Lionel I. Rebhun

Polarized Intracellular Particle Transport: Saltatory Movements and Cytoplasmic Streaming

Publisher Summary This chapter discusses the phenomenon of saltatory movement in relation to Brownian movement. It demonstrates that except for the possibility that channels of low viscosity exist in cells, it is not likely that the motive force giving rise to the movements originates from molecular kinetic processes. The most likely types of models that can account for the details of saltatory movement are those in which the motive force arises from some process outside the particle itself, which is passively moved, or one in which movement arises by an interaction of the particle with some external structure. Such an interaction may involve an ATPase mechanism, or may be electrical in nature. The chapter explores the relation of secretory processes to the particle movements, and discusses the control mechanisms involved in the process. The chapter also highlights that a detailed analysis of movement of granules in Heliozoa indicates that microtubules are necessary for the organized streaming of particles along the axopods and in the interior of the organism. It is necessary, however, to look for saltations of short path length which may escape notice after study of the extended movements of particles in intact organisms.

Cyclic nucleotides, calcium, and cell division.

Publisher Summary This chapter discusses the nature of the intracellular compounds resulting from external application of cyclic 3’,5’-adenosine monophosphate (cAMP). The chapter discusses the importance of cAMP in cell division. HTC hepatoma cells have been shown to be devoid of cAMP when grown in tissue culture situations. Because they grow and divide well, it is clear that cAMP cannot be necessary for any fundamental aspect of the physiology of cell division in cycling cells. In addition, dibutyryl cAMP (DBcAMP) added to cultures of HTC hepatoma cells (or cells of the normal rat liver line RLC1) do not inhibit growth, although in the hepatomas H35 and MH1C1 inhibition is observed. The chapter also discusses the average values of cAMP in cells as a function of environment and culture state. The cAMP levels vary depending on whether the cells show density-dependent inhibition (DDI) or growth under ordinary culture conditions (high cAMP) or whether they form a multilayer (low cAMP). Viral transformation and increased saturation density are generally associated with a decrease in cAMP levels usually correlated therefore with increased growth potential.

Cleavage inhibition in marine eggs by puromycin and 6-dimethylaminopurine

Abstract The possibility is discussed that puromycin may not inhibit cleavage in marine eggs solely by inhibiting protein synthesis. It is suggested that part of the effect of puromycin is through 6-dimethylaminopurine (DMAP) (the purine component of puromycin) a potent cleavage inhibitor which appears to enhance protein synthesis in sea urchin eggs.

Acid release following activation of surf clam (Spisula solidissima) eggs

Abstract Acid release was observed after activation of Spisula eggs with excess KCI. This acid release begins within 20 sec after the activation and continues for 9–15 min. The amount of acid released was 6.8 μmole per milliliter of packed eggs. In Ca-free or Na-free sea water, the acid release is completely inhibited; subsequent addition of the deficient ion leads to acid release and breakdown of germinal vesicles. These results suggest that Spisula eggs release protons after activation in a manner similar to that of sea urchin eggs, and that acid release with concomitant increase in cytoplasmic pH is probably a general event on activation of marine eggs.

Studies on cyclic AMP levels and phosphodiesterase activity in developing sea urchin eggs: Effects of puromycin, 6-dimethylamino purine and aminophylline

Cyclic adenosine monophosphate (CAMP) was measured in sea urchin eggs by the binding assay method of Gilman and with a radioimmune assay procedure. Intracellular concentrations of the nucleotide in unfertilized eggs were about 1.5 × 10−7 M and rose to about 3 times this value at first cleavage. Aminophylline, a known inhibitor of phosphodiesterase was shown to cause an increase in intracellular levels of CAMP by first cleavage and to inhibit phosphodiesterase activity in homogenates of both unfertilized and fertilized eggs. Puromycin and its purine component, 6-dimethylaminopurine (DMAP), did not cause an increase in intracellular CAMP levels and did not inhibit phosphodiesterase activity at concentrations an order of magnitude higher than those at which they inhibit cell division.

The binding of MAP-2 and tau on brain microtubules in vitro: implications for microtubule structure.

We have presented data that indicate that MAP-2 associates with brain microtubules at nonrandomly distributed sites, whose distribution on the microtubule polymer can best be described by the 12-dimer MAP superlattice originally described by Amos; because of the additional spacings, however, between MAP-2 projections observed on MAP-2-saturated microtubules, we suggest that the 6-dimer MAP superlattice, or what we will call the double Amos superlattice, more completely specifies the total set of MAP-binding sites on cytoplasmic microtubules. Second, we have shown that brain microtubules reassembled in vitro contain a heterogeneous population of MAP-binding sites, which differ in their affinities for the two MAPs, MAP-2 and tau. Third, we have shown that microtubule populations that differ in their MAP content have subtle, but detectable differences in their tubulin isotype composition. Based on all the data presented here, we have presented the idea of a nonrandom distribution of tubulin isotypes within a microtubule as a means by which a cell could specify both the identity and the distribution of MAP-binding sites.

