Mitotic fibers

In addition to effects on biochemical reactions, the inhibitors may influence the permeability of the various cellular membranes and through physical and chemical effects may alter the structure of other subcellular structures such as proteins, nucleic acid, and spindle fibers. Unfortunately, few definite examples can be listed. The action of colchicine and podophyllin in interfering with cell division is well known. The effect of various lactones (coumarin, parasorbic acid, and protoanemonin) on mitotic activity was discussed above. Disturbances to cytoplasmic and vacuolar structure, and the morphology of mitochondria imposed by protoanemonin, were also mentioned. Interference with protein configuration and loss of biological activity was attributed to incorporation of azetidine-2-carboxylic acid into mung bean protein in place of proline. 

It is most unlikely that the sole functions of mysoin-Il in nonmuscle cells are to provide the contractile force to bisect cells during cytokinesis and for the contractility of stress fibers. Myosin-II is present in a variety of cell types at moderate concentrations in tissues such as brain, which are almost totally non-mitotic and do... 

The chromosome structure is visible only during the mitotic portion of the cell cycle. The constituent parts of the chromosomes are nucleoprotein fibers called chromatin. When condensed, chromatin forms a microscop-ically visible chromosome-like structure. The chromosomes are composed of DNA, RNA, and proteins. The relative amounts of the three vary, but chromatin is primarily protein and DNA. 

This theory clearly predicts that the shape of the polymer length distribution curve determines the shape of the time course of depolymerization. For example Kristofferson et al. (1980) were able to show that apparent first-order depolymerization kinetics arise from length distributions which are nearly exponential. It should also be noted that the above theory helps one to gain a better feeling for the time course of cytoskeleton or mitotic apparatus disassembly upon cooling cells to temperatures which destabilize microtubules and effect unidirectional depolymerization. Likewise, the linear depolymerization kinetic model could be applied to the disassembly of bacterial flagella, muscle and nonmuscle F-actin, tobacco mosaic virus, hemoglobin S fibers, and other linear polymers to elucidate important rate parameters and to test the sufficiency of the end-wise depolymerization assumption in such cases. 

The elastic stress may be external or internal. External stresses are exerted on the chromatin during the cell cycle when the mitotic spindle separates chromosome pairs. The 30-nm fiber should be both highly flexible and extensible to survive these stresses. The in vitro experiments by Cui and Bustamante demonstrated that the 30-nm fiber is indeed very soft [66]. The 30-nm fiber is also exposed to internal stresses. Attractive or repulsive forces between the nucleosomes will deform the linkers connecting the nucleosomes. For instance, electrostatic interactions, either repulsive (due to the net charge of the nucleosome core particles) or attractive (bridging via the lysine-rich core histone tails [49]) could lead to considerable structural rearrangements. 

Colchicine is a poisonous tricyclic tropane alkaloid from the autumn crocus (Colchicum autumnale) and gloriosa lily (Gloriosa superba). This alkaloid is a potent spindle fiber poison, preventing tubulin polymerization.25 Colchicine has been used as an effective anti-inflammatory drug in the treatment of gout and chronic myelocytic leukemia, but therapeutic effects are attainable at toxic or near toxic dosages. For this reason, colchicine and its analogs are primarily used as biochemical tools in the mechanistic study of new mitotic inhibitors. 

Micronucleus Test The micronucleus test is an in vivo test usually carried out in mice. The animals are treated in vivo, and the erythrocyte stem cells from the bone marrow are stained and examined for micronuclei. Micronuclei represent chromosome fragments or chromosomes left behind at anaphase. It is basically a test for compounds that cause chromosome breaks (clastogenic agents) and compounds that interfere with normal mitotic cell division, including compounds that affect spindle fiber function. 

Figure 13.15. Inhibition of mitosis by vincristine, a The mitotic spindle consists of tubulin fibers, which are helical polymers of a,P tubulin dimers, b Structure of vincristine, an inhibitor of tubulin polymerization, c Mode of action of vincristine. The drug binds to an a,P dimer. These dimers retain the ability to associate with growing filaments however, the further attachment of dimers is inhibited. At high concentrations, vincristin-associated tubulin is also sequestrated within mis-assembled polymers.

Dopp E, Saedler J, Stopper H, et al. 1995b. Mitotic disturbances and micronucleus induction in Syrian hamster embryo fibroblast cells caused by asbestos fibers. Environ Health Perspect 103 268-271. 

Inone S. Polarization optical stndies of the mitotic spindle. I. The demonstration of spindle fibers in living cells. Chromosoma 1953 5 487-500. 

Inone S, Sato H. CeU motility by labile association of molecnles. The natnre of mitotic spindle fibers and their role in chromosome movement. J. Gen. Physiol. 1967 50 259-292. 

Figure 1 Schematic drawing of how chromosomes are formed from nucleosomes to the mitotic chromosome. Double-stranded DNA (black lines) wraps around core histones (gray cylinders) to form the nucleosome. Histone tails (curved lines) protrude from the histone core and can undergo various posttranslational modifications that regulate chromatin state. Helical arrays of nucleosomes form the 30-nm fiber. Scaffolding proteins (gray bars), such as condensin, further compact chromatin during mitosis to promote proper segregation to daughter cells.

In higher eukaryotes, a complex protein structure called the kinetochore assembles at centromeres and associates with multiple mitotic spindle fibers during mitosis. Homologs of most of the centromeric proteins found in the yeasts occur in humans and other higher eukaryotes and are thought to be components of kinetochores. The role of the centromere and proteins that bind to it in the segregation of sister chromatids during mitosis is described in Chapters 20 and 21. 

Examination of fixed samples revealed the existence of a fibrous structure, known as the mitotic spindle, which appears at each mitosis and disappears after the chromosomes have separated. One of the great challenges in the study of cell division has been to understand the organization and function of the mitotic spindle. Use of the small molecule colchicine (Fig. 2.14(b)) has contributed to our understanding of the physical properties of the spindle fibers and how they might drive chromosome movements, as well as their molecular components. 

Several issues needed to be addressed to devise a strategy to address the question of how attachment errors were corrected. First, kinase inhibition should be temporally controlled to experimentally isolate the error correction process, as Aurora kinases have been implicated in multiple mitotic processes. Second, error correction likely involves some regulation of the dynamics of the microtubule fibers that attach chromosomes to the spindle. These dynamics can be analyzed with high temporal and spatial resolution by high-resolution microscopy in living cells. Finally, the dynamics of individual microtubule fibers are difficult to analyze if that fiber is obscured by other microtubules in the spindle. The dynamics can be clearly observed, however, under conditions in which the improperly attached chromosomes are positioned away from the spindle body. 

A class of motile systems completely different from and unrelated to the actin-myosin contractile systems is used in cellular structures as diverse as the mitotic spindle, protozoan and sperm flagella, and nerve axons. These systems are constructed from microtubules, very long, tubular structures built from a helical wrapping of the protein tubulin (Figure 8.19). There are two kinds of tubulin subunits, oi and each of molecular weight 55,000. They are present in equimolar quantities in the microtubule, which can be considered a helical array of ot-/i dimers. Alternatively, we can view the microtubule as consisting of 13 rows, or protofilaments, of alternating ot and subunits. Because the oi and b units are asymmetrical proteins, with a defined and reproducible orientation in the fiber, the microtubule has a definite sense of direction. 

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