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K R Porter

Publications and source records attributed to K R Porter.

At least 55 records · Page 3Linked to original sources
PubMed1987

Release of enzymes of intermediary metabolism from permeabilized cells: further evidence in support of a structural organization of the cytoplasmic matrix.

Cultured BSC-1 cells were exposed to the mild ionic detergent, Brij 58, and the time course of the release of three enzymes of intermediary metabolism (lactate dehydrogenase, aldolase, and creatine phosphokinase) was determined spectrophotometrically. Their release correlates well with the overall decrease in structural complexity of the cytoplasmic matrix. However, each of the three enzymes tested has its own characteristic time-dependent release profile, a result suggesting enzyme-specific variability in their association with the cytomatrix. Cells lysed for 5 min in Brij 58 and then transferred to detergent-free glycolysis medium were able to produce lactate from glucose, an observation consistent with the idea that all enzymes of the glycolytic pathway remained in the cytomatrix. These observations are consistent with a structural rather than viscous organization of the cytoplasm and suggest that cytoplasmic components other than cytoskeletal filaments are able to form parafilamentous, metastable complexes.

Animals↗
PubMed1986

Microtubule polarity confers direction to pigment transport in chromatophores.

The cellular mechanisms used to direct translocating organelles are poorly understood. It is believed that the intrinsic structural polarity of microtubules may play a role in this process. We have examined the effects that differently oriented microtubules have upon the direction of pigment transport in surgically severed melanophore arms. In a previous paper (McNiven, M. A., M. Wang, and K. R. Porter, 1984, Cell, 37:753-765) we reported that after isolation, arms repolarized and reoriented their microtubules outward from their centers as if to form new "microcells." Pigment aggregation in these arms was toward a new focal point located at the arm centers. In this study we monitored pigment movement in isolated arms containing taxol-stabilized microtubules to test if the reversal in direction of pigment transport is dependent upon the repolarization of microtubules. We report that taxol delays both the microtubule reorientation and reversal in transport direction in a concentration-dependent manner. These and other presented data suggest that the polarity of the microtubule population within a melanophore confers direction on pigment transport.

Alkaloids↗
PubMed1986

Cytoskeletal changes during adhesion and release: a comparison of human and nonhuman primate platelets.

The organization of cytoskeletal proteins in whole-mount adherent platelets from African green monkeys and normal human volunteers has been studied by SEM, high vacuum electron microscopy (HVEM) and conventional (120 kV) electron microscopy. We describe three distinct organizational zones, the Central Matrix, the Trabecular Zone and the Peripheral Web in spread platelets from both sources. The Central Matrix is an ill-defined superstructure of 80-100 A filaments of short length which enshrouded the granules, dense bodies, mitochondria and elements of the open-channel and dense-tubular systems. The latter, identified through the use of peroxidase cytochemistry with the whole mounts, is an anastomosing network of elongate saccules having diameters of 600-1200 A. The Trabecular Zone, which encircles the Central Matrix, contains 165, 80-100 and 30-50 A filaments in an open lattice of irregular lattice spacing. The outermost region of the cells, the Peripheral Web, is comprised of 70 A filaments organized in a honeycomb lattice with center to center spacing in the range 150-300 A. This pattern for the spread cells is not consistently observed in cells during the early stages of adhesion; therefore, correlations of SEM and TEM observations are made for the various stages of adhesion/activation.

Animals↗
PubMed1985

Reactivation of latent murine cytomegalovirus from kidney.

Cytomegalovirus (CMV) is presumed to cause latent infection, but the sites of infection are incompletely known. We propose that latent murine cytomegalovirus is present in kidney and may be reactivated by explanation. Immunosuppressive agents and allogeneic stimuli may enhance this process. Balb/c mice were infected 11 to 14 months previously with 10(5) pfu of Smith strain murine cytomegalovirus intraperitoneally. Kidneys from 15 infected and nine uninfected mice were washed, minced into 1 to 2 mm2 explants and placed into separate tissue culture wells containing a mouse embryo fibroblast monolayer. Explants were untreated or treated with azathioprine, cyclophosphamide, anti-thymocyte serum, or allogeneic lymphocytes. Daily observations for CPE and passage of supernatant to fresh mouse embryo fibroblast were done. Standard cultures of blood, kidney, and salivary gland were negative. However, virus was isolated from the explants of 8/15 animals, with a reactivation time of 30 to 70 days. No significant difference in reactivation time was noted between treated or untreated explants. Restriction enzyme analysis of viral DNA confirmed identity with the original strain. These data show that latent murine cytomegalovirus is present in murine kidney tissue and may be reactivated by explantation.

