The role of membranes in the organization of the mitotic apparatus

Sea urchin embryos in second division have been lysed into microtubule-stabilizing buffers to yield mitotic cytoskeletons (MCSs) that consist of two mitotic spindles surrounded by a cortical array of filaments. Microtubules have been completely extracted from MCSs by incubation at 0C with Ca2l-containing buffer. An antibody to the microtubule translocator kinesin stains the spindles in MCSs and in MCSs treated with 5 mM ATP and also stains spindle-remnants of the MCSs after the microtubules have been extracted. We conclude that kinesin binds to a nonmicrotubule component in the mitotic spindle. Based on these results, we present several models of kinesin function in the spindle.Kinesin, a mechanochemical protein that mediates ATPdependent motility of microtubules (MTs) in vitro (1, 2) is a good candidate for the motor active in vesicle movement during fast axonal transport (3, 4, 32). Scholey et al. (5) have reported that kinesin localizes in mitotic spindles of sea urchin embryos as determined by staining with antibodies to sea urchin kinesin. This result raises the possibility that kinesin might be involved in some aspect of chromosome movement.Mitosis includes a complex series of movements, any one of which might be mediated by kinesin. For example, the movement of chromosomes away from the spindle poles during prometaphase (6) could result from kinesin bound to the kinetochore working on MT surfaces by a mechanism analogous to that used in bead transport along MTs in vitro (ref.Vale, J.R.M., and J.M.S., unpublished data). Kinesin might act in the overlap between half-spindles to generate shearing forces between adjacent MTs (7,8). Alternatively, kinesin may not act to move chromosomes at all but may move vesicles within the spindle (9-14).To better understand the role of kinesin in mitosis, we need to identify the spindle component(s) with which the protein interacts to generate motile force. For example, kinesin might cross-link MTs to vesicles or kinetochores to generate force for vesicle or chromosome translocation along MTs (6,15). It might cross-link MTs to MTs to generate force for MT-sliding in anaphase B, or it might cross-link MTs to a structural matrix to generate shear forces between MTs and the matrix.We therefore used antibodies to kinesin (5) to probe the location of this protein within spindles of sea urchin embryos. We found that spindles that have been depleted of MTs by extraction with Ca2+-containing buffer at 0°C contain no residual tubulin as determined by immunoblotting or immunocytochemistry. However, when these MT-depleted spindle remnants and the Ca2+ buffer extracts were examined with anti-kinesin antibodies by immunoblotting and immunocytochemistry, we found that kinesin localized to a non-MT component in the spindle and was not coextracted with tubulin. The possible significance of this observation for the role of kinesin in mitosis is discussed. MATERIALS AND METHODS Animals. Sea urchins Strongylocentrotusfranciscanus andLytechinus pictus were collected from the Santa Bar...

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Kinesin is associated with a nonmicrotubule component of sea urchin mitotic spindles.

Leslie¹,

Hird²,

Wilson³

et al. 1987

“…Permeabilized embryos (A) sequester Ca2" 27% as rapidly as homogenized ones (e), while those that are homogenized after permeabilization (n) are 84% as active as the control. (9,32,33), with some evidence (10,11,34,35), that the, system is concentrated in the very abundant membranes of the mitotic apparatus. The cycles of sequestering activity in our permeabilized cells could be influenced by the cthnging disposition of Ca-sequestering 'membranes during interphase and mitosis.…”

Section: Resultsmentioning

confidence: 99%

Fluctuation of the Ca ²⁺ -sequestering activity of permeabilized sea urchin embryos during the cell cycle

