InteractiveFly: GeneBrief

non-claret disjunctional: Biological Overview | References


Gene name - non-claret disjunctional

Synonyms -

Cytological map position - 99C1-99C2

Function - cytoskeletal motor protein

Keywords - minus end director kinesin, spindle, mitosis, meiosis

Symbol - ncd

FlyBase ID: FBgn0002924

Genetic map position - 3R: 25,630,009..25,632,849 [-]

Classification - Kinesin motor domain, Kar3 family of kinesins

Cellular location - nuclear and cytoplasmic



NCBI links: Precomputed BLAST | EntrezGene
Recent literature

Zhang, P., Dai, W., Hahn, J. and Gilbert, S. P. (2015) Drosophila Ncd reveals an evolutionarily conserved powerstroke mechanism for homodimeric and heterodimeric kinesin-14s Proc Natl Acad Sci U S A 112: 6359-6364. PubMed ID: 25941402
Summary:
Drosophila kinesin-14 Ncd cross-links parallel microtubules at the spindle poles and antiparallel microtubules within the spindle midzone to play roles in bipolar spindle assembly and proper chromosome distribution. As observed for Saccharomyces cerevisiae kinesin-14 Kar3Vik1 and Kar3Cik1, Ncd binds adjacent microtubule protofilaments in a novel microtubule binding configuration and uses an ATP-promoted powerstroke mechanism. The hypothesis tested in this study is that Kar3Vik1 and Kar3Cik1, as well as Ncd, use a common ATPase mechanism for force generationß. The presteady-state kinetics and computational modeling establish an ATPase mechanism for a powerstroke model of Ncd that is very similar to those determined for Kar3Vik1 and Kar3Cik1, although these heterodimers have one Kar3 catalytic motor domain and a Vik1/Cik1 partner motor homology domain whose interactions with microtubules are not modulated by nucleotide state but by strain. The results indicate that both Ncd motor heads bind the microtubule lattice; two ATP binding and hydrolysis events are required for each powerstroke; and a slow step occurs after microtubule collision and before the ATP-promoted powerstroke. Note that unlike conventional myosin-II or other processive molecular motors, Ncd requires two ATP turnovers rather than one for a single powerstroke-driven displacement or step. These results are significant because all metazoan kinesin-14s are homodimers, and the results presented show that despite their structural and functional differences, the heterodimeric and homodimeric kinesin-14s share a common evolutionary structural and mechanochemical mechanism for force generation.

Ito, A. and Goshima, G. (2015). Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle. J Cell Biol 211: 999-1009. PubMed ID: 26644514
Summary:
Depletion of Drosophila melanogaster Asp, an orthologue of microcephaly protein ASPM, causes spindle pole unfocusing during mitosis. However, it remains unclear how Asp contributes to pole focusing, a process that also requires the kinesin-14 motor Ncd. This study shows that Asp localizes to the minus ends of spindle microtubule (MT) bundles and focuses them to make the pole independent of Ncd. A critical domain in Asp was identified exhibiting MT cross-linking activity in vitro. Asp was also localized to, and focuses the minus ends of, intraspindle MTs that were nucleated in an augmin-dependent manner and translocated toward the poles by spindle MT flux. Ncd, in contrast, functions as a global spindle coalescence factor not limited to MT ends. A revised molecular model is proposed for spindle pole focusing in which Asp at the minus ends cross-links MTs at the pole and within the spindle. Additionally, this study provides new insight into the dynamics of intraspindle MTs by using Asp as a minus end marker.

BIOLOGICAL OVERVIEW

The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts, was developed. Green fluorescent protein (GFP)-Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic 'looping' behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 µm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. These data demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis (Oladipo, 2007).

The principal structural components of the mitotic spindle are cytoplasmic microtubules (MTs) organized into two polarized (minus ends at the center) radial arrays, located at a distance from each other. MT organization in the mitotic spindle is achieved by MT motors, kinesins and dyneins, which generate force for movement toward the plus or minus ends of MTs and that participate in the transport of chromosomes to the spindle poles, spindle elongation during anaphase, regulation of the MT turnover rates, and spindle morphogenesis (Oladipo, 2007).

A special role in the formation and maintenance of the mitotic spindle belongs to the minus-end-directed members of the kinesin-14 subfamily. Unlike conventional kinesin, kinesin-14 family members have motor domains at the carboxy terminus (for review, see Ovechkina, 2003). The best studied kinesin-14 members are Drosophila Ncd (Komma, 1991; Matthies, 1996) and Saccharomyces cerevisiae Kar3 (Meluh, 1990). These MT motors, and their mammalian (Matuliene, 1999; Mountain, 1999; Zhu, 2005) and plant (Vanstraelen, 2006) homologues, play essential roles in mitosis and meiosis. Their activities are important for the formation of the mitotic spindle poles (Goshima and Vale, 2003; Goshima, 2005; Morales-Mulia and Scholey, 2005; Zhu, 2005) and for regulation of the distance between the poles in the mitotic spindle (Saunders, 1992; Saunders, 1997; Sharp, 1999; 2000a; Troxell, 2001). Loss of a kinesin-14 function may cause different mitotic and meiotic spindle defects even within the same organism. For example, in Drosophila S2 cells and female meiosis, loss of Ncd by RNA interference (RNAi) or null mutants causes disorganization of spindle poles (Theurkauf, 1992; Goshima, 2005; Morales-Mulia, 2005), whereas in embryos, it causes changes in spindle length and a decrease in the persistence of steady-state structures (Sharp, 1999; Brust-Mascher, 2002; Oladipo, 2007 and references therein).

Evidence suggests that kinesins-14 act by driving the sliding of parallel or antiparallel MT bundles in different areas of the spindle (for review, see Sharp, 2000b;c; McIntosh, 2002; Gadde and Heald, 2004). These motors have two MT binding sites, an ATP-dependent site in the motor domain and an ATP-independent site in the tail, and they have been shown to induce MT bundling (McDonald, 1990; Chandra, 1993; Karabay, 1999; Matuliene, 1999). Recent small interfering RNA (siRNA) studies demonstrate that knockdown of the kinesin-14 family member Ncd results in the formation of splayed mitotic spindle poles, suggesting that the activity of this motor is required for focusing of the MTs at the pole (Goshima, 2003; Goshima, 2005; Morales-Mulia, 2005). It has been found that loss of kinesin-14 function in S. cerevisiae and Drosophila leads to an increase in the spindle length and that it rescues spindle pole separation defects seen in cells lacking members of the kinesin-5 family (Saunders, 1992; Saunders, 1997; Sharp, 1999; Sharp, 2000a) and that these two motors oppose each other in in vitro motility assays (Tao, 2006), suggesting that the balance of their activities is required for maintaining the correct distance between the spindle poles. It is hypothesized that kinesins-14 achieve this balance by mediating antiparallel sliding of MTs emanating from the opposite poles (Sharp, 2000b;c; McIntosh, 2002; Gadde, 2004). However, despite the abundance of indirect evidence and computational models that suggest an essential role for kinesins-14 in MT sliding (Mogilner, 2006), such sliding activity of kinesins-14 had never been demonstrated directly before (Oladipo, 2007 and references therein).

In this study, an in vivo assay was developed to test the hypothesis that kinesin-14 family member Ncd can induce sliding of MTs against each other in living cells. It was found that green fluorescent protein (GFP)-Ncd overexpressed in cultured human fibroblasts colocalized with cytoplasmic MTs, whose localization and behavior were monitored by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic 'looping' behavior never observed in control cells. Photobleaching studies demonstrated that neighboring MTs in bundles could slide against each other with the velocities of 0.1 microm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. These data confirm the hypothesis that kinesin-14 family members generate sliding forces between adjacent MTs, and they constitute the first demonstration of such sliding activity of MT motors in vivo (Oladipo, 2007).

These results demonstrate that the kinesin-14 family of MT motors Drosophila Ncd induces sliding of MTs against each other in the cytoplasm of interphase mammalian fibroblasts. It has been previously shown that Ncd exhibits motile properties in vitro and that in MT gliding assays recombinant Ncd behaves as a nonprocessive minus end-directed MT motor (McDonald, 1990; Walker, 1990; Foster, 2000). This study is the first demonstration of the motor activity of Ncd in living cells. This work developed a new in vivo assay that provides the ability to examine the motility properties of MT motors in a cellular context (Oladipo, 2007).

