Localization of cytoplasmic dynein to mitotic spindles and kinetochores

What is the origin of the forces generating chromosome and spindle movements in mitosis? Both microtubule dynamics and microtubule-dependent motors have been proposed as the source of these motor forces. Cytoplasmic dynein and kinesin are two soluble proteins that power membranous organelle movements on microtubules. Kinesin directs movement of organelles to the 'plus' end of microtubules, and is found at the mitotic spindle in sea urchin embryos, but not in mammalian cells. Cytoplasmic dynein translocates organelles to the 'minus' end of microtubules, and is composed of two heavy chains and several light chains. We report here that monoclonal antibodies to two of these subunits and to another polypeptide that associates with dynein localize the protein to the mitotic spindle and to the kinetochores of isolated chromosomes, suggesting that cytoplasmic dynein is important in powering movements of the spindle and chromosomes in dividing cells.     

Correlation between the ATPase and microtubule translocating activities of sea urchin egg kinesin

Coupling between ATP hydrolysis and microtubule movement was demonstrated several years ago in flagellar axonemes and subsequent studies suggest that the relevant microtubule motor, dynein, uses ATP to drive microtubule sliding by a cross-bridge mechanism analogous to that of myosin in muscles. Kinesin, a microtubule-based motility protein which may participate in organelle transport and mitosis, binds microtubules in a nucleotide-sensitive manner, and requires hydrolysable nucleotides to translocate microtubules over a glass surface. Recently, neuronal kinesin was shown to possess microtubule-activated ATPase activity although coupling between ATP hydrolysis and motility was not demonstrated. Here we report that sea urchin egg kinesin, prepared either with or without a 5'-adenylyl imidodiphosphate(AMPPNP)-induced microtubule binding step, also possesses significant microtubule-activated ATPase activity when Mg-ATP is used as a substrate. This ATPase activity is inhibited in a dose-dependent manner by addition of Mg-free ATP, by chelation of Mg2+ with EDTA, by addition of Na3VO4, or by addition of AMPPNP with or without Mg2+. Addition of these same reagents also inhibits the microtubule-translocating activities of sea urchin egg kinesin in a dose-dependent manner, supporting the hypothesis that kinesin-driven motility is coupled to the microtubule-activated Mg2+-ATPase activity.     

Cytoplasmic dynein is localized to kinetochores during mitosis

Recent evidence suggests that the force for poleward movement of chromosomes during mitosis is generated at or close to the kinetochores. Chromosome movement depends on motion relative to microtubules, but the identities of the motors remain uncertain. One candidate for a mitotic motor is dynein, a large multimeric enzyme which can move along microtubules toward their slow growing end. Dyneins were originally found in axonemes of cilia and flagella where they power microtubule sliding. Recently, cytoplasmic dyneins have also been found, and specific antibodies have been raised against them. The cellular localization of dynein has previously been studied with several antibodies raised against flagellar dynein, but the relevance of these data to the distribution of cytoplasmic dynein is not known. Antibodies raised against cytoplasmic dyneins have shown localization of dynein antigens to the mitotic spindles in Caenorhabditis elegans embryos (Lye et al., personal communication) and punctate cytoplasmic structures in Dictyostelium amoebae. Using antibodies that recognize subunits of cytoplasmic dyneins, we show here that during mitosis, cytoplasmic dynein antigens concentrate near the kinetochores, centrosomes and spindle fibres of HeLa and PtK1 cells, whereas at interphase they are distributed throughout the cytoplasm. This is consistent with the hypothesis that cytoplasmic dynein is a mitotic motor.     

Microtubule motors in mitosis

The mitotic spindle uses microtubule-based motor proteins to assemble itself and to segregate sister chromatids. It is becoming clear that motors invoke several distinct mechanisms to generate the forces that drive mitosis. Moreover, in carrying out its function, the spindle appears to pass through a series of transient steady-state structures, each established by a delicate balance of forces generated by multiple complementary and antagonistic motors. Transitions from one steady state to the next can occur when a change in the activity of a subset of mitotic motors tips the balance.     

Actin-dependent organelle movement in squid axoplasm.

