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D R Colman

Publications and source records attributed to D R Colman.

At least 37 records · Page 2Linked to original sources
PubMed1997

Conservation of topology, but not conformation, of the proteolipid proteins of the myelin sheath.

The proteolipid protein gene products DM-20 and PLP are adhesive intrinsic membrane proteins that make up >/=50% of the protein in myelin and serve to stabilize compact myelin sheaths at the extracellular surfaces of apposed membrane lamellae. To identify which domains of DM-20 and PLP are positioned topologically in the extracellular space to participate in adhesion, we engineered N-glycosylation consensus sites into the hydrophilic segments and determined the extent of glycosylation. In addition, we assessed the presence of two translocation stop-transfer signals and, finally, mapped the extracellular and cytoplasmic dispositions of four antibody epitopes. We find that the topologies of DM-20 and PLP are identical, with both proteins possessing four transmembrane domains and N and C termini exposed to the cytoplasm. Consistent with this notion, DM-20 and PLP contain within their N- and C-terminal halves independent stop-transfer signals for insertion into the bilayer of the rough endoplasmic reticulum during de novo synthesis. Surprisingly, the conformation (as opposed to topology) of DM-20 and PLP may differ, which has been inferred from the divergent effects that many missense mutations have on the intracellular trafficking of these two isoforms. The 35 amino acid cytoplasmic peptide in PLP, which distinguishes this protein from DM-20, imparts a sensitivity to mutations in extracellular domains. This peptide may normally function during myelinogenesis to detect conformational changes originating across the bilayer from extracellular PLP interactions in trans and trigger intracellular events such as membrane compaction in the cytoplasmic compartment.

Antibodies, Monoclonal↗
PubMed1997

The active transport of myelin basic protein into the nucleus suggests a regulatory role in myelination.

The myelin basic proteins (MBPs) are a set of membrane proteins that function to adhere the cytoplasmic leaflets of the myelin bilayer. During oligodendrocyte maturation prior to compact myelin formation, however, certain MBPs have been observed within the cell body and nucleus. We explored the parameters of the translocation of the exon II-containing MBPs (MBPexII) from the site of synthesis in the cell cytoplasm into the nucleus and in some experiments used GFP as a molecular reporter to monitor the intracellular distribution of MBP-GFP fusion proteins in living cells. We show here that the transport of MBPexII into cell nuclei is an active process, which is temperature and energy dependent, and may be regulated by phosphorylation state. Further, MBPexII can direct the entry of macromolecular complexes into cell nuclei, revealing that the exon II peptide segment may provide a nuclear localization signal (NLS), perhaps a novel one, or may induce a conformational change in the full-length protein that exposes a cryptic NLS. The MBPexII are thus very unusual in that they are plasma membrane proteins that are also targeted to the nucleus. In oligodendrocytes and Schwann cells, where the MBPs are naturally expressed, it is likely that karyophilic MBPs subserve a regulatory function in implementing the myelination program.

Biological Transport, Active↗
PubMed1997

Ischemic damage and subsequent proliferation of oligodendrocytes in focal cerebral ischemia.