It is thought that Ncd exerts its functions by bundling and sliding of MTs against each other (McDonald, 1990; Chandra, 1993; Sharp, 2000c). This MT bundling activity is explained by the simultaneous attachment to two neighboring MTs via its ATP-dependent motor domain and ATP-independent tail located on the opposite sides of the molecule (McDonald, 1990; Chandra, 1993; Karabay, 1999). In the presence of ATP such attachment would result in relative sliding of the neighboring MTs against each other. It has been previously demonstrated in in vitro assays that Ncd causes the formation of MT bundles; however, sliding of MTs in Ncd-induced bundles has never been seen in in vivo experiments. The in vivo assay allowed observation of the Ncd-dependent sliding of MTs against each other (Oladipo, 2007).

MT sliding seen in these experiments results in a remarkable pattern of MT behavior that involves the formation of dynamic MT loops with continuously changing curvature. These results indicate that the formation and expansion of MT loops do not require MT dynamics, are specific for Ncd-overexpressing cells, depend on the Ncd motor activity, and involve both MT binding domains located on opposite ends of Ncd molecule. Based on these observations, it is proposed that the looping behavior of MTs in this assay is caused by the fact that in addition to being bundled by Ncd these MTs are also anchored on the centrosome, which restricts MT movement, resulting in the bending and buckling of the MTs during Ncd-dependent MT-MT sliding. In dividing cells, MT sliding activity generated by Ncd and other members of the kinesin-14 family is thought to be involved in two aspects of mitotic and meiotic spindle morphogenesis. For one aspect, kinesin-14 family members control mitotic spindle length by generating pulling forces on the overlapping antiparallel MTs in the spindle midzone that oppose the pushing forces produced by members of the kinesin-5 family (Saunders, 1992; Sharp, 1999; 2000a;b; Kapitein, 2005; Tao, 2006). In addition, kinesins-14 are involved in the formation of the spindle poles themselves, presumably via minus-end-directed transport of MTs nucleated by noncentrosomal mechanisms in the cytoplasm (Goshima, 2005; Morales-Mulia, 2005). This study has demonstrate that kinesins-14 are indeed involved in MT-MT sliding in vivo; this result supports the hypothesis about the mitotic roles of kinesin-14 members and it provides experimental evidence for both aspects of their functioning in mitosis. In vitro reconstitution experiments are underway to confirm the sliding activity of kinesin-14 family members and to reproduce the key aspects of the mitotic spindle assembly in a purified system (Oladipo, 2007).

A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd

Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described. By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors (such as Drosophila Ncd) are less well understood. Using cryo-electron microscopy, it was demonstrated that a coiled-coil mechanical element of microtubule-bound Ncd rotates ~70° towards the minus end upon ATP binding. Extending or shortening this coiled coil increases or decreases velocity, respectively, without affecting ATPase activity. An unusual Ncd mutant that lacks directional preference shows unstable nucleotide-dependent conformations of its coiled coil, underscoring the role of this mechanical element in motility. These results show that the force-producing conformational change in Ncd occurs on ATP binding, as in other kinesins, but involves the swing of a lever-arm mechanical element similar to that described for myosins (Endres, 2006).

Cryo-EM experiments suggest that Ncd uses its neck as a lever arm to generate a minus-end-directed power stroke in a manner similar to the rotation of the light-chain-binding domain in myosin II. For a lever-arm mechanism, the velocity of the motor should be proportional to the length of its lever arm. Consistent with this prediction, a series of successive neck truncations caused a progressive decrease in velocity in a microtubule-gliding assay, but did not affect enzymatic turnover (ATPase catalytic rate constant, kcat). This finding is consistent with previous work on Ncd truncations. Truncation experiments are difficult to interpret, however, because the loss of protein structure could damage motor function in unanticipated ways. Therefore attempts were made to increase velocity by extending the length of the neck with a four-heptad leucine zipper coiled-coil motif ('LZ extension'). As expected for a lever-arm model, fusion of this LZ extension to the native Ncd neck at three different positions increased microtubule gliding velocity without changing ATPase kcat. This increase in velocity was not observed when a flexible glycine-serine linker was inserted between the LZ extension and the native Ncd neck, suggesting that the LZ extension increases Ncd velocity by extending the length of the mechanical element and not by some other mechanism. The compiled velocity data from the seven truncated or extended neck constructs show that microtubule gliding velocity is proportional to the predicted length of the neck, regardless of whether native or nonnative (LZ extension) residues were used, but ATPase kcat remains unaffected. Taken together, these data support the notion that a lever-arm rotation of the Ncd neck powers minus-end-directed motility (Endres, 2006).

A model of Ncd motility invoking the rotation of the neck suggests that the unbound head may not be necessary to generate motility. To test this notion, a single-headed Ncd heterodimer was prepared (N280_Het) in which one polypeptide consisted of an intact Ncd catalytic core and neck (residues 280-700) and the second polypeptide consisted of the neck region alone (residues 281-347). This motor elicited microtubule gliding at a velocity comparable to that of the normal two-headed Ncd homodimer with a similar ATPase kcat. Thus, although the cryo-EM data show that the unbound head rotates along with the neck, the functional data from the heterodimer indicate that contacts between the neck and unbound head are not essential for the mechanism and that the neck alone is sufficient to act as a lever arm. Studies have also shown that a naturally occurring Kinesin-14 heterodimer in yeast (the Kar3p-Cik1p complex, a motor polypeptide in complex with a motor-less coiled coil) is an active, force-producing motor (Endres, 2006).

To determine whether the lever-arm motion of the Ncd neck requires a stable coiled-coil interaction, the motility of an Ncd monomer construct (N325) was tested. This construct showed >25-fold reduced motility compared with the single-headed heterodimer, but had an ATPase activity similar to that of the other constructs. Thus, a stable coiled coil is required for optimal function of the motor, as would be expected for a lever-arm model (Endres, 2006).

On the basis of structural and functional data, the following model of Ncd motility is proposed. Ncd from solution binds to microtubules using one of its heads, triggering ADP release. The excellent fit of the Ncd/ADP structure to the nucleotide-free maps suggests that microtubule binding and ADP release do not produce large-scale conformational changes in the Ncd dimer. Cryo-EM data suggest that ATP binding leads to a ~70° rigid-body rotation of the neck that produces a minus-end-directed displacement. A subsequent protein isomerization step, possibly before phosphate release, triggers the formation of a weakly bound state and the dissociation of Ncd from the microtubule. The neck lever arm can then return to its pre-power stroke position after dissociating from the microtubule, thereby completing the cycle. Although this overall scheme is supported by the data, questions remain open about the proposed lever-arm mechanism. Specifically, although the data unequivocally show a preferred position of the lever arm in the AMPPNP and ADP-AlF4- states, the weaker density in AMPPNP maps suggests that this post-powerstroke state may not be completely rigid and fixed in position, as envisaged by classical swinging crossbridge models of myosin. Future work on this issue will require dynamic measurement of the lever-arm position in different nucleotide states with high spatial and temporal resolution (Endres, 2006).

This work shows that the mechanical event in the minus-end-directed Ncd (rotation of the coiled-coil neck) is coupled to the same step of the ATPase cycle (ATP binding) as the mechanical event in the plus-end-directed kinesins (neck linker docking). Thus, reversal of direction in Kinesin-14 motors is accomplished by the evolution of a unique mechanical element that can take advantage of existing conformational changes in the catalytic core, as is also true for direction reversal by the myosin VI motor. Unlike conventional kinesin, which is built for long-distance processive movement, Ncd is a nonprocessive motor designed for microtubule crossbridging and tension development in meiotic or mitotic spindle. In this regard, the functions of Ncd are more similar to the tension-generating myosin II motors in muscle. Thus, Ncd and muscle myosin convergently evolved a similar strategy for motility involving a large-scale rotation of an elongated lever and the primary use of only one of the two heads in the motor dimer (Endres, 2006).

