus with the kinesin-10 DNA-binding motif (helix-hairpin-helix), but it has been designated an orphan kinesin as a result of significant divergence in its structural elements and organization relative to conventional kinesins. NOD was initially identified and characterized genetically as the mutant no distributive disjunction (nod), which exhibited high frequencies of nondisjunction and chromosome loss in female meiosis. The nod gene encodes a kinesin-like protein (NOD) with an N-terminal motor domain. Achiasmate (nonexchange) chromosomes frequently failed to associate with spindles or were mispositioned near spindle poles in oocytes lacking functional NOD. The characterization of DNA binding activities in the C terminus of NOD, its N-terminal motor domain, and the misalignment phenotype of nod mutants led to the hypothesis that NOD is the PEF motor in fly oocytes, and later work in Drosophila tissue culture cells revealed a role for NOD in mitotic chromosome congression. Although NOD possesses a conserved N-terminal motor domain and MT-stimulated ATPase activity, efforts to reconstitute directional motility in vitro have failed leading to NOD being classified as a nonmotile kinesin. How could a nonmotile kinesin generate force? An alternative theory posits that, as a result of the unique mechanochemical properties of its motor domain, NOD moves chromosomes by associating with the plus ends of polymerizing MTs, although direct evidence for this mechanism is lacking. High resolution imaging of NOD-coated chromatin stretching events in living cells were suggestive of both plus end-directed motility and end-tracking coincident with EB1 comets (Ye, 2018).

NOD possesses an N-terminal motor domain and two distinct C-terminal DNA binding regions comprised of high mobility group (HMG) repeats and a helix-hairpin-helix motif that mediate chromatin-association of full length (FL) NOD-mCherry throughout the cell cycle in Drosophila S2 cells. Between the motor and DNA-binding domains, NOD is predicted to contain intrinsically disordered regions (~50%), as well as four α-helices, one of which has a low probability of forming a parallel two-stranded coiled coil (CC). To dissect NOD motor function in vivo, truncations of NOD tagged at their C termini with mCherry were expressed in GFP-α-tubulin expressing Drosophila S2 cells. The motor domain (1-324) did not bind MTs in mitosis or interphase and was diffuse in the cytosol. Thus, unlike many well-characterized motor proteins, the NOD324 motor domain exhibits weak or no MT binding activity in cells. Given the high physiological concentration of ATP, this observation is consistent with in vitro studies in which NOD motor domain exhibited significantly lower MT binding affinities in the presence of excess ATP than conventional kinesin motors (Ye, 2018).

Most kinesins with N-terminal motor domains possess a neck linker and a well-defined neck CC downstream of the motor; however, some kinesins, including NOD, have noncanonical neck regions that contribute to their functions in cells (Davies, 2015). The localization was examined of a NOD truncation (1-485) that encompasses the motor domain and the noncanonical neck extension region. Addition of the neck extension conferred MT-binding activity in cells as NOD485 uniformly coated MTs throughout the cell cycle. Inclusion of the nonconventional extension could promote MT binding through numerous nonexclusive mechanisms, including the introduction of a second MT binding site, which has been shown for hKID, posttranslational modifications, association with regulatory factors, or dimerization through the low probability CC. The MT localization of NOD485 was significantly reduced upon fixation conditions suggestive of the interaction, while stronger than NOD324, being relatively low affinity. Nonetheless, the localization of NOD485 led to the idea that the oligomeric state of NOD warranted further investigation. Several structural and functional aspects of NOD are reminiscent of the monomeric plus end-directed kinesin Unc104/KIF1A. Kinesin-10 family members have been shown to be monomeric in vitro. Furthermore, NOD and Unc104/KIF1A possess low-probability/weak CC regions adjacent to their N-terminal motors, whereas their C termini contain domains that cluster the motors on the surface of cargos: chromosomes and synaptic vesicles, respectively. Prior work on Unc104 demonstrated that constitutive dimerization of the Unc104 motor domain and its adjacent 'weak' CC converted the monomer into a processive, plus end-directed motor with physiological velocities. A comparable approach as that applied to Unc104, specifically fusion to the kinesin-1 'stalk' CC, was next used to examine how dimerization of the NOD motor domain affected its behavior (Ye, 2018).