Studies of organelle movement in axoplasm extruded from the squid giant axon have led to the basic discoveries of microtubule-dependent organelle motility and the characterization of the microtubule-based motor proteins kinesin and cytoplasmic dynein. Rapid organelle movement in higher animal cells, especially in neurons, is considered to be microtubule-based. The role of actin filaments, which are also abundant in axonal cytoplasm, has remained unclear. The inhibition of organelle movement in axoplasm by actin-binding proteins such as DNase I, gelsolin and synapsin I has been attributed to their ability to disorganize the microtubule domains where most of the actin-filaments are located. Here we provide evidence of a new type of organelle movement in squid axoplasm which is independent of both microtubules and microtubule-based motors. This movement is ATP-dependent, unidirectional, actin-dependent, and probably generated by a myosin-like motor. These results demonstrate that an actomyosin-like mechanism can be directly involved in the generation of rapid organelle transport in nerve cells.     

Active sliding between cytoplasmic microtubules

Microtubules are versatile cellular polymers that play a role in cell shape determination and mediate various motile processes such as ciliary and flagellar bending, chromosome movements and organelle transport. That a sliding microtubule mechanism can generate force has been demonstrated in highly ordered structures such as axonemes, and microtubule-based force generation almost certainly contributes to the function of mitotic and meiotic spindles. Most cytoplasmic microtubule arrays, however, do not exhibit the structural regularity of axonemes and some spindles, and often appear disorganized. Yet many cellular activities (such as shape changes during morphogenesis, axonal extension and spindle assembly) involve highly coordinated microtubule behaviour and possibly require force generated by an intermicrotubule sliding mechanism, or perhaps use sliding to move microtubules rapidly into a protrusion for stabilization. Here we show that active sliding between cytoplasmic microtubules can occur in microtubule bundles of the amoeba Reticulomyxa. A force-producing mechanism of this sort could be used by this organism to facilitate the extension of cell processes and to generate the dynamic movements of the cytoplasmic network.     

Microtubule-motor activity of a yeast centromere-binding protein complex.

During cell division, sister chromosomes segregate from each other on a microtubule-based structure called the mitotic spindle. Proteins bind to the centromere, a region of chromosomal DNA, to form the kinetochore, which mediates chromosome attachment to the mitotic spindle microtubules. In the budding yeast Saccharomyces cerevisiae, genetic analysis has shown that the 28-basepair (bp) CDEIII region of the 125-bp centromere DNA sequence (CEN sequence) is the main region controlling chromosome segregation in vivo. Therefore it is likely that proteins binding to the CDEIII region link the centromeres to the microtubules during mitosis. A complex of proteins (CBF3) that binds specifically to the CDEIII DNA sequence has been isolated by affinity chromatography. Here we describe kinetochore function in vitro. The CBF3 complex can link DNA to microtubules, and the complex contains a minus-end-directed microtubule-based motor. We suggest that microtubule-based motors form the fundamental link between microtubules and chromosomes at mitosis.     

Force generation of organelle transport measured in vivo by an infrared laser trap

Organelle transport along microtubules is believed to be mediated by organelle-associated force-generating molecules. Two classes of microtubule-based organelle motors have been identified: kinesin and cytoplasmic dynein. To correlate the mechanochemical basis of force generation with the in vivo behaviour of organelles, it is important to quantify the force needed to propel an organelle along microtubules and to determine the force generated by a single motor molecule. Measurements of force generation are possible under selected conditions in vitro, but are much more difficult using intact or reactivated cells. Here we combine a useful model system for the study of organelle transport, the giant amoeba Reticulomyxa, with a novel technique for the non-invasive manipulation of and force application to subcellular components, which is based on a gradient-force optical trap, also referred to as 'optical tweezers'. We demonstrate the feasibility of using controlled manipulation of actively translocating organelles to measure direct force. We have determined the force driving a single organelle along microtubules, allowing us to estimate the force generated by a single motor to be 2.6 x 10(-7) dynes.     

Fibrillarin redistributes to the spindle poles and partially colocalizes with NuMA during mitosis

Fibrillarin, a major protein in the nucleolus, is known to redistribute during mitosis from the nucleolus to the cytosol, and is related to the dynamics of post-mitotic reassembly of the nucleolus. To better understand the dynamic behavior and the relationship with other cytoplasmic structures, we have now expressed fibrillarin-pDsRed1 fusion protein in HeLa cells. The results showed that a part of fibrillarin was associated with mitotic spindle poles in the mitotic cells. Nocodazole-induced microtubule depolymerization resulted in fibrillarin redistribution throughout the cytoplasm, and removal of nocodazole resulted in relocalization of fibrillarin at the polar region during the mitotic spindles reassembly. In a mitotic cell free system, fibrillarin was found in the center of taxol-induced microtubule asters. Moreover, fibrillarin was found to colocalize with the nuclear mitotic apparatus protein (NuMA) at the poles of mitotic cells. Therefore, it is postulated that the polar redistribution of fibrillarin is mediated by microtubules.     

