Sharon K. Powell

Neuronal laminins and their cellular receptors

The laminins are a family of extracellular matrix glycoproteins expressed throughout developing neural tissues. The laminins are potent stimulators of neurite outgrowth in vitro for a variety of cell types, presumably reflecting an in vivo role in stimulating axon outgrowth. In recent years, the laminins have been shown to occur in several distinct isoforms; currently, the precise functional differences between the laminin variants are not well understood. A variety of neuronal surface receptors have been identified for one laminin isoform, laminin-1. These receptors include several members of the integrin family, as well as non-integrin laminin-binding proteins such as LBP-110, the 67 kDa laminin-receptor, alpha-dystroglycan, and beta 1,4 galactosyltransferase. Little is currently known about receptors for other laminin isoforms.

Identification of Cell Binding Sequences in Mouse Laminin γ1 Chain by Systematic Peptide Screening

Laminin-1, a major component of basement membranes, consists of three different chains designated α1, β1, and γ1 and has diverse biological functions. We have identified cell binding sites on the mouse laminin γ1 chain, using systematic screening of 165 overlapping synthetic peptides covering the entire chain. We identified 12 cell binding sequences using HT-1080 human fibrosarcoma and B16-F10 mouse melanoma cells in two independent assays employing peptide-conjugated Sepharose beads and peptide-coated dishes. Four peptides (C-16, C-28, C-64, and C-68) located on the globular domains of the γ1 chain were the most active and showed dose-dependent cell attachment. Cell attachment to C-68 was inhibited by EDTA and by anti-α2β1integrin antibodies. Cell attachment to C-16 and C-64 was partially inhibited by EDTA but was not inhibited by anti-integrin antibodies. EDTA and anti-integrin antibodies did not affect cell attachment to C-28. The four peptides were tested in adhesion and differentiation assays with endothelial, neuronal, and human salivary gland cells. C-16 was the most active for all of the cells, whereas the other three peptides showed cell type specificity in their activities. The active core sequences of C-16, C-28, C-64, and C-68 are YVRL, IRVTLN, TTVKYIFR, and SIKIRGTY, respectively. These sequences are highly conserved among the different species and in the laminin γ2 chain. These results suggest that the specific sequences on the laminin γ1 chain are biologically active and interact with distinct cell surface receptors.

Development of polarity in cerebellar granule neurons

Axon formation in developing cerebellar granule neurons in situ is spatially and temporally segregated from subsequent neuronal migration and dendrite formation. To examine the role of local environmental cues on early steps in granule cell differentiation, the sequence of morphologic development and polarized distribution of membrane proteins was determined in granule cells isolated from contact with other cerebellar cell types. Granule cells cultured at low density developed their characteristic axonal and dendritic morphologies in a series of discrete temporal steps highly similar to those observed in situ, first extending a unipolar process, then long, thin bipolar axons, and finally becoming multipolar, forming short dendrites around the cell body. Axonal- and dendritic-specific cytoskeletal markers were segregated to the morphologically distinct domains. The cell surface distribution of a specific class of endogenous glycoproteins, those linked to the membrane by a glycosylphosphatidyl inositol (GPI) anchor, was also examined. The GPI-anchored protein, TAG-1, which is segregated to the parallel fiber axons in situ, was found exclusively on granule cell axons in vitro; however, two other endogenous GPI-anchored proteins were found on both the axonal and somatodendritic domains. These results demonstrate that granule cells develop polarity in a cell type-specific manner in the absence of the spatial cues of the developing cerebellar cortex.

Identification of Endothelial Cell Binding Sites on the Laminin γ1 Chain

Abstract—The laminins belong to a family of trimeric basement membrane glycoproteins with multiple domains, structures, and functions. Endothelial cells bind laminin-1 and form capillary-like struc...

The Neural Cell Adhesion Molecule Expresses a Tyrosine-independent Basolateral Sorting Signal

