Biomedical subjects

Karina F Meiri

Publications and source records attributed to Karina F Meiri.

6 recordsLinked to original sources

PubMed2005

Lipid rafts and regulation of the cytoskeleton during T cell activation.

The ability of polarized cells to initiate and sustain directional responses to extracellular signals is critically dependent on direct communication between spatially organized signalling modules in the membrane and the underlying cytoskeleton. Pioneering work in T cells has shown that the assembly of signalling modules critically depends on the functional compartmentalization of membrane lipids into ordered microdomains or lipid rafts. The significance of rafts in T cell activation lies not only in their ability to recruit the signalling partners that eventually assemble into a mature immunological synapse but also in their ability to regulate actin dynamics and recruit cytoskeletal associated proteins, thereby achieving the structural polarization underlying stability of the synapse-a critical prerequisite for activation to be sustained. Lipid rafts vary quite considerably in size and visualizing the smallest of them in vivo has been challenging. Nonetheless it is now been shown quite convincingly that a surprisingly large proportion-in the order of 50%-of external membrane lipids (chiefly cholesterol and glycosphingolipids) can be dynamically localized in these liquid ordered rafts. Complementary inner leaflet rafts are less well characterized, but contain phosphoinositides as an important functional component that is crucial for regulating the behaviour of the actin cytoskeleton. This paper provides an overview of the interdependency between signalling and cytoskeletal polarization, and in particular considers how regulation of the cytoskeleton plays a crucial role in the consolidation of rafts and their stabilization into the immunological synapse.

Actins↗

PubMed2004

Membrane/cytoskeleton communication.

Accumulations of particular lipids in ordered arrays in the membrane (termed microdomains or lipid rafts) can attract proteins with specific targeting domains. Both the lipid and protein components of rafts communicate with the cytoskeleton directly thereby regulating cellular responses. Recent evidence implicating phosphoinositide 1,5 bisphosphate (PIP2) in cytoskeletal regulation shows that agonist sensitive regulation of PIP2 homoeostasis occurs specifically rafts, which appear to provide a major structural substrate for its function. The crucial role of PIP2 in generating cytoskeletal responses is chiefly achieved by regulating proteins that control actin dynamics directly. Many of these regulatory proteins are also specifically enriched in rafts either directly (by insertion into the lipid bilayer via acetylation motifs), or indirectly via interactions with other raft components. The notion that rafts form membrane platforms or modules that mediate signaling responses has been most extensively demonstrated in the immune synapse (IS) of T cells, a complex assemblage of rafts that integrates signaling cascades originating from the simultaneous activation of a wide variety of receptors. The IS is essential for both the amplification and maintenance of T-cell activation, and its assembly at the antigen presenting site depends on the interactions between rafts and the actin cytoskeleton that regulates coalescence of smaller raft components into the larger IS complex. Likewise the neuron, which represents the most highly polarized cell in the body, utilizes the regulation of actin dynamics in response to a plethora of extracellular signals to control axon pathfinding thereby sculpting nervous system cytoarchitecture with utmost precision. It is now becoming clear, that as in the T-cell, lipid rafts in the growing axon can assemble into highly specific, yet malleable and dynamic, signaling modules that regulate actin dynamics in a fashion that is also PIP2-dependent and that utilizes both familiar and novel regulatory mechanisms. It seems clear that raft mediated cytoskeletal regulation represents a highly conserved mechanism to integrate cellular responses to diverse signals.

Actins↗

PubMed2004

Failure to express GAP-43 leads to disruption of a multipotent precursor and inhibits astrocyte differentiation.

The nervous system-specific protein GAP-43 is significantly upregulated in neurons and glia that are differentiating. In P19 EC cells that do not express GAP-43, neurogenesis is inhibited; many immature neurons apoptose and the survivors do not mature morphologically. Here we show that the initial defect is in an early precursor with characteristics of a neural stem cell, which failed to respond normally to retinoic acid (RA). As a consequence, its progeny had altered cell fates: In addition to the neuronal defects previously reported, RC1-labeled radial glia failed to exit the cell cycle, accumulated, and failed to acquire GFAP immunoreactivity. However, leukemia inhibitory factor (LIF) could stimulate GFAP expression suggesting that astrocytes not derived from radial glia are less affected by absence of GAP-43. Differentiation of radial glia-derived astrocytes was also inhibited in glial cultures from GAP-43 (-/-) cerebellum, and in GAP-43 (-/-) telencephalon in vivo, differentiation of astrocytes derived from both radial and nonradial glia lineages were both affected: In the glial wedge, GFAP-labeled radial glia-derived astrocytes were reduced consistent with the interpretation that they may be unable to deflect GAP-43 (-/-) commissural axons toward the midline. At the midline, both radial and nonradial glia-derived astrocytes were also decreased although it fused normally. The results demonstrate that GAP-43 expressed in multipotent precursors is required for appropriate cell fate commitment, and that its absence affects astrocyte as well as neuronal differentiation.

Animals↗

PubMed2002

Growth-associated protein-43 is required for commissural axon guidance in the developing vertebrate nervous system.

