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A Spudich

Publications and source records attributed to A Spudich.

10 recordsLinked to original sources
PubMed1995

Large secretory structures at the cell surface imaged with scanning force microscopy.

Scanning force microscopy was used to image rat basophilic leukemia (RBL-2H3) cell surfaces under different stimulation conditions that either permit or inhibit secretion. Cross-linking the surface IgE receptors with dinitrophenol-conjugated bovine serum albumin initiates secretion in RBL cells with concomitant spreading of the cell body. Structures at the cell surface approximately 1.5 microns in diameter relate to secretion both spatially and temporally. The position of these surface pits and their sizes suggest that they may be related to the dense-core granules positioned along the cytoskeletal filaments in detergent-extracted, unactivated RBL cell processes. Topographic scanning force microscopy images of RBL cell surfaces at 2, 5, and 35 min after activation show that these structures persist and change in cross-sectional profile with time after activation. These structures may be related to the membrane retrieval mechanism of cells after intense stimulation.

Animals↗
PubMed1994

Myosin reorganization in activated RBL cells correlates temporally with stimulated secretion.

Rat basophilic leukemia cells secrete histamine and serotonin in response to cross-linking of the IgE receptor by multivalent antigen [Metzger et al., 1986: Ann. Rev. Immunol. 4:419-470]. Receptor crosslinking also induces phosphorylation of the light and heavy chains of myosin II with kinetics similar to that of secretion [Ludowyke et al., 1989: J. Biol. Chem. 264:12492-12501]. Here we show that myosin II localization changes after activation with similar kinetics. Furthermore, these changes are coincident with changes in cell shape and increase in motile activity induced by activation. Within 2 min, activated cells begin to flatten, spread on their substratum, and extend lamellipodia which show active ruffling. Quantitation of the extent of cell spreading from video micrographs shows that 48% of the cells increase significantly in surface area by 5 min and 71% by 15 min. Myosin II is uniformly distributed in unactivated cells but is deficient in newly formed lamellipodia that start to appear at 2 min after activation. In contrast these lamellipodia show strong staining for actin. Further changes in myosin organization are detected by 15 min after activation when myosin reappears in the cell periphery, is concentrated in the perinuclear area, and is also organized in punctate linear arrays that extend from the nucleus to the cell periphery. The kinetics of the early cell shape changes and formation of the myosin-deficient lamellipodia correlate well with, and may relate to, the increase in the level of myosin II phosphorylation reported by Ludowyke et al. [1989: J. Biol. Chem. 264:12492-12501]. Changes in the distribution of cell surface-bound IgE also occur upon antigen activation, and they correlate with the myosin distribution in a manner that suggests that they may be driven by myosin II.

Actins↗
PubMed1994

Structure and activation dynamics of RBL-2H3 cells observed with scanning force microscopy.

Surface and subsurface dynamics of Rat Basophilic Leukemia cells, a model system of stimulated secretion, were imaged using Scanning Force Microscopy (SFM) at a rate of 50-60 s/image. Cytoskeletal elements and organelles were tracked within quiescent cells and those activated after IgE receptor crosslinking. In addition, surface waves were observed moving within the plasma membrane. The structures seen in quiescent and activated cells can be correlated with those seen in electron micrographs and topographic SFM images of fixed detergent-extracted cells. Furthermore, images of the detergent-extracted nuclei reveal the presence of numerous nuclear pore complexes. High-magnification images of the nuclear pore complexes show evidence of subunit structure and exhibit dimensions consistent with those reported previously using electron microscopy. The behavior and overall change in morphology of cells observed during activation was consistent with that observed under similar conditions with Differential Interference Contrast microscopy. This study demonstrates that SFM, unlike other techniques, can be used to provide high-resolution information in both fixed and living cells.

Animals↗
PubMed1993

Patterns in Dictyostelium discoideum: the role of myosin II in the transition from the unicellular to the multicellular phase.

