Myr 8, a novel unconventional myosin expressed during brain development associates with the protein phosphatase catalytic subunits 1alpha and 1gamma1.

Directed neuronal, astroglial, and oligodendroglial cell migrations comprise a prominent feature of mammalian brain development. Because molecular motor proteins have been implicated in a wide spectrum of processes associated with cell motility, we initiated studies to define the pool of myosins in migrating cerebellar granule neurons and type-1 neocortical astrocytes. Our analyses identified two isoforms of a novel unconventional myosin, which we have cloned, sequenced, and designated myr 8a and 8b (eighth unconventional myosin from rat). Phylogenetic analysis indicates that myr 8 myosins comprise a new class of myosins, which we have designated class XVI. The head domain contains a large N-terminal extension composed of multiple ankyrin repeats, which are implicated in mediating an association with the protein phosphatase 1 (PP1) catalytic subunits 1alpha and 1gamma. The motor domain is followed by a single putative light-chain binding domain. The tail domain of myr 8a is comparatively short with a net positive charge, whereas the tail domain of myr 8b is extended, bears an overall neutral charge, and reveals several stretches of poly-proline residues. Neither the myr 8a nor the myr 8b sequence reveals alpha-helical coiled-coil motifs, suggesting that these myosins exist as monomers. Both immunoblot and Northern blot analyses indicate that myr 8b is the predominant isoform expressed in brain, principally at developmental time periods. The structural features and restricted expression patterns suggest that members of this novel class of unconventional myosins comprise a mechanism to target selectively the protein phosphatase 1 catalytic subunits 1alpha and/or 1gamma in developing brain.

A comparison of caveolae and caveolin-1 to folate receptor alpha in retina and retinal pigment epithelium.

Caveolae are flask-shaped membrane invaginations present in most mammalian cells. They are distinguished by the presence of a striated coat composed of the protein, caveolin. Caveolae have been implicated in numerous cellular processes, including potocytosis in which caveolae are hypothesized to co-localize with folate receptor alpha and participate in folate uptake. Our laboratory has recently localized folate receptor alpha to the basolateral surface of the retinal pigment epithelium (RPE). It is present also in many other cells of the retina. In the present study, we asked whether caveolae were present in the RPE, and if so, whether their pattern of distribution was similar to folate receptor alpha. We also examined the distribution pattern of caveolin-1, which can be a marker of caveolae. Extensive electron microscopical analysis revealed caveolae associated with endothelial cells. However, none were detected in intact or cultured RPE. Laser scanning confocal microscopical analysis of intact RPE localized caveolin-1 to the apical and basal surfaces, a distribution unlike folate receptor alpha. Western analysis confirmed the presence of caveolin-1 in cultured RPE cells and laser scanning confocal microscopy localized the protein to the basal plasma membrane of the RPE, a distribution like that of folate receptor alpha. This distribution was confirmed by electron microscopic immunolocalization. The lack of caveolae in the RPE suggests that these structures may not be essential for folate internalization in the RPE.

Developmental expression, pattern of distribution, and effect on cell aggregation implicate a neuron-glial junctional domain protein in neuronal migration.

We developed a panel of monoclonal antibodies to cerebellar astroglial cells and selected for study those that revealed microdomain structures on the cell surface of neocortical and cerebellar astrocytes. One antibody, 15D7-AD7, recognized the approximately 72 kDa polypeptide doublet that was identified previously by the polyclonal antibody D4 as a component of the microdomain structure formed between migrating neurons and radial glial cell processes (Cameron and Rakic [1994] J. Neurosci. 14:3139-3155). Immunofluorescent localization studies reveal a spatial and temporal pattern of 15D7 immunoreactivity in multiple brain regions that correlates well with time periods when neuronal cell migration is a prominent morphogenetic event. In areas where the process of migration is underway, 15D7 immunoreactivity is detected simultaneously in both radial glial cells and cells that have the positional and morphologic features characteristic of migrating neurons. Subsequent to the completion of migration, immunoreactivity is detected in the transitional forms of radial glial cells and mature astrocytes, but not in neurons. Cell aggregation analyses reveal that 15D7 antibodies perturb the rate of aggregation for astrocyte-astrocyte, neuron-neuron, and mixed cell-cell combinations. Taken together, the present studies suggest that the polypeptides recognized by the 15D7 antibodies likely participate in an adhesive process, principally within the ventricular and subventricular zones, that is essential at the onset of the cell migration process.

Identification of caveolin and caveolin-related proteins in the brain.

