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Biomedical subjects

D G Jay

Publications and source records attributed to D G Jay.

At least 19 recordsLinked to original sources
PubMed1997

Essential functions of ezrin in maintenance of cell shape and lamellipodial extension in normal and transformed fibroblasts.

BACKGROUND: Changes in cell shape and motility are important manifestations of oncogenic transformation, but the mechanisms underlying these changes and key effector molecules in the cytoskeleton remain unknown. The Fos oncogene induces expression of ezrin, the founder member of the ezrin/radixin/moesin (ERM) protein family, but not expression of the related ERM proteins, suggesting that ezrin has a distinct role in cell transformation. ERM proteins have been suggested to link the plasma membrane to the actin-based cytoskeleton and are substrates and anchoring sites for a variety of protein kinases. Here, we examined the role of ezrin in cellular transformation. RESULTS: Fos-mediated transformation of Rat-1 fibroblasts resulted in an increased expression and hyperphosphorylation of ezrin, and a concomitant increased association of ezrin with the cortical cytoskeleton. We tagged ezrin with green fluorescent protein and examined its distribution in normal and Fos-transformed fibroblasts: ezrin was concentrated at the leading edge of extending pseudopodia of Fos-transformed Rat-1 cells, and was mainly cytosolic in normal Rat-1 cells. Functional ablation of ezrin by micro-CALI (chromophore-assisted laser inactivation) blocked plasma-membrane ruffling and motility of Fos-transformed fibroblasts. Ablation of ezrin in normal Rat-1 cells caused a marked collapse of the leading edge of the cell. CONCLUSIONS: Ezrin plays an important role in pseudopodial extension in Fos-transformed Rat-1 fibroblasts, and maintains cell shape in normal Rat-1 cells. The increased expression, hyperphosphorylation and subcellular redistribution of ezrin upon fibroblast transformation coupled with its roles in cell shape and motility suggest a critical role for ezrin in oncogenic transformation.

Animals↗
PubMed1996

Chromophore-assisted laser inactivation of a repulsive axonal guidance molecule.

BACKGROUND: The axons of retinal ganglion neurons from a precise topographic map in the optic tectum in the midbrain, and the guidance of retinal axons by directional cues in the tectum is crucial in this process. Several in vitro systems have been developed in order to identify the molecular basis of these directional cues. Temporal, but not nasal, retinal axons avoid posterior tectal membranes and grow on anterior membranes as a result of repellent guidance activities that are linked by glycosylphosphatidylinositol (GPI) anchors to the posterior membranes. A putative GPI-anchored repulsive guidance molecule with a molecular weight of 33 kDa has previously been characterized. Indirect results from experiments in vitro support the hypothesis that this 33 kDa molecule guides temporal retinal axons. RESULTS: To assess whether the 33 kDa protein is involved in axon guidance in vitro, we raised monoclonal antibodies against molecules that had been removed from tectal membranes by treatment with phosphatidylinositol-specific phospholipase C, which cleaves GPI anchors. A monoclonal immunoglobulin M, F3D4, recognized the 33 kDa molecule. In combination with chromophore-assisted laser inactivation, F3D4 caused a loss of the repellent activity from posterior tectal membranes in vitro. As a result, temporal retinal fibers were no longer repelled by posterior tectal membranes. This demonstrates that the F3D4 antigen, which we name RGM (repulsive guidance molecule) is involved in the guidance of retinal axons in an assay in vitro. In vivo, the expression of RGM increases from the anterior to the posterior pole of the optic tectum. CONCLUSIONS: These findings not only support the hypothesis that retinal axons are guided by gradients of repulsive guidance molecules but, in combination with earlier studies of receptor kinases and their ligands that act during guidance, argue for the presence of several repellent guidance molecules with similar functions in vitro and expression patterns in vivo.

Animals↗
PubMed1996

Function of myosin-V in filopodial extension of neuronal growth cones.

