Judith M. White

Structure, function and evolutionary relationship of proteins containing a disintegrin domain.

Disintegrins are soluble integrin ligands from snake venoms that disrupt cell-matrix interactions. Recently, the nuclear magnetic resonance structures of two disintegrins were determined, providing provocative molecular insight into how a disintegrin may engage an integrin. In addition, it has recently been realized that disintegrins are derived from larger multifunctional proteins, and that there is a family of membrane-anchored, disintegrin domain-containing proteins that may promote important cell-cell interactions.

Studies of HIV-1 envelope glycoprotein-mediated fusion using a simple fluorescence assay.

ObjectiveTo study HIV envelope glycoprotein (Env)-mediated entry using a sensitive fusion assay. Design and methodsCD4+ lymphocytes or T-cell lines were labelled with fluorescent cytoplasm or membrane markers. Fusion with Env-expressing adherent cells was monitored by observing dye transfer from CD4+ cells to Env cells. ResultsCell-cell fusion began 20–30 min after co-cultivation at 37°C. Pre-binding at 4°C was observed not to decrease the lag phase before fusion. Cells expressing envelope glycoproteins from non-syncytium-inducing (NSI) HIV strains showed dye transfer between two cells without progression to syncytia. A glycosylphosphatidylinositol anchored Env was found to be incapable of mediating membrane fusion, as measured either by lipid or cytoplasm contents mixing. Primary mouse cells expressing human CD4 and mouse 3T3 cells stably expressing both human CD4 and human CD26 did not support fusion with our Env-expressing cells. ConclusionsEnv-mediated cell-cell fusion is a relatively slow process, probably reflecting a multi-step process occurring after CD4 binding and requiring the trans-membrane domain of gp41. Env proteins are able to mediate cell-cell fusion at least under some experimental conditions, indicating that lack of a syncytia phenotype does not rule out the possibility of fusion occurring between only two or a few cells.

Delivery of liposome-encapsulated RNA to cells expressing influenza virus hemagglutinin

Publisher Summary This chapter focuses on the delivery of liposome encapsulated RNA to cells expressing influenza virus hemagglutinin. The virus binds to sialic acid-containing receptors on the target cell surface and fuses when it encounters low pH in the endosome. In this manner, the influenza virus RNA genome gains access to the cell interior for replication. In the RNA delivery scheme, HA is expressed on the target cell surface and RNA-containing liposomes are prepared with the red blood cell sialoglycoprotein, glycophorin, which provides a specific attachment site for the HA. After binding the liposomes to HA-expressing cells, fusion is induced (at the cell surface) by a brief drop in medium pH. The result is synchronous delivery of RNA into the cells. The method has been used in the laboratory for studying the replication of hepatitis delta virus, a virus with an RNA genome. This chapter also expect the method to be helpful in investigating other aspects of viral life cycles, such as packaging of the viral genome and budding of virus particles. The HA-mediated liposome delivery technique may be particularly well suited for studying the intracellular effects of antisense RNA and ribozymes, agents designed to destroy specific RNA molecules.

Chapter 9 Delivery of Macromolecules into Cells Expressing a Viral Membrane Fusion Protein

Publisher Summary This chapter reviews a macromolecular delivery technique that employs cells that express a potent membrane fusion protein, the influenza virus hemagglutinin (HA). Briefly, the protein or nucleic acid of interest is loaded into either red blood cells (RBC) or liposomes. After binding of the delivery vesicles to the HA-expressing cells, fusion is induced by briefly treating the cultures at pH 4.8. The cells are then returned to normal growth medium. As a result of the fusion step, the contents of the RBC or liposomes are delivered into the cell cytoplasm. This chapter discusses the methods for generating HA-expressing cell lines, for loading RBC and liposomes, and for fusing the delivery vesicles to target cells. The chapter evaluates the advantages and disadvantages of this method. There are several advantages to the technique: (1) nearly all of the cells in a population receive macromolecules, (2) large numbers of molecules can be delivered per cell (3) the same general approach and recipient cells can be used for the delivery of proteins, nucleic acids, and other substances. A limitation of the technique is the need to obtain cells that express high levels of the HA. However, several HA-expressing cell lines suitable for delivery are currently available.

Antibodies to 100- and 60-kDa surface proteins inhibit substratum attachment and differentiation of rodent skeletal myoblasts

A rabbit polyclonal antiserum was raised against membrane vesicles shed from the surface of fusing L6 rat myoblasts. In immunoblots the antiserum recognized fibronectin, a protein of approximately 100,000 Da (100-kDa), and a protein of approximately 60,000 Da (60 kDa). If added prior to cellular alignment, immunoglobulins from this serum inhibited fusion of both rat (L6) and mouse (C2) myoblasts in a dose-dependent fashion. To determine which component of this serum was responsible for fusion inhibition, antibodies against fibronectin, the 100- and 60-kDa proteins were microaffinity purified and tested, individually, for their effects on myoblast fusion. Antibodies against fibronectin had no effect on fusion. Antibodies against the 100-kDa protein released most cells from the substratum. Antibodies against the 60-kDa protein completely inhibited fusion. Fusion inhibition was accompanied by a corresponding inhibition of expression of two differentiation markers, creatine phosphokinase and the acetylcholine receptor. The 60-kDa protein was found, by immunoblot analysis, in smooth muscle-like cells (BC3H1 cells) and in variant L6 cells that do not differentiate and do not fuse. However, in the differentiation incompetent cells, the 60-kDa antigen appeared to be present in reduced amount. Indirect immunofluorescence of unpermeabilized L6 cells revealed alterations in the distribution of all three antigens during development. Fibronectin first appeared in long fibrillar arrays above the surface of cells that were beginning to align and fuse; fibronectin was not present on myotubes. The 100-kDa protein was seen initially in prominent fibrillar projections at the tips of prefusion myoblasts. During fusion the antigen was observed at sites of cell-cell contact and on extracellular vesicles. The 100-kDa protein appeared to be less abundant on myotubes. The 60-kDa protein first appeared in regions of cell-cell contact on cells that were beginning to align and fuse. As. fusion progressed, the 60-kDa protein was also found in extracellular vesicles. The 60-kDa protein was not observed on myotubes. As a result of this study we have identified two previously undescribed cell surface proteins involved in rodent skeletal myogenesis. The first is an approximately 100-kDa protein involved in early interactions of skeletal myoblasts with their substratum. The second is an approximately 60-kDa protein involved in myoblast differentiation. Both proteins are shed from the myoblast surface during myotube formation.