αTubulin 67C and Ncd are essential for establishing a cortical microtubular network and formation of the Bicoid mRNA gradient in Drosophila

The Bicoid (Bcd) protein gradient in Drosophila serves as a paradigm for gradient formation in textbooks. To explain the generation of the gradient, the ARTS (active RNA transport and synthesis) model, which is based on the observation of a bcd mRNA gradient, proposes that the bcd mRNA, localizes at the anterior pole at fertilization, migrates along microtubules (MTs) at the cortex to the posterior to form a bcd mRNA gradient which is translated to form a protein gradient. To fulfill the criteria of the ARTS model, an early cortical MT network is thus a prerequisite. This study reports hitherto undiscovered MT activities in the early embryo important for bcd mRNA transport: (1) an early and omnidirectional MT network exclusively at the anterior cortex of early nuclear cycle embryos showing activity during metaphase and anaphase only, (2) long MTs up to 50 microm extending into the yolk at blastoderm stage to enable basal-apical transport. The cortical MT network is not anchored to the actin cytoskeleton. The posterior transport of the mRNA via the cortical MT network critically depends on maternally-expressed αTubulin67C and the minus-end motor Ncd. In either mutant, cortical transport of the alphaTubulin67C mRNA does not take place and the mRNA migrates along another yet undisclosed interior MT network, instead. These data strongly corroborate the ARTS model and explain the occurrence of the alphaTubulin67C mRNA gradient (Fahmy, 2014).

Mechanisms for focusing mitotic spindle poles by minus end-directed motor proteins

During the formation of the metaphase spindle in animal somatic cells, kinetochore microtubule bundles (K fibers) are often disconnected from centrosomes, because they are released from centrosomes or directly generated from chromosomes. To create the tightly focused, diamond-shaped appearance of the bipolar spindle, K fibers need to be interconnected with centrosomal microtubules (C-MTs) by minus end-directed motor proteins. This study characterized the roles of two minus end-directed motors, dynein and Ncd, in such processes in Drosophila S2 cells using RNA interference and high resolution microscopy. Even though these two motors have overlapping functions, Ncd is primarily responsible for focusing K fibers, whereas dynein has a dominant function in transporting K fibers to the centrosomes. A novel localization of Ncd to the growing tips of C-MTs is reported, that is shown is mediated by the plus end-tracking protein, EB1. Computer modeling of the K fiber focusing process suggests that the plus end localization of Ncd could facilitate the capture and transport of K fibers along C-MTs. From these results and simulations, a model is proposed on how two minus end-directed motors cooperate to ensure spindle pole coalescence during mitosis (Goshima, 2005; full text of article).

EB1 is a highly conserved microtubule plus end-tracking protein that binds various cargo proteins (e.g., APC [adenomatous polyposis coli protein]). Ncd also was recently observed to bind to an EB1 affinity column. It was therefore of interest investigate whether the plus end accumulation of Ncd-GFP is mediated by EB1. After EB1 depletion by RNAi in Ncd-GFP-NES cell line, no plus end accumulation of Ncd-GFP-NES was seen; instead the microtubules were evenly labeled with this protein. After EB1 RNAi, microtubules become less dynamic and frequently enter a pause state where they exhibit minimal growth or shrinkage. However, even the subset of growing microtubule never accumulated Ncd-GFP-NES at their tips (Goshima, 2005).

Next, an in vitro interaction between purified Ncd and EB1 was tested using a GST pull-down assay. It was found that the nonmotor 'tail' domain (aa 1-290) of Ncd can bind directly to the COOH terminus domain of EB1 (EB1-C; aa 208-278), albeit weakly. This binding was competed by addition of a fragment of human APC protein (2744-2843 aa) that binds to EB1's COOH-terminal domain, suggesting that the Ncd tail and APC bind to the same site on EB1. However, expression of the Ncd tail domain (1-290 aa) fused to GFP did not track along microtubule plus ends in vivo, suggesting that the motor domain may augment affinity for the microtubules and thereby aid plus end localization (Goshima, 2005).

No specific mutagenesis strategy was availabe for eliminating plus end tracking of Ncd while retaining its other critical mitotic activities such as microtubule cross-bridging. However, the consequences were tested of pole focusing and Ncd-GFP localization in mitosis after RNAi of EB1. Time-lapse imaging of mitotic EB1 RNAi cells showed no plus end microtubule enrichment, as expected from the observed interphase results. However, Ncd-GFP still strongly and dynamically (revealed by FRAP) localized to spindle MTs, indicating that K fiber binding does not require EB1. In this setting of EB1 RNAi where Ncd was mislocalized from microtubule plus ends but not the spindle, centrosome detachment and K fiber focusing was examined. Qualitative study of EB1 RNAi found both centrosome detachment and pole defocusing. When these phenotypes were examined quantitatively, it was found that the EB1 phenotype consists of pronounced K fiber defocusing and has less pronounced centrosome detachment, which is more similar to the Ncd RNAi than the dynein RNAi phenotype. Although not definitive proof because EB1 RNAi causes microtubule dynamics defects in addition to displacing Ncd from the microtubule plus end, this result is consistent with a functional link between EB1-dependent localization of Ncd to microtubule tips and K fiber coalescence (Goshima, 2005).

Based on live cell imaging and computer simulation analyses, it is proposed that the coalescence of spindle poles involves the following steps: (1) inter-K fiber cross-linking, (2) 'search and capture' of K fibers by the tip of growing C-MTs, and (3) K fiber transport on C-MTs. RNAi analysis of dynein and Ncd as well as live cell imaging of Ncd-GFP has provided insight into the roles of these minus end-directed motor proteins in these processes. By quantitatively comparing Ncd and dynein knockdown phenotypes in the same cell type, it was found that these Ncd and dynein have distinct but overlapping functions in the three steps of pole focusing (Goshima, 2005).

RNAi results suggest that Ncd plays a role in K fiber focusing through three mechanisms described above. Ncd's major role is likely to be in inter-K fiber cross-linking, as evidenced by the splaying of K fibers after Ncd RNAi and the prominent localization of Ncd-GFP to K fibers. This process most likely involves cross-linking of microtubules by Ncd's force generating motor domain and its positively charged 'tail' domain that also binds to microtubules independently of the motor. This process occurs in the absence of centrosomes and C-MTs, because acentrosomal spindles created by centrosomin RNAi also show severe K fiber unfocusing when Ncd is also knocked down by RNAi. It is also showm that the process of lateral K fiber interactions is highly dynamic, because K fibers are continually splaying and coalescing. Such observations are also consistent with FRAP measurements of Ncd-GFP, which show that these motors are associating and dissociating with K fibers on a rapid time scale and thus are not behaving as static cross-linkers. Albeit less efficient than dynein, Ncd also likely contributes to minus end-directed transport of K fibers along C-MTs, because the centrosome to K fiber distance is somewhat greater in the Ncd/Dhc64C double RNAi compared with Dhc64C alone. Finally, it is believed that Ncd at the tips of C-MTs may act to capture K fibers and facilitate subsequent minus end transport of the K fiber (Goshima, 2005).

Cytoplasmic dynein in S2 cells plays a dominant role in transporting K fibers along microtubules, as evidenced by finding that Dhc64C RNAi causes detachment of centrosomes from the minus ends of K fibers. Although secondary to Ncd, dynein also contributes to the focusing of the minus ends of K fibers. This role of dynein becomes particularly clear after Ncd depletion, since Ncd/Dhc64c double RNAi causes very severe splaying of K fibers. It is believed that this K fiber focusing effect also primarily involves dynein's role as a transporter of K fiber bundles along C-MTs, which causes the coalescence of most peripheral K fibers toward the centrosome as shown in computer simulations. However, dynein may have other roles in K fiber coalescence, such as potentially transporting and concentrating cross-linking proteins at minus ends of K fibers. Indeed, synthetic effects of kinesin-14 and dynein motors on pole focusing have been reported in centrosome-free spindles reconstituted in Xenopus egg extract (Goshima, 2005).

The molecular properties of kinesin-14/Ncd and cytoplasmic dynein are well designed to support the above proposed functions of these two motors in pole focusing. Cytoplasmic dynein is a fast, processive motor. Thus, small numbers of dyneins could rapidly transport K fibers along C-MTs. In contrast, Ncd is nonprocessive, slow motor that is not designed for cargo transport. Instead, its ability to bind two microtubules and its slow motor activity makes it an effective cross-bridger between microtubules in the spindle. These properties are likely to be advantageous for inter-K fiber cross-linking, as well as for crossbridging of C-MTs plus ends to K fiber. However, the rapid on-off rates of these cross-bridges, as shown by FRAP data, would still enable dynein to effectively transport the K fibers along the C-MTs (Goshima, 2005).

