Poleward transport of Eg5 by dynein-dynactin in Xenopus laevis egg extract spindles

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein–dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.     

The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles

Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This "poleward flux" of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose-responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.     

Microtubule plus-end tracking proteins in differentiated mammalian cells

Differentiated mammalian cells are often characterized by highly specialized and polarized structure. Its formation and maintenance depends on cytoskeletal components, among which microtubules play an important role. The shape and dynamic properties of microtubule networks are controlled by multiple microtubule-associated factors. These include molecular motors and non-motor proteins, some of which accumulate specifically at the growing microtubule plus-ends (the so-called microtubule plus-end tracking proteins). Plus-end tracking proteins can contribute to the regulation of microtubule dynamics, mediate the cross-talk between microtubule ends, the actin cytoskeleton and the cell cortex, and participate in transport and positioning of structural and regulatory factors and membrane organelles. Malfunction of these proteins results in various human diseases including some forms of cancer, neurodevelopmental disorders and mental retardation. In this article we discuss recent data on microtubule dynamics and activities of microtubule plus-end binding proteins important for the physiology and pathology of differentiated mammalian cells such as neurons, polarized epithelia, muscle and sperm cells.     

Mechanism and function of poleward flux in Xenopus extract meiotic spindles

In Xenopus extract meiotic spindles, microtubules slide continuously towards their minus ends, a process called poleward flux. This article discusses recent progress in determining the mechanism of poleward flux, and its functions in spindle organization and generating force on chromosomes. Bipolar organization is required for flux and inhibition of the mitotic kinesin Eg5 inhibits flux, suggesting the sliding force for flux is generated by Eg5 pushing anti-parallel microtubules apart. An important function of flux in spindle organization may be to transport minus ends nucleated at chromatin towards the pole. By pulling microtubules through attachment sites at kinetochores, flux may generate poleward force on metaphase chromosomes.     

Studying nuclear disassembly in vitro using Xenopus egg extract

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.     

Microtubule dynamics in a cytosolic extract of fetal rat brain

Brain microtubule dynamics were studied by video-enhanced differential interference contrast microscopy in a cytosolic extract from fetal rat brain, prepared under conditions designed to produce minimal alterations in microtubule stability. With urchin sperm axoneme fragments as nucleation seeds, the extract was shown to support cellular-like microtubule dynamics. Microtubules elongated from one end of the axonemes, and did not spontaneously self-assemble in the absence of axonemes. The following microtubule kinetic parameters were measured in the extract: velocity of elongation (8.1 mm/min), velocity of rapid shortening (5.8 mm/min), catastrophe frequency (0.17 min-1), and rescue frequency (1 min-1). These parameters were in close agreement with reported values for growth cones of living neurons. Microtubule properties in the fetal brain extract were shown to be affected by agents with known effects on the cytoskeleton. pp60c-src, a tyrosine kinase important in cell adhesion molecule-dependent axon growth, caused small increases in the frequency of microtubule catastrophe (0.31 min-1) and rescue (2 min-1) without changing the velocities of elongation or rapid-shortening. Although pp60c-src phosphorylated purified porcine brain tubulin in vitro, it did not elicit significant changes in its polymerization properties, suggesting that other cytoskeletal proteins in the brain extract are involved in modulating microtubule dynamics. The cytosolic extract of fetal rat brain provides a useful system for studying regulation of microtubule assembly in neuronal growth cones.     

A versatile multivariate image analysis pipeline reveals features of Xenopus extract spindles

Imaging datasets are rich in quantitative information. However, few cell biologists possess the tools necessary to analyze them. Here, we present a large dataset of Xenopus extract spindle images together with an analysis pipeline designed to assess spindle morphology across a range of experimental conditions. Our analysis of different spindle types illustrates how kinetochore microtubules amplify spindle microtubule density. Extract mixing experiments reveal that some spindle features titrate, while others undergo switch-like transitions, and multivariate analysis shows the pleiotropic morphological effects of modulating the levels of TPX2, a key spindle assembly factor. We also apply our pipeline to analyze nuclear morphology in human cell culture, showing the general utility of the segmentation approach. Our analyses provide new insight into the diversity of spindle types and suggest areas for future study. The approaches outlined can be applied by other researchers studying spindle morphology and adapted with minimal modification to other experimental systems.     

