Biomedical subjects

Matthew J Hayes

Publications and source records attributed to Matthew J Hayes.

6 recordsLinked to original sources

PubMed2006

Regulation of actin dynamics by annexin 2.

Annexin 2 is a ubiquitous Ca(2+)-binding protein that is essential for actin-dependent vesicle transport. Here, we show that in spontaneously motile cells annexin 2 is concentrated in dynamic actin-rich protrusions, and that depletion of annexin 2 using siRNA leads to the accumulation of stress fibres and loss of protrusive and retractile activity. Cells co-expressing annexin 2-CFP and actin-YFP exhibit Ca(2+)-dependent fluorescense resonance energy transfer throughout the cytoplasm and in membrane ruffles and protrusions, suggesting that annexin 2 may directly interact with actin. This notion was supported by biochemical studies, in which we show that annexin 2 reduces the polymerisation rate of actin monomers in a dose-dependent manner. By measuring actin polymerisation rates in the presence of barbed-end and pointed-end cappers, we further demonstrate that annexin 2 specifically inhibits filament elongation at the barbed ends. These results show that annexin 2 has an essential role in maintaining the plasticity of the dynamic membrane-associated actin cytoskeleton, and that its activity in this context may be at least partly explained through direct interactions with polymerised and monomeric actin.

Actin Cytoskeleton↗

PubMed2004

Annexins and disease.

The annexins are a family of closely related calcium- and membrane-binding proteins expressed in most eukaryotic cell types. Despite their structural and biochemical similarities annexins have diverse functions, in cellular activities that include vesicle trafficking, cell division, apoptosis, calcium signalling, and growth regulation. To date there is no evidence to suggest that any individual member of the annexin family is a disease-causing gene, i.e., a gene that through loss, mutation, translocation or amplification leads to a known human disease. However, there is good evidence that in certain clinical conditions, changes in annexin expression levels or localisation may contribute to the pathological consequences and sequelae of disease. In this way, annexins are indirectly linked to some of the most serious human disease classes including cardiovascular disease and cancer. In this review we consider the roles played by annexins in disease and examine the molecular basis for anomalous annexin behaviour that may contribute to disease pathophysiology.

Animals↗

PubMed2004

Annexin 2 binding to phosphatidylinositol 4,5-bisphosphate on endocytic vesicles is regulated by the stress response pathway.

Annexin 2 is a Ca(2+)-binding protein that has an essential role in actin-dependent macropinosome motility. We show here that macropinosome rocketing can be induced by hyperosmotic shock, either alone or synergistically when combined with phorbol ester or pervanadate. Rocketing was blocked by inhibitors of phosphatidylinositol-3-kinase(s), p38 mitogen-activated protein (MAP) kinase, and calcium, suggesting the involvement of phosphoinositide signaling. Since various phosphoinositides are enriched on inwardly mobile vesicles, we examined whether or not annexin 2 binds to any of this class of phospholipid. In liposome sedimentation assays, we show that recombinant annexin 2 binds to phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5P(2)) but not to other poly- and mono-phosphoinositides. The affinity of annexin 2 for PtdIns-4,5P(2) (K(D) approximately 5 microm) is comparable with those reported for a variety of PtdIns-4,5P(2)-binding proteins and is enhanced in the presence of Ca(2+). Although annexin 1 also bound to PtdIns-4,5P(2), annexin 5 did not, indicating that this is not a generic annexin property. To test whether annexin 2 binds to PtdIns-4,5P(2) in vivo, we microinjected rat basophilic leukemia cells stably expressing annexin 2-green fluorescent protein (GFP) with fluorescently tagged antibodies to PtdIns-4,5P(2). Annexin 2-GFP and anti-PtdIns-4,5P(2) IgG co-localize at sites of pinosome formation, and annexin 2-GFP relocalizes to intracellular membranes in Ptk cells microinjected with Arf6Q67L, which has been shown to stimulate PtdIns-4,5P(2) synthesis on pinosomes through activation of phosphatidylinositol 5 kinase. These results establish a novel phospholipid-binding specificity for annexin 2 consistent with a role in mediating the interaction between the macropinosome surface and the polymerized actin tail.

Actins↗

PubMed2004

Annexin-actin interactions.

The actin cytoskeleton is a malleable framework of polymerised actin monomers that may be rapidly restructured to enable diverse cellular activities such as motility, endocytosis and cytokinesis. The regulation of actin dynamics involves the coordinated activity of numerous proteins, among which members of the annexin family of Ca2+- and phospholipid-binding proteins play an important role. Although the roles of annexins in actin dynamics are not understood at a mechanistic level, annexins have the requisite properties to integrate Ca2+-signaling with actin dynamics at membrane contact sites. In this review we discuss the current state of knowledge on this topic, and consider how and where annexins may fit into the complex molecular machinery that regulates the actin cytoskeleton.

Actins↗

PubMed2004

Basement membrane-dependent modification of phenotype and gene expression in human retinal pigment epithelial ARPE-19 cells.

PURPOSE: To use porcine lens capsule (PLC) as basement membrane for ARPE-19 cells and to characterize its effects on cell differentiation and gene expression. METHODS: Postconfluent cultures of ARPE-19 cells were established on either porous polyester filters or PLC membranes and characterized by electron microscopy, immunocytochemistry, and transepithelial electrical resistance measurements. Metabolic activity was assessed by measuring phagocytosis of rod outer segments. mRNA populations of ARPE-19 cells grown on polyester and PLC membranes were compared by suppressive subtractive hybridization. Differentially regulated messages were subsequently identified by DNA sequencing and their altered expression confirmed by Northern or virtual Northern blot analysis. RESULTS: Culture of ARPE-19 cells on PLC membrane induced the formation of apical microvilli and the ability to phagocytose rod outer segments. These culture conditions also led to enhanced junctional distribution of ZO-1 and occludin, the formation of polarized membrane domains, and a significant increase in transepithelial resistance. Gene expression was significantly altered by growth on PLC membranes and 29 differentially expressed transcripts were identified. CONCLUSIONS: Culture of ARPE-19 cells on PLC membranes resulted in a more differentiated phenotype and in expression of a specific set of transcripts encoding protein products that may affect epithelial differentiation, polarity and survival.

Animals↗

PubMed2002

RSC2, encoding a component of the RSC nucleosome remodeling complex, is essential for 2 microm plasmid maintenance in Saccharomyces cerevisiae.

The stable maintenance of the 2 microm circle plasmid depends on its ability to overcome intrinsic maternal inheritance bias, which in yeast normally results in the failure to transmit DNA molecules efficiently to daughter cells. In addition to the plasmid proteins Rep1 and Rep2 acting on the plasmid DNA locus STB, it is likely that other chromosomally encoded yeast proteins are required. We have isolated mutants of yeast unable to maintain 2 microm and found that RSC2 is essential for 2 microm to overcome maternal inheritance bias. Rsc2 is part of a multisubunit RSC chromatin remodeling complex, and we show that in the absence of Rsc2 the chromatin structure of the STB region is significantly altered and the Rep1 protein loses its normal localization to subnuclear foci. Rsc1, a closely related homolog of Rsc2 present in an alternative form of the RSC complex, is not required for 2 microm maintenance and does not replace the requirement for Rsc2 when overexpressed. This represents the first specific role for Rsc2 that has been related to a change in chromatin structure, as well as the first direct evidence linking chromatin structure to 2 microm segregation.

Chromatin↗