In order to achieve a better understanding of the pathophysiology of ischemic white matter lesions, oligodendrocytic degeneration and subsequent proliferation were examined in the mouse model of middle cerebral artery occlusion. In situ hybridization histochemistry for proteolipid protein messenger RNA was employed as a sensitive and specific marker of oligodendrocytes, and immunohistochemistry for myelin basic protein was used as a compact myelin marker. Immunohistochemistry for microtubule-associated protein 2 and albumin was employed to monitor neuronal degeneration and the breakdown of the blood brain barrier, respectively. In the ischemic core of the caudoputamen, the immunoreactivity for microtubule-associated protein 2 disappeared and massive albumin extravasation occurred several hours after vessel occlusion, while proteolipid protein messenger RNA signals remained relatively strong at this time. The messenger RNA signals began to attenuate 12 h after ischemia and were hardly detectable 24 h after ischemia in the whole ischemic lesion. In situ end-labeling of fragmented DNA showed some cells with proteolipid protein messenger RNAs to have DNA fragmentation at this period. In contrast to proteolipid protein messenger RNA signals, the immunoreactivity for myelin basic protein was detected as long as five days after ischemia. An apparent increase in the cells possessing strong proteolipid protein messenger RNA signals was found five days after ischemia, mainly in the corpus callosum and the cortex bordering the infarcted areas. A double simultaneous procedure with in situ hybridization for proteolipid protein messenger RNA and immunohistochemistry for glial fibrillary acid protein or lectin histochemistry for macrophages/microglia showed proliferating oligodendrocytes to be co-localized with reactive astrocytes and macrophages/microglia. These findings show that oligodendrocytic damage occurred following ischemic neuronal damage and the breakdown of the blood brain barrier, but preceded the breakdown of myelin proteins in the ischemic lesion, that an apoptosis-like process was involved in ischemic oligodendrocytic death, and that surviving oligodendrocytes responded and proliferated in the outer border of the infarcted area.

Animals↗
PubMed1996

Cytoplasmic and nuclear localization of myelin basic proteins reveals heterogeneity among oligodendrocytes.

Myelin basic proteins (MBPs) are major proteins of central nervous system (CNS) myelin, where they facilitate the apposition of cytoplasmic faces of myelin lamellae. Myelin-bearing oligodendrocytes transport MBP mRNA to myelin, where newly translated protein is directly inserted into the myelin sheath. An apparent absence of MBPs in oligodendrocyte perikarya has suggested that MBP localized to the soma is translationally inert. We now demonstrate by confocal immunofluorescence microscopy that not only are MBPs present in the majority of oligodendrocyte perikarya but oligodendrocytes are heterogeneous with respect to their localization of MBPs; MBPs are concentrated in some cells at the plasmalemma and distributed in others throughout the cytoplasm and, surprisingly, the nucleus. MBPs are present in the nuclei of over half of oligodendrocytes in the adult, but in almost all MBP+oligodendrocytes during myelinogenesis. Transport of MBPs into nuclei appears to be a regulated process since some cells exhibit robust MBP accumulation in their cytoplasm but exclude MBPs from their nuclei. We show that oligodendrocyte nuclei contain all four major MBP isoforms, but that in transgenic mice, the epitope-tagged 14 kD MBP isoform preferentially segregates to the plasmalemma. Our data demonstrate that oligodendrocytes are not required to exclude MBPs from their perikarya and suggest that MBPs have a specific function in the oligodendrocyte perikarya and nucleus.

Animals↗
PubMed1996

Membrane adhesion and other functions for the myelin basic proteins.

The myelin basic proteins are a set of peripheral membrane polypeptides which play an essential role in myelination. Their most well-documented property is the unique ability to 'seal' the cytoplasmic aspects of the myelin membrane, but this is probably not the only function for these highly charged molecules. Despite extensive homology, the individual myelin basic proteins (MBPs) exhibit different expression patterns and biochemical properties, and so it is now believed that the various isoforms are not functionally equivalent in myelinating cells. We now think that while the major MBPs are intracellular adhesion molecules, some of the quantitatively less abundant isoforms that are expressed very early in development may have regulatory effects on the myelination program.

Animals↗
PubMed1996

Parallel evolution and coexpression of the proteolipid proteins and protein zero in vertebrate myelin.

Vertebrate myelin contains two proteins that mediate compaction: protein zero (P0), an immunoglobulin gene superfamily member, or proteolipid proteins, 4-hydrophobic domain-motif proteins biogenetically unrelated to P0. The prevailing view has been that expression of P0 and proteolipid proteins is mutually exclusive; P0, which mediates myelin compaction in fish, is thought to be completely replaced by the newer proteolipid proteins in the terrestrial vertebrate CNS. However, we now find that proteolipid proteins are actually major myelin constituents in bony fish and amphibia, and so are coexpressed with P0. Clearly, myelin proteolipids are not new additions to the myelin protein repertoire, but instead were ancestral sheath components, expressed approximately 440 million years ago in the first myelinated fish that existed at least approximately 100 million years before the origin of amphibians. In conclusion, P0 and the proteolipid proteins are evolving in parallel in myelinating cells of most vertebrate species.

