cytochalasin-d and 7-nitrobenz-2-oxa-1-3-diazole-phallacidin

The content of filamentous actin in individual platelets was measured by flow cytometry, using a fluorescent probe specific for filamentous actin (F-actin), 7-nitrobenz-2-oxa-1,3-phallacidin (NBD-phallacidin). NBD-phallacidin binding to fixed platelets was specific in that either pretreatment of platelets with unlabeled phallacidin or absorption of NBD-phallacidin by rabbit skeletal F-actin, but not globular actin (G-actin), resulted in a significant loss in the bound fluorescent probe. Mean NBD-phallacidin binding to fixed platelets varied with the agonist and paralleled the changes in F-actin reported with the DNAse I inhibition assay. (1) NBD-phallacidin binding increased with stimulation by ADP, U46619 (a prostaglandin H2 analogue), or collagen and paralleled shape change. (2) Epinephrine did not increase NBD-phallacidin binding. (3) Platelets treated at 4 degrees C contained more F-actin than did platelets kept at 37 degrees C. (4) Cytochalasin D (10 mumol/L) inhibited the increase of phallacidin binding to individual platelets stimulated by either ADP or U46619. In measurements of cytosolic free calcium concentration ([Ca2+]i) by flow cytometry in Indo-1-loaded platelets, ADP's dose-response for actin polymerization was similar to that for calcium mobilization. As shown by flow cytometry, a tail population that had a minimal increase in F-actin upon stimulation with ADP or U46619 also contained the platelets with the least forward and right angle light scattering, which are functions of platelet size and shape. When platelets treated with NBD-phallacidin were incubated with S12-murine monoclonal antibody (a marker of alpha-granule secretion detected by phycoerythrin-conjugated antimouse IgG second antibody), phallacidin fluorescence paralleled S12 binding. Thus, human blood platelets are heterogeneous in regard to actin polymerization at rest and in association with platelet activation; different degrees of phallacidin binding may identify functionally different platelet populations.

Topics: Actins; Adenosine Diphosphate; Amanitins; Animals; Blood Platelets; Calcium; Collagen; Cytochalasin D; Cytoplasmic Granules; Epinephrine; Flow Cytometry; Fluorescent Dyes; Humans; Light; Prostaglandin Endoperoxides, Synthetic; Rabbits; Scattering, Radiation

The process of spermiation and sperm transport was studied using specific inhibitors of cytoskeletal elements. Within 12-24 hr after the intratesticular injection of taxol, a compound that acts to stabilize microtubules and inhibit microtubule-related processes, an unusually large number of microtubules was seen within the body of the Sertoli cell. At the same time, transport of elements within the seminiferous epithelium was affected. At the end of stage VI of the cycle, step 19 spermatids were maintained in the deep recesses of the Sertoli cell and not transported to the rim of the seminiferous tubule lumen. At stage VIII, residual bodies remained at, or near, the rim of the tubule and were not transported to the base of the tubule. They underwent only partial degradation at this site, indicating that there may have been two phases involved in their dissolution--one autophagic and one phagocytic, but the latter did not occur since the residual bodies were not transported to Sertoli lysosomes at the base of the tubule. The observations suggest that microtubules are involved in transport processes within the seminiferous epithelium. Within 1-12 hr after the intratesticular injection of 500 microM cytochalasin D, a compound which interferes with actin-related processes, normal appearing tubulobulbar complexes were not present. The tubular portion (distal tube) of the complex did not initiate development. It was assumed that filaments (which were identified as such using NBD-phallacidin and the S-1 fragment of myosin) played an important role in the development of this portion of the complex. Cells did not eliminate cytoplasm normally, as evidenced by an enlarged cytoplasmic droplet, further emphasizing the published role for tubulobulbar complexes in cytoplasmic elimination. Although sperm were released normally from stage VIII tubules, many remained within the tubular lumen and did not traverse the duct system. Cytochalasin did not inhibit fluid secretion by the Sertoli cell, as demonstrated by efferent duct ligation, but did alter myoid cell actin cytoskeletal organization, suggesting that myoid cell contractility is primarily responsible for transport of sperm. Overall, the observations suggest that cytoskeletal activity of the Sertoli cell is important for several aspects of the spermiation process as well as sperm transport.