Phenotypic RNAi analyses may account for differences in the pole unfocusing phenotypes of Ncd or dynein depletions that have been described in the literature. Specifically, spindle architecture in the given system (e.g., the presence or absence of centrosome and differences in microtubule dynamics) may determine whether Ncd or dynein acts as the essential contributor to pole coalescence. For example, Ncd/kinesin-14 function, is particularly important for K fiber focusing, may become more crucial when centrosomes are detached or absent from the spindle. Consistent with this idea, the most dramatic pole unfocusing phenotypes for kinesin-14 mutations/depletions have been described in plant mitosis and animal meiosis, systems in which spindle assembly occurs through a centrosome-independent mechanism and in which interactions between C-MTs and K fibers are simply absent. However, dynein also is likely to play crucial roles in pole coalescence in some acentrosomal spindles, as shown convincingly in Xenopus extract system. In contrast, somatic animal cell mitosis utilizes centrosomes, and kinesin-14 is less important for pole focusing in such cells (e.g., Ncd is nonessential in fly development). Microtubule dynamics, specifically the relative number of K- to C-MTs in the bipolar spindle, also may alter the relative contribution of the two motors. For example, if C-MTs are very abundant, the high probability of close approximation of K- and C-MTs may enable dynein to easily link these two networks without any assistance from kinesin-14 motors at microtubule plus ends (Goshima, 2005).

An unexpected finding of this study is the microtubule plus end tracking of Ncd. Localization of a kinesin-14 motor protein to the plus end of interphase microtubules has been recently reported in plants. The accumulation of kinesin-14 at the plus end overlap zone in mitotic spindle has been shown but whether this localization reflects localization of plus ends of individual microtubules is not known. Nevertheless, this work does suggest that the plus end tracking in mitotic microtubules might be a broadly conserved feature of kinesin-14 motors. Yeast Kar3p also was shown to accumulate at the plus ends of microtubules at the shmoo, but it is more enriched on the depolymerizing microtubules, which is not observed for Ncd. Even though it was found that C-MTs are still dynamic after Ncd RNAi, it is possible that Ncd at the plus end also modulates microtubule dynamics, as does EB1 or other tip-localized proteins (Goshima, 2005).

The enhanced K fiber unfocusing in EB1 RNAi-treated cells, which displaces Ncd from plus ends but not K fibers, suggests that plus end tracking of Ncd may serve a function in pole focusing. It is proposed that plus end tracking of Ncd on newly nucleated C-MTs, as a 'capture factor,' facilitates their connection to K fibers, possibly using its second microtubule binding site located in its NH2-terminal tail domain. This idea is analogous to a 'search and capture' model for how C-MTs find chromosomes. In this case, the tip-localized motor Ncd enables C-MTs to 'search' for and then 'capture' a second major microtubule network in the spindle, the K fibers. Ncd may generate a connection between K fiber and C-MTs temporary, and thereby facilitate the recruitment of minus end-directed transporter (primarily dynein but Ncd contributing as well) for the transport of K fibers. Additionally, Ncd at the plus end may act as a K fiber transporter once it binds, although this transport would be less efficient than that produced by fast and processive dynein motors. It is also noted that the simulations are two-dimensional and encounters between C-MTs and K fibers would become less likely in three dimensions, and one might expect the effect of a C-MT-mediated capture/transport mechanism to become more important under such circumstances (Goshima, 2005).

The microtubule plus end search-and-capture mechanism might apply to other aspects of metazoan cell division. For example, cross-linking interactions between antiparallel microtubules occurs at overlap zone of microtubules, and genetic study demonstrates that Ncd produces an inward force on antiparallel microtubules during early mitosis. Ncd at the tips of growing microtubules may act to capture microtubules that arise from the opposite pole. Another possible target of tip-localized Ncd may be free microtubules, which are either released from centrosomes or generated de novo in cytoplasm and are eventually incorporated into the spindle by a dynein-dependent transport process (Goshima, 2005).

Early spindle assembly in Drosophila embryos: Role of a force balance involving cytoskeletal dynamics and nuclear mechanics

Mitotic spindle morphogenesis depends upon the action of microtubules (MTs), motors and the cell cortex. It has been proposed that cortical- and MT-based motors acting alone can coordinate early spindle assembly in Drosophila embryos. This model was tested using microscopy of living embryos to analyze spindle pole separation, cortical reorganization, and nuclear dynamics in interphase-prophase of cycles 11-13. This study shows that actin caps remain flat as they expand and that furrows do not ingress. As centrosomes separate, they follow a linear trajectory, maintaining a constant pole-to-furrow distance while the nucleus progressively deforms along the elongating pole-pole axis. These observations are incorporated into a model in which outward forces generated by zones of active cortical dynein are balanced by inward forces produced by nuclear elasticity and during cycle 13, by Ncd, which localizes to interpolar MTs. Thus, the force-balance driving early spindle morphogenesis depends upon MT-based motors acting in concert with the cortex and nucleus (Cytrynbaum, 2005).

The task of constructing a mechanistic and quantitative understanding of mitosis remains a major challenge. This study used a combination of experimental and modeling tools to understand the coordinated behavior of motors, MTs, actin, and the nucleus in spindle morphogenesis. Quantitative data gathered from observations of successive cycles in the syncytial blastoderm stage of the Drosophila early embryo combined with modeling allows leads to the conclusion that the nucleus plays a crucial role in determining the size of the nascent spindle at the end of prophase. In addition, it is observed that the nucleus deforms and aligns with the spindle axis, so it is suggested that nuclear elasticity (assisted by Ncd in cycle 13) develops a spring-like inward force growing with the pole-to-pole separation. Evidence is provided that dynein is spread more or less uniformly throughout the actin cortex, in the shape of solid and flat, not hollowed out, caps expanding in synchrony with the separating centrosomes. Interestingly, cap expansion is not accompanied by furrow ingression during the pre-NEB stage. Moreover, the subcortical linear trajectories of the centrosomes imply an outward force on the spindle originating at the cortex (Cytrynbaum, 2005).

Comparing successive cycles in the blastoderm stage, it is concluded that Ncd, which colocalizes with the interpolar MT bundles, does not play as significant a role in pre-NEB centrosome separation before cycle 13 as it does in cycle 13 (the cycle studied in Sharp, 2000a). This observation is consistent with the fact that it is only after cycle 12, when the cortex is crowded with hundreds of buds, that the embryo must implement additional means of preventing inappropriate MT-kinetochore connections between buds. It seems more likely that NCD affects the final spindle length rather than the centrosomes' ability to separate or their separation rate. For example, it could be that Ncd is one of the cross-linking agents responsible for interpolar MT bundling and thus it indirectly regulates the outward force acting on the spindle poles. Also, Ncd action could be interpolar MT-length dependent due to plausible collective motor effects. To conclude, Ncd is probably not essential during spindle assembly and nuclear elasticity is in fact equally or more important in determining spindle size, particularly in the Ncd-null embryo (Cytrynbaum, 2005).

A computational model leads to the suggestion that the actin cap growth at the cortex is regulated by the centrosomes with possible involvement of astral MTs, and that dynein at the cortex generates a constant outward force. Of course, other structures in the vicinity of the centrosomes, such as the nucleus, are alternative candidate sources of kinases generating the diffusible signal. The model predicts that to generate sufficient outward force, either dynein activity has to be locally inhibited by the centrosomes, or astral MTs have to be depleted at the cortex between the centrosomes by cross-linking or other mechanisms, or both; otherwise, the dynein force would have a significant inward component, and pole separation could not proceed. The simplest interpretation of these results is that cap expansion is directly affected by centrosome- or astral-MT-mediated regulation of actin. Furthermore, actin polymerization dynamics has to be at least as fast as centrosome separation, or otherwise, actin cap growth would be rate limiting and centrosomes would not be able to move any faster in the Ncd null embryo than in wild type (Cytrynbaum, 2005). On a more quantitative front, the computational model predicts that the total dynein force is of the order of tens of piconewtons, indicating that tens of dynein motors at the cortex function by pulling on tens of astral MTs. This is in agreement with previously estimated numbers of MTs (Piehl, 2003) and of pulling dynein motors in other cells. An unexpectedly large value of the effective viscous drag coefficient of the centrosome would be predicted. One plausible explanation is a dynamic cross-linking of astral MTs to either the nuclear envelope, F-actin, or the hypothetical spindle matrix. Alternatively, motor-mediated sliding may have effective friction associated with it. Also, in contrast with earlier modeling attempts, the time series for pole separation can be fit without the MT polymerization force that was invoked in an the initial model (Cytrynbaum, 2003). There is no convincing proof, however, that this force does not contribute to spindle morphogenesis; future research is needed to resolve this issue (Cytrynbaum, 2005).

To summarize, this study has significantly improved the spindle elongation model by resolving existing uncertainties and proposing new hypotheses. The resulting quantitative model successfully explains the obtained data, including the kinetics of pole separation before NEB, through the balance of effective drag, dynein, and nuclear elastic forces and of the Ncd force in cycle 13 (Cytrynbaum, 2005).