Amino Acid Sequence↗
PubMed1996

A model for central synaptic junctional complex formation based on the differential adhesive specificities of the cadherins.

Cadherins control critical developmental events through well-documented homophilic interactions. In epithelia, they are hallmark constituents of junctions that mediate intercellular adhesion. Brain tissue expresses several cadherins, and we now show that two of these, neural (N)- and epithelial (E)-cadherin, are localized to synaptic complexes in mutually exclusive distributions. In cerebellum, N-cadherin is frequently found associated with synapses, some of which are perforated, and in hippocampus, N- and E-cadherin-containing synapses are found aligned along dendritic shafts within the stratum lucidum of CA3. We propose that the cadherins function as primary adhesive moieties between pre- and postsynaptic membranes in the synaptic complex. According to this model, once neurites have been guided to the vicinity of their cognate targets, it is the differential distribution of cadherins along the axonal and dendritic plasma membranes, and ultimately cadherin self-association, that "locks in" nascent synaptic connections.

Animals↗
PubMed1996

Crystal structure of the extracellular domain from P0, the major structural protein of peripheral nerve myelin.

P0, the major protein of peripheral nerve myelin, mediates membrane adhesion in the spiral wraps of the myelin sheath. We have determined the crystal structure of the extracellular domain from P0 (P0ex) at 1.9 A resolution. P0ex is folded like a typical immunoglobulin variable-like domain; five residues at the C-terminus are disordered, suggesting a flexible linkage to the membrane. The requirements for crystallization of P0ex are similar to those for maintaining the native extracellular spacing of adjacent myelin lamellae; thus, given the self-adhesive character of P0ex, the crystal itself may reveal some of the natural interactions that occur between P0 molecules in myelin. The structure leads to the suggestion that P0 extracellular domains may emanate from the membrane surface as tetramers that link to tetramers on the opposing membrane surface, to result in the formation of networks of molecules. We report analytical ultracentrifugation data for P0ex that support this idea.

Animals↗
PubMed1995

Myelin basic protein mRNA localization and polypeptide targeting.

Myelin basic proteins (MBPs), the major peripheral membrane proteins of central nervous system (CNS) myelin, are encoded by mRNAs that are selectively segregated to the myelinating processes of oligodendrocytes. In order to test whether the intracellular mechanisms responsible for MBP mRNA translocation are oligodendrocyte-specific, or alternatively, are present in other cell types and may therefore be more general, we have studied the localization of the 14 kD MBP mRNA and its encoded polypeptide (MBP14) in transiently transfected HeLa cells (a cervical carcinoma cell line) and in the rat pheochromocytoma cell line PC12. Unlike the situation in oligodendrocytes in situ, where MBP mRNAs are translocated and become "centrifugally" distributed, in both of the non-glial cells MBP mRNA was primarily detected in the perinuclear region. The MBP14 polypeptide was found associated with intracellular membranes, and not exclusively with the plasma membrane. Our results indicate that the inability of HeLa and PC12 cells to correctly target MBP mRNAs to the cell periphery leads to a failure to incorporate MBP polypeptides directly into the plasma membrane. Further, the data lend credence to the concept that MBP mRNA segregation appears to be a specific feature of myelin-forming cells which is required for the precise delivery of the encoded polypeptides to the forming myelin membrane.

Animals↗
PubMed1995

Considerations on the folding topology and evolutionary origin of cadherin domains.

