Biomedical subjects

V Lemmon

Publications and source records attributed to V Lemmon.

At least 37 records · Page 2Linked to original sources

PubMed1997

Growth cone interactions with purified cell and substrate adhesion molecules visualized by interference reflection microscopy.

The migration of growth cones on substrates consisting of naturally occurring cell adhesion molecules has been extensively studied in cell culture. However, relatively little is known about how growth cones contact the substrate or how the patterns of contact change as growth cones move forward. We have examined the interactions of chick retinal ganglion cell growth cones with laminin, merosin, N-cadherin, L1 and poly-L-lysine by time-lapse interference reflection microscopy (IRM) using a laser scanning confocal microscope. In images obtained by IRM, areas of a cell that are closely apposed to the substrate appear dark whereas areas that are farther away appear light. Growth cones on Jaminin and merosin were almost uniformly light, indicating that very little of the membrane was in close contact with the substrate. Growth cones on N-cadherin had a mottled appearance with some relatively large dark gray areas. The proximal portions of filopodia often were dark, in contrast to those on laminin and merosin which were light. In addition, growth cones on N-cadherin had numerous dark gray punctate regions of close association with the substrate. Growth cones on L1 had darker regions than growth cones on other substrates and these comprised a larger fraction of their area. There also were differences in the temporal dynamics of growth cone interactions with different substrates and these differences correlated with differences in rates of growth. None of the contacts observed in growth cones were as dark or stable as focal contacts of fibroblasts.

Animals↗

PubMed1997

The role of cell adhesion molecule L1 in axonal extension, growth cone motility, and signal transduction.

Axonal pathfinding is a complex process dependent on cell-cell and cell-matrix interactions. L1 is a cell adhesion molecule that is abundant in the nervous system and that is concentrated on axons. As a culture substrate, L1 is a potent promoter of neurite outgrowth and elicits specific growth cone behavior. It interacts with the actin cytoskeleton via an ankyrin linkage and promotes specific distribution of F-actin within the growth cone. In addition, L1 has been implicated in signal transduction. For example, L1 is associated with kinases, L1-L1 binding regulates second messenger systems, and mutations in the L1 gene in humans result in abnormalities in the development of the corticospinal tract and corpus callosum. In this short review, recent advances in understanding the way in which L1 regulates growth cone behavior will be discussed.

Animals↗

PubMed1997

CRASH syndrome: mutations in L1CAM correlate with severity of the disease.

X-linked hydrocephalus, MASA syndrome and certain forms of X-linked spastic paraplegia and agenesis of corpus callosum are now known to be due to mutations in the gene for the neural cell adhesion molecule L1 (19, 30). As a result, these syndromes have recently been reclassified as CRASH syndrome, an acronym for Corpus callosum hypoplasia, Retardation, Adducted thumbs, Spasticity and Hydrocephalus (8). A comparison of existing case reports with molecular genetic analysis reveals a striking correlation between the type of mutation in the L1CAM gene and the severity of the disease. Mutations that produce truncations in the extracellular domain of the L1 protein are more likely to produce severe hydrocephalus, grave mental retardation or early death than point mutations in the extracellular domain or mutations affecting only the cytoplasmic domain of the protein. While less severe than extracellular truncations, point mutations in the extracellular domain do produce more severe neurologic problems than mutations in just the cytoplasmic domain.

Abnormalities, Multiple↗

PubMed1996

N-cadherin expression and function in cultured oligodendrocytes.