Dictyostelium discoideum amoebae which lack the myosin II gene are motile and aggregate to form rudimentary mounds, but do not undergo further morphological development (Manstein et al., 1989). Here we use scanning electron microscopy, light microscopy, immunofluorescence and computer analysis of time-lapse video films to study how D. discoideum myosin null cells of strains HS2205 and HS2206 aggregate. Myosin null cells are sufficiently coordinated in their movements to form two-dimensional aggregation streams, although mutant cells within streams lack the elongated shape and parallel orientation of wild-type strains. In the wild-type, cell movements are coordinated, cells usually joining streams that spiral inwards and upwards as the mound extends into the standing papilla. In the aggregates of mutant strains, cell movements are chaotic, only occasionally forming short-term spirals that rotate at less than half the speed of wild-type spirals and frequently change direction. Unlike the situation in the wild-type where spirals continue with mound elongation, cells within the mutant mound eventually cease translocation altogether as the terminal shape of the mound is reached and only intracellular particle movement is observed. Scanning electron micrographs show that the surface of the wild-type mound consists of flattened cells which fit neatly together. The myosin null cell mound has an uneven surface, the orientation of the cells is chaotic and no tip is formed. This is consistent with the results of synergy experiments in which myosin null cells were absent from the tips of chimeric HS2205/AX2 slugs and pre-culminates. Immunofluorescence microscopy using prespore and spore cell markers reveals that a prestalk/prespore pattern forms within the mutant mound but that terminal spore differentiation is incomplete. These results are discussed in relation to the role of myosin II in aggregation and morphogenesis.

Animals↗
PubMed1992

Association of the beta isoform of protein kinase C with vimentin filaments.

Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The beta isoform of PKC is translocated and degraded much more rapidly than the alpha isoform in phorbolester-stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC alpha and beta are strikingly different in antigen-activated RBL cells. PKC beta associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC alpha concentrates in regions of the cell periphery. This distribution of PKC beta is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC beta and to the intermediate filament protein vimentin are almost identical, indicating that PKC beta associates with vimentin filaments. These bundles of 100 A filaments may provide docking sites for interactions of PKC beta with its substrates and thus confer specificity to the actions of this isoform.

Animals↗
PubMed1988

Unfertilized sea urchin eggs contain a discrete cortical shell of actin that is subdivided into two organizational states.

Changes in the distribution and organizational state of actin in the cortex of echinoderm eggs are believed to be important events following fertilization. To examine the initial distribution and form of actin in unfertilized eggs, we have adapted immunogold-labeling procedures for use with eggs of Strongylocentrotus purpuratus. Using these procedures, as well as fluorescence microscopy, we have revealed a discrete 1-micron-thick concentrated shell of actin in the unfertilized egg cortex. This actin is located in the short surface projections of unfertilized eggs and around the cortical granules in a manner that suggests it is associated with the cortical granule surface. The actin in the short surface projections appears to be organized into filaments. However, most if not all of the actin surrounding the cortical granules is organized in a form that does not bind phalloidin, even though it is accessible to actin antibody. The lack of phalloidin binding is consistent with either the presence of nonfilamentous actin associated with the cortical granules or the masking of actin-filament phalloidin-binding sites by some cellular actin-binding component. In addition to the concentrated shell of actin found in the cortex, actin was also found to be concentrated in the nuclei of unfertilized eggs.

Actins↗
PubMed1983

Ca2+-sensitive isolation of a cortical actin matrix from Dictyostelium amoebae.

A cortical actin matrix has been isolated from amoebae of Dictyostelium discoideum grown in liquid culture. The existence of this actin matrix in whole cells is indicated in electron micrographs as an area free of cytoplasmic organelles. The actin beneath the membrane is more clearly visible in sections of cells that are lysed gently with 0.5% Triton X-100 and fixed with 1% glutaraldehyde. Such Triton-lysed cells have fragments of plasma membrane associated with the cortical actin matrix. Isolation of the actin matrix, which sediments at 400 g, is inhibited by Ca2+. As much as 50% of the actin of the cell and about 12% of the total protein is found in the matrix isolated in lysis buffer containing no added Ca2+ and 2.5 mM EGTA, whereas less than 15% of the actin of the cell is recovered in a 400 g pellet when cells are lysed in buffer containing 2.5 mM Ca2+ and 2.5 mM EGTA. A 40 000 molecular weight protein that fragments F-actin in a Ca2+-dependent manner is not found in the isolated cortical actin matrix.

Actins↗
PubMed1979

Actin in triton-treated cortical preparations of unfertilized and fertilized sea urchin eggs.

Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.

Actins↗