The molecular mechanisms underlying directed motility of growth cones have not been determined. The role of myosin-V, an unconventional myosin, in growth cone dynamics was examined by chromophore-assisted laser inactivation (CALI). CALI of purified chick brain myosin-V absorbed onto nitrocellulose-coated cover slips inhibited the ability of myosin-V to translocate actin filaments. CALI of myosin-V in growth cones of chick dorsal root ganglion neurons resulted in rapid filopodial retraction. The rate of filopodial extension was significantly decreased, whereas the rate of filopodial retraction was not affected, which suggests a specific role for myosin-V in filopodial extension.

Adenosine Triphosphate↗
PubMed1996

Talin and vinculin play distinct roles in filopodial motility in the neuronal growth cone.

Filopodial motility is critical for many biological processes, particularly for axon guidance. This motility is based on altering the F-actin-based cytoskeleton, but the mechanisms of how this occurs and the actin-associated proteins that function in this process remain unclear. We investigated two of these proteins found in filopodia, talin and vinculin, by inactivating them in subregions of chick dorsal root ganglia neuronal growth cones and by observing subsequent behavior by video-enhanced microscopy and quantitative morphometry. Microscale chromophore-assisted laser inactivation of talin resulted in the temporary cessation of filopodial extension and retraction. Inactivation of vinculin caused an increased incidence of filopodial bending and buckling within the laser spot but had no effect on extension or retraction. These findings show that talin acts in filopodial motility and may couple both extension and retraction to actin dynamics. They also suggest that vinculin is not required for filopodial extension and retraction but plays a role in the structural integrity of filopodia.

Animals↗
PubMed1996

Molecular mechanisms of directed growth cone motility.

Establishing molecular mechanisms of axon guidance presents one of the greatest challenges in understanding the development of the nervous system. There are many neurons, and each neuron by virtue of its location, biochemistry, and time of development, may generate a unique axon morphology in its response to environmental cues that may also change during development. The context dependence and combinatorial nature of these interactions make analysis of axon guidance particularly difficult. This article will focus on the neuronal growth cone as axon guidance is controlled by interaction of the growth cone with its environment. I present here an overview of growth cone motility from the perspective of cytoskeletal dynamics. I conclude with a discussion of our application of regional laser inactivation of growth cone proteins to address what proteins might be involved in locally modulating the cytoskeleton and how they affect growth cone motility.

Animals↗
PubMed1995

Asymmetric retraction of growth cone filopodia following focal inactivation of calcineurin.

The neuronal growth cone is thought to be the site of decision making in nerve growth and guidance. One likely mechanism of how the growth cone translates various extracellular cues into directed motility involves rises in intracellular calcium. A variety of physiological cues, such as adhesion molecules and neurotransmitters, increases intracellular calcium, and artificial manipulations of growth cone calcium levels affect growth cone morphology and neurite outgrowth. The molecular events downstream of calcium fluxes are incompletely understood. Here we show that calcineurin, a protein phosphatase enriched in growth cones that is dependent on calcium ions and calmodulin, functions in neurite outgrowth and directed filopodial motility in cultured chick dorsal root ganglia neurons. Cyclosporin A and FK506, inhibitors of calcineurin, delayed neuritogenesis and inhibited neurite extension. Chromophore-assisted laser inactivation of calcineurin in regions of growth cones causes localized filopodial and lamellipodial retraction and influences the direction of subsequent outgrowth. We suggest that a spatial distribution of calcineurin activity within the growth cone can regulate motility and direct outgrowth.

Animals↗
PubMed1995

Chromophore-assisted laser inactivation of subunits of the T-cell receptor in living cells is spatially restricted.