N-Cadherin is a major cell adhesion molecule that is expressed in the developing nervous system where it has been implicated in neural migration and axon growth. Recently, a role for N-cadherin in oligodendrocyte differentiation has been identified [23]. Oligodendrocyte precursors adhere to N-cadherin and mature rapidly to produce myelin sheets. Since this implies that oligodendrocytes express N-cadherin, we examined the expression of N-cadherin by oligodendrocytes in culture. N-Cadherin was expressed by O-2A progenitors, immature oligodendrocytes and mature oligodendrocytes, but at a lower level than in type 1 astrocytes in the same cultures. On mature oligodendrocytes, the N-cadherin was concentrated on the major processes emerging from the soma. The ability of N-cadherin and merosin to promote oligodendrocyte precursor migration was also studied. Average migration rates were significantly higher on merosin (11.2 microns/h) than on N-cadherin (5.6 microns/h). These results suggest that N-cadherin is not likely to function predominantly as a substrate that stimulates migration of O-2A progenitors, but may be more important in initiating early oligodendrocyte-axon interactions that promote the process of myelination.

Animals↗

PubMed1996

Involvement of p90rsk in neurite outgrowth mediated by the cell adhesion molecule L1.

L1 is a neural cell adhesion molecule that has been shown to help guide nascent axons to their targets. This guidance is based on specific interactions of L1 with its binding partners and is likely to involve signaling cascades that alter cytoskeletal elements in response to these binding events. We have examined the phosphorylation of L1 and the role it may have in L1-directed neurite outgrowth. Cytosolic extracts from nerve growth factor-stimulated PC12 cells were fractionated by anion-exchange chromatography, and an activity was found that phosphorylated the cytoplasmic domain of L1. This activity was then assayed using a battery of L1-derived synthetic peptides. Based on these peptide assays and sequencing of radiolabeled L1 proteolytic fragments, the phosphorylation site was determined to be Ser1152. Western blot analysis demonstrated that the L1 kinase activity from PC12 cells that phosphorylated this site was co-eluted with the S6 kinase, p90(rsk). Moreover, S6 kinase activity and p90(rsk) immunoreactivity co-immunoprecipitate with L1 from brain, and metabolic labeling studies have demonstrated that Ser1152 is phosphorylated in vivo in the developing rat brain. The phosphorylation site is located in a region of high conservation between mammalian L1 sequences as well as L1-related molecules in vertebrates from fish to birds. We performed studies to investigate the functional significance of this phosphorylation. Neurons were loaded with peptides that encompass the phosphorylation site, as well as the flanking regions, and their effects on neurite outgrowth were observed. The peptides, which include Ser1152, inhibit neurite outgrowth on L1 but not on a control substrate, laminin. A nonphosphorylatable peptide carrying a Ser to Ala mutation did not affect neurite outgrowth on either substrate. These data demonstrate that the membrane-proximal 15 amino acids of the cytoplasmic domain of L1 are important for neurite outgrowth on L1, and the interactions it mediates may be regulated by phosphorylation of Ser1152.

Amino Acid Sequence↗

PubMed1996

L1, N-cadherin, and laminin induce distinct distribution patterns of cytoskeletal elements in growth cones.

Growth cones are highly motile extensions of growing neuronal processes that have a well-characterized cytoskeleton, which is necessary for motility and directed growth. In culture, neurons have been observed to extend processes on a variety of substrates made of cell adhesion molecules (CAMs) or extracellular matrix molecules (ECM molecules). We have previously shown that retinal ganglion cell (RGC) growth cones exhibit characteristic morphologies dependent on the substrate on which they are grown (Payne et al., 1992: Cell Motil. Cytoskel. 21:65-73). Upon contact with a sharp border between two substrates, the growth cones display rapid changes in morphology (Burden-Gulley et al., 1995: J. Neurosci. 15:4370-4381) that may result from extensive restructuring of the cytoskeleton. In the present study, immunocytochemical methods were used to examine the distribution of three cytoskeletal elements in RGC growth cones growing on L1, N-cadherin, or laminin as well as on dishes coated with alternating lanes of these substrates. Distinct distribution patterns of f-actin, microtubules (MTs), and neurofilaments (NFs) were observed in growth cones growing on individual substrates. At border regions between two substrates, growth cones with extensive lamellipodial contact with the second substrate were observed to have f-actin and MT distribution patterns appropriate for the new substrate encountered. Contact via filopodia alone did not evoke this change. Redistribution of NFs was observed only after the majority of the growth cone had crossed onto the second substrate. These results suggest that actin and MTs, but probably not NFs, are directly influenced by CAMs and ECM molecules to produce changes in growth cone morphology. The distribution of two members of the protein tyrosine kinase family, pp60arc and p59fyn, and phosphorylated tyrosine residues was also examined. No differences were observed in the distribution patterns of the kinases and phosphorylated tyrosine residues in growth cones on any of the substrate molecules tested. In addition, the distribution patterns were unchanged in growth cones that contacted and crossed borders between two substrates. These results suggest that redistribution of pp60arc or p59fyn is not required to produce alterations in growth cone morphology induced by contact with L1, N-cadherin or laminin.