Inhibition of mitosis by an antibody to the mitotic calcium transport system

Calcium ions are important in the regulation of mitotic apparatus assembly and in the control of chromosome movement. Changes in intracellular free calcium concentration, [Ca2+]i are achieved by an intracellular calcium-transport system which is highly conserved in different cell types. A membrane-bound protein of relative molecular mass (Mr) 46,000 (46K) is part of this transport system and has been implicated in the regulation of the [Ca2+]i changes associated with the course of mitosis. A monoclonal antibody against this 46K protein inhibits Ca2+-uptake into isolated Ca2+-sequestering membranes and specifically labels membranes associated with the mitotic apparatus of sea urchin embryos. Here we investigate the relationship between the intracellular calcium transport system and mitosis by injection of this monoclonal antibody into living mitotic sea urchin embryos. We find that after injection the intracellular free calcium increases up to 10(-6) M, the mitotic apparatus is rapidly destroyed and the cell is irreversibly blocked in its development.     

Bead movement by single kinesin molecules studied with optical tweezers

Kinesin, a mechanoenzyme that couples ATP hydrolysis to movement along microtubules, is thought to power vesicle transport and other forms of microtubule-based motility. Here, microscopic silica beads were precoated with carrier protein, exposed to low concentrations of kinesin, and individually manipulated with a single-beam gradient-force optical particle trap ('optical tweezers') directly onto microtubules. Optical tweezers greatly improved the efficiency of the bead assay, particularly at the lowest kinesin concentrations (corresponding to approximately 1 molecule per bead). Beads incubated with excess kinesin moved smoothly along a microtubule for many micrometres, but beads carrying from 0.17-3 kinesin molecules per bead, moved, on average, only about 1.4 microns and then spontaneously released from the microtubule. Application of the optical trap directly behind such moving beads often pulled them off the microtubule and back into the centre of the trap. This did not occur when a bead was bound by an AMP.PNP-induced rigor linkage, or when beads were propelled by several kinesin molecules. Our results are consistent with a model in which kinesin detaches briefly from the microtubule during a part of each mechanochemical cycle, rather than a model in which kinesin remains bound at all times.     

Retrograde transport by the microtubule-associated protein MAP 1C

Microtubules are involved in several forms of intracellular motility, including mitosis and organelle movement. Fast axonal transport is a highly ordered form of organelle motility that operates in both the anterograde (outwards from the cell body) and retrograde (from the periphery towards the cell body) direction. Similar microtubule-associated movement is observed in non-neuronal cells, and might be involved in secretion, endocytosis and the positioning of organelles within the cell. Kinesin is a mechanochemical protein that produces force along microtubules in an anterograde direction. We recently found that the brain microtubule-associated protein MAP 1C (ref. 7) is a microtubule-activated ATPase and, like kinesin, can translocate microtubules in an in vitro assay for microtubule-associated motility. MAP 1C seemed to be related to the ciliary and flagellar ATPase, dynein, which is thought to produce force in a direction opposite to that observed for kinesin. Here we report that MAP 1C, in fact, acts in a direction opposite to kinesin, and has the properties of a retrograde translocator.     

Myosin-V is a processive actin-based motor.

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.     

Alp7/TACC is a crucial target in Ran-GTPase-dependent spindle formation in fission yeast

Microtubules are essential intracellular structures involved in several cellular phenomena, including polarity establishment and chromosome segregation. Because the nuclear envelope persists during mitosis (closed mitosis) in fission yeast (Schizosaccharomyces pombe), cytoplasmic microtubules must be reorganized into the spindle in the compartmentalized nucleus on mitotic entry. An ideal mechanism might be to take advantage of an evolutionarily conserved microtubule formation system that uses the Ran-GTPase nuclear transport machinery, but no targets of Ran for spindle formation have been identified in yeast. Here we show that a microtubule-associated protein, Alp7, which forms a complex with Alp14, is a target of Ran in yeast for spindle formation. The Ran-deficient pim1 mutant (pim1-F201S) failed to show mitosis-specific nuclear accumulation of Alp7. Moreover, this mutant exhibited compromised spindle formation and early mitotic delay. Importantly, these defects were suppressed by Alp7 that was artificially targeted to the nucleus by a Ran-independent and importin-alpha-mediated system. Thus, Ran targets Alp7-Alp14 to achieve nuclear spindle formation, and might differentiate its targets depending on whether the organism undergoes closed or open mitosis.     

