The polymerization dynamics of microtubules are central to their biological functions. Polymerization dynamics allow microtubules to adopt spatial arrangements that can change rapidly in response to cellular needs and, in some cases, to perform mechanical work. Microtubules utilize the energy of GTP hydrolysis to fuel a unique polymerization mechanism termed dynamic instability. In this review, we first describe progress toward understanding the mechanism of dynamic instability of pure tubulin and then discuss the function and regulation of microtubule dynamic instability in living cells.

/pdf/microtubule-polymerization-dynamics-27938flntt.pdf

Microtubule polymerization dynamics

Taxol, a potent inhibitor of human HeLa and mouse fibroblast cell replication, blocked cells in the G2 and M phase of the cell cycle and stabilized cytoplasmic microtubules. The cytoplasmic microtubules of taxol-treated cells were visualized by transmission electron microscopy and indirect immunofluorescence microscopy. More than 90% of the cells treated with 10 micro M taxol for 22 hr at 37 degrees C displayed bundles of microtubules that appeared to radiate from a common site (or sites), in addition to their cytoplasmic microtubules. Untreated cells that were kept in the cold (4 degrees C) for 16 hr lost their microtubules, whereas cells that were pretreated with taxol for 22 hr at 37 degrees C continued to display their microtubules and bundles of microtubules in the cold. Taxol inhibited the migration behavior of fibroblast cells, but these cells did not lose their ability to produce mobile surface projections such as lamellipodia and filopodia.

https://www.pnas.org/content/pnas/77/3/1561.full.pdf

Taxol stabilizes microtubules in mouse fibroblast cells.

Heavy meromyosin (HMM) forms characteristic arrowhead complexes with actin filaments in situ. These complexes are readily visualized in sectioned muscle. Following HMM treatment similar complexes appear in sectioned fibroblasts, chondrogenic cells, nerve cells, and several types of epithelial cells. Thin filaments freshly isolated from chondrogenic cells also bind HMM and form arrowhead structures in negatively stained preparations. HMM-filament complexes are prominent in the cortex of a variety of normal metaphase and Colcemid-arrested metaphase cells. There is no detectable binding of HMM with other cellular components such as microtubules, 100-A filaments, tonofilaments, membranes, nuclei, or collagen fibrils. The significance of HMM-filament binding is discussed in view of the finding that arrowhead complexes form in types of cells not usually thought to contain actin filaments.

https://rupress.org/jcb/article-pdf/43/2/312/1069195/312.pdf

Formation of arrowhead complexes with heavy meromyosin in a variety of cell types.

Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules.

This article summarizes our current views on the dynamic structure of the mitotic spindle and its relation to mitotic chromosome movements. The following statements are based on measurements of birefringence of spindle fibers in living cells, normally developing or experimentally modified by various physical and chemical agents, including high and low temperatures, antimitotic drugs, heavy water, and ultraviolet microbeam irradiation. Data were also obtained concomitantly with electron microscopy employing a new fixative and through measurements of isolated spindle protein. Spindle fibers in living cells are labile dynamic structures whose constituent filaments (microtubules) undergo cyclic breakdown and reformation. The dynamic state is maintained by an equilibrium between a pool of protein molecules and their linearly aggregated polymers, which constitute the microtubules or filaments. In living cells under physiological conditions, the association of the molecules into polymers is very weak (absolute value of Δ F 25°C S 25°C ≃ 100 eu) or by the addition of heavy water. The spindle proteins tend to polymerize with orienting centers as their geometrical foci. The centrioles, kinetochores, and cell plate act as orienting centers successively during mitosis. Filaments are more concentrated adjacent to an orienting center and yield higher birefringence. Astral rays, continuous fibers, chromosomal fibers, and phragmoplast fibers are thus formed by successive reorganization of the same protein molecules. During late prophase and metaphase, polymerization takes place predominantly at the kinetochores; in metaphase and anaphase, depolymerization is prevalent near the spindle poles. When the concentration of spindle protein is high, fusiform bundles of polymer are precipitated out even in the absence of obvious orienting centers. The shift of equilibrium from free protein molecules to polymer increases the length and number of the spindle microtubules or filaments. Slow depolymerization of the polymers, which can be brought about by low concentrations of colchicine or by gradual cooling, allows the filaments to shorten and perform work. The dynamic equilibrium controlled by orienting centers and other factors provides a plasusible mechanism by which chromosomes and other organelles, as well as the cell surface, are deformed or moved by temporarily organized arrays of microtubules or filaments.

https://rupress.org/jgp/article-pdf/50/6/259/1244119/259.pdf

Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement.

Using polarized light we have studied the effects of various mitotic poisons on mitotic spindles of living Pectinaria oocytes; we have studied fixed specimens with phase and electron microscopy. Vinblastine caused attrition and eventual disappearance of spindle structure as rapidly as did colcemid, and subsequent recovery from this treatment was at least as fast as that from colcemid. Griseofulvin, however, was easily the best agent for rapid, reversible, and repeated dissolution of the spindle. Agents that arrest metaphase may act on nondividing cells by interfering with the organization of other gelated structures.

https://science.sciencemag.org/content/sci/160/3829/770.full.pdf

Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle.

Meiotic spindle and chromosome movements were studied during spermatocyte divisions in Chrysochraon japonicus and Trilophidia annulata with a sensitive polarizing microscope. When the nuclear envelope disintegrated, birefringent astral fibers invaded the karyoplasm pushing some chromosomes toward the mid-region of the developing spindle. During prometaphase the birefringence (BR) of the kinetochore fibers fluctuated, and fibers at this period often exhibited lateral oscillations. By full metaphase, the kinetochore fibers BR became stable. BR of the continuous fibers was rather weak throughout meiosis. During anaphase in most cells, the kinetochore fibers shortened at a constant rate of 0.7 μm per minute at 28°C. In some cells the rate of shortening slowed down in late anaphase. The overall spindle length increased during anaphase, although considerable fluctuations were observed. The number of microtubules per kinetochore fiber estimated from measured BR and fiber thickness using the Wiener equation was consistent with the direct count of microtubules on electron micrographs.

/pdf/the-behavior-of-spindle-fibers-and-movement-of-chromosomes-ospynljvdt.pdf

Hidemi Sato

Papers

Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement.

Vinblastine and griseofulvin reversibly disrupt the living mitotic spindle.

The Behavior of Spindle Fibers and Movement of Chromosomes in Dividing Grasshopper Spermatocytes