The model underscores additional uncertainties that should be addressed in future studies. For example, cortical myosin II has been identified as a critical motor for spindle assembly in some cells (Rosenblatt, 2004). However, this study maintains that myosin II is unlikely to play an important role in the interphase-prophase force-balance analyzed in these studies for three reasons: (1) based on direct observations of syncytial embryos; (2) because myosin II was found to act only after NEB, which is not relevant to studies addressing pre-NEB spindle assembly, and (3) because the suggested coordinated cortical expansion and contraction model of Rosenblatt seems to require a circumferential cortex surrounding the entire spindle, which is also not relevant to the syncytial blastoderm where the cortex is adjacent to only one surface of the spindle. Given the conservation of spindle-associated force-generating mechanisms, however, directly testing the role of actin-myosin II sliding in the force balance will be worthwhile. In addition, rapid motor-mediated transport of regulatory molecules along astral MTs may influence the growth of the actin cap edges and the activity of cortical dynein, Pav-KLP being one candidate, but other mechanisms, including transport of F-actin and MT cross-linking, are also possible. Specifying the nature of such mechanisms is of utmost importance. Measurements of MT nucleation and dynamic instability parameters are needed to verify the model assumptions. It would be very important to obtain a spatial-temporal map of dynein activity. Direct biophysical measurements of the nuclear mechanics as well as the numbers, force-velocity relationships and collective force-generating properties of the multiple molecular motors would make the understanding of spindle morphogenesis more precise. Another open biophysical question is the nature and magnitude of the effective viscous drag on the centrosomes. The crowding effects of the synchronous division of thousands of nuclei near the embryo's surface have not yet been explored and will likely uncover interesting interbud interactions. Finally, extending the quantitative model to describe later stages of mitosis is necessary for understanding the role of forces and movements in spindle morphogenesis (Cytrynbaum, 2005).

The current study also seems to be relevant to the currently intriguing question of whether the mitotic spindle contains an additional mechanical component, the spindle matrix, whose activity augments those of microtubules and mitotic motors in driving spindle morphogenesis and chromosome motility. Although no definitive evidence for the existence of such a matrix exists, proposed candidates include actin, NuMA/Asp, the elastic 'microtrabecular matrix' the Drosophila skeletor-megator-chromator matrix and poly-(ADP-ribose). In an initial model for early spindle morphogenesis (Cytrynbaum, 2003), it was argued that such a spindle matrix is not required because a force-balance involving only MT polymer ratchets, mitotic motors, and the cortex could account for the dynamics of spindle pole separation. However, it could be argued that the augmentation of the force-generating properties of MTs and motors by cortical and nuclear dynamics fulfills some of the functions proposed for the matrix. Indeed, in some respects the inward elastic restoring force exerted by nuclear deformation on the spindle poles acts in the manner proposed for the hypothetical elastic microtrabecular matrix. It will therefore be interesting to determine whether this mechanical function of the nucleus in spindle morphogenesis is transferred to another spindle component such as the skeletor-megator-chromator complex after NEB at which times the nuclear envelope cannot serve as an underlying mechanical substrate (Cytrynbaum, 2005).

The current findings have general cell biological implications, because many crucial phenomena suggest self-organizing interactions between microtubules, actin, and molecular motors. These include maintaining the polarity of migrating cells and the formation of a contractile ring and cleavage furrow in cytokinesis. An important feature of all such interactions is cross-talk between mechanical and force- and movement-generating molecular machines and biochemical regulation mechanisms. In this study, MTs, the nucleus, and dynein and Ncd motors play the role of the former, whereas the proposed actin and dynein regulators, potentially delivered by MT-mediated pathways, play the role of the latter (Cytrynbaum, 2005).

Assembly pathway of the anastral Drosophila oocyte meiosis I spindle

Oocyte meiotic spindles of many species are anastral and lack centrosomes to nucleate microtubules. Assembly of anastral spindles occurs by a pathway that differs from that of most mitotic spindles. This study analyzed assembly of the Drosophila oocyte meiosis I spindle and the role of the Nonclaret disjunctional (Ncd) motor in spindle assembly using wild-type and mutant Ncd fused to GFP. Unexpectedly, motor-associated asters were observed at germinal vesicle breakdown that migrate towards the condensed chromosomes, where they nucleate microtubules at the chromosomes. Newly nucleated microtubules are randomly oriented, then become organized around the bivalent chromosomes. The meiotic spindle forms by lateral associations of microtubule-coated chromosomes into a bipolar spindle. Lateral interactions between microtubule-associated bivalent chromosomes may be mediated by microtubule crosslinking by the Ncd motor, based on analysis of fixed oocytes. Spindle assembly occurs in an ncd mutant defective for microtubule motility, but lateral interactions between microtubule-coated chromosomes are unstable, indicating that Ncd movement along microtubules is needed to stabilize interactions between chromosomes. A more severe ncd mutant that probably lacks ATPase activity prevents formation of lateral interactions between chromosomes and causes defective microtubule elongation. Anastral Drosophila oocyte meiosis I spindle assembly thus involves motor-associated asters to nucleate microtubules and Ncd motor activity to form and stabilize interactions between microtubule-associated chromosomes during the assembly process. This is the first complete account of assembly of an anastral spindle and the specific steps that require Ncd motor activity, revealing new and unexpected features of the process (Sköld, 2005).

Nucleation of microtubules for spindle assembly is typically by centrosomes, which form asters at the poles and act as microtubule organizing and nucleating centers. However, some spindles lack centrosomes and asters. Classical examples are the anastral meiotic spindles of Drosophila, Xenopus and mouse oocytes. The mechanism by which anastral spindles form is poorly understood. Although significant advances have been made, including the first live imaging of meiosis I spindle assembly in Drosophila oocytes and the remarkable development of in vitro spindle assembly assays using Xenopus egg extracts, major questions remain regarding the assembly mechanism and proteins involved (Sköld, 2005).

Minus-end spindle motor, Ncd, when expressed as a GFP fusion protein, binds to spindles of Drosophila oocytes and early embryos, and can be used to image spindle assembly during meiosis II and mitosis (Endow, 1997; Endow, 1998; Endow, 1996). The observation that Ncd does not bind to oocyte cytoplasmic microtubules (Hatsumi, 1992a; Matthies, 1996) led to an examination of immature late stage 13 oocytes to determine whether germinal vesicle breakdown and meiosis I spindle assembly could be followed in ncdgfp* oocytes (* refers to a hybrid protein). The studies reported here using NcdGFP* binding to meiotic spindle microtubules significantly extend the pioneering work of Matthies (Matthies, 1996), who observed meiosis I spindle assembly in live wild-type and cand mutant oocytes by injecting rhodamine-tubulin into the oocytes prior to germinal vesicle breakdown. The use of NcdGFP* fluorescence to follow spindle assembly is an advance, as it is less disrupting to oocytes and much easier in terms of specimen preparation than performing the injections (Sköld, 2005).

Based on this analysis of meiosis I spindle assembly, a model is proposed for assembly of anastral meiosis I spindles in Drosophila oocytes. Following nuclear envelope breakdown, spindle assembly can be divided into seven phases: (1) formation of asters containing Ncd and microtubules; (2) migration of asters to the endobody; (3) microtubule nucleation at the endobody; (4) microtubule elongation in random directions; (5) bivalent association with microtubules; (6) lateral associations between bivalents owing to microtubule crosslinking; and (7) elongation into a bipolar spindle (Sköld, 2005).

The initial event of microtubule nucleation is attributed to NcdGFP*-associated foci or asters that migrate towards the karyosome of condensed meiotic chromosomes, or endobody, and nucleate microtubules at the endobody. The specific mechanism by which microtubules are nucleated by the asters requires further work to elucidate. One possibility is that the Ncd motor crosslinks and bundles small microtubules, allowing them to serve as foci for microtubule growth. Nucleation at the endobody and outward growth implies that microtubule minus ends are associated with the chromosomes. The asters are larger in ncdNKgfp* oocytes than in wild-type ncdgfp* oocytes, presumably owing to tighter binding by the NcdNKGFP* motor to microtubules. The tighter binding by NcdNKGFP* than NcdGFP* to spindle microtubules in live oocytes was measured in FLIP experiments and is probably associated with increased bundling activity by the mutant motors, causing the asters to be larger. The asters are also larger in APL10/+ and APL10 oocytes mutant for a γ-Tub37C allele that affects a residue that may be involved in lateral associations between γ-tubulin subunits, implying that the asters may containγ-tubulin as well as the NcdGFP* motor.γ-Tub37C has not been localized to developing Drosophila egg chambers or mature oocytes, despite several attempts. Mutants ofγ-Tub37C have been reported previously to disrupt oocyte meiosis I spindles, although the abnormal meiotic figures in one of these studies may have been caused by inadvertent activation of the oocytes, based on their reported absence in ovulated oocytes or embryos. The role ofγ-tubulin37C in meiosis I spindle assembly in Drosophila oocytes remains an open question that will probably require expression and careful analysis of a fluorescently labeledγ-tubulin protein to answer definitively (Sköld, 2005).