The motor domain alone (1-324) was first dimerized. NOD324CC evenly coated interphase MTs and robustly associated with spindle MTs during mitosis, exhibiting a slight enrichment toward the plus ends of kinetochore fibers in the vicinity of aligned chromosomes with some evident MT bundling. NOD485CC exhibited a striking localization pattern in which it was highly enriched near MT plus ends in mitosis and interphase. Significant MT bundling was also observed throughout the cell cycle especially in cells with high NOD485CC levels, which was not observed in the NOD485-expressing cells. The localization patterns and behavior of the dimerized NOD truncations were not attributable to the kinesin-1 CC because CC-mCherry only weakly associated with a subset of MTs in some cells and exhibited no obvious or consistent localization pattern. Furthermore, the neck extension region, while necessary, was not sufficient for MT binding, as the localization patterns of NOD325-485 and NOD325-485CC were identical to mCherry and CC-mCherry, respectively (Ye, 2018).

The localization pattern of NOD485CC suggested dimerized NOD could possess plus end-directed motility in cells. This possibility was further examined by imaging cells using total internal reflection fluorescence (TIRF) microscopy to visualize NOD on the MTs closest to the cell cortex. Puncta of both NOD485 and NOD324CC were visible on MTs, but motility was not obvious. The behavior of NOD485CC was markedly different from the other truncations as NOD puncta were clearly observed moving processively on MTs toward the cell periphery. The distribution of fluorescence intensities of motile NOD485CC-mCherry puncta was indistinguishable from that of motile kinesin-1-mCherry dimers, demonstrating that NOD485CC was assembling into motile dimers. Measurements of motile NOD485CC dimers in cells yielded a mean velocity of 8.70 ± 3.61 micrometers/min. These data demonstrate that dimerization of the NOD motor domain alone (NOD324CC) promotes MT binding but not motility, most likely because it lacks a functional neck linker, which plays an important role in coordinating motor head functions (Vale and Milligan, 2000), whereas dimerization of the motor domain and nonconventional neck extension region converts NOD into a directional motor (Ye, 2018).

To further characterize NOD motility in vitro, TIRF-based imaging was applied to cell extracts in which NOD485CC was the only fluorescently tagged component. Using this technique, NOD motility could be studied in near physiological conditions, but in a chamber in which the state of the MTs, which are attached to the cover-glass, as well as the buffering conditions can be controlled. In cell lysates prepared from NOD485CC-expressing cells, NOD walked unidirectionally on taxol stabilized MTs in an ATP-dependent manner. The mean velocity of 8.62 2.32 micrometers/min measured for motile NOD485CC dimers in cell lysates was indistinguishable from live-cell measurements (Ye, 2018).

NOD motility has never been reconstituted in vitro and, like others,this study could not purify active NOD485CC from bacteria. Furthermore, NOD485CC activity in cell lysates was labile and sensitive to buffer conditions. Thus, NOD485CC-mCherry was purified from Drosophila S2 cells using a C-terminal Strep tag that allows for affinity purification on a streptavidin-based matrix and gentle elution using biotin-containing buffers to better preserve protein activity. Silver staining of the purification revealed a unique band, when compared with a mock purification from wild-type cell extracts, at the predicted size of NOD485CC-mCherry (~110 kD), which was confirmed by Western blot using an mCherry antibody. Although it is probable that NOD-associated proteins were copurified in the preparation, the majority of background bands were likely biotinylated proteins or nonspecific because a nearly identical banding pattern was observed in mock purifications from wild-type cell extracts. In TIRF assays, the affinity purified NOD485CC-mCherry exhibited ATP-dependent, unidirectional motility, although the mean velocity of 5.79 ± 1.56 micrometers/min was slower than velocities measured in cells and lysates (Ye, 2018).

NOD485CC dimers often moved toward the cell periphery where the MT plus ends are typically oriented during interphase. To define the directionality of NOD485CC motility, purified GFP-labeled human kinesin-1 (Kif5B) motor, a plus end-directed motor, was added to lysates from NOD485CC-mCherry expressing cells. NOD485CC and kinesin-1 walked in the same direction, establishing that NOD is a plus end-directed motor. Collectively, the data in cells and in vitro demonstrate that dimerized NOD is a plus end-directed motor with velocities similar to those measured for Xkid and hKID, which in combination with the presence of a C-terminal helix-hairpin-helix DNA-binding motif warrants consideration of NOD as the Drosophila kinesin-10 orthologue (Ye, 2018).

