Bruce S. Jacobson

Interaction of the plasma membrane with the cytoskeleton: An overview

The intent of this review was to point out the diversity of cellular functions thought to be mediated by PM-cytoskeleton interactions. Based upon possible molecular mechanism, the functions were categorized into those involving PM proteins which are dispersed and those involving clustered proteins. Functions associated with dispersed proteins are thought to mediate the stabilization and shape of the PM. Clustering of PM proteins provides the driving force inducing their interaction with the cytoskeleton. Clustering by external ligands, pH or ionic exchanges, etc., is also a means of transmembrane signalling. Various methods used to explore cytoskeletal-PM mediated functions were evaluated. The methods were considered separately under biophysical, morphological and biochemical headings. This made it easier to point out current and potential values of the methods as well as their limitations. Each method taken separately is insufficient to elucidate molecular mechanisms regulating cytoskeletal-PM reactions, but combined they hold great promise of future solutions.

Improved method for isolation of plasma membrane on cationic beads. Membranes from Dictyostelium discoideum

The plasma membrane from Dictyostelium discoideum was routinely purified 35-fold by an improved technique using beads coated with positively charged polymers. Cells were attached to the beads and bare regions between the cells were neutralized with a polyanion. The neutralization decreased contamination of the bare regions by intracellular proteins released when cells were disrupted to leave behind beads coated by plasma membrane. The neutralization increased the purification as measured by membrane-bound 125I-labeled concanavalin A. Contamination by markers for various intracellular components was markedly decreased. Various bare-site neutralization reagents were evaluated and gave different results depending upon their charge density and molecular weight. The pH of the neutralization was critical. The optimum pH for cell attachment to beads, 5.0, had little effect as regard bare-site neutralization. A new procedure is given that optimizes the essential features for the plasma membrane isolation on beads.

MACRO- AND MICROVASCULAR ENDOTHELIAL CELLS IN VITRO: MAINTENANCE OF BIOCHEMICAL HETEROGENEITY DESPITE LOSS OF ULTRASTRUCTURAL CHARACTERISTICS

SummaryMicrovascular endothelial cells from bovine adrenal medulla and brain and macrovessel endothelial cells from bovine aorta were isolated and cultured under similar conditions in order to determine morphologic and biochemical heterogeneity in vitro. All three cell types exhibited nearly identical ultrastructural morphology and two-dimensional gel protein patterns of35S-methionine-labeled whole cells. Two-dimensional gel analysis of35S-methionine-labeled plasma membrane proteins however, revealed two-dimensional gel protein patterns unique to the tissue type from which the endothelial cells were isolated. This suggests that the functional significance of these specific endothelial cell types is manifested primarily in surface-associated proteins and that many of the differences are sustained in culture. To determine the potential of aorta, brain, and adrenal medulla endothelial cell (EC) cultures to respond to developmentally significant signals, morphology, growth pattern, and cell surface proteins were monitored in the presence and absence of growth factors. A 17 to 26% increase in cell density as well as an increase in the number of elongated and overlapping cells resulted when all three EC types were exposed to a mitogenic medium. Additionally, expression of specific glycoprotein profiles, as determined by Concanavalin A Western blotting of two-dimensional gels, was dependent on the presence or absence of growth factors in the medium. The ability to induce this morphologic and biochemical variation in the three endothelial cell types was maintained into later passage. Taken together, these data imply that endothelial cells isolated from different tissues exhibit and maintain biochemical heterogeneity and do not completely dedifferentiate into a common endothelial cell type in culture. Furthermore, expression of specific subsets of cell surface proteins is dependent on environmental conditions, and in some cases is both cell-type and media-type dependent. Thus, even though endothelial cells are considered terminally differentiated cells, there exists additional or “latent” heterogeneity in the ability of these different cells to respond to “developmental signals” (i.e. mitogenic medium) in vitro.

Pure gelatin microcarriers: Synthesis and use in cell attachment and growth of fibroblast and endothelial cells

SummaryA new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S3 cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally.

Arachidonate initiated protein kinase C activation regulates HeLa cell spreading on a gelatin substrate by inducing F-actin formation and exocytotic upregulation of β1 Integrin

HeLa cell spreading on a gelatin substrate requires the activation of protein kinase C (PKC), which occurs as a result of cell‐attachment‐induced activation of phospholipase A2 (PLA2) to produce arachidonic acid (AA) and metabolism of AA by lipoxyginase (LOX). The present study examines how PKC activation affects the actin‐ and microtubule‐based cytoskeletal machinery to facilitate HeLa cell spreading on gelatin. Cell spreading on gelatin is contingent on PKC induction of both actin polymerization and microtubule‐facilitated exocytosis, which is based on the following observations. There is an increase in the relative content of filamentous (F)‐actin during HeLa cell spreading, and treating HeLa cells with PKC‐activating phorbol esters such as 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) further increases the relative content of F‐actin and the rate and extent to which the cells spread. Conversely, inhibition of PKC by calphostin C blocked both cell spreading and the increase of F‐actin content. The increased F‐actin content induced by PKC activators also was observed in suspension cells treated with TPA, and the kinetics of F‐actin were similar to that for PKC activation. In addition, PKCϵ, which is the PKC isoform most involved in regulating HeLa cell spreading in response to AA production, is more rapidly translocated to the membrane in response to TPA treatment than is the increase in F‐actin. Blocking the activities of either PLA2 or LOX inhibited F‐actin formation and cell spreading, both of which were reversed by TPA treatment. This result is consistent with AA and a LOX metabolite of AA as being upstream second messengers of activation of PKC and its regulation of F‐actin formation and cell spreading. PKC appears to activate actin polymerization in the entire body of the cell and not just in the region of cell‐substrate adhesion because activated PKC was associated not only with the basolateral plasma membrane domain contacting the culture dish but also with the apical plama membrane domain exposed to the culture medium and with an intracellular membrane fraction. In addition to the facilitation of F‐actin formation, activation of PKC induces the exocytotic upregulation of β1 integrins from an intracellular domain to the cell surface, possibly in a microtubule‐dependent manner because the upregulation is inhibited by Nocodazole. The results support the concept that cell‐attachment‐induced AA production and its metabolism by LOX results in the activation of PKC, which has a dual role in regulating the cytoskeletal machinery during HeLa cell spreading. One is through the formation of F‐actin that induces the structural reorganization of the cells from round to spread, and the other is the exocytotic upregulation of collagen receptors to the cell surface to enhance cell spreading. J. Cell. Physiol. 173:361–370, 1997.