Cell-cell adhesion in zonula adherens and desmosomal junctions is mediated by cadherins, and recent crystal structures of the first domain from murine N-cadherin provide a plausible molecular basis for this adhesive action. A structure-based sequence analysis of this adhesive domain indicates that its fold is common to all extracellular cadherin domains. The cadherin folding topology is also shown to be similar to immunoglobulin-like domains and to other Greek-key beta-sandwich structures, as diverse as domains from plant cytochromes, bacterial cellulases, and eukaryotic transcription factors. Sequence similarities between cadherins and these other molecules are very low, however, and intron patterns are also different. On balance, independent origins for a favorable folding topology seem more likely than evolutionary divergence from an ancestor common to cadherins and immunoglobulins.

Amino Acid Sequence↗
PubMed1995

Structural basis of cell-cell adhesion by cadherins.

Crystal structures of the amino-terminal domain of N-cadherin provide a picture at the atomic level of a specific adhesive contact between cells. A repeated set of dimer interfaces is common to the structure in three lattices. These interactions combine to form a linear zipper of molecules that mirrors the linear structure of the intracellular filaments with which cadherins associate. This cell-adhesion zipper may provide a mechanism to marshal individual molecular adhesive interactions into strong bonds between cells.

Amino Acid Sequence↗
PubMed1995

Novel E-cadherin-mediated adhesion in peripheral nerve: Schwann cell architecture is stabilized by autotypic adherens junctions.

Previous studies (Blank, W. F., M. B. Bunge, and R. P. Bunge. 1974. Brain Res. 67:503-518) showed that Schwann cell paranodal membranes were disrupted in calcium free medium suggesting that cadherin mediated mechanisms may operate to maintain the integrity of the paranodal membrane complex. Using antibodies against the fifth extracellular domain of E-cadherin, we now show by confocal laser and electron immunomicroscopy that E-cadherin is a major adhesive glycoprotein in peripheral nervous system Schwann cells. E-Cadherin is not found, however, in compact myelin bilayers. Rather, it is concentrated at the paranodes, in Schmidt-Lanterman incisures, and at the inner and outer loops. At these loci, E-cadherin is associated with subplasmalemmal electron densities that coordinate in register across several cytoplasmic turns of a single Schwann cell. F-Actin and beta-catenin, two proteins implicated in cellular signaling, also co-localize to E-cadherin positive sites. These complexes are autotypic adherens-type junctions that are confined to the plasma membrane synthesized by a single Schwann cell; E-cadherin was never observed between two Schwann cells, nor between Schwann cells and the axon. Our findings demonstrate that E-cadherin and its associated proteins are essential components in the architecture of the Schwann cell cytoplasmic channel network, and suggest that this network has specialized functions in addition to those required for myelinogenesis.

Amino Acid Sequence↗
PubMed1995

Protein zero, a nervous system adhesion molecule, triggers epithelial reversion in host carcinoma cells.

Protein zero (P(o)) is the immunoglobulin gene superfamily glycoprotein that mediates the self-adhesion of the Schwann cell plasma membrane that yields compact myelin. HeLa is a poorly differentiated carcinoma cell line that has lost characteristic morphological features of the cervical epithelium from which it originated. Normally, HeLa cells are not self-adherent. However, when P(o) is artificially expressed in this line, cells rapidly aggregate, and P(o) concentrates specifically at cell-cell contact sites. Rows of desmosomes are generated at these interfaces, the plasma membrane localization of cingulin and ZO-1, proteins that have been shown to be associated with tight junctions, is substantially increased, and cytokeratins coalesce into a cohesive intracellular network. Immunofluorescence patterns for the adherens junction proteins N-cadherin, alpha-catenin, and vinculin, and the desmosomal polypeptides desmoplakin, desmocollin, and desmoglein, are also markedly enhanced at the cell surface. Our data demonstrate that obligatory cell-cell adhesion, which in this case is initially brought about by the homophilic association of P(o) molecules across the intercellular cleft, triggers pronounced augmentation of the normally sluggish or sub-basal cell adhesion program in HeLa cells, culminating in suppression of the transformed state and reversion of the monolayer to an epithelioid phenotype. Furthermore, this response is apparently accompanied by an increase in mRNA and protein levels for desmoplakin and N-cadherin which are normally associated with epithelial junctions. Our conclusions are supported by analyses of ten proteins we examined immunochemically (P(o), cingulin, ZO-1, desmoplakin, desmoglein, desmocollin, N-cadherin, alpha-catenin, vinculin, and cytokeratin-18), and by quantitative polymerase chain reactions to measure relative amounts of desmoplakin and N-cadherin mRNAs. P(o) has no known signaling properties; the dramatic phenotypic changes we observed are highly likely to have developed in direct response to P(o)-induced cell adhesion. More generally, the ability of this "foreign" membrane adhesion protein to stimulate desmosome and adherens junction formation by augmenting well-studied cadherin-based adhesion mechanisms raises the possibility that perhaps any bona fide cell adhesion molecule, when functionally expressed, can engage common intracellular pathways and trigger reversion of a carcinoma to an epithelial-like phenotype.