Microtubule-associated protein 1C from brain is a two-headed cytosolic dynein

Dynein, an ATPase, is the force-generating protein in cilia and flagella. It has long been speculated that cytoplasmic microtubules contain a related enzyme involved in cell division or in intracellular organelle transport. A 'cytoplasmic dynein' has been described in sea urchin eggs, but because the egg stockpiles precursors for both cytoplasmic and ciliary microtubules, the role of this enzyme in the cell has remained unresolved. We recently found that the microtubule-associated protein (MAP) 1C (ref. 6) from brain is a microtubule-activated ATPase that produces force in the direction corresponding to retrograde organelle transport in the cell. MAP 1C has several similar properties to ciliary and flagellar dynein. Here we show directly, using scanning transmission electron microscopy, that MAP 1C is structurally equivalent to the ciliary and flagellar enzyme and is the long-sought cytoplasmic analogue of this enzyme.     

Cell division

In creating the mitotic spindle and the contractile ring, natural selection has engineered fascinating precision machines whose movements depend upon forces generated by ensembles of cytoskeletal proteins. These machines segregate chromosomes and divide the cell with high fidelity. Current research on the mechanisms and regulation of spindle morphogenesis, chromosome motility and cytokinesis emphasizes how ensembles of dynamic cytoskeletal polymers and multiple motors cooperate to generate the forces that guide the cell through mitosis and cytokinesis.     

The Drosophila claret segregation protein is a minus-end directed motor molecule

A product encoded at the claret locus in Drosophila is needed for normal chromosome segregation in meiosis in females and in early mitotic divisions of the embryo. The predicted amino-acid sequence of the segregation protein was shown recently to be strikingly similar to Drosophila kinesin heavy chain. We have expressed the claret segregation protein in bacteria and have found that the bacterially expressed protein has motor activity in vitro with several novel features. The claret motor is slow (4 microns min-1), unlike either kinesin or dyneins. It has the directionality, the ability to generate torque and the sensitivity to inhibitors reported previously for dyneins. The finding of minus-end directed motor activity for a protein with sequence similarity to kinesin suggests that the dynein and kinesin motor domains are ancestrally related. The minus-end directed motor activity of the claret motor is consistent with a role for this protein in producing chromosome movement along spindle microtubules during prometaphase and/or anaphase.     

Interaction of brain cytoplasmic dynein and MAP2 with a common sequence at the C terminus of tubulin

Two main types of microtubule-associated proteins (MAPs) have been identified in neuronal cells. The fibrous MAPs, including MAP2 and tau, serve to organize and regulate the assembly of microtubules. A second distinct class of force-producing MAPs, including kinesin, dynein and dynamin, are involved in microtubule-based movement. These proteins are mechanochemical ATPases which seem to be responsible for the bidirectional transport of organelles and perhaps also the movement of chromosomes. Here we report that MAP2 inhibits microtubule gliding on dynein-coated coverslips, as well as the microtubule-activated ATPase of dynein, indicating that MAP2 and other fibrous MAPs could be important modulators of microtubule-based motility in vivo. By proteolytic modification of tubulin, we found that dynein interacts with microtubules at the C termini of alpha- and beta-tubulin, the regions previously reported to be the sites for the interaction of MAP2. The use of site-directed antibodies implicates a small region of alpha- and beta-tubulin, containing the sequence Glu-Gly-Glu-Glu, as the site of the interaction of dynein and MAP2 with the microtubule.     

Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.     

Mitotic spindle organization by a plus-end-directed microtubule motor.

Intracellular microtubule motor proteins may direct the motile properties and/or morphogenesis of the mitotic spindle (reviewed in ref. 3). The recent identification of kinesin-like proteins important for mitosis or meiosis indicates that kinesin-related proteins may play a universal role in eukaryotic cell division, but the precise function of such proteins in mitosis remains unknown. Here we use an in vitro assay for spindle assembly, derived from Xenopus egg extracts, to investigate the role of Eg5, a kinesin-like protein in Xenopus eggs. Eg5 is localized along spindle microtubules, and particularly enriched near spindle poles. Immunodepletion of Eg5 from egg extracts markedly reduces the extent of spindle formation in extracts, as does direct addition of anti-Eg5 antibodies. We also demonstrate that Eg5 is a plus-end-directed microtubule motor in vitro. Our results suggest a novel mechanism for the dynamic self-organization of spindle poles in mitosis.