In the work by Matthies (1996), microtubule asters were occasionally observed but were suggested not to have a role in spindle assembly. Visualization of asters in the Matthies study after injection of rhodamine-tubulin supports the interpretation that the asters contain microtubules, as well as NcdGFP*. In this study multiple asters were frequently seen in close proximity to the germinal vesicle. One or more of these asters moved towards the endobody at the time of germinal vesicle breakdown and associated with it, after which spindle microtubules began to nucleate. The timing of these events and the apparent nucleation of microtubules from the asters imply that the asters are involved in microtubule nucleation at the endobody, although further studies will be required to establish this (Sköld, 2005).

A previous model for anastral spindle assembly, based on in vitro studies of spindle assembly around DNA-coated beads, proposes that microtubules nucleate at the chromatin with random polarity and form bundles of mixed polarity that undergo assortment and focusing to form a bipolar array (Heald, 1996). These workers suggested that microtubule nucleation is due to the influence of chromatin on the 'local state of the cytoplasm to favor microtubule nucleation and stabilization'. The model presented here differs from this previous study in that it is proposed that Ncd motor-associated asters migrate towards the chromosomes and nucleate microtubules, based on observations in live ncdgfp* oocytes. The bundled microtubules project in various orientations from the endobody and then undergo reorganization around the chromosomes in a manner similar to that proposed previously (Heald, 1996), except the chromosomes are the separated bivalents that subsequently interact laterally, presumably via motor-mediated microtubule crosslinking, to form a single bipolar array. Differences in the initial steps of microtubule nucleation may be species specific, but this possibility will require further study to establish (Sköld, 2005).

The subsequent steps of bivalent chromosome association with microtubules and lateral association of the microtubule-coated chromosomes are reported here as intermediates in the assembly of an anastral spindle. Separated spindles associated with bivalent chromosomes were reported previously in ncd null mutant oocytes (Wald, 1936; Kimble, 1983; Hatsumi, 1992b), but were interpreted to arise as a consequence of loss of microtubule crosslinking by the Ncd motor (Hatsumi, 1992a; Matthies, 1996) which caused them to detach from the spindle and become associated with microtubules, rather than disruption of an essential intermediate in the spindle assembly process. The previous lack of a complete pathway of meiosis I spindle assembly, including the initial steps of microtubule nucleation and intermediate steps that culminate in the appearance of a bipolar spindle, means that the effects of mutants could not be accurately determined. This study presents the first complete account of the assembly of an anastral spindle. The effects on spindle assembly of Ncd motility are further separated from the ability of the motor to hydrolyze ATP by analyzing an immobile mutant motor, ncdNK, and a severe loss-of-function mutant, ncd2, which is probably defective in ATP hydrolysis. The effects of specific motor activities on spindle assembly have not been reported previously (Sköld, 2005).

The requirement for Ncd motility in meiosis I spindle assembly was tested using a mutant motor, NcdNK, which is defective in microtubule gliding in vitro (Song, 1998). The NcdNK mutant motor binds microtubules to the coverslip, but does not move on microtubules in coverslip gliding assays. When assayed in vitro, the NcdNK mutant hydrolyzes ATP at the basal level, but shows no microtubule-stimulated ATPase activity and binds more tightly to microtubules than wild-type Ncd by ~two- to threefold. The mutant NcdNKGFP* motor showed a slower rate of release from spindle microtubules in the present study, indicating tighter binding to microtubules in live oocytes, although the increase over wild-type Ncd that was observed was less than in vitro, only ~1.5-fold. This may be due to the fact that the entire protein was present in the live oocytes, including the microtubule-binding tail region, whereas a truncated form of the motor that contained only the conserved motor domain was used in the in vitro biochemical assays. Nonetheless, these data show that the mutant NcdNKGFP* motor binds to microtubules more tightly than wild-type NcdGFP* in live cells, as well as in microtubule pelleting assays in vitro (Sköld, 2005).

The NcdNKGFP* mutant motor is defective in meiosis I spindle assembly, primarily in maintaining stable lateral interactions between the microtubule-associated bivalent chromosomes. Lateral interactions between the bivalent chromosomes are probably due primarily to crosslinking of microtubules by the motor but also require motor movement along microtubules for stability, based on the effects of ncdNKgfp*. The absence of stable lateral interactions prolongs or prevents bipolar spindle formation and causes the ncdNKgfp* mutant oocytes to typically exhibit multiple or multipolar spindles. The abnormal spindles of the mutant indicate that the basal ATPase of the motor partially rescues spindle assembly, but Ncd motility is needed to stabilize lateral interactions of the chromosomes for bipolar spindle formation. Presumably, minus-end movement of wild-type Ncd along microtubules helps to hold the dynamically moving spindle-associated chromosomes together, which would otherwise move apart in the absence of a mobile Ncd motor. The requirement for Ncd motility in spindle assembly has not been demonstrated previously. Here, the effects of motor movement on microtubules were separated from microtubule binding by analysis of a mutant defective in motility, but not microtubule binding (Sköld, 2005).

These conclusions from analysis of the ncdNKgfp* mutant are supported by analysis of the more severe ncd2gfp* mutant, which is probably defective for both the motor basal and microtubule-stimulated ATPase, based on the nature of the mutation and its location in the motor domain (Endow, 1997). The ncd2gfp* mutant is defective in formation of lateral interactions between microtubule-associated bivalent chromosomes, which in turn affects pole formation and bipolar spindle assembly, causing them to be defective. Effects of the cand deletion mutant of ncd on pole formation in meiosis I spindles have been reported previously (Wald, 1936; Kimble, 1983; Hatsumi, 1992b). The defect in an early step of the spindle assembly pathway greatly prolongs the process and prevents the appearance of normal bipolar spindles. The ncd2gfp* mutant is not defective in microtubule association with bivalent chromosomes, although the microtubules do not form a sheath around the chromosomes as in wild-type ncdgfp* oocytes, indicating that hydrolysis of ATP by the motor may also be required for the motor to stabilize microtubule elongation. These effects of the Ncd2 motor could reflect the requirement for binding and releasing microtubules, which is thought to be coupled to the motor ATPase. A motor defective in ATPase activity could bind tightly to microtubules with both its microtubule-binding tail and motor domain during the initial stages of spindle assembly, and fail to release and rebind microtubules associated with an adjacent bivalent chromosome, blocking subsequent steps of assembly (Sköld, 2005).

These observations significantly extend those of Matthies (1996), who reported the effects of cand as prolonging assembly of bipolar spindles and causing spindles that did form to disorganize transiently and then reform. Matthies inferred from these observations that microtubule bundling by the Ncd motor 'promotes assembly of a stable bipolar spindle', but did not identify the steps in the spindle assembly process that were defective, largely because the assembly pathway the study reported was incomplete and lacked key initial and intermediate steps. Based on the current time-lapse analysis of live wild-type and mutant oocytes, the defects of the ncd2gfp* severe loss-of-function mutant are thought to be at an early stage of spindle assembly: the formation of lateral interactions between microtubule-associated bivalent chromosomes and stabilization of elongating microtubules. The spindles observed in ncd2gfp* mutant oocytes did not disassemble and reform as reported for cand (Matthies, 1996), although this may be a difference between the cand null mutant and ncd2gfp* loss-of-function mutant, in which a mutated Ncd motor capable of binding to spindle microtubules is expressed that may partially stabilize initial spindle structures (Sköld, 2005).