Growth-associated protein-43 (GAP-43) is a major growth cone protein whose phosphorylation by PKC in response to extracellular guidance cues can regulate F-actin behavior. Here we show that 100% of homozygote GAP-43 (-/-) mice failed to form the anterior commissure (AC), hippocampal commissure (HC), and corpus callosum (CC) in vivo. Instead, although midline fusion was normal, selective fasciculation between commissural axons was inhibited, and TAG-1-labeled axons tangled bilaterally into Probst's bundles. Moreover, their growth cones had significantly smaller lamellas and reduced levels of F-actin in vitro. Likewise, 100% of GAP-43 (+/-) mice with one disrupted allele also showed defects in HC and CC, whereas the AC was unaffected. Individual GAP-43 (+/-) mice could be assigned to two groups based on the amount that PKC phosphorylation of GAP-43 was reduced in neocortical neurons. In mice with approximately 1% phosphorylation, the HC and CC were absent, whereas in mice with approximately 10% phosphorylation, the HC and CC were smaller. Both results suggest that PKC-mediated signaling in commissural axons may be defective. However, although Probst's bundles formed consistently at the location of the glial wedge, both GAP-43 (-/-) and GAP-43 (+/+) cortical axons were still repulsed by Slit-2 in vitro, precluding failure of this deflective signal from the glial wedge as the source of the phenotype. Nonetheless, the data show that a functional threshold of GAP-43 is required for commissure formation and suggests that failure to regulate F-actin in commissural growth cones may be related to inhibited PKC phosphorylation of GAP-43.

Agenesis of Corpus Callosum↗

PubMed2002

Changes in protein kinase C (PKC) activity, isozyme translocation, and GAP-43 phosphorylation in the rat hippocampal formation after a single-trial contextual fear conditioning paradigm.

The hippocampus plays an important role in spatial learning and memory. However, the biochemical alterations that subserve this function remain to be fully elucidated. In this study, rats were subjected to a single-trial contextual fear conditioning (CFC) paradigm; the activation of different protein kinase C (PKC) subtypes and the levels and phosphorylation of the plasticity-associated protein GAP-43 were assayed in the hippocampus at varying times after training. We observed a rapid activation of hippocampal PKC (15 min through 24 h), with differential translocation of the PKC isotypes studied. At early times after CFC (15-90 min), PKCalpha and PKCgamma translocated to the membrane, while PKCbetaII and PKCepsilon moved more transiently (15 to 30 min) to the cytosol. These PKC isotypes returned to the membrane at later time points after CFC. Correlating with these changes in PKC translocation and activity, there was an early decrease in GAP-43 phosphorylation followed by a more sustained increase from 1.5-72 h. GAP-43 protein levels were also increased after 3 h, and these levels remained elevated for at least 72 h. These changes in PKC and GAP-43 were specific to the CFC trained animals and no changes were seen in animals exposed to the same stimuli in a non-associative fashion. Comparison of translocation of different PKC isotypes with the changes in GAP-43 phosphorylation suggested that PKCbetaII and PKCepsilon may mediate both the early changes in the phosphorylation of this protein and the increases in GAP-43 expression at later times after CFC.

Animals↗

PubMed2002

Isolation and characterization of detergent-resistant microdomains responsive to NCAM-mediated signaling from growth cones.

It is still largely unclear how cell adhesion molecule (CAM)-mediated signaling evokes responses from the growth cone cytoskeleton. Here we used TX-114 extraction of growth cones followed by equilibrium gradient centrifugation to isolate subfractions of detergent-resistant microdomains (DRMs) that could be structurally and functionally distinguished on the basis of localization and activation of components of CAM-mediated signaling pathways. DRMs enriched in cholesterol, caveolin, NCAM140, GPI-linked NCAM120, fyn, and GAP-43, all conventional markers of microdomains or rafts, were located in areas 2 and 3 of the gradient. Coimmunoprecipitation of specific components of CAM signaling pathways by GAP-43 then identified distinct subpopulations of DRMs. GAP-43 from area 2 DRMs coprecipitated GPI-linked NCAM120 and was inactive, i.e., PKC phosphorylation had not been stimulated. In contrast the GAP-43 from area 3 DRMs coprecipitated both transmembrane NCAM140 and caveolin and was active, i.e., highly phosphorylated by PKC. A different subset of DRMs from both area 2 and area 3 contained fyn that could not be coprecipitated with GAP-43 antibodies. In this case area 2 DRMs contained activated fyn that was phosphorylated on Y415. In contrast area 3 DRMs contained inactive fyn. Hence fyn and GAP-43, both targets of NCAM signaling, are located in distinct populations of DRMs, and their activated forms are reciprocally distributed on the gradient. A detergent-resistant membrane fraction recovered from area 4 was enriched in NCAM140, phosphorylated GAP-43, and actin, but not cholesterol, caveolin, or fyn. Immunoelectron microscopy revealed that phosphorylated GAP-43 was localized where the membranes and F-actin interacted. Our results provide evidence for NCAM-mediated signaling in DRMs and suggest that the DRMs responsible for fyn and PKC/GAP-43-mediated NCAM signaling are structurally distinct and differentially distributed in growth cones.

Actins↗