Next, it was tested if chemically induced dimerization was sufficient to support NOD motility by building cell lines coexpressing dark (no fluorescent tag) NOD485-FRB and NOD485-FKBP-EGFP. Like NOD485, NOD485-FKBP-EGFP localized uniformly to MTs in the absence of rapamycin. Upon addition of 100 nM rapamycin, motile puncta of NOD485-FKBP-EGFP moved on MTs toward the cell periphery. Extracts were next prepared from NOD485-FKBP-EGFP, NOD485-FRB-expressing cells to visualize NOD on MTs by TIRF microscopy in vitro. Without rapamycin, nonmotile puncta of NOD485-FKBP-EGFP associated with MTs. Unidirectional motility of NOD485-FKBP-EGFP puncta was achieved within minutes of adding 100 nM rapamycin to the same cell extracts that did not exhibit motility in the absence of rapamycin. Cell lines coexpressing NOD485-FKBP-EGFP and NOD485-FRB-mCherry were next visualized. In the absence of rapamycin, the mCherry and EGFP-tagged NOD uniformly coated the MTs similar to NOD485, but NOD localization changed upon addition of 100 nM rapamycin as EGFP- and mCherry-tagged, chemically dimerized NOD485 accumulated toward MT plus ends at the cell periphery similar to NOD485CC. Altogether, the kinesin-1 CC and chemically induced dimerization data support the conclusion that multiple modes of NOD485 dimerization can support directional motility (Ye, 2018).

Although earlier work visualizing MT-associated stretching of NOD-coated chromatin was indicative of plus end-directed motility (Cane, 2013), it is technically challenging to assess the activity of nonartificially dimerized NOD in cells as a result of its constitutive chromatin localization. Fortuitously, NOD contains an endogenous nuclear export signal (NES) computationally predicted and borne out by the localization of various NOD truncations. Although NOD possesses an NES, it is not typically observed in the cytosol because it is tightly associated with chromatin throughout the cell cycle. It was reasoned that the affinity of NOD for chromatin could be reduced by deleting one of its two chromosome-associating domains (HMG14/17 repeats), a truncation called NODΔHMG. In ~10% of cells expressing NODΔHMG, possibly as a result of reduced affinity for the chromatin and the presence of an NES, cytosolic NOD puncta were visible. Therefore, it was possible to visualize nearly FL cytosolic NOD that was not artificially dimerized with CC or FKBP-FRB domains. Importantly, the cytosolic NODΔHMG exhibited plus end-directed motility toward the cell periphery along interphase MTs. Compared with the kinesin-1-mCherry standard, motile NODΔHMG puncta spanned a range of fluorescence intensities corresponding to ~5-200 NOD molecules with a mean of 52 motors per puncta, suggesting that NOD may function as clusters of dimers or more complex oligomers. Nevertheless, the velocity of NODΔHMG clusters was comparable to the velocities measured for NOD485CC dimers in cells and cell lysates in vitro (7.6 micrometers/min for NODΔHMG in cells vs. 8.6-8.7 micrometers/min for NOD485CC in cells and lysates) (Ye, 2018).

Prior work characterized NOD-coated chromatin stretch events that colocalized with EB1 comets, as well as occasional cytoplasmic NOD fragments that tracked polymerizing and depolymerizing MT plus ends (Cane, 2013), and NODΔHMG puncta also tracked growing plus ends. Spinning disc confocal imaging of cells coexpressing NOD485CC-EGFP and EB1-TagRFP-T revealed that a subset of NOD485CC puncta colocalized with EB1 tracks on MT plus ends. It is worth noting that NOD often remained associated with depolymerizing MTs through a mechanism that is presently unclear, but that could be similar to the bidirectional tracking activity of CENP-E because NOD, like hKID, may possess low-affinity MT binding regions outside of the motor domain. Plus end-directed NOD485CC dimers were evident behind tip-tracking NOD puncta in kymographs from NOD485CC-, EB1-expressing cells. Given the utility of cell lysates in visualizing NOD motility, extracts from NOD485CC-EGFP-expressing cells were added to dynamic rather than taxol-stabilized MTs in an effort to better visualize NOD end tracking by TIRF microscopy in vitro. The cell-based observations were fully validated in vitro using dynamic MTs as NOD485CC clearly walked toward the faster growing plus ends while a subpopulation of NOD485CC tip tracked on growing MT plus ends (Ye, 2018).