Both of the ncd mutants examined in this study express defective Ncd motor proteins, whereas mutant cand oocytes produce no Ncd protein but can assemble abnormal multipolar meiotic spindles, requiring a much longer time. These results indicate that there is a dominant pathway of meiosis I spindle assembly in Drosophila oocytes that depends on the Ncd motor. In the absence of Ncd, spindles can initiate assembly, but the process is much less efficient. Initiation of microtubule nucleation may eventually occur by chance encounters of small microtubules with the karyosome, rather than being facilitated in their association by the Ncd motor. Assembly is then blocked or destabilized at an early step of the pathway, resulting in abnormal spindles. This parallels findings for mitotic spindle assembly in C. elegans, which depends onγ-tubulin, and fails in its absence or loss of function (Hannak, 2002). The findings present in this study define a pathway for anastral spindle assembly, including key features of the process: microtubule nucleation at the chromosomes, formation of microtubule-associated bivalent chromosomes and their lateral association into a bipolar spindle, and identify the steps dependent on Ncd motility and ability to hydrolyze ATP. This pathway provides an essential framework for further analysis of the assembly process and the proteins involved (Sköld, 2005).

Novel nuclear defects in KLP61F-deficient mutants in Drosophila are partially suppressed by loss of Ncd function

KLP61F in Drosophila and other BimC kinesins are essential for spindle bipolarity across species; loss of BimC function generates high frequencies of monopolar spindles. Concomitant loss of Kar3 kinesin function increases the frequency of bipolar spindles although the underlying mechanism is not known. Recent studies raise the question of whether BimC kinesins interact with a non-microtubule spindle matrix rather than spindle microtubules. This study presents cytological evidence that loss of KLP61F function generates novel defects during M-phase in the organization and integrity of the nuclear lamina, an integral component of the nuclear matrix. Larval neuroblasts and spermatocytes of klp61F mutants showed deep involutions in the nuclear lamina extending toward the centrally located centrosomes. Repositioning of centrosomes to form monopolar spindles probably does not cause invaginations as similar invaginations formed in spermatocytes lacking centrosomes entirely. Immunofluorescence microscopy indicated that non-claret disjunctional (Ncd) is a component of the nuclear matrix in somatic cells and spermatocytes. Loss of Ncd function increases the frequency of bipolar spindles in klp61F mutants. Nuclear defects were incompletely suppressed; micronuclei formed near telophase at the poles of bipolar spindle in klp61F ncd spermatocytes. These results are consistent with a model in which KLP61F prevents Ncd-mediated collapse of a nonmicrotubule matrix derived from the interphase nucleus (Wilson, 2004).

This study present cytological evidence that loss of KLP61F function generates spindle defects as well as novel defects in organization of the nuclear matrix during M-phase in somatic cells and spermatocytes. These results also show that Ncd is nuclear during interphase and spindle-associated in M-phase in the soma and male germ line. Loss of Ncd function increases the frequency of biastral spindles in klp61F mutants, but fails or incompletely restores nuclear matrix defects. These findings raise new questions about the molecular basis of genetic interactions between KLP61F and Ncd (Wilson, 2004).

Somatic cells in klp61F and klp61F ncd mutants with monopolar spindles show deep invaginations in the nuclear lamina that extended toward centrally located centrosomes. Similar involutions were found in klp61F mutant spermatocytes judged to be near prometaphase, irrespective of the presence or absence of centrosomes. These observations suggest that the driving force in forming invaginations in the nuclear lamina is associated with nuclear and/or cytoplasmic material rather than with centrosomes or centrosome organized microtubules. A contribution of nuclear forces to repositioning of centrosomes has precedence in yeast; spindle-pole bodies in preassembled spindles move through the nuclear envelope to side-by-side positions when temperature-dependent BimC function is inactivated at non-permissive temperatures. Because spindle pole bodies assume face-to-face positions when microtubules are depolymerized, side-by-side positions suggests that nuclear forces contribute to spindle defects in BimC-deficient yeast as well (Wilson, 2004).

Nuclear defects in somatic cells differed from those in spermatocytes, raising the question of whether KLP61F function in somatic cells and spermatocytes is mediated by a common mechanism or two different mechanisms. Arguments can be made for and against a common mechanism. The strongest argument for a common mechanism is the striking similarity of spindle defects in somatic cells and spermatocytes. Another argument is the ability of ncd mutants to suppress the klp61F mutant phenotype in both cell types. At first glance, other aspects of the mutant phenotype are not consistent with a common function. Somatic cells in KLP61F-deficient animals showed extensive disorganization of the nuclear lamina, including cells showing bipolar positioning of centrosomes and metaphase alignment of chromosomes. In contrast to somatic cells, the nuclear lamina appeared to collapse around bivalents near prometaphase and form micronuclei in klp61F mutant spermatocytes. These differences could reflect different functions in somatic cells and spermatocytes. Alternatively, the difference may reflect cell cycle regulation; the spindle assembly checkpoint is active in somatic cells, but inactive or severely abrogated in spermatocytes. This view is consistent with the disorganized state of the nuclear lamina in cultured clone 8 cells that were delayed in mitosis with an inhibitor of APC. Thus, disorganization of the nuclear lamina in somatic cells and formation of micronuclei in spermatocytes in KLP61F-deficient mutants could reflect a common underlying defect in different cell types (Wilson, 2004).

KLP61F shows overlapping, but differential localization during mitosis and male meiosis. In somatic cells, KLP61F is highly enriched near centrosomal asters during prophase, spindle-associated in metaphase and located in midbodies in telophase. In meiotic spermatocytes, KLP61F fails to show centrosomal enrichment or spindle association, but in late anaphase/early telophase KLP61F localizes to a sphere that bisects the entire spermatocyte and then follows the ingressing cleavage furrow. Similar localization in proliferating germ cells in telophase was found to reflect interactions, directly or indirectly, with components of fusomes. It is not possible to draw firm conclusions from the failure to detect KLP61F localization to centrosomal asters or to spindles as a small pool could escape detection methods. However, given static positioning of Eg5 in spindles assembled in Xenopus egg extracts, the failure to detect KLP61F localization to male meiotic spindles may indicate that KLP61F is not associated with spindle microtubules, but with non-microtubule binding partners. In most cell types, BimC kinesins are diffusely distributed throughout the cytoplasm during interphase. Localization of BimC kinesins to spindles in vertebrate cells has been linked to phosphorylation of a Cdk1 target site in the conserved BimC Box near the carboxyl tail region of these kinesins, postulated to elicit or strengthen intrinsic microtubule binding activity and spindle localization. However, phosphorylation of the BimC Box of Cut7 in Saccharomyces pombe is not required for spindle association or for Cut7 function in assembly of a bipolar spindle. It is possible that KLP61F and other BimC kinesins crosslink microtubules and non-microtubule binding partners during interphase. BimC Box phosphorylation may downregulate microtubule binding activity and allow interactions with non-microtubule binding partners to direct KLP61F localization during M-phase. Identification of non-microtubule binding partners and genetic analysis of BimC Box function in KLP61F localization could test these possibilities (Wilson, 2004).

Similar to Kar3 kinesins in vertebrates, Ncd is nuclear during interphase in spermatocytes and in somatic cells as well as in somatically derived clone 8 cells. It is not clear why Ncd fails to show nuclear localization in early embryos, but the rapidity of the cell cycle in early embryos may preclude complete reorganization of the nuclear envelope and nuclear entry of Ncd through nuclear pores. Two lines of cytological evidence suggest that Ncd may be associated with the nuclear matrix during interphase in somatic and male germ line cells. First, Ncd shows subnuclear enrichment near heterochromatin attached to the nuclear envelope. Given that fibrillar components of the nuclear matrix connect chromatin to the inner nuclear membrane, subnuclear enrichment could reflect localization of Ncd to fibrillar components of the nuclear matrix and/or localization to chromatin-associated material at these sites. Second, Ncd localizes to fibers extending between the poles of metaphase spindles in somatic cells and cultured clone 8 cells, reminiscent of Ncd localization in embryos (Endow, 1997). The functional significance of these fibers is not clear since pole to pole fibers have not been reported in female meiotic spindles and this study did not detect strong immunostaining of similar fibers in meiotic spermatocytes. However, there is a precedence for localization of other nuclear matrix proteins to spindle fibers. The nuclear matrix protein NuSAP is localized to spindle-associated fibers in cultured vertebrate cells and loss of its function generates defects in spindle organization and chromosome segregation. The nuclear matrix protein Skeletor also localizes to fibers in embryonic spindles although the fibers do not extend the full distance between spindle poles. Loss of Ncd function did not appreciably alter Skeletor distribution in spermatocytes, indicating that Ncd is not necessary or plays only a very limited role in Skeletor localization. Nonetheless, the findings are consistent with the view that Ncd is a component of the nuclear matrix in somatic cells and spermatocytes (Wilson, 2004).

