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C S Gillespie

Publications and source records attributed to C S Gillespie.

15 recordsLinked to original sources
PubMed2007

Modelling the checkpoint response to telomere uncapping in budding yeast.

One of the DNA damage-response mechanisms in budding yeast is temporary cell-cycle arrest while DNA repair takes place. The DNA damage response requires the coordinated interaction between DNA repair and checkpoint pathways. Telomeres of budding yeast are capped by the Cdc13 complex. In the temperature-sensitive cdc13-1 strain, telomeres are unprotected over a specific temperature range leading to activation of the DNA damage response and subsequently cell-cycle arrest. Inactivation of cdc13-1 results in the generation of long regions of single-stranded DNA (ssDNA) and is affected by the activity of various checkpoint proteins and nucleases. This paper describes a mathematical model of how uncapped telomeres in budding yeast initiate the checkpoint pathway leading to cell-cycle arrest. The model was encoded in the Systems Biology Markup Language (SBML) and simulated using the stochastic simulation system Biology of Ageing e-Science Integration and Simulation (BASIS). Each simulation follows the time course of one mother cell keeping track of the number of cell divisions, the level of activity of each of the checkpoint proteins, the activity of nucleases and the amount of ssDNA generated. The model can be used to carry out a variety of in silico experiments in which different genes are knocked out and the results of simulation are compared to experimental data. Possible extensions to the model are also discussed.

Cell Cycle↗
PubMed2001

Specific disruption of a schwann cell dystrophin-related protein complex in a demyelinating neuropathy.

Dystroglycan-dystrophin complexes are believed to have structural and signaling functions by linking extracellular matrix proteins to the cytoskeleton and cortical signaling molecules. Here we characterize a dystroglycan-dystrophin-related protein 2 (DRP2) complex at the surface of myelin-forming Schwann cells. The complex is clustered by the interaction of DRP2 with L-periaxin, a homodimeric PDZ domain-containing protein. In the absence of L-periaxin, DRP2 is mislocalized and depleted, although other dystrophin family proteins are unaffected. Disruption of the DRP2-dystroglycan complex is followed by hypermyelination and destabilization of the Schwann cell-axon unit in Prx(-/-) mice. Hence, the DRP2-dystroglycan complex likely has a distinct function in the terminal stages of PNS myelinogenesis, possibly in the regulation of myelin thickness.

Animals↗
PubMed2000

Peripheral demyelination and neuropathic pain behavior in periaxin-deficient mice.

The Prx gene in Schwann cells encodes L- and S-periaxin, two abundant PDZ domain proteins thought to have a role in the stabilization of myelin in the peripheral nervous system (PNS). Mice lacking a functional Prx gene assemble compact PNS myelin. However, the sheath is unstable, leading to demyelination and reflex behaviors that are associated with the painful conditions caused by peripheral nerve damage. Older Prx-/- animals display extensive peripheral demyelination and a severe clinical phenotype with mechanical allodynia and thermal hyperalgesia, which can be reversed by intrathecal administration of a selective NMDA receptor antagonist We conclude that the periaxins play an essential role in stabilizing the Schwann cell-axon unit and that the periaxin-deficient mouse will be an important model for studying neuropathic pain in late onset demyelinating disease.

Animals↗
PubMed1998

Two PDZ domain proteins encoded by the murine periaxin gene are the result of alternative intron retention and are differentially targeted in Schwann cells.

Periaxin was first described as a 147-kDa protein that was suggested to have a potential role in the initiation of myelin deposition in peripheral nerves based upon its abundance, cell type specificity, pattern of developmental expression, and localization (Gillespie, C. S., Sherman, D. L., Blair, G. E., and Brophy. P. J. (1994) Neuron 12, 497-508). Here we show that the murine periaxin gene spans 20.6 kilobases and encodes two mRNAs of 4.6 and 5.2 kilobases that encode two periaxin isoforms, L-periaxin and S-periaxin of 147 and 16 kDa respectively. The larger mRNA is produced by a retained intron mechanism that introduces a stop codon and results in a truncated protein with an intron-encoded C terminus of 21 amino acids. Both proteins possess a PDZ domain at the N terminus; nevertheless, they are targeted differently in Schwann cells. Like other proteins that contain PDZ domains, L-periaxin is localized to the plasma membrane of myelinating Schwann cells: in contrast, S-periaxin is expressed diffusely in the cytoplasm. This suggests that proteins that contain this protein-binding module may also participate in protein-protein interactions at sites other than the cell cortex.