NOD's end-tracking activity combined with the observed colocalization of NOD with EB1 comets led to an investigation of whether NOD and EB1 directly interact in pulldown assays using purified components. GST-EB1-TagRFP-T pulled down with MBP-NOD485CC-EGFP, but not the MBP-EGFP control protein. To further characterize the NOD-EB1 interaction, attention turned to amino acids 325-485 because NOD485CC exhibited MT plus end-tracking activity, and NOD324CC did not. NOD contains multiple regions that are predicted to be disordered, centered around proline (P) and threonine (T) residues between residues 430-480. These motifs are similar to unconventional EB1-binding motifs recently identified in fungal species in the Saccharomyces cerevisiae Kar9 motor and Schizosaccharomyces pombe Dis1/Tog MAP. To identify EB1-interacting regions in NOD, overlapping peptide SPOT arrays encompassing what were deemed the 'PT' motifs were synthesized onto a cellulose membrane, and their interactions with purified EB1 were probed. The SPOT arrays comprised 15 amino acid peptides spanning NOD residues 430-480 with an offset of two residues and included a 'perfect' SxIP aptamer as a positive control peptide. The NOD peptides exhibiting the strongest association with purified Drosophila EB1-TagRFP-T centered on the PT motifs (Ye, 2018).

The amino acids surrounding SxIP motifs impact their affinities for EB1. To examine how PT-flanking amino acids contributed to EB1 binding, the peptides that exhibited the strongest association with EB1 on the array were subjected to alanine scanning. In each peptide, mutation of a surrounding arginine (R) residue approximately three to six amino acids upstream or downstream of the PT sequence eliminated EB1 binding. The results suggest that, like the SxIP motif, neighboring basic residues contribute to the affinity of NOD PT motifs for EB1. During the preparation of this manuscript, the unconventional Ka9-EB1 interaction was further characterized, leading to the designation of LxxPTPh as a novel tip-localization motif. Although the PT motifs in NOD resemble the LxxPTPh motif, the compositional differences may indicate that the Drosophila EB1 PT motif has diverged somewhat and/or that variability in the motif still supports EB1 interactions (Ye, 2018).

Microscale thermophoresis (MST) was used to measure the binding affinity of the NOD PT motif-1 for EB1. The N terminus of either the PT motif-1 or a bona fide 'perfect' SxIP peptide (positive control) was labeled with fluorescein (FITC) dye, and the movement of each fluorescently labeled peptide (fixed at 50 nM) in a temperature gradient was measured by MST while varying concentrations of unlabeled Drosophila GST-EB1. The binding affinity measured by MST for the SxIP motif was 807 nM, which is relatively close to the Kd (~570 nM) measured by isothermal titration calorimetry using the 'perfect' SxIP peptide and Drosophila EB1. The affinity of the PT motif-1 for Drosophila EB1 was measured to be 725 nM, slightly higher than the Kd of the positive control SxIP aptamer. Thus, the NOD PT motif-1 possesses an affinity for Drosophila EB1 that is comparable to other bona fide SxIP motifs and higher than affinities measured for EB1-CAP-Gly interactions (Ye, 2018).

How the Drosophila kinesin-10 NOD generates PEFs has long been a mystery. Prior work led to a hypothesis that NOD possesses two force producing activities: (1) MT plus end-directed motility, which had never been directly demonstrated and (2) end tracking on polymerizing MTs, the mechanism of which was speculative. This study demonstrates, for the first time, that NOD exhibits plus end-directed motility in cells and in vitro and that NOD directly interacts with the tip-tracking protein EB1 via a new type of conserved MT tip localization sequence. It is noteworthy that both MT polymerization and motor activity were originally proposed as sources of the PEF when the phenomenon was first described, more than 30 years later the current findings reveal NOD as a molecular nexus of both force-producing mechanisms (Ye, 2018).

Elevated polar ejection forces stabilize kinetochore-microtubule attachments

Chromosome biorientation promotes congression and generates tension that stabilizes kinetochore-microtubule (kt-MT) interactions. Forces produced by molecular motors also contribute to chromosome alignment, but their impact on kt-MT attachment stability is unclear. A critical force that acts on chromosomes is the kinesin-10-dependent polar ejection force (PEF). PEFs are proposed to facilitate congression by pushing chromosomes away from spindle poles, although knowledge of the molecular mechanisms underpinning PEF generation is incomplete. This study describes a live-cell PEF assay in which tension was applied to chromosomes by manipulating levels of the chromokinesin NOD (no distributive disjunction; Drosophila melanogaster kinesin-10). NOD stabilized syntelic kt-MT attachments in a dose- and motor-dependent manner by overwhelming the ability of Aurora B to mediate error correction. NOD-coated chromatin stretched away from the pole via lateral and end-on interactions with microtubules, and NOD chimeras with either plus end-directed motility or tip-tracking activity produced PEFs. Thus, kt-MT attachment stability is modulated by PEFs, which can be generated by distinct force-producing interactions between chromosomes and dynamic spindle microtubules (Cane, 2013).