Base Sequence↗
PubMed1994

Proteolipid protein interactions in transfectants: implications for myelin assembly.

The proteolipid proteins (PLP and DM20) are major constituents of CNS myelin, but how they are delivered to and organized within the oligodendrocyte plasma membrane is incompletely understood. We have expressed both PLP and DM20 singly or together in a host cell line, HeLa. In either DM20 or PLP transfectants, at early time points (24 hours), the expressed proteins are found within intracellular compartments. In DM20 transfectants, the protein is delivered to the plasma membrane by 48 hours. In HeLa cells, PLP remains intracellular when expressed in the absence of DM20; only when it is coexpressed with DM20 is it transported to the plasma membrane. In cotransfectants, PLP can also be localized to organelles involved in both the protein biosynthetic and the endocytic pathways. Since, in HeLa cells at least, the delivery of PLP to the plasma membrane is facilitated by the coexpression of DM20, we suggest that the two proteins interact intracellularly to form a complex. In some PLP/DM20 cotransfectants, the proteolipids are concentrated in regions of cell-cell contact. The regional accumulation of these proteins at cell-cell interfaces is highly reminiscent of the behavior in transfected cells of another myelin protein, P0, and certain cadherin polypeptides, both of which have readily demonstrable membrane adhesive properties. Our data suggests that at certain stoichiometric ratios, proteolipids can become stabilized at cell surfaces to form adhesive bonds.

Animals↗
PubMed1994

Tissue lipoproteins revisited: new proteolipid protein gene family members in elasmobranchs.

The proteolipids (PLPs) are abundant components of mammalian CNS myelin. Recombinant DNA methodologies have enabled us to search for evolutionary antecedents of PLP/DM20. Polymerase chain reactions of Torpedo and Squalus brain cDNA were performed with degenerate primers designed according to the mammalian PLP/DM20 sequence. Three DM20-related products (DM alpha, DM beta, and DM gamma) were amplified; no cDNAs containing the PLP-specific segment were found. Regions of the DM alpha and DM gamma are similar to the pore-forming segments of certain ligand-gated channels. In embryonic Squalus CNS, DM alpha and DM gamma appear to be co-expressed with P0. Antiserum raised against Torpedo DM alpha recognizes a protein in mouse CNS myelin, demonstrating that at least one of the newly recognized fish DMs is also in mammals. Our data, as well as that of other laboratories, supports the existence of a ubiquitously expressed proteolipid gene family.

Amino Acid Sequence↗
PubMed1993

The distribution of myelin basic protein mRNAs within myelinating oligodendrocytes.

The nervous system contains oligodendrocytes with processes that are greatly extended in space. It is now clear that there are numerous complex, poorly understood mechanisms by which polypeptides are synthesized and delivered to their sites of function in these cells. One mechanism is by the active positioning of mRNAs encoding certain proteins to restricted intracellular subdomains. Perhaps the best studied example of this in the vertebrate CNS is the translocation of myelin basic protein mRNAs to the forming myelin sheath, where the newly synthesized polypeptides, which avidly associate with membranes, can be directly incorporated into the myelin membrane. Evidence for this conclusion is presented here in the context of related work on the general phenomenon of mRNA translocation that is under analysis in other systems.

Animals↗