At this point, it is only possible to speculate on the relationship between spindle and nuclear defects in klp61F mutants and the functional significance of Ncd-mediated suppression. With few exceptions, cooperation between BimC and Kar3 kinesins in spindle assembly has been ascribed to application of antagonistic motive forces to spindle microtubules to establish or maintain centrosome separation. According to this view, nuclear defects in KLP61F-deficient animals could be secondary to primary defects in spindle organization; increasing the frequency of bipolar spindles in klp61F ncd mutants results in a decreased frequency of nuclear defects. However, this explanation does not easily explain formation of micronuclei at the poles of bipolar spindles in klp61F ncd spermatocytes. Moreover, collapse of the nuclear lamina about bivalents cannot be ascribed to spindle defects since similar defects are not found in meiotic spermatocytes of ß2tn mutants that lack microtubules and spindle structures owing to loss of an essential testis specific ß-tubulin. An alternative interpretation of the findings is that spindle defects are secondary; spindle defects reflect collapse of a nonmicrotubule spindle matrix that is derived from the nuclear matrix and attached to centrosomes and/or spindle microtubules. According to this view, interactions between KLP61F and nonmicrotubule binding partners prevent collapse of a compressible spindle matrix when nuclear and cytoplasmic contents mix at prometaphase, whether or not KLP61F is spindle associated (Wilson, 2004).

The results of this study are in part unexpected because they question the assumed relationship between localization and function of a microtubule-dependent motor protein. KLP61F is required for spindle bipolarity, but its function in male meiosis does not require spindle association. Conversely, KLP61F localizes to cleavage furrows, but it is not required for cytokinesis. With these contradictions in mind, further work must address the mechanism of KLP61F function in spindle organization and the functional significance of nuclear localization of Ncd (Wilson, 2004).

In female meiosis, subito, encoding a member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes, have a similar phenotype to ncd

The female meiotic spindle lacks a centrosome or microtubule-organizing center in many organisms. During cell division, these spindles are organized by the chromosomes and microtubule-associated proteins. Previous studies in Drosophila have implicated at least one kinesin motor protein, NCD, in tapering the microtubules into a bipolar spindle. A second Drosophila kinesin-like protein, Sub, has been identified that is required for meiotic spindle function. At meiosis I in males and females, sub mutations affect only the segregation of homologous chromosomes. In female meiosis, sub mutations have a similar phenotype to ncd; even though chromosomes are joined by chiasmata they fail to segregate at meiosis I. Cytological analyses have revealed that sub is required for bipolar spindle formation. In sub mutations, spindles were observed that were unipolar, multipolar, or frayed with no defined poles. On the basis of these phenotypes and the observation that sub mutations genetically interact with ncd, it is proposed that Sub is oe member of a group of microtubule-associated proteins required for bipolar spindle assembly in the absence of the centrosomes. sub is also required for the early embryonic divisions but is otherwise dispensable for most mitotic divisions (Giunta, 2002).

The sub genetic and cytological mutant phenotypes are similar to those previously described for mutants in ncd. Most important, both mutants cause nondisjunction of homologous chromosomes at the first meiotic division but have no effect on the second meiotic division. On the basis of a live analysis, it has been proposed that Ncd is required in the acentrosomal spindle to taper the microtubules into a pole with its minus-end-directed motor moving outward from the chromosomes, bundling together microtubules in the process. It has also been proposed that at least one additional motor is involved in the process because poles can still form in the absence of Ncd. Thus, one possible function of Sub is to bundle microtubules to form that portion of the poles that is not handled by Ncd. This model predicts that a sub; ncd double mutant would have a more severe defect in spindle pole formation than would either single mutant. Double mutant analysis showed this is not the case; the double mutant is able to make spindle poles with a similar array of defects as the single mutants. Therefore, it is concluded that both ncd and sub are involved in the same process of spindle formation (Giunta, 2002).

The subDub mutation changes a highly conserved amino acid in the motor domain. The original study of the dominant subDub mutation did not distinguish between an antimorph or neomorph (Moore, 1994). The subDub meiotic phenotypes are almost identical to the null alleles, arguing that it is an antimorph. The kinesin motor nod also has a dominant antimorphic allele (nodDTW) that is associated with a single amino acid change in a highly conserved region of the ATP-binding domain. Similar to subDub, nodDTW dominantly affects chiasmate and achiasmate chromosomes. Both the subDub and nodDTW proteins could have altered microtubule-binding activities that lead to interference with other proteins on the meiotic spindle. Both dominant mutations also cause lethality due to mitotic defects, and this phenotype is lessened by the presence of wild-type gene activity, suggesting that both sub and nod gene products interact with the mitotic spindle. The observation that ncd1 and subnull alleles are synthetically lethal also argues that sub has a function in mitotic cells (Giunta, 2002).

The Drosophila female meiotic spindle must organize in the absence of centrosomes and segregate homologs at the reductional division. Genes likes sub that are not required for the typical mitotic division or meiosis II may be required for these unique properties of the meiotic spindle. The simplest hypothesis is that SUB is a kinesin that interacts with spindle microtubules. This is supported by the specific genetic interactions with the ncdD and nod mutations. Interestingly, the Sub homologs MKLP1 and Pav have been shown to localize at centrosomes in mitotic metaphase. In addition, MKLP1 is known to bundle microtubules and be a plus-end-directed motor. Indeed, most of the members in the MKLP1 group have this property, although one, RB6K, has been reported to associate with the Golgi. Although a more recent report demonstrates that RB6K has an important role in cytokinesis, an indirect role for Sub in spindle formation cannot be ruled out (Giunta, 2002) (Sköld, 2005).

Spindle assembly in the absence of centrosomes can be divided into four stages. Stage 1: the nucleation or capture of microtubules by the chromosomes. Stage 2: the microtubules are bundled together by proteins that can form bridges between parallel and/or antiparallel microtubules. Stage 3: extension of the spindle by antiparallel microtubule sliding or a 'polar ejection force' exerted by motors associated with the chromosomes. Stage 4: the minus-ends of the microtubules are focused to produce defined spindle poles. Although these stages probably overlap and share genetic requirements, the function of Sub appears to be most important for the last stage of bipolar spindle formation. In sub mutants, microtubule arrays of wild-type length are able to form, but they fail to be focused into only two poles. It has been proposed that an inherent product of the microtubule bundling process is the formation of a single axis and therefore at least a crude bipolar spindle. A relationship between maintaining the integrity of the poles and constructing a spindle with only two poles would explain why in sub mutants the spindle poles are often frayed and/or they are tripolar or monopolar (Giunta, 2002).

One aspect of the sub mutation phenotype could be the inability to generate poleward forces. In sub mutants the position of the chromosomes within the karyosome is abnormal. Thus, Sub could facilitate interactions between the chromosomes and microtubules that are part of the process that organizes meiotic spindles. As was argued from an analysis of alphaTubulin67C mutants, defects in sub could result in a disruption of poleward forces, leading to a failure in centromere positioning. This would provide a link between the chromosomes and spindle pole organization, which is plausible considering that the chromosomes have a role in organizing the spindle (Giunta, 2002).

In addition to female spindle formation, sub is required for at least two other cell divisions: male meiosis and the early embryonic cleavage divisions. There are significant differences between the Drosophila male and female meiotic divisions; for example, in male meiosis crossing over does not occur and centrosomes are present. In both male and female meiosis, however, there is a reductional division involving the segregation of homologs. Thus, the importance of SUB may not be for spindle pole formation, but to organize a spindle where bivalents must be oriented and segregated. It is noteworthy that the sub alleles are the only Drosophila mutants that are defective at the first meiotic division of both males and females without affecting the segregation of sister chromatids (Giunta, 2002).

The null alleles of sub are female sterile due to a failure in the early embryonic cell divisions. The early defects in sub mutation embryos have some similarities to mutants in other genes with a variety of roles in spindle function such as Klp3A, alpha-Tubulin67C, polo, and wispy. In all these cases, it was suggested that the embryonic arrest was due to a defect in pronuclear migration, although the variety of defects observed in sub and the other mutants make it difficult to define a precise function for these proteins. In addition, pronuclear fusion may be a sensitive point for a wide variety of defects in microtubule-based processes, leading to the arrest prior to pronuclear fusion in many different mutants. It will be interesting to determine the nature of the sub function that is required for reductional meiotic divisions and an early event in embryogenesis (Giunta, 2002).


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