Gerard J. Bourguignon

Capping and the Cytoskeleton

Publisher Summary This chapter summarizes the results obtained in capping/cytoskeleton research. There are two types of capping phenomena: ligand-dependent and ligand-independent processes. A number of different reagents, such as colchicine and hypertonic media, can cause the formation of cap structures in the absence of any externally added ligand. Morphological studies, using both transmission and scanning electron microscopy, of cells forming caps in hypertonic medium have identified a strong association between cap structures and microvilli. External ligand (antibody, lectin, or some hormones) mediated receptor patching and capping is responsible for important immune-related responses: ( 1) proliferation and differentiation of the cells into antibody-secreting plasma cells, (2) the development of an increased number of precursor cells which are responsible for immunological memory, and (3) the activation and proliferation of T cells which carry out cell-mediated cytotoxic killing. In nonimmune cells, capping may also be involved in important cellular processes, such as endocytosis, chemotaxis, mitogenesis, and general cell–cell recognition.

Electric stimulation of protein and DNA synthesis in human fibroblasts.

Human fibroblast cell cultures were employed as a model system to rapidly examine several potentially important variables involved in the use of high‐voltage, pulsed galvanic stimulation (HVPGS) to increase the healing rate of soft tissue injuries. Fibroblasts were grown on Millipore filters and exposed to HVPGS of various voltages and pulse rates for 20 min in a rectangular, plastic chamber filled with growth medium. Filters with attached cells were placed either in the center of the chamber or close to the positive or negative electrode. Protein synthesis and DNA synthesis were monitored after stimulation using the radioactively labeled precursors, [3H]proline and [3H]thymidine, respectively. The major results obtained in this study are as follows: 1) the rates of both protein and DNA synthesis can be significantly increased by specific combinations of HVPGS voltage and pulse rate; 2) maximum stimulation of protein and DNA synthesis was obtained at 50 and 75 V, respectively, with a pulse rate of 100 pulses/s and the cells located near the negative electrode; and 3) exposure to HVPGS intensities greater than 250 V (at all pulse rates and locations within the chamber) is inhibitory for both protein and DNA synthesis. In view of the results obtained in preliminary clinical studies on the use of HVPGS for the treatment of dermal ulcers, it appears that similar voltages, pulse rates, and relative electrode location may be required for maximum acceleration of human skin wound healing.— Bourguignon, G. J.; Bourguignon, L. Y. W. Electric stimulation of protein and DNA synthesis in human fibroblasts. FASEB J. 1: 398‐402; 1987.

Immunocytochemical localization of intermediate filament proteins during lymphocyte capping.

Using double immuno-fluorescence techniques on frozen-thick sections, we have examined the fate of intermediate filaments during Con A receptor capping in lymphoid cells. Our results indicate that during capping intermediate filaments are preferentially aggregated between the surface receptor cap structure and the cell nucleus. It is possible, therefore, that intermediate filaments are directly involved in lymphocyte capping.

Role of Ca2+ in the regulation of hormone receptor exposure during lymphocyte activation

Ca2+ is known to be required for mitogen-mediated lymphocyte activation. In order to further define the regulatory role of Ca2+, we have examined the activation events which occur following treatment with ionomycin (a Ca2+ ionophore), as compared to those occurring following concanavalin A (Con A) treatment of mouse splenic T-lymphocytes. Our results indicate that ionomycin and Con A induce the exposure of both interleukin-2 (IL-2) and insulin receptors on the surface of the lymphocytes within the first 5 min of treatment. The exposed insulin and IL-2 receptors have the following properties: (1) they consist of both high- and low-affinity receptors; and (2) they appear on the cell surface in small clusters (i.e., patches) or, occasionally, a large aggregate (i.e., cap). c-myc gene expression and DNA synthesis occur in both the ionomycin and Con A-treated lymphocytes when either IL-2 or insulin is present in the culture medium. Furthermore, the exposure of both hormone receptors can be inhibited by either EGTA (a Ca2+ chelator), bepridil (a Ca2+ channel blocker), W-7 (a calmodulin antagonist) or cytochalasin D (a microfilament inhibitor). Treatment with these inhibitors also blocks the expression of c-myc gene and DNA synthesis which occur at later times during IL-2 and insulin-induced activation of ionomycin- and Con A-treated lymphocytes. These findings suggest that a Ca2+ and calmodulin-mediated contractile system is involved in the exposure of certain hormone receptors which appear to be required for complete lymphocyte activation.

Ca2+ Signaling and Cytoskeleton Reorganization in Endothelial Cells Stimulated by Bradykinin

In this study we have examined the activation events which occur following the binding of bradykinin (BK) to its receptor on cultured bovine endothelial cells. Using Fura-2 and a fluorescence spectrophotometer, we have found that intracellular Ca2+ mobilization occurs within seconds after the addition of BK to the cells and reaches a maximal level within 20 seconds. Subsequently, the [Ca2+]i decreases to a constant level after about 3 min following the addition of BK to the cells. After Ca2+ mobilization the cytoskeletal protein, ankyrin, is readily reorganized and accumulated in the perinuclear region. This event is followed by the onset of DNA synthesis. Cytoskeletal protein inhibitors, such as cytochalasin D (known to impair microfilament function) and W-7 (a calmodulin antagonist) have no effect on BK-induced Ca2+ mobilization, but block BK-induced cytoskeleton reorganization and the onset of BK-induced DNA synthesis. These findings suggest that the ankyrin-associated microfilament system and a calmodulin-dependent event are required for BK-stimulated endothelial cell proliferation.