Transmembrane isoforms of the neural cell adhesion molecule, N-CAM (N-CAM-140 and N-CAM-180), are vectorially targeted from the trans-Golgi network to the basolateral domain upon expression in transfected Madin-Darby canine kidney cells (Powell, S. K., Cunningham, B. A., Edelman, G. M., and Rodriguez-Boulan, E. (1991) Nature 353, 76-77). To localize basolateral targeting information, mutant forms of N-CAM-140 were constructed and their surface distribution analyzed in Madin-Darby canine kidney cells. N-CAM-140 deleted of its cytoplasmic domain shows a non-polar steady state distribution, resulting from delivery from the trans-Golgi network to both the apical and basolateral surfaces. This result suggests that entrance into the basolateral pathway may occur without cytoplasmic signals, implying that apical targeting from the trans-Golgi network is not a default mechanism but, rather, requires positive sorting information. Subsequent construction and analysis of a nested set of C-terminal deletion mutants identified a region of 40 amino acids (amino acids 749-788) lacking tyrosine residues required for basolateral targeting. Addition of these 40 amino acids is sufficient to restore basolateral targeting to both the non-polar cytoplasmic deletion mutant of N-CAM as well as to the apically expressed cytoplasmic deletion mutant of the p75 low affinity neurotrophin receptor (p75NTR), indicating that this tyrosine-free sequence is capable of functioning independently as a basolateral sorting signal. Deletion of both cytoplasmic and transmembrane domains resulted in apical secretion of N-CAM, demonstrating that the ectodomain of this molecule carries recessive apical sorting information.

Laminin‐like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro

The basement membrane glycoprotein laminin‐1 is a potent stimulator of neurite outgrowth. Although a variety of laminin isoforms have been described in recent years, the role of alternative laminin isoforms in neural development remains largely uncharacterized. We found that a polyclonal antibody raised against the α1, β1, and γ1 chains of laminin‐1 and a monoclonal antibody raised against the α2 chain of laminin‐2 detect immunoreactive material in neuronal cell bodies in the developing mouse cerebellum. In addition, laminin‐1‐like immunoreactivity was found in cell types throughout the cerebellum, but laminin‐α2‐like immunoreactivity was restricted to the Purkinje cells. Purified laminin‐1 and laminin‐2 stimulated neurite outgrowth in primary cultures of mouse cerebellar granule neurons to a similar extent, whereas the synthetic peptides tested appeared to be active only for cell adhesion and not for stimulation of neurite outgrowth. The E8 proteolytic fragment of laminin‐1 contained full neurite outgrowth activity. The identity of laminins expressed in granule neurons was also examined by Western blotting; laminin‐like complexes were associated with the cell and appeared to have novel compositions. These results suggest that laminin‐like complexes play important roles in cerebellar development. J. Neurosci. Res. 54:233–247, 1998.

Neural cell response to multiple novel sites on laminin‐1

The basement membrane protein laminin‐1 is a potent stimulator of neurite outgrowth for a variety of neuronal cell types. Previous studies have identified neurite outgrowth activity in several distinct regions of the laminin‐1 molecule. In this study, 545 overlapping 12‐ to 14‐mer synthetic peptides, corresponding to most of the amino acid sequence of the α1, β1, and γ1 chains of laminin‐1, were screened for cell attachment and neurite outgrowth activity using primary cultures of mouse cerebellar granule neurons and two neuronal cell lines. We identified 48 peptides derived from novel regions of the laminin‐1 molecule that were positive for neural cell adhesion activity. Only the cerebellar cells were found to have true neurite outgrowth activity with certain of the peptides, whereas some peptides induced short spike‐like process with the cell lines. Although 23 of these peptides were active on all 3 cell types screened, 25 others showed cell‐type specificity in their activity. These studies show that (1) there are multiple and distinct sites on laminin‐1 for cell adhesion and neurite‐like outgrowth and (2) that there are neural cell‐type‐specific active domains. The multiple active sites found explains, in part, the potent activity of laminin‐1 on neurite outgrowth. J. Neurosci. Res. 61:302–312, 2000. Published 2000 Wiley‐Liss, Inc.

The Generation of Polarity in Neuronal Cells

Publisher Summary This chapter reviews a number of recent developments that have provided clues to the molecular mechanisms underlying the generation of polarity in neuronal cells. Neurons are divided broadly into two domains, the axonal domain and the somato-dendritic domain. These two domains differ in a number of features. Usually only one axon extends from the cell body; this long process is often unbranched or infrequently branched until it reaches its synaptic target, where it divides into fine branches with swellings at the pre-synaptic terminals that are the specialized sites for neurotransmitter release. Neurons contain many proteins known to play a role in intracellular targeting in other cell types, but for the most part the function of these proteins in neurons has not been directly tested. This is largely because of technical difficulties in obtaining sufficient amounts of purified populations of primary neurons for biochemical assays. Studies of neuronal polarity are dependent on molecular-biological approaches, such as the use of anti-sense oligonucleotides to inhibit the expression of known membrane-trafficking components and the use of viral vectors to express exogenous proteins.