Over 40 years ago, Nicklas and Koch (1969) stabilized erroneous kt-MT attachments in grasshopper spermatocytes by artificially creating tension with microneedles. It is proposed that NOD overexpression is the molecular equivalent of Nicklas' microneedles and that elevated PEFs produced by NOD overexpression stabilize syntelic attachments by introducing tension at kinetochores. The pioneering spermatocyte studies provided the first direct evidence that tension regulates interactions between chromosomes and the spindle. However, the use of microneedles is technically challenging, requires significant time investment per cell/experiment, and is restricted to a small number of manipulatable cell types that are not genetically tractable. The PEF assay developed in this study overcomes these previous limitations because (a) force application simply requires the addition of CuSO4 to the growth media, (b) proteins of interest in Drosophila S2 cells can be readily manipulated by RNAi, overexpression, and molecular engineering, and (c) the assay is scalable because many cells can be examined in one experiment. Consequently, it is envisioned that the PEF assay will provide a powerful tool for studying tension-dependent regulation of kt-MT attachment stability in living cells (Cane, 2013).

Since its discovery, the PEF has been implicated in chromosome positioning via regulation of both chromosome oscillation and congression. During chromosome oscillations, movement is driven by the poleward moving or leading kinetochore. The poleward moving kinetochore remains attached to its depolymerizing k-fiber and pulls the lagging sister kinetochore, which must elongate its k-fiber by microtubule polymerization. A change in direction has been hypothesized to be triggered by the introduction of tension at the leading kinetochore as it approaches the pole and experiences increasing levels of opposing PEFs. Current observations support this model and are in agreement with recent cell-based examinations of the contribution of PEFs to chromosome behavior as well as the finding that the application of tension to MT-associated kinetochore particles inhibited catastrophes and promoted rescues. Thus, emerging evidence supports chromosome oscillation models where the introduction of tension by PEFs at the leading kinetochore promotes a directional switch by rescuing depolymerizing kt-MTs (Cane, 2013).

The fact that ~80% of the attachments in high NOD-expressing cells are syntelic suggests that most chromosomes establish improper attachments before becoming bioriented. This mirrors a recent characterization of chromosome biorientation in meiosis I mouse oocytes, where ~90% of chromosomes experienced at least one round of Aurora kinase-dependent error correction before biorientation. Thus, transient formation of incorrect attachments is commonplace during cell division. Interestingly, improperly attached chromosomes often move to the spindle poles where they remain until error correction occurs. Misoriented chromosomes must experience increasingly higher levels of PEFs as they move poleward. Hence, the fact that elevated PEFs counteract error correction presents a conundrum: the spindle pole, where error correction often takes place, is also where PEFs are highest. Over time, baseline error correction mechanisms may win out over the stabilizing effects of the PEFs. Alternatively, other kt-MT attachment destabilizing activities may exist to counter the stabilizing effects of PEFs (Cane, 2013).

The findings also bear upon the interplay between force-dependent stabilization of kt-MT attachments and Aurora B-mediated error correction. Application of force to reconstituted kinetochore particles stabilized kt-MT attachments in the absence of Aurora B. The application of force to kinetochores in living cells stabilizes kt-MT attachments even in the presence of active Aurora B. Thus, kinetochore tension is capable of overpowering the ability of Aurora B to mediate error correction without compromising its activity (Cane, 2013).

The data support the hypothesis that NOD end tracks on polymerizing microtubules. But what is the molecular basis of NOD end tracking? That NOD fragments associate with paused and depolymerizing microtubule plus ends, when EB1 is absent, suggests that NOD could track nonpolymerizing microtubule ends in an EB1-independent manner although tracking on polymerizing ends by NOD may require EB1. NOD end tracking has been envisioned as an EB1-independent phenomenon although it has never been directly demonstrated. Thus, it will be important to determine whether NOD behaves like budding yeast dynein, which is targeted to microtubule plus ends independent of EB1, or like MCAK (kinesin-13), which contains an S/TxIP motif and exhibits EB1-dependent tip tracking (Cane, 2013).

Because NOD has never been shown to possess plus end-directed motility in vitro, it is currently classified as a nonmotile kinesin. However, the observation of persistent chromatin stretching events that moved along the sides of microtubules toward the plus ends provides compelling evidence that NOD could exhibit plus end-directed motility in cells. This work is thought yo strongly support the NOD end tracking hypothesis but does not rule out plus end-directed motility as another potential source of force production by NOD. It will be worthwhile to further test the hypothesis that NOD possesses two force-producing activities (Cane, 2013).