Chromophore-assisted laser inactivation (CALI) is a molecular photoablation technique that has been used to elucidate the in vivo roles of specific proteins in neural development. The interpretation of its effects on proteins in living cells relies on knowing how spatially restricted the CALI-induced damage is in vivo. To determine the spatial specificity of CALI in living cells, we have applied CALI to individual subunits of the T-cell receptor (TCR) complex on the surface of 2B4 hybridoma cells in culture and have examined the consequent structural and functional integrity of the TCR-alpha, TCR-beta and CD3-epsilon. The CALI of TCR-beta resulted in the disruption of the beta subunit and also resulted in a small effect on antibody binding alone to the neighboring TCR-alpha but caused no effect on another subunit, CD3-epsilon. Reciprocal experiments directing CALI to TCR-alpha and CD3-epsilon gave consistent results. No effects other than a simple loss of function were observed for any of these CALI experiments. These data demonstrate the extent of CALI-induced damage within a multisubunit complex in living cells and provide greater confidence for the future application of this technique to understanding in vivo function of proteins during complex cellular processes.

Animals↗
PubMed1994

Chromophore-assisted laser inactivation of proteins is mediated by the photogeneration of free radicals.

Chromophore-assisted laser inactivation (CALI) is a technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions and an understanding of its mechanism is required for its judicious application. We report here that CALI is not mediated by photoinduced thermal denaturation but by photogenerated free radicals. Thermal diffusion calculations suggest that the temperature changes resulting from CALI are too small to cause thermal denaturation, and Arrhenius plots of CALI are inconsistent with a photothermal mechanism. CALI shows an energy dose reciprocity above a threshold and can be inhibited by free-radical quenchers, thus demonstrating a photochemical mechanism of protein inactivation. The type of quenchers that are effective in inhibiting CALI indicates that the active species is a hydrogen abstractor which is not derived from molecular oxygen. We suggest that the active free-radical species is the hydroxyl radical and its very short lifetime explains the spatial specificity of CALI such that half-maximal damage is effected within 15 A from the dye moiety and no significant damage occurs at 34 A. The data are consistent with free-radical formation resulting from a sequential two-photon process.

Coloring Agents↗
PubMed1994

Chromophore-assisted laser inactivation of patched protein switches cell fate in the larval visual system of Drosophila.

The Drosophila segment-polarity gene patched (ptc) is an integral component of the segmentation gene cascade acting in the early embryo. At later stages of embryogenesis, ptc is expressed in the primordia of epithelial placodes of a specific portion of the brain, the optic lobes. Mutant analysis shows that the lack of ptc activity alters the fate of optic-lobe primordia precursors. In ptc mutants they give rise to supernumerary neurons in the larval light-sensory system, termed Bolwig organ, which is derived from precursor cells next to the optic-lobe anlagen. We specifically eliminated ptc protein by chromophore-assisted laser inactivation (CALI) in late wild-type embryos. Such embryos show a normal segment pattern, but they develop phenocopies equivalent to the phenotype of ptc mutant Bolwig organs. Our results demonstrate that the CALI technique can be applied to separate genetic functions at different developmental stages of a living organism and that the segment-polarity gene ptc is redeployed to functionally discriminate between distinct developmental pathways in adjacent pools of precursor cells.

Animals↗
PubMed1993

Fasciclin I and II have distinct roles in the development of grasshopper pioneer neurons.

We have used a new technique, micro-CALI (chromophore-assisted laser inactivation), to investigate the function of the neural cell adhesion molecules fasciclin I and II in the development of the grasshopper Ti1 neurons. Micro-CALI of fasciclin I results in defasciculation of the Ti1 axons similar to that achieved using large scale CALI (Jay and Keshishian, 1990). The initial point of axon separation corresponds to the site of laser irradiation, and defasciculation always continues distal to this point. Micro-CALI of fasciclin II prevents the initiation of Ti1 axon outgrowth but has no effect on fasciculation. This effect is restricted to a 3 hr interval between cytokinesis and growth cone emergence.

Animals↗
PubMed1993

Methods for ablating neurons.

This past year, laser ablation has been applied to investigations of neuromuscular connectivity in Drosophila, neuronal function in nematode, and mammalian central nervous system development. Ablation by targeted gene expression has been refined and applied to questions of neural development. Chromophore-assisted laser inactivation has been used to demonstrate distinct functions for two proteins during grasshopper neural development.