Alternative Splicing↗
PubMed1998

Transient expression of neurofascin by oligodendrocytes at the onset of myelinogenesis: implications for mechanisms of axon-glial interaction.

Cell adhesion molecules (CAMs) must play a crucial role in both the initiation and signalling of axon-glial contact. However, the proteins that permit myelinating oligodendrocytes to recognize the axons that they ensheath in the developing CNS are unknown. By a subtractive cDNA library strategy, we have identified neurofascin as a powerful candidate for such a molecule. Neurofascin is strongly but transiently up-regulated in oligodendrocytes at the onset of myelinogenesis. Once oligodendrocytes have engaged their target axons the protein plays no further part, since the expression of the gene declines precipitously, in contrast to that of the major myelin component proteolipid protein, which remains elevated. After the initial surge of neurofascin expression in oligodendrocytes, there is a shift to a predominantly neuronal localization that persists into adulthood. Hence neurofascin in oligodendrocytes is unlikely to serve a function in the stabilization of the multilamellar sheath around the axon. The major neurofascin isoform of oligodendrocytes contains the third fibronectin type 3 (FNIII) repeat but lacks the mucin-like domain which supports the view that neurofascin isoforms are differentially expressed in the nervous system. Among the genes that are up-regulated during the terminal differentiation of the oligodendrocyte, neurofascin is unique in displaying a transient pattern of expression at the early stages of myelination. We propose that this CAM not only has a role in mediating axon recognition but also signals axonal contact through its links with the actin cytoskeleton.

Animals↗
PubMed1997

Transient expression of the neurofilament proteins NF-L and NF-M by Schwann cells is regulated by axonal contact.

Expression of the genes that encode neurofilament proteins is considered to be confined normally to neurons. However, in demyelinating peripheral nerves Schwann cells upregulate the mRNA for the medium-sized neurofilament protein (NF-M), and cultured Schwann cells of the myelin-forming phenotype can also synthesize and incorporate NF-M protein into their intermediate filament (IF) cytoskeleton. The purpose of this study was to establish how axonal contact might influence glial neurofilament gene expression and regulate the synthesis of neurofilament proteins. We show that the gene encoding NF-M is expressed at early stages of differentiation in myelin-forming Schwann cells in vivo; nevertheless, little NF-M protein can be detected in these cells. The transient induction of NF-M mRNA is also apparent in dedifferentiating Schwann cells during Wallerian degeneration. In these Schwann cells the mRNAs for NF-M and NF-L (the smallest polypeptide), but not NF-H (the largest neurofilament subunit), are coordinately expressed. In contrast to differentiating myelin-forming Schwann cells, the cells of degenerating nerves express both NF-M and NF-L polypeptides. Restoration of axonal contact in the growing nerve stimulates the recapitulation of Schwann cell differentiation including the elevation of NF-M and NF-L mRNA expression. These results demonstrate that the transient induction of neurofilament mRNAs in Schwann cells is a feature of both differentiation and dedifferentiation. However translation of these mRNAs is confined to Schwann cells deprived of axonal contact either by nerve injury or by culture in the absence of axons. These findings suggest that the expression of the NF-M and NF-L polypeptides is an important characteristic of those Schwann cells that will contribute to the repair of damaged peripheral nerves.

Animals↗
PubMed1994

Periaxin, a novel protein of myelinating Schwann cells with a possible role in axonal ensheathment.

We report the cloning and subcellular localization of a novel Schwann cell-specific protein of 147 kd that we have named periaxin. Periaxin has a remarkable domain of repetitive pentameric units in the primary sequence. It is expressed in the first uncompacted whorls of membrane that ensheathe the axon, and further synthesis of the protein in the rat sciatic nerve parallels the deposition of myelin. In mature myelin, periaxin colocalizes with the myelin-associated glycoprotein in the cytoplasm-filled periaxonal regions of the sheath but is excluded from compact myelin. We propose that periaxin has a role in axon-glial interactions, possibly by interacting with the cytoplasmic domains of integral membrane proteins such as myelin-associated glycoprotein in the periaxonal regions of the Schwann cell plasma membrane.

Amino Acid Sequence↗
PubMed1992

Schwann cells of the myelin-forming phenotype express neurofilament protein NF-M.