Caveolae are 50-100 nm, nonclathrin-coated, flask-shaped plasma membrane microdomains that have been identified in most mammalian cell types, except lymphocytes and neurons. To date, multiple functions have been ascribed to caveolae, including the compartmentalization of lipid and protein components that function in transmembrane signaling events, biosynthetic transport functions, endocytosis, potocytosis, and transcytosis. Caveolin, a 21-24 kDa integral membrane protein, is the principal structural component of caveolae. We have initiated studies to examine the relationship of detergent-insoluble complexes identified in astrocytes to the caveolin-caveolae compartment detected in cells of peripheral tissues. Immunolocalization studies performed in astrocytes reveal caveolin immunoreactivity in regions that correlate well to the distribution of caveolae and caveolin determined in other cell types, and electron microscopic studies reveal multiple clusters of flask-shaped invaginations aligned along the plasma membrane. Immunoblot analyses demonstrate that detergent-insoluble complexes isolated from astrocytes are composed of caveolin-1alpha, an identification verified by Northern blot analyses and by the cloning of a cDNA using reverse transcriptase-PCR amplification from total astrocyte RNA. Using a full-length caveolin-1 probe, Northern blot analyses suggest that the expression of caveolin-1 may be regulated during brain development. Immunoblot analyses of detergent-insoluble complexes isolated from cerebral cortex and cerebellum identify two immunoreactive polypeptides with apparent molecular weight and isoelectric points appropriate for caveolin. The identification of caveolae microdomains and caveolin-1 in astrocytes and brain, as well as the apparent regulation of caveolin-1 expression during brain development, identifies a cell compartment not detected previously in brain.

Colocalization of synaptophysin with transferrin receptors: implications for synaptic vesicle biogenesis.

We have reported previously that the synaptic vesicle (SV) protein synaptophysin, when expressed in fibroblastic CHO cells, accumulates in a population of recycling microvesicles. Based on preliminary immunofluorescence observations, we had suggested that synaptophysin is targeted to the preexisting population of microvesicles that recycle transferrin (Johnston, P. A., P. L. Cameron, H. Stukenbrok, R. Jahn, P. De Camilli, and T. C. Südhof. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:2863-2872). In contrast to our results, another group reported that expression of synaptophysin in cells which normally do not express SV proteins results in the generation of a novel population of microvesicles (Leube, R. E., B. Wiedenmann, and W. W. Franke. 1989. Cell. 59:433-446). We report here a series of morphological and biochemical studies conclusively demonstrating that synaptophysin and transferrin receptors are indeed colocalized on the same vesicles in transfected CHO cells. These observations prompted us to investigate whether an overlap between the distribution of the two proteins also occurs in endocrine cell lines that endogenously express synaptophysin and other SV proteins. We have found that endocrine cell lines contain two pools of membranes positive for synaptophysin and other SV proteins. One of the two pools also contains transferrin receptors and migrates faster during velocity centrifugation. The other pool is devoid of transferrin receptors and corresponds to vesicles with the same sedimentation characteristics as SVs. These findings suggest that in transfected CHO cells and in endocrine cell lines, synaptophysin follows the same endocytic pathway as transferrin receptors but that in endocrine cells, at some point along this pathway, synaptophysin is sorted away from the recycling receptors into a specialized vesicle population. Finally, using immunofluorescent analyses, we found an overlap between the distribution of synaptophysin and transferrin receptors in the dendrites of hippocampal neurons in primary cultures before synapse formation. Axons were enriched in synaptophysin immunoreactivity but did not contain detectable levels of transferrin receptor immunoreactivity. These results suggest that SVs may have evolved from, as well as coexist with, a constitutively recycling vesicular organelle found in all cells.

Synaptic vesicles immunoisolated from rat cerebral cortex contain high levels of glutamate.

L-Glutamate is regarded as the major excitatory neurotransmitter in the mammalian CNS. However, whether the released transmitter originates from a cytosolic pool or is discharged from synaptic vesicles by exocytosis (vesicle hypothesis) remains controversial. A problem with the general acceptance of the vesicle hypothesis is that the enrichment of glutamate in synaptic vesicles has not been convincingly demonstrated. In the present study, we have analyzed the glutamate content of synaptic vesicles isolated from rat cerebral cortex by a novel immunobead procedure. A large amount of glutamate was present in these vesicles when a proton electrochemical gradient was maintained across the vesicle membrane during isolation. Compared with the starting fraction, glutamate was enriched more than 10-fold relative to other amino acids. Addition of N-ethylmaleimide prevented glutamate loss during isolation. Isotope exchange experiments revealed that exchange or re-uptake of glutamate after homogenization is negligible. We conclude that rat brain synaptic vesicles contain high levels of glutamate in situ.

Synaptophysin is targeted to similar microvesicles in CHO and PC12 cells.

Synaptophysin, an integral membrane protein of small synaptic vesicles, was expressed by transfection in fibroblastic CHO-K1 cells. The properties and localization of synaptophysin were compared between transfected CHO-K1 cells and native neuroendocrine PC12 cells. Both cell types similarly glycosylate synaptophysin and sort it into indistinguishable microvesicles. These become labeled by endocytic markers and are primarily concentrated below the plasmalemma and at the area of the Golgi complex and the centrosomes. A small pool of synaptophysin is transiently found on the plasma membrane. In CHO-K1 cells synaptophysin co-localizes with transferrin that has been internalized by receptor-mediated endocytosis. These findings suggest that synaptophysin in transfected CHO-K1 cells and neuroendocrine PC12 cells is directed into a pathway of recycling microvesicles which, in CHO cells, is shown to coincide with that of the transferrin receptor. They further indicate that fibroblasts have the ability to sort a synaptic vesicle membrane protein. Our results suggest a pathway for the evolution of small synaptic vesicles from a constitutively recycling organelle which is normally present in all cells.

A common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands.

A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues--pancreatic, lacrimal, and submandibular--from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.