Actins↗

PubMed1996

Transient coupling of Ng-CAM expression to NgCAM-dependent calcium signaling during migration of new neurons in the adult songbird brain.

The adult avian forebrain continues to generate neurons from subependymal zone (SZ) precursor cells, whose neuronal progeny migrate into the brain upon radial guide fibers. These neurons express the immunoglobulin-family adhesion molecule NgCAM, and their migration in culture is disrupted by anti-NgCAM Fab. Confocal imaging of adult zebra finch SZ loaded with the calcium indicator fluo-3, as well as ratio imaging with the indicator fura-2, revealed that migrating new neurons responded to microgram amounts of NgCAM with reversible increments in cytosolic calcium. The calcium response to NgCAM antigen was developmentally restricted, in that it was only manifested by neurons for roughly the 3- to 4-day period between 6 and 9 DIV, even though NgCAM expression persisted tonically thereafter. The period during which NgCAM elicited a calcium signal corresponded to the postmitotic age at which new, bipolar neurons leave the adult SZ to enter the brain parenchyma in vivo. Accordingly, the calcium response to NgCAM was largely limited to morphologically bipolar cells. Anti-NgCAM IgG also evoked a neuronal calcium signal over the same restricted period that NgCAM protein exerted its effect. These findings suggest a dynamic coupling and uncoupling of calcium-dependent signal transduction pathways to a stably expressed surface adhesion molecule, whose function in a given neuron may therefore evolve with cellular maturation.

Animals↗

PubMed1996

Casein kinase II phosphorylates the neural cell adhesion molecule L1.

L1 is an axonal cell adhesion molecule found primarily on projection axons of both the CNS and PNS. It is a phosphorylated membrane-spanning glycoprotein that can be immunoprecipitated from rat brain membranes in association with protein kinase activities. Western blot analysis demonstrates that casein kinase II (CKII), a ubiquitous serine/threonine kinase enriched in brain, is present in these immunoprecipitates. CKII preparations partially purified from PC12 cells are able to phosphorylate recombinant L1 cytoplasmic domain (L1CD), which consists of residues 1,144-1,257. Using these as well as more highly purified kinase preparations, phosphorylation assays of small peptides derived from the L1CD were performed. CKII was able to phosphorylate a peptide encompassing amino acids (aa) 1,173-1,185, as well as a related peptide representing an alternatively spliced nonneuronal L1 isoform that lacks aa 1,177-1,180. Both peptides were phosphorylated with similar kinetic profiles. Serine to alanine substitutions in these peptides indicate that the CKII phosphorylation site is at Ser1,181. This is consistent with experiments in which L1CD was phosphorylated by these kinase preparations, digested, and the radiolabeled fragments sequenced. Furthermore, when L1 immunoprecipitates were used to phosphorylate L1CD, one of the residues phosphorylated is the same residue phosphorylated by CKII. Finally, in vivo radiolabeling indicates that Ser1,181 is phosphorylated in newborn rat brain. These data show that CKII is associated with and able to phosphorylate L1. This phosphorylation may be important in regulating certain aspects of L1 function, such as adhesivity or signal transduction.

Amino Acid Sequence↗

PubMed1995

The cytoplasmic domain of the cell adhesion molecule L1 is not required for homophilic adhesion.