Microtubule polymerization and molecular motors have long been proposed as possible sources of the PEF and the focus has rightfully been placed on molecular motors since the discovery of chromokinesins. This study reports that PEFs can be generated not only by plus end-directed chromokinesins but also by chromosome-associated factors that associate with polymerizing plus ends. Thus, it may be time to look beyond the motility of kinesin-10 motors and consider chromosome-based tip-tracking factors as potential mediators of PEF production (Cane, 2013).

The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding

During mitosis and meiosis, the bipolar spindle facilitates chromosome segregation through microtubule sliding as well as microtubule growth and shrinkage. Kinesin-14, one of the motors involved, causes spindle collapse in the absence of kinesin-5, participates in spindle assembly and modulates spindle length. However, the molecular mechanisms underlying these activities are not known. This study reports that Drosophila melanogaster kinesin-14 (Ncd) alone causes sliding of anti-parallel microtubules but locks together (that is, statically crosslinks) those that are parallel. Using single molecule imaging this study shows that Ncd diffuses along microtubules in a tail-dependent manner and switches its orientation between sliding microtubules. These results show that kinesin-14 causes sliding and expansion of an anti-parallel microtubule array by dynamic interactions through the motor domain on the one side and the tail domain on the other. This mechanism accounts for the roles of kinesin-14 in spindle organization (Fink, 2009).

DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein

The Drosophila no distributive disjunction (nod) gene encodes a kinesin-like protein that has been proposed to push chromosomes toward the metaphase plate during female meiosis. This paper reports that the nonmotor domain of the Nod protein can mediate direct binding to DNA. Using an antiserum prepared against bacterially expressed Nod protein, it was shown that during prometaphase Nod protein is localized on oocyte chromosomes and is not restricted to either specific chromosomal regions or to the kinetochore. Thus, motor-based chromosome-microtubule interactions are not limited to the centromere, but extend along the chromosome arms, providing a molecular explanation for the polar ejection force (Afshar, 1995).

The lethal(1)TW-6cs mutation of Drosophila melanogaster is a dominant antimorphic allele of nod and is associated with a single base change in the putative ATP-binding domain

The l(1)TW-6cs mutation is a cold-sensitive recessive lethal mutation in Drosophila melanogaster, that affects both meiotic and mitotic chromosome segregation. This paper reports the isolation of three revertants of this mutation. All three revert both the meiotic and mitotic effects as well as the cold sensitivity, demonstrating that all three phenotypes are due to a single lesion. It was further shown that these revertants fail to complement an amorphic allele of the nod (no distributive disjunction) locus, which encodes a kinesin-like protein. These experiments demonstrate that l(1)TW-6cs is an antimorphic allele of nod, and it was renamed nodDTW. Sequencing of the nod locus on a nodDTW-bearing chromosome reveals a single base change in the putative ATP-binding region of the motor domain of nod. Recessive, loss-of-function mutations at the nod locus specifically disrupt the segregation of nonexchange chromosomes in female meiosis. At 23.5 degrees, the meiotic defects in nodDTW/+ females are similar to those observed in nod/nod females; that is, the segregation of nonexchange chromosomes is abnormal. However, in nodDTW/nodDTW females, or in nodDTW/+ females at 18 degrees, a more severe meiotic defect was observed that apparently affects the segregation of both exchange and nonexchange chromosomes. In addition, nodDTW homozygotes and hemizygous males have previously been shown to exhibit mitotic defects including somatic chromosome breakage and loss. It is proposed that the defective protein encoded by the nodDTW allele interferes with proper chromosome movement during both meiosis and mitosis, perhaps by binding irreversibly to microtubules (Rasooly, 1991).

Genetic analysis of microtubule motor proteins in Drosophila: a mutation at the ncd locus is a dominant enhancer of nod

The nod (no distributive disjunction) and the ncd (non-claret disjunctional) mutations are both female-specific, recessive meiotic mutations in Drosophila melanogaster. Mutations at either locus show high frequencies of nondisjunction at meiosis I and both have been shown to encode kinesin-like proteins. Unlike the ncd mutation, which affects all chromosome pairs, the nod mutation affects only the disjunction of nonexchange chromosomes. Although both the nod and ncd mutations are fully recessive, females doubly heterozygous for nod and ncd mutations show levels of X and fourth chromosome nondisjunction that are 6- to 35-fold above those observed in control females. Exchange between chromosomes can suppress this effect; thus, only nonexchange chromosomes segregating via the distributive system are sensitive in double heterozygotes. Since the phenotype of double heterozygotes mimics that of the nod mutation, it is infered that ncd is a dominant enhancer of nod. Failure of ncd to fully complement nod reveals the chromosome segregation machinery to be dosage sensitive. The probability that the distributive system will fail is enhanced in females simultaneously haploinsufficient at the nod and ncd loci (Knowles, 1991).