Topics: Actins; Alkaloids; Amanitins; Animals; Cytochalasin D; Cytoskeleton; Injections; Male; Microscopy, Electron; Microtubules; Myosin Subfragments; Paclitaxel; Rats; Rats, Inbred Strains; Sertoli Cells; Sperm Transport; Staining and Labeling; Testis

Ectoplasmic specializations (ES) containing packed actin microfilaments are associated with the numerous parallel rows of occluding junctions which form the Sertoli cell (blood-testis) barrier. To determine if ES regulate the structure of the occluding junctions and/or barrier permeability, we experimentally disrupted ES microfilaments in vivo with intratesticularly injected cytochalasin D (CD). Electron microscopic observations of seminiferous tubules from CD-treated (150-500 microM CD; 0.5-12 hr) animals indicated that ES was absent from regions where the Sertoli cell barrier is located. Seminiferous epithelial sheets from uninjected or vehicle-injected animals (1 DMSO: 1 saline) stained with NBD-phallacidin demonstrated the presence of patterned ES actin surrounding the basolateral regions of adjacent Sertoli cells. After exposure to CD, epithelial sheets exhibited increasingly patchy fluorescence indicating progressive F-actin disruption. Freeze-fracture replicas of CD-injected testes revealed numerous focal alterations in the region of occluding junctions which included disorganization of the parallel arrangement of junctional rows, the presence of free-ending rows, clustering of intramembranous particles (IMPs) between rows, reduction in the number of rows, and loss of IMPs on both the P-face and E-face. Tracer experiments, following CD exposure, were conducted to test the integrity of occluding junctions: lanthanum hydroxide, dextrose, or filipin was added, in separate experiments, to the fixative during perfusion-fixation. In another study, serum containing an antibody against adluminal germ cells was injected intratesticularly, and frozen sections were processed for immunofluorescence study. A final study consisted of simultaneous intratesticular infusions of CD and radiolabelled inulin with subsequent intraluminal and peritubular fluid sampling. In animals which were injected with CD, lanthanum was found to enter the adluminal compartment; fixative made hypertonic by addition of dextrose caused germ cells within the adluminal compartment to shrink and produce exaggerated intercellular spaces; filipin-cholesterol perturbations were present between some Sertoli cell junctional rows and on spermatid plasma membranes; and IgG was detected within the adluminal compartment of many seminiferous tubules. None of these adluminal manifestations was noted in control animals or those which received vehicle. Quantitatively, in the in vivo micropuncture exper

Topics: Actin Cytoskeleton; Actins; Amanitins; Animals; Blood-Testis Barrier; Cell Membrane Permeability; Cytochalasin D; Cytochalasins; Intercellular Junctions; Male; Microscopy, Electron; Rats; Rats, Inbred Strains; Sertoli Cells

Aggregation-competent amoeboid cells of Dictyostelium discoideum are chemotactic toward cAMP. Video microscopy and scanning electron microscopy were used to quantitate changes in cell morphology and locomotion during uniform upshifts in the concentration of cAMP. These studies demonstrate that morphological and motile responses to cAMP are sufficiently synchronous within a cell population to allow relevant biochemical analyses to be performed on large numbers of cells. Changes in cell behavior were correlated with F-actin content by using an NBD-phallacidin binding assay. These studies demonstrate that actin polymerization occurs in two stages in response to stimulation of cells with extracellular cAMP and involves the addition of monomers to the cytochalasin D-sensitive (barbed) ends of actin filaments. The second stage of actin assembly, which peaks at 60 sec following an upshift in cAMP concentration, is temporally correlated with the growth of new pseudopods. The F-actin assembled by 60 sec is localized in these new pseudopods. These results indicate that actin polymerization may constitute one of the driving forces for pseudopod extension in amoeboid cells and that nucleation sites regulating polymerization are under the control of chemotaxis receptors.

Topics: Actins; Amanitins; Animals; Chemotactic Factors; Cytochalasin D; Cytochalasins; Dictyostelium; Fluorescent Dyes; Kinetics; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Polymers; Pseudopodia