L1 is a highly conserved cell adhesion molecule with complete homology of the cytoplasmic domain between the known mammalian protein sequences. Since the cytoplasmic domains of other adhesion molecules have been shown to influence adhesion, we have investigated the effects of deletion of the cytoplasmic domain on the ability of L1 to mediate homophilic adhesion. Full length L1 and a truncated L1, lacking 95% of the cytoplasmic domain, were expressed in myeloma cells. Independent stable transfectants were assayed for the ability to form aggregates. Myelomas expressing L1 lacking the cytoplasmic domain were able to form cell aggregates as well as the myelomas expressing full length L1. Cell aggregate formation was correlated with the level of L1 expression, and the aggregation could be blocked by anti-L1 Fabs. Similar results were obtained in adhesion assays of the myeloma cells to substrate-bound L1. These results indicate that the cytoplasmic domain of L1 is not required for homophilic interactions.

Amino Acid Sequence↗

PubMed1995

Müller glia stabilizes cell columns during retinal development: lateral cell migration but not neuropil growth is inhibited in mixed chick-quail retinospheroids.

Radial columnar organization of cell clones is a characteristic feature of vertebrate retinae that is structurally not understood. Here we provide in vitro evidence that Müller glia processes stabilize cells within columns. Dissociated embryonic chick retinal plus pigmented cells regenerate in vitro into fully laminated stratospheroids. After reaggregating chick and quail cells, quail-derived spheroid areas are detected as isolated sectors, as shown by a quail-specific antibody. Each sector contains one or multiple cell columns. The radial borders separating chick and quail sectors are fully congruent with the extension of 3A7-labelled Müller glia processes. While cell somata do not show any lateral interspecies mixing, quail-derived neuropil extends within the inner plexiform areas far into chick sectors. After selective damage of Müller cells by the gliotoxin DL-alpha-aminoadipic acid, the columnar organization is destabilized, as evidenced by a decrease in vimentin expression and by the migration of individual neurons out of their cell column. These data demonstrate that Müller cells actively stabilize cells within their columns, while neuritic growth is not hindered.

Animals↗

PubMed1995

Two chondroitin sulfate proteoglycans differentially expressed in the developing chick visual system.

Two monoclonal antibodies, 2B9 and 9BA12, were used to identify and characterize two different chondroitin sulfate proteoglycans (CSPGs) associated with the embryonic chick visual system. Monoclonal antibody 2B9 recognizes a carbohydrate epitope of collagen type IX proteoglycan. Immunohistochemistry showed that collagen type IX proteoglycan was abundant in the vitreous body and meninges, but absent in brain and retina. In developing trunk regions, collagen type IX proteoglycan is segmentally distributed in the somites, appearing only in the posterior sclerotome. Monoclonal antibody 9BA12 recognizes collagen type IX proteoglycan from vitreous body and an unidentified chondroitin sulfate proteoglycan in retina and brain, herein referred to as 9BA12 CSPG. Immunohistochemistry showed that 9BA12 CSPG is present in the optic fiber layer of the retina, coinciding temporally and spatially with the onset and cessation of ganglion cell axon growth. In the trunk region, 9BA12 immunostaining appears in the developing spinal cord and throughout the sclerotome. In culture, neither the collagen type IX proteoglycan nor the brain-derived 9BA12 CSPG were able to support neurite outgrowth from retinal ganglion cell explants. In combination with basal lamina proteins, collagen type IX proteoglycan slightly inhibited neurite outgrowth and led to a stronger fasciculation of retinal axons. In contrast, 9BA12 CSPG had no inhibitory effect on the outgrowth of retinal axons and had no effect on their fasciculation. Our study demonstrates the existence of two chondroitin sulfate proteoglycans in the developing visual system of the chick. Based on the developmental expression and the results from neurite outgrowth experiments, it was concluded that the 9BA12 CSPG does not operate as a neurite outgrowth inhibitor for retinal axons.

Animals↗

PubMed1995

Unique changes of ganglion cell growth cone behavior following cell adhesion molecule perturbations: a time-lapse study of the living retina.