Suprynowicz

Mazia

1985

We have followed the sequestration of Ca2+ by intracellular compartments in sea urchin embryos through the first cell cycles. To gain biochemical access to these compartments, the embryos were permeabilized by brief exposure to an intense electric field. Sequestration was determined as the retention of tracer, 45Ca, after filtration of aliquots on Millipore filters. The permeabilized cells sequester Ca2+ at a constant rate for at least 20 min, with the following characteristics: (i) ATP is required. (it) Sequestration occurs at Ca2+ levels corresponding to those estimated in vivo.(ii) The Ca2+ concentration dependence of sequestration and its insensitivity to mitochondrial poisons imply that the activity derives from a single, nonmitochondrial transport system. The Ca2_-sequestering activities of embryos that are permeabiized at successive stages of the first cell cycle (one-cell stage) progressively increase to 5 times the initial level. The rate of sequestration is maximal during telophase and, in some populations of zygotes, is nearly as great throughout prophase. Over the course of the second cell cycle (two-cell stage), the activity undergoes a 2-fold oscillation that bears the same temporal relationship to mitosis as the previous fluctuation.The hypothesis that cytosolic Ca2+ levels undergo cyclical fluctuation during the cell cycle has been put forth to account for a variety of events. A local increase in Ca2+ may regulate the poleward migration of chromosomes via the disassembly of spindle microtubules (see refs. 1 and 2; reviewed in ref.3). There is evidence that the transition from metaphase to anaphase is triggered by changing concentrations of intracellular Ca2' (4-6). The inactivation of a protein responsible for arresting cells in meiotic metaphase has been attributed to a transient increase in cytosolic Ca2+ (7, 8).The abundant membranous components of the mitotic spindle (reviewed in ref. 9) are known to sequester Ca2t in vivo (10) and in vitro (11). In many cells, the relative activity of a Ca2+-activated ATPase, which appears to be associated with these membranes, differs during specific stages of the cell cycle (reviewed in ref. 12).We anticipate that direct measurement of Ca2+ levels ih living cells will, in the future, characterize or refute the proposed Ca2+ cycles. In the present work, we deal with oscillations in the activity of the Ca2'-sequestering system of fertilized sea urchin eggs as they proceed through several cleavage divisions. The intracellular transport of Ca2+ is measured in embryos that have been permeabilized at successive stages of the cell cycle by brief administration of an intense electric field. Such treatment causes localized dielectric breakdown of the plasma membrane (13-16) without damaging intracellular membranes (13,14). MATERIALS AND METHODSAll manipulations were done at 18'C, unless otherwise indicated.Handling of Gametes. Eggs and sperm were obtained from the sea urchin Lytechinus pictus, after intracoelomic injection of 0.5 M KCl. Sperm was collect...

“…The Ca2+-ATPase described in this paper is probably one of the main components of this system, Its solubility properties indicate that the enzyme is membrane bound. It is concentrated in the isolated mitotic apparatus (18) and most probably localized in the vesicles that have been described to exist in the spindle (26,27).-The en'zyme has functional similarities to the Ca2+-ATPase of the sarcoplasmic reticulum. It is inhibited in vitro by SH-oxidizing agents like p-chloromercuribenzoate (Petzelt, unpublished) and diamide (28).…”

Section: Discussionmentioning

confidence: 99%

Synthesis and activation of mitotic Ca2+-adenosinetriphosphatase during the cell cycle of mouse mastocytoma cells.

Petzelt¹,

Auel²

1977

The activity of a Ca2+-adenosinetriphosphatase (ATP phosphohydrolase, EC 3.6.1.3) increases during the mitotic phase of synchronized mouse mastocytoma P-815X2 cells. The enzyme is synthesized mainly during a distinct period of the interphase and is activated at mitosis. It is thought to regulate the formation of the mitotic apparatus by controlling the concentration of Ca2+ ions at the site of formation of the mitotic spindle.Ca2+ ions have been implicated as regulating factors in mitosis (1). They can interfere with microtubule assembly (2-4) and a system capable of controlling the concentration of these ions at the place of spindle formation has been tentatively identified (1,5).In sea urchins a Ca2+-adenosinetriphosphatase (ATPase; ATP phosphohydrolase, EC 3.6.1.3) exists which shows in early development cyclic fluctuations during the cell cycle; one peak of activity occurs in the first half of the cell cycle and another one at the time of mitosis (6, 7). Whereas the meaning of the first peak is as yet unclear, some evidence has accumulated that the activity increase of the Ca2+-ATPase at mitosis is involved in the regulation of mitosis. If the length of the cell cycle is altered experimentally, the increase of the enzymatic activity still remains linked with mitosis (7,8). In parthenogenetically activated eggs the activity of the enzyme rises whenever a spindle-like figure is formed (5). If only parts of the cell cycle are turned on, which in sea urchins can be performed by NH3 treatment of unfertilized eggs (9, 10), the Ca2+-ATPase cycle is also turned on, reaching its maximum of activity when the chromosomes show a mitosis-like configuration (11).These and the following results suggest that the enzyme is indeed involved in the regulation of mitosis. Therefore, we propose to name this enzyme the "mitotic Ca2+-ATPase."The experiments so far described were carried out on sea urchin eggs, in which cell divisions follow each other without cell growth during interphases. There, fluctuations of an enzyme activity are likely to represent the activation of pre-existing proteins rather than production of additional enzyme molecules. The same Ca2+-ATPase has also been found in growing cells (mouse fibroblasts) and a cyclic change of activity with a peak at mitosis has been observed (12). The purified enzyme promotes the polymerization of brain tubulin onto isolated mitotic apparatus from L cells (12). The interaction of tubulin from nonneuronal cells with isolated spindles has been reported to be enhanced in the presence of the enzyme (13). In the present work we measure the synthesis and the activity of the mitotic Ca2+-ATPase through the entire cell cycle in synchronized mouse mastocytoma cells. The synthesis of new enzyme proteins takes place during interphase. The peak of the enzymatic activity occurs during mitosis, as in sea urchin, eggs, and must reflect the activation of preexisting enzyme molecules. 1610MATERIAL AND METHODS Cells. Mouse mastocytoma P-815X2 cells (14) were grown in medium I supple...