Microtubule-dependent assembly of the nuclear envelope in Xenopus laevis egg extract

Microtubules take part in several mechanisms of intracellular motility, including organelle transport and mitosis. We have studied the ability of Xenopus egg extract to support nuclear membrane and pore complex formation when microtubule dynamics are manipulated. In this report we show that the formation of a nuclear envelope surrounding sperm chromatin requires polymerized microtubules. We have observed that microtubule-depolymerizing reagents, and AS-2, a known inhibitor of the microtubule motor protein kinesin, do not inhibit the formation of a double nuclear membrane. However these double membranes contain no morphologically identifiable nuclear pore complexes and do not support the accumulation of karyophilic proteins. In contrast, the assembly of annulate lamellae, cytoplasmic structures containing a subset of pore complex proteins, was not affected. Our data show that not only polymerized microtubules, but also the microtubule motor protein kinesin, are involved in the formation of the nuclear envelope. These results support the conclusion that multiple nuclear envelope-forming mitotic vesicle populations exist, that microtubules play an essential and selective role in the transport of nuclear envelope-forming vesicle population(s), and that separate mechanisms are involved in nuclear envelope and annulate lamellae formation.     

Roles of polymerization dynamics, opposed motors, and a tensile element in governing the length of Xenopus extract meiotic spindles

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.     

The microtubule lattice and plus-end association of Drosophila Mini spindles is spatially regulated to fine-tune microtubule dynamics

Individual microtubules (MTs) exhibit dynamic instability, a behavior in which they cycle between phases of growth and shrinkage while the total amount of MT polymer remains constant. Dynamic instability is promoted by the conserved XMAP215/Dis1 family of microtubule-associated proteins (MAPs). In this study, we conducted an in vivo structure-function analysis of the Drosophila homologue Mini spindles (Msps). Msps exhibits EB1-dependent and spatially regulated MT localization, targeting to microtubule plus ends in the cell interior and decorating the lattice of growing and shrinking microtubules in the cell periphery. RNA interference rescue experiments revealed that the NH(2)-terminal four TOG domains of Msps function as paired units and were sufficient to promote microtubule dynamics and EB1 comet formation. We also identified TOG5 and novel inter-TOG linker motifs that are required for targeting Msps to the microtubule lattice. These novel microtubule contact sites are necessary for the interplay between the conserved TOG domains and inter-TOG MT binding that underlies the ability of Msps to promote MT dynamic instability.     

Microtubule organization during maturation of Xenopus oocytes: assembly and rotation of the meiotic spindles.

Assembly of the meiotic spindles during progesterone-induced maturation of Xenopus oocytes was examined by confocal fluorescence microscopy using anti-tubulin antibodies and by time-lapse confocal microscopy of living oocytes microinjected with fluorescent tubulin. Assembly of a transient microtubule array from a disk-shaped MTOC was observed soon after germinal vesicle breakdown. This MTOC-TMA complex rapidly migrated toward the animal pole, in association with the condensing meiotic chromosomes. Four common stages were observed during the assembly of both M1 and M2 spindles: (1) formation of a compact aggregate of microtubules and chromosomes; (2) reorganization of this aggregate resulting in formation of a short bipolar spindle; (3) an anaphase-B-like elongation of the prometaphase spindle, transversely oriented with respect to the oocyte A-V axis; and (4) rotation of the spindle into alignment with the oocyte axis. The rate of spindle elongation observed in M1 (0.7 microns min-1) was slower than that observed in M2 (1.8 microns min-1). Examination of spindles by immunofluorescence with antitubulin revealed numerous interdigitating microtubules, suggesting that prometaphase elongation of meiotic spindles in Xenopus oocytes results from active sliding of antiparallel microtubules. A substantial number of maturing oocytes formed monopolar microtubule asters during M1, nucleated by hollow spherical MTOCs. These monasters were subsequently observed to develop into bipolar M1 spindles and proceed through meiosis. The results presented define a complex pathway for assembly and rotation of the meiotic spindles during maturation of Xenopus oocytes.     

Microtubule dynamics

A combination of biochemical, structural, and morphological analyses during the last 2 decades has shown that the cytoplasm of a cell is not a disorganized mass of jelly but a highly structured cell compartment formed of a cytoskeleton, one of which principal components are the microtubules. More recently, studies have revealed that microtubule cytoskeleton is not only well organized but highly dynamic, and that microtubule dynamics may be responsible for several cell functions such as chromosome segregation, cell morphogenesis, or intracytoplasmic organization.     

Site-specific initiation of DNA replication in Xenopus egg extract requires nuclear structure.