In the mammalian retina, multiple mechanisms are responsible for guiding retinal ganglion cell axons to the optic fissure. In the present study we have used time-lapse videomicroscopy to show that, within the center of the retinal neuroepithelium, growth cones use a scaffold of previously formed axons as a substrate for guidance. High magnification time-lapse videomicroscopy of normal growth cones in the midretina have shown that they have the ability to alter their shape from long, streamlined forms that hug other axons to more flattened forms that move between axons or neuroepithelial endfeet. In studies on the role of specific cell interactions in these events, Fab fragments against L1 and NCAM, administered either alone or in combination, were found to have dramatic and distinct effects on retinal ganglion cell growth cones. Anti-L1 Fab fragments severely disrupted radial growth cone orientation and rate of outgrowth. The anti-L1-treated growth cones initially stalled for 2 h, then changed direction and, thereafter, resumed an elongation rate twice as fast as in control preparations. By contrast, anti-NCAM Fab did not affect growth cone direction, but caused subsets of growth cones to speed up initially, then to dramatically increase in size, stall, and eventually halt. These results imply that L1 and NCAM play different roles in the promotion and direction of axon growth and, along with repulsive molecules and physical channels, provide essential information for the unidirectional growth of retinal axons into the optic fissure.

Animals↗

PubMed1995

Mutations in the cell adhesion molecule L1 cause mental retardation.

Recently, studies in the usually disparate fields of human genetics and developmental neurobiology have converged to reveal that some types of human mental retardation and brain malformations are due to mutations that affect the neural cell adhesion molecule L1. L1 has a very complex biology, interacting with a variety of ligands, and functioning in migration of neurons and growth of axons. Over the past few years, it has also become clear that L1 is able to influence intracellular second messengers. The identification of a number of different mutations in L1, some of which alter the extracellular portion of the molecule, and others that change only the cytoplasmic tail, confirm that L1 is a crucial player in normal brain development. The information gained from genetic analysis of human L1 is giving new insights into how L1 functions in the formation of major axon pathways, but it also raises unanticipated questions about how L1 participates in the development of cortical and ventricular systems.

Cell Adhesion Molecules↗

PubMed1995

CRASH syndrome: clinical spectrum of corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraparesis and hydrocephalus due to mutations in one single gene, L1.

L1 is a neuronal cell adhesion molecule with important functions in the development of the nervous system. The gene encoding L1 is located near the telomere of the long arm of the X chromosome in Xq28. We review here the evidence that several X-linked mental retardation syndromes including X-linked hydrocephalus (HSAS), MASA syndrome, X-linked complicated spastic paraparesis (SP1) and X-linked corpus callosum agenesis (ACC) are all due to mutations in the L1 gene. The inter- and intrafamilial variability in families with an L1 mutation is very wide, and patients with HSAS, MASA, SP1 and ACC can be present within the same family. Therefore, we propose here to refer to this clinical syndrome with the acronym CRASH, for Corpus callosum hypoplasia, Retardation, Adducted thumbs, Spastic paraplegia and Hydrocephalus.

Abnormalities, Multiple↗

PubMed1995

Growth cones are actively influenced by substrate-bound adhesion molecules.

As axons advance to appropriate target tissues during development, their growth cones encounter a variety of cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM molecules). Purified CAMs and ECM molecules influence neurite outgrowth in vitro and are thought to have a similar function in vivo. For example, when retinal ganglion cell (RGC) neurons are grown on different CAM and ECM molecule substrates in vitro, their growth cones display distinctive morphologies (Payne et al., 1992). Similarly, RGC growth cones in vivo have distinctive shapes at different points in the pathway from the eye to the tectum, suggesting the presence of localized cues that determine growth cone behaviors such as pathway selection at choice points. In this report, time-lapse video microscopy was utilized to examine dynamic transformations of RGC growth cones as they progressed from L1/8D9, N-cadherin, or laminin onto a different substrate. Contact made by the leading edge of a growth cone with a new substrate resulted in a rapid and dramatic alteration in growth cone morphology. In some cases, the changes encompassed the entire growth cone including those regions not in direct contact with the new substrate. In addition, the growth cones displayed a variety of behavioral responses that were dependent upon the order of substrate contact. These studies demonstrate that growth cones are actively affected by the substrate, and suggest that abrupt changes in the molecular composition of the growth cone environment are influential during axonal pathfinding.

