Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest–deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.

/pdf/probing-spindle-assembly-mechanisms-with-monastrol-a-small-7mau3egu9o.pdf

Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.

Analysis of the kinesin superfamily: insights into structure and function.

During cell division, mitotic spindles are assembled by microtubule-based motor proteins. The bipolar organization of spindles is essential for proper segregation of chromosomes, and requires plus-end-directed homotetrameric motor proteins of the widely conserved kinesin-5 (BimC) family. Hypotheses for bipolar spindle formation include the 'push-pull mitotic muscle' model, in which kinesin-5 and opposing motor proteins act between overlapping microtubules. However, the precise roles of kinesin-5 during this process are unknown. Here we show that the vertebrate kinesin-5 Eg5 drives the sliding of microtubules depending on their relative orientation. We found in controlled in vitro assays that Eg5 has the remarkable capability of simultaneously moving at approximately 20 nm s(-1) towards the plus-ends of each of the two microtubules it crosslinks. For anti-parallel microtubules, this results in relative sliding at approximately 40 nm s(-1), comparable to spindle pole separation rates in vivo. Furthermore, we found that Eg5 can tether microtubule plus-ends, suggesting an additional microtubule-binding mode for Eg5. Our results demonstrate how members of the kinesin-5 family are likely to function in mitosis, pushing apart interpolar microtubules as well as recruiting microtubules into bundles that are subsequently polarized by relative sliding.

The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks

Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding only exceedingly small compressive forces. This discrepancy calls into question the structural role of microtubules, and highlights our lack of quantitative knowledge of the magnitude of the forces they experience and can withstand in living cells. We show that intracellular microtubules do bear large-scale compressive loads from a variety of physiological forces, but their buckling wavelength is reduced significantly because of mechanical coupling to the surrounding elastic cytoskeleton. We quantitatively explain this behavior, and show that this coupling dramatically increases the compressive forces that microtubules can sustain, suggesting they can make a more significant structural contribution to the mechanical behavior of the cell than previously thought possible.

/pdf/microtubules-can-bear-enhanced-compressive-loads-in-living-11gktjfmzy.pdf

Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement

In eukaryotic cells, microtubules and their associated motor proteins can be organized into various large-scale patterns. Using a simplified experimental system combined with computer simulations, we examined how the concentrations and kinetic parameters of the motors contribute to their collective behavior. We observed self-organization of generic steady-state structures such as asters, vortices, and a network of interconnected poles. We identified parameter combinations that determine the generation of each of these structures. In general, this approach may become useful for correlating the morphogenetic phenomena taking place in a biological system with the biophysical characteristics of its constituents.

https://science.sciencemag.org/content/sci/292/5519/1167.full.pdf

Physical Properties Determining Self-Organization of Motors and Microtubules

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bi- polar kinesin, KLP61F, cross-links and slides microtu- bules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluo- rescence and immunoelectron microscopy (immu- noEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These inter- polar MT bundles are parallel near the poles and anti- parallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphory- lated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of orga- nizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle mid- zone. Thus we propose that bipolar kinesin motors and MTs interact by a "sliding filament mechanism" during the formation and function of the mitotic spindle.

/pdf/the-bipolar-kinesin-klp61f-cross-links-microtubules-within-4lz0tmhxce.pdf

The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles

We have investigated the intracellular roles of an Xklp2-related kinesin motor, KRP180, in positioning spindle poles during early sea urchin embryonic cell division using quantitative, real-time analysis. Immunolocalization reveals that KRP180 concentrates on microtubules in the central spindle, but is absent from centrosomes. Microinjection of inhibitory antibodies and dominant negative constructs suggest that KRP180 is not required for the initial separation of spindle poles, but instead functions to transiently position spindle poles specifically during prometaphase.

/pdf/a-kinesin-related-protein-krp-180-positions-prometaphase-2uu4rt6oq2.pdf

A kinesin-related protein, KRP(180), positions prometaphase spindle poles during early sea urchin embryonic cell division

High-pressure freezing (HPF) followed by freeze-substitution is a valuable method for specimen preservation for transmission electron microscopy (TEM) in Drosophila. However, not all projects require this level of precision. In addition, some tissues are too large to fit into the HPF specimen carriers, and some fly tissues such as eyes and ovaries do not freeze well. This protocol describes a trialdehyde fixation procedure for embryos, to be used in situations where optimal preservation is not required or when HPF is not an option. Because the vitelline membrane is impermeable to aqueous solvents, it is necessary to either mechanically disrupt it or render it permeable by treatment with organic solvents. Good ultrastructural preservation has been achieved by puncturing embryos immersed in fixative with extremely sharp tungsten needles, as described here.

Transmission electron microscopy of thin sections of Drosophila: conventional chemical fixation of embryos using trialdehyde.

The state of the art in fine-structure preservation for thin sectioning can be achieved by using fast-freezing technology followed by freeze substitution and embedding in resin. Samples prepared by high-pressure freezing are estimated to be "fixed" in 20-50 msec. Fast freezing also freezes every cell component regardless of its chemistry. Once frozen, tissues can be processed in a variety of ways before viewing in the electron microscope; here we describe only freeze substitution. In freeze substitution, cells are dehydrated at very low temperatures and cell water is replaced with organic solvent at -80°C to -90°C. At this temperature, large molecules such as proteins are immobilized, yet smaller molecules such as water (ice) can be dissolved and replaced with organic solvents, e.g., acetone. The ideal way to do freeze substitution is with a dedicated freeze-substitution device such as the Leica AFS2 system. These devices allow programming of the times and temperatures needed. Alternatively, if this equipment is not available, freeze substitution can still be performed using items commonly found around the laboratory, as is described here. This protocol is useful for preparing thin sections of Drosophila when the best possible preservation of ultrastructure and antigenicity is required.

Transmission Electron Microscopy of Thin Sections of Drosophila: High-Pressure Freezing and Freeze-Substitution

There is no single, simple procedure for fixing and embedding all tissues for transmission electron microscopy (TEM). The chemistry of different cell types is to some extent unique, and this affects the way each cell type reacts to the wide array of fixatives, buffers, organic solvents, and resins used in TEM specimen preparation. A recurring theme in those organisms or cell types that are difficult to fix is the presence of a diffusion barrier that prevents the free diffusion of fixative and other chemicals in and out of the cell or tissue. This in turn means that fixation takes a relatively long time (measured in minutes or tens of minutes in some cases), during which the cells begin autolysis or are otherwise degraded from their original state. Drosophila requires specific preparation methods for TEM because most fly tissues are surrounded by significant diffusion barriers. In the embryo, it is the vitelline envelope, and in larvae and adults, it is the cuticle. In this article, we discuss methods that have evolved to cope with these barriers to achieve reasonable preservation of ultrastructure.

Preparation of Drosophila Specimens for Examination by Transmission Electron Microscopy

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