Patricia Harris

The role of membranes in the organization of the mitotic apparatus

Abstract The relationship between the microtubules and a massive system of vesicles associated with the sea urchin mitotic apparatus was examined by light and electron microscopy. Astral rays made up of elongated vesicles and associated tracts of microtubules continue to grow toward the cell surface during late anaphase and telophase at the same time the aster center appears to be breaking down in preparation for the next division. On the basis of current knowledge of the requirements for microtubule polymerization and the known presence of a calcium-dependent ATPase in the mitotic apparatus, it is proposed that the vesicle system functions to control the polymerization and depolymerization of microtubules by calcium ion regulation. A model for such a system is proposed.

A spiral array of microtubules in the fertilized sea urchin egg cortex examined by indirect immunofluorescence and electron microscopy

Abstract A transient spiral system of fibers in the cortex of fertilized eggs of the sea urchin Strongylocentrotus purpuratus was examined with indirect immunofluorescence microscopy and found to contain tubulin. Electron microscopy identified the tubulin-containing bands as bundles of up to 40 or more microtubules. These cortical microtubules, which are initially radial, form a spiral array about the time of pronuclear fusion. This basket-like structure, at a depth of 10–15 μm below the cell surface, reaches a peak of development about 45 min after fertilization and disappears before the streak stage at 70 min, in a division cycle of slightly more than 2 h. Possible functions of the cortical microtubules, which appear to be independent of the interphase asters, are discussed.

Cytology and immunocytochemistry.

Publisher Summary This chapter describes techniques that worked out primarily for use with Strongylocentrotus purpuratus and Lytechinus pictus . The chapter discusses whole mounts of fixed and embedded material, sectioned and stained material for bright-field light microscopy, and indirect immunofluorescence microscopy and other fluorochrome probes. Whole mounts are useful for establishing a developmental timetable, where paired samples are taken for other studies. They do not provide good resolution, however are adequate to establish the timing of such developmental markers as pronuclear fusion, streak stage, nuclear membrane breakdown, furrowing, and in later stages, cell number, cilia formation, and gastrulation. A more time-consuming method for monitoring stages of development, but one which provides considerably more accuracy, utilizes stained sections of epoxy-embedded material. The success of immunofluorescence microscopy will depend largely on the quality of the antibodies that are used—that is, the specificity and affinity of the antibody for the antigen, the titer of the elicited antibody, and nonspecific staining. The fluorochrome probes including bisbenzimide (hoechst 33258) stain for DNA, NBD-phallacidin and rhodamine-phalloidin stains for actin are discussed.

Intermediate filaments, microtubules and microfilaments in epidermis of sea urchin tube foot

SummaryTube foot epidermal cells of the sea urchin Strongylocentrotus purpuratus were examined by transmission electron microscopy and fluorescence microscopy to identify the chemical nature of prominent bundles of cytoplasmic filaments. Cross sections revealed filaments of roughly 7–8 nm in diameter closely packed into dense bundles. These bundles, in turn, were each surrounded by a loose sheath of microtubules. The filament size and negative reaction with the fluorescent F-actin binding drug NBD-phallacidin indicated that they were not actin. Indirect immunofluorescence microscopy of whole tissues and frozen sections revealed a strong reaction of the filaments with a monoclonal antibody prepared against porcine stomach desmin. In SDS-polyacrylamide gels of whole tube foot protein, a band of apparent molecular weight around 50 000 daltons reacted with the anti-desmin monoclonal antibody. The combined data provide evidence that the epidermal filament bundles are related to vertebrate intermediate filaments, but further biochemical studies will be necessary to assign them to a particular class of filament proteins.

A spiral cortical fiber system in fertilized sea urchin eggs.

Abstract Fiber systems of fertilized eggs of the sea urchin Strongylocentrotus purpuratus become aggregated and thus visible in phase-contrast light microscopy, when cells are fixed in 2% glutaraldehyde in 0.45 M Na-acetate buffer at pH 6.0 and embedded in epoxy. Studies of whole mounts and of 1-μm stained sections of the first-division cycle revealed a spiral array of subcortical fibers that apparently grow inward from the cell surface shortly after sperm entry and disappear prior to streak stage. They are independent of the microtubule system associated with the sperm aster, amphiaster, and mitotic apparatus. Their chemical identity is not known, but they may very likely be actin.

Caffeine-induced monaster cycling in fertilized eggs of the sea urchin Strongylocentrotus purpuratus☆

Caffeine has several highly visible effects on mitosis in eggs of the sea urchin Strongylocentrotus purpuratus. It increases the cell cycle length in a dose-dependent manner and at a concentration between 5 and 10 mM it prevents the normal separation and function of the mitotic centers, resulting in periodic monaster formation rather than division. If fertilized eggs are placed in seawater containing 10 mM caffeine at prometaphase, the spindle rapidly shrinks, moving the mitotic centers to the middle of the metaphase plate in less than 10 min. Within 30 min the eggs recover their ability to polymerize microtubules to form a large monaster, and from this time on they continue to cycle monasters in the presence of caffeine, with a period of about 150 min, where the normal second division time is 55 min. Other cell cycle functions, such as chromosome condensation, nuclear membrane breakdown and reformation, attempted furrowing and surface membrane addition continue for at least several cycles, but at the slower rate. Although the evidence strongly suggests that the rapid breakdown of the prometaphase spindle results from a calcium release induced by caffeine, these experiments do not rule out other physiological effects of caffeine on the longer-term changes in cycle length, centriole function, and ultimately on cell survival.

Structural effects of mercaptoethanol during mitotic block of sea urchin eggs.

Abstract 1. (1) The first visual effect of mercaptoethanol on dividing sea urchin eggs is a collapse of the asters and spindle into a central clear region containing many membranes and vesicles. 2. (2) Microtubules are present in blocked cells. Chromosomal microtubules and probably also aster microtubules shorten in the block, retaining the structure of a mitotic apparatus, but with decreasing spindle length and aster size. Radial orientation is retained around the aster centers. 3. (3) The ability of the centers to separate in the block increases as the blocking time approaches metaphase and appears related to the number of polymerized microtubules present at the time of the block. Centers separate with formation of vesicle and microtubule tracts between them. 4. (4) Mercaptoethanol apparently prevents the normal increase in the pool of available microtubule protein during mitosis, but does not prevent microtubule depolymerization or new polymerization from an existing pool, which may be derived in part from depolymerization of existing microtubules.