Cell Motility by Labile Association of Molecules

Abstract

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 AF25.c < 1 kcal), and the equilibrium is readily shifted to dissociation by low temperature or by high hydrostatic pressure. The equilibrium is shifted toward formation of polymer by increase in temperature (with a large increase in entropy: AS~soc ~---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 259

Mechanisms of Mitotic Spindle Assembly

“…Two key characteristics define spindle MTs. First, early perturbation and fluorescence recovery after photobleaching studies revealed that spindle MTs exchange very quickly within tens of seconds with the soluble tubulin pool (61, 62). Second, speckle microscopy and photomarking experiments uncovered that spindle MTs continuously slide toward the poles, termed MT flux (6).…”

A Brief History of Research on Mitotic Mechanisms

“…Quantification of spindle birefringence under various physical and pharmacological conditions provided key insights into the reversible assembly of the mitotic spindle (reviewed in [135,136,137]). A sharp dependency of spindle birefringence on temperature suggested a low energy bonding between aligned subunits of the birefringent spindle fibers and provided compelling evidence for the self-assembly of fibers at physiological temperatures [135].…”

“…A sharp dependency of spindle birefringence on temperature suggested a low energy bonding between aligned subunits of the birefringent spindle fibers and provided compelling evidence for the self-assembly of fibers at physiological temperatures [135]. Inoue and Sato [135] proposed the dynamic equilibrium model of spindle assembly in which aligned subunits forming birefringent spindle fibers were in equilibrium with a pool of unaligned, non-birefringent subunits. Consistent with Inoue’s equilibrium model, Edwin Taylor [138,139] showed that the protein synthesis required for spindle assembly is completed well before the onset of prophase.…”

“…Inoue and Sato [135] described a model for both chromosome to pole and pole-from-pole motions based on a labile association of the subunits that formed spindle fibers. They posited a dynamic equilibrium between soluble and assembled states of spindle subunits, using the formulation: $A_0-B\rightleftharpoons B$ where A 0 represented the total amount of polymerizable material and B the amount in polymer at any given time and temperature.…”

Dark-field light scattering imaging of living cancer cell component from birth through division using bioconjugated gold nanoprobes

“…The mitosis of eukaryotic cells is a com- plex process involving chromosome condensation, nuclear envelope and lamina disassembly, spindle formation, and movement of varied nuclear proteins. [59][60][61] Although fluorescence live-cell imaging techniques facilitate the detection of chromosomes and various cellular proteins, the study of dynamical properties of centrosomes and kinetochores, and gain us insights on the molecular mechanisms of tubulin, 14-17 the disadvantages of photobleaching and phototoxicity limit the applications in long-term imaging. The light scattering imaging technique assisted by Au NP contrast agents could be a potential alternative for imaging the complicated process of mitosis in real time.…”