Biomedical subjects

B H Clubb

Publications and source records attributed to B H Clubb.

6 recordsLinked to original sources

PubMed2000

The 300-kDa intermediate filament-associated protein (IFAP300) is a hamster plectin ortholog.

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin.

Amino Acid Sequence↗

PubMed1998

Peripheral nuclear matrix actin forms perinuclear shells.

Perinuclear actin shells have been reported in a variety of organisms. The shells have been identified by staining perinuclear material with fluorescently-labelled phalloidin, but have not been localized to a specific subcellular compartment at the ultrastructural level. We show here that the shells of 3T3 cells lie in the peripheral nuclear matrix. Nuclear shells and matrix actin in other parts of the nucleus are not usually detected by immunohistochemical staining because they are inaccessible to antibodies or to phalloidin. Immunohistochemical detection of nuclear actin is only possible during its deposition at the end of mitosis, or in interphase nuclei that have been extracted with detergent, digested with nucleases and washed with high salt buffers.

3T3 Cells↗

PubMed1998

3T3 cells have nuclear invaginations containing F-actin.

Nuclear envelope invaginations occur in many kinds of cell. Double-labeling of 3T3 cells with Hoechst 33342 strain for DNA and phalloidin-rhodamine for F-actin, show that some nuclei appear to contain tangled knots of F-actin. Concanavalin A-fluorescein staining for membranes shows that the knots are continuations of the nuclear envelope. Although they contain F-actin, the knots appear by electron microscopy to be cytoplasmic invaginations lacking microfilaments. Since we have shown previously that nuclear-membrane associated actin forms perinuclear shells in 3T3 cells, we propose that nuclear knots also are composed of actin associated with the nuclear membrane. 3T3 nuclei also contain nuclear invaginations of a second kind. These invaginations lie perpendicular to the first type and lack F-actin.

3T3 Cells↗

PubMed1996

F-actin forms transient perinuclear shells at the mitosis-interphase transition.

Intermediate filaments and microtubules are known to be involved in establishing and maintaining nuclear shape. F-actin may also be involved in determining nuclear shape, since we have found it associated with reforming nuclei very briefly after cell division. We stained cells from vertebrate tissue cultures (3T3 and NRK-49F) and epidermal cells from an insect with rhodamine-phalloidin and Hoechst #33342 to localize F-actin in relation to the nucleus. We found that F-actin forms shells only around nuclei during reorganization in late mitosis and early interphase. We suggest that perinuclear F-actin shells may be generally present in eukaryotes, but that they are easily missed because of their delicacy and transience.

3T3 Cells↗

PubMed1996

Extracellular matrix regulates microfilament and vinculin organization in C6-glioma cells.

Since the extracellular matrix (ECM) has a role in regulating cell proliferation it has been hypothesized that unregulated growth of transformed cells results from an inability of cells to interpret the constraints on growth as dictated by the ECM. The most likely candidate to communicate the restraint on growth to the nucleus is the cytoskeleton because it is the only cytoplasmic structure to physically connect the nucleus and plasma membrane. The purpose of this study was to determine whether the cytoskeleton, specifically the microfilaments (MF), of the C6-glioma cell line possesses the ability to respond to changes in the ECM. The C6-glioma cell line was chosen as a model because it exhibits the characteristics of a tumor cell, both in vivo and in vitro. In this study cells were grown on bare glass, Matrigel, laminin and type IV collagen prior to staining with phalloidin tetramethylrhodamine B isothiocyanate or antibodies specific to vinculin to visualize MF and adhesion plaques, respectively. On the basis of MF orientation, network complexity and location of adhesion plaques, this study reports that the cytoskeletal arrangements of C6 cells grown on various substrates are distinctly different. Each distinct organization pattern may reflect a change of cell behavior promoted by the different culture conditions. The significance of this study is that it demonstrates that the process of transformation in C6-glioma cells has not interfered with the cells ability to recognize, interpret and adapt to changes in the ECM.

Actin Cytoskeleton↗

PubMedDate not supplied

Glycerol disrupts tight junction-associated actin microfilaments, occludin, and microtubules in Sertoli cells.

Intratesticular injections of glycerol have been shown to result in a marked and prolonged reduction of spermatogenesis, accompanied by increased permeability of the blood-testis barrier. Because the permeability of the blood-testis barrier is regulated by Sertoli cell tight junctions, and tight junction organization is regulated by the cytoskeleton, we undertook to examine the effects of glycerol treatment on cytoskeletal actin microfilaments and microtubules, and on the tight junction protein, occludin, in Sertoli cells. Adult rats received a single intratesticular injection of either saline (controls) or a 10% glycerol solution. At 24 hours and 7, 15, and 21 days after injection, testes were collected and prepared for routine histology, cryosectioning, or whole seminiferous tubule immunohistochemical staining; and the preparations were viewed by light and confocal microscopy. In saline-injected testes, Sertoli cells had a cytoskeletal and junctional organization that resembled that of normal testes. F-actin microfilaments, located in the basal region, were arranged in regular bundles or chords that circumscribed the perimeter of each Sertoli cell at the level of the tight junction. Occludin colocalized with tight junction-associated actin filament distribution and microtubules formed a geometric array associated with spermatogenic cells. In contrast, in glycerol-treated Sertoli cells, microfilament and microtubule organization and occludin distribution were partially or completely disrupted. From these results we conclude that glycerol treatment either directly or indirectly disrupts tight junction-associated F-actin and occludin and tubulin organization in rat Sertoli cells. Perturbation of the tight junction-associated proteins could explain the increase in permeability of the blood-testis barrier observed after glycerol treatment. Impaired spermatogenesis following glycerol treatment is likely a consequence of a leaky blood testis barrier and disrupted Sertoli cell cytoskeleton. Glycerol injections may serve as a useful tool in studying the relationship between cytoskeletal organization and the stabilization of Sertoli-Sertoli cell junctions.

Actins↗