Previous studies have shown that Xenopus egg extract can initiate DNA replication in purified DNA molecules once the DNA is organized into a pseudonucleus. DNA replication under these conditions is independent of DNA sequence and begins at many sites distributed randomly throughout the molecules. In contrast, DNA replication in the chromosomes of cultured animal cells initiates at specific, heritable sites. Here we show that Xenopus egg extract can initiate DNA replication at specific sites in mammalian chromosomes, but only when the DNA is presented in the form of an intact nucleus. Initiation of DNA synthesis in nuclei isolated from G1-phase Chinese hamster ovary cells was distinguished from continuation of DNA synthesis at preformed replication forks in S-phase nuclei by a delay that preceded DNA synthesis, a dependence on soluble Xenopus egg factors, sensitivity to a protein kinase inhibitor, and complete labeling of nascent DNA chains. Initiation sites for DNA replication were mapped downstream of the amplified dihydrofolate reductase gene region by hybridizing newly replicated DNA to unique probes and by hybridizing Okazaki fragments to the two individual strands of unique probes. When G1-phase nuclei were prepared by methods that preserved the integrity of the nuclear membrane, Xenopus egg extract initiated replication specifically at or near the origin of bidirectional replication utilized by hamster cells (dihydrofolate reductase ori-beta). However, when nuclei were prepared by methods that altered nuclear morphology and damaged the nuclear membrane, preference for initiation at ori-beta was significantly reduced or eliminated. Furthermore, site-specific initiation was not observed with bare DNA substrates, and Xenopus eggs or egg extracts replicated prokaryotic DNA or hamster DNA that did not contain a replication origin as efficiently as hamster DNA containing ori-beta. We conclude that initiation sites for DNA replication in mammalian cells are established prior to S phase by some component of nuclear structure and that these sites can be activated by soluble factors in Xenopus eggs.     

Microtubule dynamics.

Although compelling evidence has been obtained for heterogeneity in the structure of subunits in microtubules, it has not been possible to prove that this results from the presence of tubulin-GDP and tubulin-GTP in polymers. There are reasons to exclude the existence of even a monolayer of tubulin-GTP subunits at microtubule ends. Dynamic behavior appears to be best accounted for by a mechanism in which tubulin-GDP in microtubules exists in two conformations. The mechanism of microtubule-associated protein binding to microtubules and the role of phosphorylation on this reaction are discussed.     

Microtubule flux and sliding in mitotic spindles of Drosophila embryos

We proposed that spindle morphogenesis in Drosophila embryos involves progression through four transient isometric structures in which a constant spacing of the spindle poles is maintained by a balance of forces generated by multiple microtubule (MT) motors and that tipping this balance drives pole-pole separation. Here we used fluorescent speckle microscopy to evaluate the influence of MT dynamics on the isometric state that persists through metaphase and anaphase A and on pole-pole separation in anaphase B. During metaphase and anaphase A, fluorescent punctae on kinetochore and interpolar MTs flux toward the poles at 0.03 microm/s, too slow to drive chromatid-to-pole motion at 0.11 microm/s, and during anaphase B, fluorescent punctae on interpolar MTs move away from the spindle equator at the same rate as the poles, consistent with MT-MT sliding. Loss of Ncd, a candidate flux motor or brake, did not affect flux in the metaphase/anaphase A isometric state or MT sliding in anaphase B but decreased the duration of the isometric state. Our results suggest that, throughout this isometric state, an outward force exerted on the spindle poles by MT sliding motors is balanced by flux, and that suppression of flux could tip the balance of forces at the onset of anaphase B, allowing MT sliding and polymerization to push the poles apart.     

Microtubule dynamics in cultured cells

The behavior of microtubules in cultured cells in a cooled matrix after the microinjection of fluorescent tubulin was studied using a frame recording by a digital camcorder. In the cell lamella, thepositive ends of individual microtubules extend and shorten at random. The histograms of rate distribution have an almost normal distribution with a mode around 0. The maximum rate of lengthening and shortening reaches 30 and 50 microns/min, respectively. The positive ends of microtubules in PtK1 cells were in an equilibrium state, while in murine embryonic fibroblasts and Vero cells, they were displaced, usually, to the cell edge. Free microtubules were present in the cells of all three cultures. In the epithelial cells, they were numerous and relatively stable, while in the fibroblasts, they occurred rarely and were depolymerized at the proximal end. Free microtubules in PtK1 cells appeared, mostly due to spontaneous assembly in the cytoplasm, not in the relationship with the preexisting microtubules, and, more rarely, due to breakage of long microtubules. Separation of microtubules from the centrosome is a very rare event. Unlike positive ends that were characterized by dynamic instability, negative ends were stable and were sometimes depolymerized. When long microtubules were broken, new negative ends were formed that were, as a rule, stable, while in the lamella of fibroblasts (in murine embryonic fibroblasts and Vero cells), new negative ends were immediately depolymerized: free microtubules existed in these cells no more than 1-2 min. A diffusion model has been proposed where the behavior of microtubule ends is considered as unidimensional diffusion. The coefficient of diffusion of positive ends in the epithelial cells is several times less than in the fibroblasts, thus suggesting a higher rate of tubulin metabolism in the fibroblasts, as compared to the epithelium. The results obtained indicate that for the exchange of long microtubules, the dynamic instability is not sufficient. In the fibroblasts, their exchange takes place, mostly, at the expense of depolymerization of the liberating negative ends, which agrees with the previously proposed conveyer hypothesis of microtubule assembly on the centrosome.