Animals↗
PubMed1985

The cytoplasmic matrix: its volume and surface area and the diffusion of molecules through it.

In this work we look into the problem of why proteins, unlike small molecules, diffuse in the cytoplasm much more slowly than in aqueous solutions. In order to examine whether the cytoplasmic matrix could, by simple obstruction, retard protein diffusion to such an extent, we developed a method to measure semiquantitatively the fractional volume occupied by the cytoplasmic matrix (which includes the microfilaments, intermediate filaments, microtubules, and the microtrabeculae of the cytoplasmic matrix). This method yielded values in the range of only 16-21%. Thus, a more elaborate model is suggested in which the diffusing proteins bind and dissociate constantly from the surfaces in the cytoplasmic matrix. From this model, the diffusion coefficients and the measured values of the fractional volumes, we calculated the corresponding binding constants. These values indicate that most of the diffusing proteins are bound to the matrix at any given time, in spite of the possibility that they may bind and dissociate very rapidly. In addition, from our measurements, we estimate the surface area of structures within the cytoplasmic matrix to be in the range of 69,000-91,000 micron 2 per cell.

Animals↗
PubMed1985

High voltage electron microscopy of sperm axoneme.

The axoneme of sea urchin spermatozoon has been examined in whole preparation by high voltage electron microscopy. Radial spokes appear arranged in triplets with intratriplet distance of 40 nm, distance between spoke 1 and 2 of 32 nm and between spoke 2 and 3 of 24 nm. Spoke 1 is thicker than others; the complex of the triplets around the 9 microtubule doublets forms a helix of 96 nm pitch. Nexin links connect the A tubule of a doublet with the B of the adjacent one, and also form a helix of 96 nm pitch.

Animals↗
PubMed1984

Interaction of frog virus 3 with the cytomatrix. II. Structure and composition of the virus assembly site.

We have described the structure of virus assembly sites in frog virus 3-infected tissue culture cells based on an examination of sectioned and whole cells by conventional and high voltage (1 000 kV) electron microscopy (HVEM), respectively. We have also attempted to identify the cellular and viral components within the assembly sites using immunofluorescence and a combination of DNase digestion and EM autoradiography. Immunofluorescence studies showed that the sites do not contain tubulin, vimentin, actin, or myosin, confirming EM and HVEM studies which showed the absence of these cytoskeletal filaments from these sites. Enzymatic digestion and autoradiographic studies identified viral DNA in clumps of electron-dense material which are suspended within the matrix of the assembly site. Examination of whole, virus-infected cells by HVEM revealed that the matrix of the assembly site appears as a compaction of the cytoplasmic matrix and appears to be continuous with it. The fine strands of cytomatrix, equated with the microtrabeculae of other cells, are covered with or contain granules measuring 6-8 nm; similar granules also populate the virus assembly sites. Disruption of purified FV 3 with chloroform yielded capsomeres measuring 6-8 nm. Based on all these observations, we postulate that the virus assembly sites are regions of the cytomatrix specialized for virus assembly and that the viral components are transported along the cytomatrix to the assembly sites.

Actins↗
PubMed1984

Microtubule polarity and the direction of pigment transport reverse simultaneously in surgically severed melanophore arms.

The transport of pigment through the long cytoplasmic extensions (arms) of teleost melanophores is a microtubule-dependent event. We have severed the arms from melanophores to test whether microtubules isolated from the centrosome maintain their original polarity and disposition. In addition, we have tested whether arms containing microtubules of mixed polarities alter the direction of pigment transport. We find that microtubules within severed arms eventually change their polarity and reorganize from the arm center as if to form a new minicell. Concomitant with this change is a reversal in the direction of pigment transport.

Animals↗
PubMed1984

A tumor promoter induces rapid and coordinated reorganization of actin and vinculin in cultured cells.