Animals↗
PubMed1992

Spatial specificity of chromophore assisted laser inactivation of protein function.

Chromophore assisted laser inactivation (CALI) is a new technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions. An understanding of aspects of the mechanism of CALI is required for its judicious application. A critical concern for CALI is its spatial specificity because nonspecific inactivation of neighboring unbound proteins by CALI is a possibility. We show here that CALI is very dependent on the distance between the chromophore and the protein such that there is no significant effect beyond 60 A. CALI using antibodies can inactivate other proteins through a complex but its efficacy decreases approximately fourfold for each intervening protein. These data imply that CALI is spatially specific and damage to neighboring proteins is unlikely.

Acetylcholinesterase↗
PubMed1990

Laser inactivation of fasciclin I disrupts axon adhesion of grasshopper pioneer neurons.

A molecular mechanism for selective axonal adhesion is a central question of neural development. Cell adhesion molecules have been identified, but it has been difficult to ascribe functions for these proteins in vivo. Here we show that the neuronal membrane glycoprotein fasciclin I has a role in the adhesion of sister axons during the development of the grasshopper limb bud. To do this we used a new technique, chromophore-assisted laser inactivation (CALI), which causes the precisely timed thermal denaturation of specific proteins by laser light targeted through a dye-labelled antibody, without any other observable damage to living cells. This can be achieved by relaxation of the laser-excited dye which releases heat to denature the bound protein; the rapid dissipation of heat with distance insulates unbound proteins from damage. CALI is a molecular analogue of cellular laser ablation and provides an unprecedented level of spatial and temporal resolution. Using dye-labelled antibodies that recognize fasciclin I, CALI disrupts fasciculation of the pioneer neurons without affecting their growth or guidance.

Animals↗
PubMed1988

Selective destruction of protein function by chromophore-assisted laser inactivation.

Chromophore-assisted laser inactivation of protein function has been achieved. After a protein binds a specific ligand or antibody conjugated with malachite green (C.I. 42,000), it is selectively inactivated by laser irradiation at a wavelength of light absorbed by the dye but not significantly absorbed by cellular components. Ligand-bound proteins in solution and on the surfaces of cells can be denatured without other proteins in the same samples being affected. Chromophore-assisted laser inactivation can be used to study cell surface phenomena by inactivating the functions of single proteins on living cells, a molecular extension of cellular laser ablation. It has an advantage over genetics and the use of specific inhibitors in that the protein function of a single cell within the organism can be inactivated by focusing the laser beam.

Chemical Phenomena↗
PubMed1987

Basic protein enhances the incorporation of DNA into lipid vesicles: model for the formation of primordial cells.

DNA can be encapsulated into lipid vesicles formed by sonication. The presence of a basic protein, lysozyme, enhances the incorporation 100-fold above the level expected by random trapping. This is demonstrated by the ability of the lipid vesicles to protect DNA from digestion with DNase. Such an enhancement of nuclei acid incorporation into vesicles by basic polypeptides and the sharply increased concentration of these macromolecules in the internal volume may have been advantageous in prebiotic evolution.

Biological Evolution↗
PubMed1986

Glycosylation site of band 3, the human erythrocyte anion-exchange protein.

The band 3 protein has a single glycosylation site on the carboxy-terminal 55 000-dalton tryptic fragment that defines a sequence of the polypeptide on the extracytoplasmic surface of the cell. To locate this site, a novel procedure involving end labeling of the 55 000-dalton tryptic fragment was used. Peptides resulting from partial proteolysis of the end radiolabeled glycoprotein were separated by lectin-Sepharose chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The smallest fragment observed defined the distance between the glycosylation site and the amino terminus. The procedure was first tested on a protein for which the location of the glycosylation site is known, HLA-B7 antigen. It was then used to show that the glycosylation site of human band 3 is 28 000 +/- 3000 daltons from the carboxy terminus of the protein.

Anion Exchange Protein 1, Erythrocyte↗