Biomedical subjects

Philippe Collas

Publications and source records attributed to Philippe Collas.

At least 19 recordsLinked to original sources

PubMed2007

Targeted gene delivery to differentiated skeletal muscle: a tool to study dedifferentiation.

Cellular dedifferentiation is required for functional regeneration in salamanders. Dedifferentiating multinucleate skeletal muscle gives rise to mononucleate cells during limb regeneration. Efficient methods and tools must be developed in order to understand the molecular cues underlying dedifferentiation. Here we describe a non-viral method to express extra-chromosomal DNA exclusively in terminally differentiated muscle without the need for cell purification steps. After cytoplasmic injection of various expression vectors into myotubes or myofibres, we detect long-lasting mRNA and protein expression in up to 70% of the injected cells. The combination of the transfection protocol with live imaging allows a time- and cost-effective screen of candidate genes in terminally differentiated muscle cells of both amphibian and mammalian origin.

Animals↗

PubMed2006

The KH-Tudor domain of a-kinase anchoring protein 149 mediates RNA-dependent self-association.

A-Kinase anchoring proteins (AKAPs) control the subcellular localization and temporal specificity of protein phosphorylation mediated by cAMP-dependent protein kinase. AKAP149 (AKAP1) is found in mitochondria and in the endoplasmic reticulum-nuclear envelope network where it anchors protein kinases, phosphatases, and a phosphodiesterase. AKAP149 harbors in its COOH-terminal part one KH and one Tudor domain, both known to be involved in RNA binding. We investigated the properties of the COOH-terminal domain of AKAP149. We show here that AKAP149 is a self-associating protein with RNA binding features. The KH domain of AKAP149 is sufficient for self-association in a RNA-dependent manner. The Tudor domain is not necessary for self-association, but it is required together with the KH domain for targeting to well-defined nuclear foci. These foci are spatially closely related to nucleolar subcompartments. We also show that the KH-Tudor-containing domain of AKAP149 binds RNA in vitro and in RNA coprecipitation experiments. AKAP149 emerges as a scaffolding protein involved in the integration of intracellular signals and possibly in RNA metabolism.

A Kinase Anchor Proteins↗

PubMed2006

CpG methylation profiles of endothelial cell-specific gene promoter regions in adipose tissue stem cells suggest limited differentiation potential toward the endothelial cell lineage.

In vivo endothelial commitment of adipose stem cells (ASCs) has scarcely been reported, and controversy remains on the contribution of ASCs to vascularization. We address the epigenetic commitment of ASCs to the endothelial lineage. We report a bisulfite sequencing analysis of CpG methylation in the promoters of two endothelial-cell-specific genes, CD31 and CD144, in freshly isolated and in cultures of ASCs before and after induction of endothelial differentiation. In contrast to adipose tissue-derived endothelial (CD31(+)) cells, freshly isolated ASCs display a heavily methylated CD31 promoter and a mosaically methylated CD144 promoter despite basal transcription of both genes. Methylation state of both promoters remains globally stable upon culture. Endothelial stimulation of ASCs in methylcellulose elicits phenotypic changes, marginal upregulation of CD31, and CD144 expression and restrictive induction of a CD31(+)CD144(+) immunophenotype. These events are accompanied by discrete changes in CpG methylation in CD31 and CD144 promoters; however, no global demethylation that marks CD31(+) cells and human umbilical vein endothelial cells occurs. Immunoselection of CD31(+) cells after endothelial stimulation reveals consistent demethylation of one CpG immediately 3' of the transcription start site of the CD31 promoter. Adipogenic or osteogenic differentiation maintains CD31 and CD144 methylation patterns of undifferentiated cells. Methylation profiles of CD31 and CD144 promoters suggest a limited commitment of ASCs to the endothelial lineage. This contrasts with the reported hypomethylation of adipogenic promoters, which reflects a propensity of ASCs toward adipogenic differentiation. Analysis of CpG methylation at lineage-specific promoters provides a robust assessment of epigenetic commitment of stem cells to a specific lineage.

Adipose Tissue↗

PubMed2006

Stable CpG hypomethylation of adipogenic promoters in freshly isolated, cultured, and differentiated mesenchymal stem cells from adipose tissue.