The genetic analysis of distributive segregation in Drosophila melanogaster. II. Further genetic analysis of the nod locus

In Drosophila melanogster females the segregation of nonexchange chromosomes is ensured by the distributive segregation system. The mutation noda specifically impairs distributive disjunction and induces nonexchange chromosomes to undergo nondisjunction, as well as both meiotic and mitotic chromosome loss. This report describes the isolation of seven recessive X-linked mutations that are allelic to noda. As homozygotes, all of these mutations exhibit a phenotype that is similar to that exhibited by noda homozygotes. These mutations were also used to demonstrate that nod mutations induce nonexchange chromosomes to nondisjoin at meiosis II. The data demonstrate that the effects of noda on meiotic chromosome behavior are a general property of mutations at the nod locus. Several of these mutations exhibit identical phenotypes as homozygotes and as heterozygotes with a deficiency for the nod locus; these likely correspond to complete loss-of-function or null alleles. None of these mutations causes lethality, decreases the frequency of exchange, or impairs the disjunction of exchange chromosomes in females. Thus, either the nod locus defines a function that is specific to distributive segregation or exchange can fully compensate for the absence of the nod+ function (Zhang, 1990).

A meiotic mutant defective in distributive disjunction in Drosophila melanogaster

The female meiotic mutant no distributive disjunction (symbol: nod) reduces the probability that a nonexchange chromosome will disjoin from either a nonexchange homolog or a nonhomolog; the mutant does not affect exchange or the disjunction of bivalents that have undergone exchange. Disjunction of nonexchange homologs was examined for all chromosome pairs; nonhomologous disjunction of the X chromosomes from the Y chromosome in XXY females, of compound chromosomes in females bearing attached-third chromosomes with and without a Y chromosome, and of the second chromosomes from the third chromosomes were also examined. The results suggest that the defect in nod is in the distributive pairing process. The frequencies and patterns of disjunction from a trivalent in nod females suggest that the distributive pairing process involves three separate events-pairing, orientation, and disjunction. The mutant nod appears to affect disjunction only (Carpenter, 1973).

The balance between KIFC3 and EG5 tetrameric kinesins controls the onset of mitotic spindle assembly

One of the first steps in mitotic spindle assembly is the dissolution of the centrosome linker followed by centrosome separation driven by EG5, a tetrameric plus-end-directed member of the kinesin-5 family. However, even in the absence of the centrosome linker, the two centrosomes are kept together by an ill-defined microtubule-dependent mechanism. This study shows that KIFC3, a minus-end-directed kinesin-14, provides microtubule-based centrosome cohesion. KIFC3 forms a homotetramer that pulls the two centrosomes together via a specific microtubule network. At mitotic onset, KIFC3 activity becomes the main driving force of centrosome cohesion to prevent premature spindle formation after linker dissolution as it counteracts the increasing EG5-driven pushing forces. KIFC3 is eventually inactivated by NEver in mitosis-related Kinase 2 (NEK2) to enable EG5-driven bipolar spindle assembly. It was further shown that persistent centrosome cohesion in mitosis leads to chromosome mis-segregation. These findings reveal a mechanism of spindle assembly that is evolutionary conserved from yeast to humans (Hata, 2019).

Microtubule minus-end stability is dictated by the tubulin off-rate

Dynamic organization of microtubule minus ends is vital for the formation and maintenance of acentrosomal microtubule arrays. In vitro, both microtubule ends switch between phases of assembly and disassembly, a behavior called dynamic instability. Although minus ends grow slower, their lifetimes are similar to those of plus ends. The mechanisms underlying these distinct dynamics remain unknown. This study used an in vitro reconstitution approach to investigate minus-end dynamics.Minus-end lifetimes were not defined by the mean size of the protective GTP-tubulin cap. Rather, it is concluded that the distinct tubulin off-rate is the primary determinant of the difference between plus- and minus-end dynamics. Further, the results show that the minus-end-directed kinesin-14 HSET/KIFC1 suppresses tubulin off-rate to specifically suppress minus-end catastrophe. HSET maintains its protective minus-end activity even when challenged by a known microtubule depolymerase, kinesin-13 MCAK. These results provide novel insight into the mechanisms of minus-end dynamics, essential for understanding of microtubule minus-end regulation in cells (Strothman, 2019).