REGULATION OF THE IN VIVO MITOTIC APPARATUS BY GLYCOLS AND METABOLIC INHIBITORS

In order to partition the chromosomes properly after DNA replication, most eukaryotic cells construct a transient organelle, the mitotic apparatus (MA), which both orients and separates the chromosomes. Microtubules are major structural components of the MA, which give rise to most of its birefringence,’, and it is therefore of considerable interest to understand the state of this material in the MA. Thus it is possible that the microtubule forms, and once assembled remains intact until it is no longer needed, whereupon it is disassembled. On the other hand, it may exist in a dynamic state in which its building blocks continually cycle through the MA, and disassembly of the MA results from inhibition of the assembly of microtubules, rather than from initiation of some special disassembly process. This dynamic state need not be coupled to any specific mechanism by which chromosomes are separated, and so a demonstration of its occurrence in the cell would not necessarily allow us to distinguish between sliding models,R assembly-disassembly models;’ or “zipper” model^,^ although its compatibility with assembly-disassembly models is clear. Nevertheless, its demonstration would restrict the kinds of three-dimensional models one could construct for an MA functional in vivo. If assemblydisassembly models for chromosome separation do indeed survive further experimental attacks, the relevance of subunit cycling through the MA is clear. If some other theory more tied to the concept of fixed microtubules (such as a sliding theory) survives, then the importance and function of cycling is not obvious although speculative suggestions are not difficult to conceive. In this paper we will describe the effects of certain mitotic inhibitors, which are most simply interpreted in terms of the cycling of subunits through the MA. The details of the morphological effects of the inhibitors and the nature of the inhibitors will allow some suggestions on which physiological regulators may and which may not be involved in the control of assembly and disassembly of the MA in vivo.

Disassembly of the nuclear envelope of spisula oocytes in a cell-free system

Nuclei isolated from oocytes of the surf clam Spisula solidissima are disassembled when exposed to extracts from maturing oocytes. In the course of this process the nuclear lamina undergoes a marked reduction in size and the nuclear membrane appears to be fragmented into vesicles. These events are accompanied by extensive phosphorylation of the oocyte 67-kDa lamin and its solubilization. The changes observed are similar to those which occur in vivo in activated Spisula oocytes. Nuclear envelope breakdown in vitro requires ATP and Mg2+, but not Ca2+. It is not affected by protease inhibitors and is inhibited by alkaline phosphatase.

Studies on the uptake and metabolism of adenosine 3':5'-cyclic monophosphate and N6,O2-dibutyryl 3':5'-cyclic adenosine monophosphate in sea urchin eggs.

Abstract Application of adenosine 3′: 5′-cyclic monophosphate (CAMP), N 6 , O 2′ -dibutyryl cyclic AMP (DBCAMP) or N 6 -monobutyryl cyclic AMP (N6-MBCAMP), from concentrations of 10 −7 to 10 −2 M had no effect on fertilization or cleavage rates in sea urchin eggs up to early gastrula stages. In order to study the permeability of CAMP and DBCAMP in sea urchin eggs, they were incubated with the nucleotides. CAMP was readily taken up by the eggs. There was no significant breakdown of the nucleotide in the extracellular medium, indicating that CAMP entered the cell as such and was not resynthesized from a metabolic breakdown product. When eggs were incubated with DBCAMP, no dibutyryl derivative could be recovered from egg-extracts, the compound having been converted to N 6 MBCAMP. Homogenates of the eggs similarly were able to deacylate DBCAMP, indicating the presence of a potent esterase activity. The intracellular levels of CAMP and N 6 -MBCAMP reached concentrations greater than 10 −5 M, at least two orders of magnitude higher than the CAMP level normally found in sea urchin eggs. These results indicate that CAMP does not participate in regulation of mitosis in early embryogenesis in sea urchin eggs.

Concanavalin A-stimulated Ca2+ uptake in rat splenocytes.

SummaryCommercially available concanavalin A binds Ca2+ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca2+ uptake (defined as an increased association of 45Ca2+ with cells) in rat splenocytes and Ca2+ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in 45Ca2+ uptake over control. The concanavalin A stimulated uptake of 45Ca2+ occurred within minutes, and required concentrations of concanavalin A which promoted [3H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca2+ uptake than concanavalin A. Sodium periodate inhibited Ca2+ uptake at concentrations which promoted 3H-thymidine incorporation into splenocytes.It is concluded that con canavalin A promotes Ca2+ uptake which is not due to binding of 45Ca2+ to concanavalin A. Although the concanavalin A-promoted Ca2+ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by 3H-thymidine incorporation, the role of Ca2+ in this event remains unclear.