Mesenchymal stem cells from adipose tissue can differentiate into mesodermal lineages. Differentiation potential, however, varies between clones of adipose stem cells (ASCs), raising the hypothesis that epigenetic differences account for this variability. We report here a bisulfite sequencing analysis of CpG methylation of adipogenic (leptin [LEP], peroxisome proliferator-activated receptor gamma 2 [PPARG2], fatty acid-binding protein 4 [FABP4], and lipoprotein lipase [LPL]) promoters and of nonadipogenic (myogenin [MYOG], CD31, and GAPDH) loci in freshly isolated human ASCs and in cultured ASCs, in relation to gene expression and differentiation potential. Uncultured ASCs display hypomethylated adipogenic promoters, in contrast to myogenic and endothelial loci, which are methylated. Adipogenic promoters exhibit mosaic CpG methylation, on the basis of heterogeneous methylation between cells and of variation in the extent of methylation of a given CpG between donors, and both between and within clonal cell lines. DNA methylation reflects neither transcriptional status nor potential for gene expression upon differentiation. ASC culture preserves hypomethylation of adipogenic promoters; however, between- and within-clone mosaic methylation is detected. Adipogenic differentiation also maintains the overall CpG hypomethylation of LEP, PPARG2, FABP4, and LPL despite demethylation of specific CpGs and transcriptional induction. Furthermore, enhanced methylation at adipogenic loci in primary differentiated cells unrelated to adipogenesis argues for ASC specificity of the hypomethylated state of these loci. Therefore, mosaic hypomethylation of adipogenic promoters may constitute a molecular signature of ASCs, and DNA methylation does not seem to be a determinant of differentiation potential of these cells.

Adipogenesis↗

PubMed2006

Association of PP1 with its regulatory subunit AKAP149 is regulated by serine phosphorylation flanking the RVXF motif of AKAP149.

Reformation of the nuclear envelope at the end of mitosis involves the recruitment of the B-type lamin phosphatase PP1 to nuclear membranes by A-kinase anchoring protein AKAP149. PP1 remains associated to AKAP149 throughout G1 but dissociates from AKAP149 when AKAP149 is phosphorylated at the G1/S transition. We examine here the role of phosphorylation of serines flanking the RVXF PP1-binding motif of AKAP149, on PP1 anchoring. The use of AKAP149 peptides encompassing the RVXF motif and five flanking serines, either wild type (wt) or bearing S-->A or S-->D mutations, specifically shows that phosphorylation of S151 or S159 abolishes PP1 binding to immobilized AKAP149. Peptides with S151 or S159 as the only wt serine residue trigger dissociation of PP1 from immunoprecipitated AKAP149, whereas S151/159D mutants are ineffective. Furthermore, immunoprecipitated AKAP149 from purified G1-phase nuclear envelopes binds PKA and PKC in overlay assays. PKA binding to AKAP149 in vitro is unaffected by the presence of PKC or PP1, and similarly, PKC binding is independent of PKA or PP1. The immunoprecipitated AKAP149 complex contains PKA and PKC activities. Both AKAP149-associated PKA and PKC serine-phosphorylate immunoprecipitated AKAP149 in vitro; however, only PKC-mediated phosphorylation promotes dissociation of PP1 from the AKAP. The results suggest a putative temporally and spatially controlled mechanism promoting release of PP1 from AKAP149. AKAP149 emerges as a scaffolding protein for multiple protein kinases and phosphatases that may be involved in the integration of intracellular signals that converge at the nuclear envelope.

A Kinase Anchor Proteins↗

PubMed2006

In vitro reprogramming of nuclei and cells.

Directly turning a somatic cell type into another would be beneficial for producing replacement cells for therapeutic purposes. To this end, novel cell reprogramming strategies are being developed. We describe here methods for functionally reprogramming a somatic cell using an extract derived from another somatic cell type. The procedure involves reversible permeabilization of 293T fibroblasts, incubation of the permeabilized cells in a nuclear and cytoplasmic extract of T-cells, resealing of the "reprogrammed" cells, and culture for assessment of reprogramming. Reprogramming has been evidenced by nuclear uptake and assembly of transcription factors, induction of activity of a chromatin remodeling complex, changes in chromatin composition, activation of lymphoid cell-specific genes, and expression of T-cell-specific surface molecules. The system is likely to constitute a powerful tool to examine the processes of nuclear reprogramming, at least as they occur in vitro.

Bacterial Proteins↗

PubMed2006

On the way to reprogramming cells to pluripotency using cell-free extracts.

The functional reprogramming of a differentiated cell to pluripotency may present beneficial applications in regenerative medicine. Somatic cell nuclear transfer may offer this possibility, but technical hurdles and ethical guidelines currently prevent application of this technology in several countries. As a result, alternative approaches are being developed for altering cell fate. Recent non-nuclear transfer-based approaches for reprogramming somatic cells are discussed as well as ways to enhance their differentiation potential. These approaches include the fusion of differentiated cells with embryonic stem cells and the use of extract from pluripotent cells to reprogramme differentiated cells into multipotent or pluripotent cells.