Mitotic Motor KIFC1 Is an Organizer of Microtubules in the Axon

KIFC1 (also called HSET or kinesin-14a) is best known as a multifunctional motor protein essential for mitosis. The present studies are the first to explore KIFC1 in terminally postmitotic neurons. Using RNA interference to partially deplete KIFC1 from rat neurons (from animals of either gender) in culture, pharmacologic agents that inhibit KIFC1, and expression of mutant KIFC1 constructs, critical roles were demonstrated for KIFC1 in regulating axonal growth and retraction as well as growth cone morphology. Experimental manipulations of KIFC1 elicit morphological changes in the axon as well as changes in the organization, distribution, and polarity orientation of its microtubules. Together, the results indicate a mechanism by which KIFC1 binds to microtubules in the axon and slides them into alignment in an ATP-dependent fashion and then cross-links them in an ATP-independent fashion to oppose their subsequent sliding by other motors (Muralidharan, 2019).

Search PubMed for articles about Drosophila Nod

Afshar, K., Barton, N. R., Hawley, R. S. and Goldstein, L. S. (1995). DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein. Cell 81(1): 129-138. PubMed ID: 7720068

Cane, S., Ye, A. A., Luks-Morgan, S. J. and Maresca, T. J. (2013). Elevated polar ejection forces stabilize kinetochore-microtubule attachments. J Cell Biol 200(2): 203-218. PubMed ID: 23337118

Carpenter, A. T. (1973). A meiotic mutant defective in distributive disjunction in Drosophila melanogaster. Genetics 73(3): 393-428. PubMed ID: 4633612

Davies, T., Kodera, N., Kaminski Schierle, G. S., Rees, E., Erdelyi, M., Kaminski, C. F., Ando, T. and Mishima, M. (2015). CYK4 promotes antiparallel microtubule bundling by optimizing MKLP1 neck conformation. PLoS Biol 13(4): e1002121. PubMed ID: 25875822

Fink, G., Hajdo, L., Skowronek, K. J., Reuther, C., Kasprzak, A. A. and Diez, S. (2009). The mitotic kinesin-14 Ncd drives directional microtubule-microtubule sliding. Nat Cell Biol 11(6): 717-723. PubMed ID: 19430467

Hata, S., Pastor Peidro, A., Panic, M., Liu, P., Atorino, E., Funaya, C., Jakle, U., Pereira, G. and Schiebel, E. (2019). The balance between KIFC3 and EG5 tetrameric kinesins controls the onset of mitotic spindle assembly. Nat Cell Biol 21(9): 1138-1151. PubMed ID: 31481795

Knowles, B. A. and Hawley, R. S. (1991). Genetic analysis of microtubule motor proteins in Drosophila: a mutation at the ncd locus is a dominant enhancer of nod. Proc Natl Acad Sci U S A 88(16): 7165-7169. PubMed ID: 1908090

Muralidharan, H. and Baas, P. W. (2019). Mitotic motor KIFC1 is an organizer of microtubules in the axon. J Neurosci 39(20): 3792-3811. PubMed ID: 30804089

Nicklas, R. B. and Koch, C. A. (1969). Chromosome micromanipulation. 3. Spindle fiber tension and the reorientation of mal-oriented chromosomes. J Cell Biol 43(1): 40-50. PubMed ID: 5824068

Rasooly, R. S., New, C. M., Zhang, P., Hawley, R. S. and Baker, B. S. (1991). The lethal(1)TW-6cs mutation of Drosophila melanogaster is a dominant antimorphic allele of nod and is associated with a single base change in the putative ATP-binding domain. Genetics 129(2): 409-422. PubMed ID: 1743485

Strothman, C., Farmer, V., Arpag, G., Rodgers, N., Podolski, M., Norris, S., Ohi, R. and Zanic, M. (2019). Microtubule minus-end stability is dictated by the tubulin off-rate. J Cell Biol 218(9): 2841-2853. PubMed ID: 31420452

Ye, A. A., Verma, V. and Maresca, T. J. (2018). NOD is a plus end-directed motor that binds EB1 via a new microtubule tip localization sequence. J Cell Biol. PubMed ID: 29899040

Zhang, P. and Hawley, R. S. (1990). The genetic analysis of distributive segregation in Drosophila melanogaster. II. Further genetic analysis of the nod locus. Genetics 125(1): 115-127. PubMed ID: 2111262

date revised: 26 October 2019

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