Treatment of epithelial African green monkey kidney (BSC-1) cells with the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) induces a rapid and reversible redistribution of actin and vinculin that is detectable after only 2 min of treatment. Within 20-40 min, stress fibers disappear, while at the same time large actin-containing ribbons resembling ruffles develop both at the cell periphery and in more central regions. Vinculin is associated with these actin ribbons or bands in a punctate or patchy staining pattern. Adhesion to the substratum is changed from predominantly focal contacts associated with stress fiber ends in untreated cells to broad zones of close contact after TPA treatment. High voltage electron microscopic observations disclose the ribbons to consist of highly cross-linked actin filament networks. Thus, association of vinculin with filament networks, rather than (the ends of) filament bundles, is demonstrated. The integrity of microtubules and vimentin filaments is not affected by TPA treatment, but their distribution is altered to conform with the highly distorted cell shape. The response to TPA is neither prevented nor modified by nocodazole-induced depolymerization or taxol-induced stabilization of microtubules. An intact intermediate filament network seems not required either since colcemid-induced collapse of vimentin filaments towards the nucleus does not affect the cell's response to TPA. Rapid redistribution of actin and vinculin also takes place in enucleated cells and in the presence of cycloheximide, but is prevented by dinitrophenol or oligomycin. TPA-induced cytoskeletal alterations are independent of fibronectin expression and not mimicked, modified, or prevented by calmodulin inhibitors or experimentally elevated levels of calcium and cyclic AMP. Thus the morphological response to TPA involves rapid redistribution of actin and vinculin independent of transcription and translation, fluctuations in the levels of calcium or cyclic AMP, or changes in the organization of microtubules, intermediate filaments, and fibronectin.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Three-dimensional organization of the platelet cytoskeleton during adhesion in vitro: observations on human and nonhuman primate cells.

Adhesion of platelets in vitro resulted in rapid polymerization of the amorphous cytoplasmic ground substance into an organized cytoskeletal superstructure. This cytoskeleton, characterized through the use of whole-mount and stereo (3-D), high-voltage microscopy in conjunction with morphometrics and cytochemistry, comprised four major size classes of filaments organized in distinctive zones. The central matrix, or granulomere, at the center of the cell mass, was an ill-defined meshwork of 80-100-A filaments which enshrouded granules, dense bodies, and elements of the dense tubular system as identified through peroxidase cytochemistry. Demarcating this central matrix was a trabecular zone containing 30-50, 80-100, and 150-170 A filaments in an open and rigid-appearing lattice. Circumscribing the trabecular zone and extending to the margins of the hyalomere was the third region, the peripheral web, in which 70-A filaments were arranged in a tight honeycomb lattice. This organizational pattern was retained in cytoskeletons prepared by Triton x-100 extraction of the adherent cells, and was observed in basally located cells of aggregates which formed subsequent to adhesion. Our observations are consistent with biochemical studies of cytoskeletons prepared from suspended platelets and suggest a contractile protein composition for the superstructure during adhesion.

Animals↗
PubMed1983

Analysis of the role of microtubules and actin in erythrophore intracellular motility.

The Holocentrus erythrophore, a red pigment cell, represents a model system for the study of organized intracellular transport. We have investigated the possibility that microtubules and actin are integral components of the pigment translocating motility machine. By creating cells that have total or partial loss of the microtubule framework we have demonstrated that the presence of microtubules is essential for organized, radial transport of the pigment granules. However, in the absence of microtubules, some undirected movement of the pigment can be stimulated; this suggests that a nonmicrotubular component of the cytoplast is responsible, at least in part, for the generation of motive force. In order to test the hypothesis that this component consists of actin or actomyosin, we examined the effects of probes for these classical motility proteins. Neither microinjection of phalloidin, DNase I or N-ethylmaleimide-modified heavy meromyosin nor exogenous application of cytochalasin B has any effect on pigment motion, although these materials do block the actin-mediated motility of other systems in our hands. Therefore, intracellular particle transport in erythrophores does not appear to be actin or actomyosin-based.

Actins↗
PubMed1983

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes.

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.

Actins↗
PubMed1982

The cytoplast: a unit structure in chromatophores.

We followed the translocation of identifiable pigment granules in living erythrophores through normal aggregation and dispersion and observed that they always return in dispersion to the same location relative to the whole pigment complex. This is interpreted to mean that each granule occupies a fixed position within a unit structure, the cytoplast. This position is retained even though the cytoplast undergoes dramatic reversals in form from ellipsoid to spheroid and back again with each aggregation and dispersion. The major structural components of the cytoplast, besides pigment granules, are microtubules and microtrabeculae. The latter constitute an irregular lattice that is confluent with microtubules and contains the pigment granules. In aggregation, the microtrabeculae shorten and seemingly contribute to the contraction of the entire cytoplast plus pigment. In dispersion, the microtrabeculae elongate in an apparent restructuring of the ellipsoidal cytoplast. The microtubules, however, persist in the cell cortex and appear to give radial direction to the pigment motion.

Animals↗