Animals↗

PubMed1994

N-cadherin and Ng-CAM/8D9 are involved serially in the migration of newly generated neurons into the adult songbird brain.

In the adult avian forebrain, neurons continue to be produced in the subependymal zone (SZ), from which they migrate upon radial fibers. To identify ligands regulating this process, we studied N-cadherin and Ng-CAM/8D9 expression in HVC, a neurogenic region of the canary neostriatum. N-cadherin was relatively restricted to the SZ and was expressed by dividing, [3H]thymidine-labeled precursor cells. However, cellular N-cadherin was down-regulated prior to neuronal migration from the SZ. Addition of anti-N-cadherin Fab hastened neuronal migration from adult SZ explants, without influencing neuronal number. Unlike N-cadherin, Ng-CAM/8D9 was expressed by migrating neurons. Anti-8D9 Fab inhibited neuronal migration upon cultured ependymoglia, which did not express Ng-CAM/8D9. Thus, the departure of new neurons from the adult SZ may require their suppression of N-cadherin, whereas their subsequent migration and survival may depend upon neuronal expression of Ng-CAM/8D9 and its interaction with a heterophilic radial cell receptor.

Animals↗

PubMed1993

Molecular characterization of a multi-promoter gene encoding a chicken filamin protein.

We report the cloning and sequencing of cDNA encoding a chicken filamin protein. The 2,567 amino acid protein contains an NH2-terminal 267 amino acid actin-binding domain followed by a series of 24 repeating units that are each approximately 95 amino acids in length. The overall primary structure of filamin closely resembles that of human actin-binding protein (ABP). However, filamin lacks a 24-amino-acid insertion prior to repeat 16 that is contained within ABP. This region of human ABP is a site of calpain cleavage and is thought to confer flexibility on the molecule. Hence, it is possible that the properties of actin gels formed with either human ABP or filamin reflect the presence or absence of this insertion. Filamin is encoded within a multi-promoter transcription unit. A downstream filamin promoter (Fil1) resembles those of certain housekeeping genes and has a putative binding site for the transcription factor E2F. Thus, transcription from this promoter may be linked to the cell cycle. A second filamin promoter (Fil2) is located at least 8 kilobases upstream from the Fil1 promoter. This structural arrangement suggests that regulation of filamin gene expression is likely to be complex.

Amino Acid Sequence↗

PubMed1993

Migration of newly generated neurons upon ependymally derived radial guide cells in explant cultures of the adult songbird forebrain.

The adult songbird forebrain undergoes neuronal production throughout adulthood, with the production of new neurons in discrete regions of the neostriatal ventricular zone. Upon mitogenesis, these new neurons migrate into the subjacent brain parenchyma along radially directed guide fibers. In long-term ventricular zone explant cultures, derived from the higher vocal center of the adult canary, newly migratory neurons were found to associate preferentially with a characteristic substrate cell type. These small, parvonuclear substrate cells formed tightly packed epithelioid sheets, in which ciliated ependymal cells were common, as recognized by both live observation and electron microscopy. A subpopulation of these cells was immunostained by monoclonal antibody 3A7, which preferentially stains the guide fiber network of the adult avian brain. These 3A7+ cells included ependymal cells and bipolar radial cells, as well as morphologically defined astrocytes. As they matured in vitro, the 3A7+ bipolar radial cells extended long, unbranching fibers, which ultimately traversed the culture substrate. Like ependymal cells, they supported neuronal migration. These cells were likely homologous to radial guide cells in vivo. Thus, neuronal migration in adult avian forebrain culture occurred upon guide cells of ependymal derivation.

Animals↗