Immature Schwann cells of the rat sciatic nerve can differentiate into myelin-forming or non-myelin-forming cells. The factors that influence this divergent development are unknown but certain markers such as galactocerebroside distinguish the two cell populations at an early stage of Schwann cell differentiation. Because myelination requires extensive changes in cell morphology, we have investigated the composition and structure of the Schwann cell cytoskeleton at a time when these cells become committed to myelination. Here we show that Schwann cells express a cytoskeletal protein of M(r) 145 before diverging into the myelin-forming path, i.e., before they acquire cell-surface galactocerobroside. The p145 protein has the characteristics of an intermediate filament (IF) protein and immunoelectron microscopy shows that it colocalizes with vimentin, which suggests that these two proteins can coassemble into IFs. Elevated intracellular cAMP levels, which can mimic some of the early effects of axons on Schwann cell differentiation, induced p145 synthesis, therefore, we conclude that myelin-forming Schwann cells express this protein at a very early stage in their development. Immunological comparisons with other IF proteins revealed a close similarity between p145 and the neurofilament protein NF-M; the identification of p145 as NF-M was confirmed by isolating and sequencing a full-length clone from a Schwann cell cDNA library. These data demonstrate that Schwann cells remodel their IFs by expressing NF-M before acquiring the myelin-forming phenotype and that IF proteins of the neurofilament-type are not restricted to neurons in the vertebrate nervous system.

Amino Acid Sequence↗
PubMed1990

Protein zero of peripheral nerve myelin: biosynthesis, membrane insertion, and evidence for homotypic interaction.

Protein zero (P0), an integral membrane glycoprotein synthesized by Schwann cells, is the major glycoprotein of peripheral nerve myelin. The predicted disposition of P0 with respect to the membrane bilayer postulates the existence of extracellular and intracellular domains, that mediate compaction of the myelin lamellae. We used in vitro translations programmed with sciatic nerve mRNA and cells transfected with a P0 cDNA construct to study the biosynthesis and topology of P0 in the bilayer. The behavior of P0 at the cell surface, when expressed under physiological conditions, was also examined. We have verified the topological predictions of an earlier model, derived from analysis of a P0 cDNA, and provide evidence that the extracellular domain of P0 mediates homotypically cell-cell interactions in the transfectants.

Amino Acid Sequence↗
PubMed1990

Distribution of myelin basic protein and P2 mRNAs in rabbit spinal cord oligodendrocytes.

Myelin basic protein (MBP) and P2 protein are small positively charged proteins found in oligodendrocytes of rabbit spinal cord. Both proteins become incorporated into compact myelin. We have begun investigations into the mechanisms by which MBP and P2 become incorporated into the myelin membrane. We find that P2, like the MBPs, is synthesized on free polysomes in rabbit spinal cord. Cell fractionation experiments reveal that rabbit MBP mRNAs are preferentially segregated to the peripheral myelinating regions whereas P2 mRNAs are predominantly localized within the perikaryon of the cell. In vitro synthesized rabbit MBP readily associates with membranes added to translation mixtures, whereas P2 protein does not. It is possible that P2 requires a "receptor" molecule, perhaps a membrane-anchored protein, for association with the cytoplasmic face of the myelin membrane.

Animals↗
PubMed1990

Biosynthesis of the myelin 2',3'-cyclic nucleotide 3'-phosphodiesterases.

We have investigated the site of synthesis of the 2',3'-cyclic nucleotide 3'-phosphodiesterases (CNPs I and II) in rat brain. Rapid kinetics of incorporation of CNPs into oligodendrocyte plasma membrane in the intact brain are consistent with their synthesis on free polysomes. This hypothesis was confirmed by the translation in vitro of RNA isolated from free and bound polysomes, respectively. Unlike myelin basic protein (MBP) mRNAs, CNP mRNAs are not enriched in a myelin-associated pool of RNA. MBPs, but not CNPs, were found to readily associate in vitro with membrane vesicles derived from rough endoplasmic reticulum. The avidity of MBPs in binding to membranes is probably related to the previously observed spatial segregation of MBP mRNAs into actively myelinating cellular processes of the oligodendrocyte. Such a segregation would ensure that newly synthesized MBPs are immediately incorporated into myelin. In contrast, the CNPs probably associate with the cytoplasmic surface of the oligodendrocyte plasma membrane through interaction with a membrane-bound receptor.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1989

Characterization of a cytoskeletal matrix associated with myelin from rat brain.

Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.

Animals↗
PubMed1985

The effect of polar head group substitution on phospholipid methylation and the beta-adrenergic response in C6 glial cells.

The membranes of intact C6 cells were enriched with phosphatidyldimethylethanolamine or phosphatidylmonomethylethanolamine. These cells showed enhanced rates of phospholipid methylation but this was not accompanied by an increased beta-adrenergic response. We conclude that phospholipid methylation is not coupled to the activation of adenylate cyclase in the beta-adrenergic response of C6 glial cells.

Animals↗