The detection of melanocyte autoantibodies in the Smyth chicken model for vitiligo.

Smyth line (SL) chickens are phenotypically characterized by a posthatch depigmentation (vitiligo) of the feathers. The destruction of melanocytes in the feather follicle as well as in other tissues such as the choroid is genetically determined. Previous studies have shown that bursectomy or treatment with immunosuppressive agents decreases the incidence and severity of SL depigmentation (1). These observations implicate a role for the immune system, specifically the humoral component, in melanocyte destruction. In this study we show that there are circulating melanocyte-specific autoantibodies in the sera of depigmented SL chicks which are not present in sera from Light Brown Leghorn (LBL) control chicks. By immunoblots and by immunoprecipitation of radiolabeled melanocyte proteins, SL autoantibodies were shown to bind to multiple melanocyte proteins between 65 and 80 kDa. These proteins are not detected in SL fibroblasts. By immunoblotting, the incidence of autoantibodies for these 65- to 80-kDa proteins was determined to be 95% in depigmented SL chicks (n = 20), 0% in normally pigmented SL chicks (n = 8), and 5% in LBL chicks (n = 20). Melanocyte autoantibodies are detectable in the sera of affected chicks at or several weeks prior to the expression of depigmentation. This information, plus previously published data, implicate melanocyte autoantibodies in the depigmentary phenomenon of vitiligo observed in Smyth line chickens.

Fast and efficient purification of yeast plasma membranes using cationic silica microbeads

A fast and efficient procedure for the purification of plasma membranes of Saccharomyces cerevisiae is described. Protoplasts served as starting material. They were coated with cationic silica microbeads. After lysis, the plasma membranes were washed free from debris and cell organelles. This procedure resulted in a high yield (about 85%) of plasma membranes, as judged by measuring vanadate-sensitive ATPase as a plasma membrane marker. The enzyme was enriched 12-fold relative to the homogenate after lysis. Its specific activity was 1.5--2.0 micromol/min per mg protein, the pH optimum was 6.5, and 10 microM vanadate was sufficient to obtain maximum inhibition. Based on the assay of internal markers and electron microscopic studies, we found our preparation essentially free of contamination from other cell organelles.

Actin binding to the cytoplasmic surface of the plasma membrane isolated from Dictyosteliumdiscoideum

Summary Actin was dissociated from the cytoplasmic surface of the plasma membrane from Dictyostelium discoideum . The cytoplasmic surface was selectively exposed by isolating the membrane by a new method using cationic beads. When purified actin was added back to the membrane the amount that bound was proportional to the actin concentration. Trypsinization of the cytoplasmic surface abolished binding while chymotrypsin was only partly effective. Far more F-actin bound to the membrane than G-actin and the binding was not inhibited by cytochalasin B. The results support the hypothesis that actin specifically interacts with unique proteins exposed on the cytoplasmic surface of the plasma membrane.

Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate

The plasma membrane is a complex organelle responsible for many cellular functions. In addition to mediating the exchange of components with the extracellular fluid, the plasma membrane is involved in cell adhesion to matrix proteins in vivo and in vitro. In vitro, adherent cells have three distinct plasma membrane domains to carry out these functions: one attached to the substrate (ventral); another exposed to the media (dorsal); and an intracellular domain involved in endocytosis and secretion. A technique has been developed for the rapid isolation of these specific domains from HeLa cells immediately following adhesion to a gelatin substrate. The isolation procedure utilizes the tight binding of cationic colloidal silica to the dorsal plasma membrane domain of attached cells. Following silica binding and cell lysis, the silica-coated dorsal plasma membrane domain is readily separated from intracellular plasma membrane components by virtue of the high density of the silica pellicle, and the intact ventral plasma membrane domain remains attached to the gelatin substrate. Fluorescence and electron microscopy and biochemical studies using 125I-lactoperoxidase labeling, 125I-labeled wheat germ agglutinin binding, and [3H]-fucose incorporation into plasma membrane glycoproteins confirmed the separation of these three topologically distinct plasma membrane domains. The fractions isolated by the technique contained essentially all of the plasma membrane components present in intact cells. This unique membrane-isolation procedure is now being used to analyze membrane flow during plasma membrane domain formation accompanying cell adhesion to an extracellular matrix.

Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: sedimentation versus magnetic isolation.

A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to alpha-methylmannoside in concanavalin A-carbohydrate competition studies.