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PubMed1988

Subcellular compartmentalization by local differentiation of cytoplasmic structure.

The compartmentalization of eukaryotic cells by internal membranes and the subcellular localization of endogenous macromolecules by specific binding mechanisms are familiar concepts. In this report we present evidence that the cytoplasmic ground substance, which surrounds and contains the membrane-bound compartments, may also be compartmentalized by local differentiations of its submicroscopic structure that sort subcellular particles on the basis of size. The subcellular distribution of size-fractionated, fluorescent tracer particles was studied in living cells by ratio imaging and fluorescence recovery after photobleaching (FRAP). Large and small particles showed different distributions within the cytoplasmic volume, suggesting that the large particles were relatively excluded from some domains. While the structural basis for this phenomenon is not yet understood in detail, ratio imaging of large and small particles can be used as an empirical tool to identify cytoplasmic compartments for further study. The cytoplasmic diffusion coefficient (Dcyto) and % mobile fraction of the large particles showed considerable spatial variation over the projected area of the cell, while Dcyto and % mobile fraction of the small particles did not. A model is presented to account for this difference. Based on this model, a method is proposed by which FRAP can be used to detect sol-gel transitions in the cytoplasmic ground substance of living cells.

Animals↗
PubMed1996

Localization of the Us protein kinase of equine herpesvirus type 1 is affected by the cytoplasmic structures formed by the noval IR6 protein.

Previous work revealed that the Us (unique short) segment of equine herpesvirus type-1 (EHV-1), like that of other alphaherpesviruses, encodes a serine/threonine protein kinase (PK). Experiments were carried out to identify the PK encoded by the EHV-1 EUS2 gene (ORF 69) and to ascertain its time course of synthesis and cellular localization. Western blot and immunoprecipitation analyses of EHV-1-infected cell extracts using a PK-specific polyclonal antibody generated against a bacterially expressed TrpE/PK fusion protein identified the Us PK as a 42- to 45-kDa phosphoprotein. The PK protein is first synthesized at 3 hr postinfection, is produced throughout the infection cycle, and is incorporated into EHV-1 virions. Interestingly, immunoprecipitation analyses revealed that the PK protein within the cytoplasm is associated with the 33-kDa IR6 novel protein of EHV-1, is expressed abundantly as an early protein, and is present in the large rod-like structures formed by the IR6 protein (ORF67 protein) within the cytoplasm of infected cells. Confocal microscopic examination of cells stained with fluorescein-labeled antibody clearly showed that the PK protein colocalized with the cytoplasmic IR6 rod-like structures and remained associated with these unique structures during infection. In contrast, in cells infected with the EHV-1 RacM strain in which the IR6 protein harbors four amino acid substitutions that prevent formation of the rod-like structures (Osterrieder et al., 1996, Virology 217, 442-451), the PK protein localized predominantly to the nucleus. The possible significance of the association of the IR6 and PK proteins in EHV-1 replication is discussed.

Animals↗
PubMed1983

Poly(A) RNA of the egg cytoplasm: structural resemblance to the nuclear RNA of somatic cells.

This paper concerns the structural characteristics of the poly(A) RNA stored in unfertilized amphibian and echinoderm eggs. Though located in the egg cytoplasm, at least two-thirds of these maternal transcripts display an interspersed sequence organization similar to that of nuclear RNA. In Xenopus laevis interspersed poly(A) RNA molecules are synthesized and deposited in the oocyte cytoplasm throughout the main growth phase of oogenesis. Regions of the sea urchin genome that are represented by interspersed maternal transcripts have been recovered from recombinant clone libraries. In one case the same single-copy sequence is found both in an abundant message-sized 1.6 kilobase (kb) maternal transcript and in a 7.5 kb maternal transcript that structurally resembles a precursor form and is not found in embryonic polysomes. In a second example considered, a 9.5 kb transcript was identified in embryo nuclear RNA that may be identical in structure with an interspersed maternal poly(A) RNA derived from the same transcription unit. Transcription of this sequence appears to be constitutive in somatic cell nuclei, though no homologous cytoplasmic RNAs are found after early cleavage. This may be a widespread form of regulation for transcription units expressed in female germ cells, and represented in the maternal poly(A) RNA pools of unfertilized eggs.

Animals↗
PubMed1975

Concanavalin A receptors, immunoglobulins, and theta antigen of the lymphocyte surface. Interactions with concanavalin A and with Cytoplasmic structures.

The effect of concanavalin A (Con A) on the capping of mouse lymphocyte surface immunoglobulin (surface Ig), cross-linked by rabbit anti-mouse Ig antibody, and on the capping of mouse thymocyte theta antigen, cross-linked by anti-theta alloantibody and rabbit anti-mouse Ig antibody, has been studied by immunofluorescence, using fluorescein conjugated Con A and rhodamine-conjugated anti-mouse Ig antibody, and by electron microscopy, using native or fluorescein-conjugated Con A and ferritin-conjugated anti-mouse Ig antibody. Prior incubation of the cells with Con A inhibited only partially capping os surface Ig, whereas it blocked almost completely capping of theta antigens. Both on cells with rings and on cells with caps the staining for surface Ig or theta antigen was superimposed to the staining for Con A. When Con A receptors on spleen cells were capped by Con A at concentrations of 10 mug/ml or higher, and the distribution of surface Ig was examined under noncapping conditions, all detectable surface Ig were found in the caps. As shown by electron microscopy, surface Ig remained dispersed in a layer of Con A. The ability of Con A to cap surface Ig was not altered by the presence of cohchicine or vinblastine. These results suggest that surface Ig are cross-linked by Con A to other Con A receptors. In these conditions surface Ig behave essentially as Con A receptors, as for example, in their sensitivity to cytochalasin B during inhibition or reversal of capping induced by this drug. The behavior of surface Ig parallels that of Con A receptors also in the presence of vinblastine. It is concluded that in the presence of Con A, antimitotic drugs do not modify directly the interaction between Con A receptors and surface Ig, but probably influence the capping ability of the Con A receptors or, more in general, affect the ability to elicit movements over the cell surface. The role in capping of cytochalasin-sensitive and vinblastine-sensitive structures is discussed. Both types of structures appear to play an active role in the formation of a cap, although the former probably corresponds to the main mechanical system responsible for the active displacement of cytoplasmic and surface material.

Animals↗
PubMed1960

Permanganate fixation of the Golgi complex and other cytoplasmic structures of mammalian tests.

Observations on the fine structure of KMnO(4)-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO(4). Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO(4)-fixed testes in view of what has already been reported for mammalian testes fixed in OsO(4).

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