Animals↗

PubMed2006

Isolation of stromal stem cells from human adipose tissue.

The stromal compartment of mesenchymal tissues is thought to harbor stem cells that display extensive proliferative capacity and multilineage potential. Stromal stem cells offer a potentially large therapeutic potential in the field of regenerative medicine. Adipose tissue contains a large number of stromal stem cells, is relatively easy to obtain in large quantities, and thus constitutes a very convenient source of stromal stem cells. Importantly, the number of stem cells obtained is compatible with extensive analyses of the cells in an uncultured, freshly isolated, form. This chapter describes procedures for isolating millions of highly purified stromal stem cells from human adipose tissue and methods of establishing polyclonal and monoclonal cultures of adipose tissue-derived stem cells.

Adipose Tissue↗

PubMed2006

Modulation of cell fate using nuclear and cytoplasmic extracts.

The direct transformation of one somatic cell type into another somatic cell type would be beneficial for producing isogenic replacement cells for therapeutic use. Various approaches for altering cell fate are being developed, including methods for differentiating stem cells isolated from somatic tissues. This chapter describes a procedure for turning one somatic cell type (the "donor" cell) into another somatic "target" cell type using cellular extracts. The method also can be used to promote differentiation of a somatic stem cell along a specific pathway. The procedure involves permeabilization of the donor cell, incubation of the permeabilized cell in a nuclear and cytoplasmic extract derived from the target cell type, the resealing of donor cell membrane, and culture. Cells can be analyzed for induction of new gene and protein expression, as well as for the establishment of cellular functions specific to the target cell type. We also describe a slight modification of the procedure to allow analysis of extract-induced chromatin remodeling in nuclei purified from somatic cells. Because large numbers of cells and nuclei can be treated in cell extracts and because extracts can be fractionated or supplemented with various agents, this system constitutes a powerful tool to examine the molecular mechanisms of nuclear reprogramming and of cell differentiation, at least as they take place in vitro.

Animals↗

PubMed2005

Induction of dedifferentiation, genomewide transcriptional programming, and epigenetic reprogramming by extracts of carcinoma and embryonic stem cells.

Functional reprogramming of a differentiated cell toward pluripotency may have long-term applications in regenerative medicine. We report the induction of dedifferentiation, associated with genomewide programming of gene expression and epigenetic reprogramming of an embryonic gene, in epithelial 293T cells treated with an extract of undifferentiated human NCCIT carcinoma cells. 293T cells exposed for 1 h to extract of NCCIT cells, but not of 293T or Jurkat T-cells, form defined colonies that are maintained for at least 23 passages in culture. Microarray and quantitative analyses of gene expression reveal that the transition from a 293T to a pluripotent cell phenotype involves a dynamic up-regulation of hundreds of NCCIT genes, concomitant with down-regulation of 293T genes and of indicators of differentiation such as A-type lamins. Up-regulated genes encompass embryonic and stem cell markers, including OCT4, SOX2, NANOG, and Oct4-responsive genes. OCT4 activation is associated with DNA demethylation in the OCT4 promoter and nuclear targeting of Oct4 protein. In fibroblasts exposed to extract of mouse embryonic stem cells, Oct4 activation is biphasic and RNA-PolII dependent, with the first transient rise of Oct4 up-regulation being necessary for the second, long-term activation of Oct4. Genes characteristic of multilineage differentiation potential are also up-regulated in NCCIT extract-treated cells, suggesting the establishment of "multilineage priming." Retinoic acid triggers Oct4 down-regulation, de novo activation of A-type lamins, and nestin. Furthermore, the cells can be induced to differentiate toward neurogenic, adipogenic, osteogenic, and endothelial lineages. The data provide a proof-of-concept that an extract of undifferentiated carcinoma cells can elicit differentiation plasticity in an otherwise more developmentally restricted cell type.

Carcinoma↗

PubMed2005

PNUTS enhances in vitro chromosome decondensation in a PP1-dependent manner.

PP1 (protein phosphatase-1) is a serine/threonine phosphatase involved in mitosis exit and chromosome decondensation. In the present study, we characterize the subcellular and subnuclear localization of PNUTS (PP1 nuclear targeting subunit), a nuclear regulatory subunit of PP1, and report a stimulatory role of PNUTS in the decondensation of prometaphase chromosomes in two in vitro systems. In interphase, PNUTS co-fractionates, together with a fraction of nuclear PP1, primarily with micrococcal nuclease-soluble chromatin. Immunofluorescence analysis shows that PNUTS is targeted to the reforming nuclei in telophase following the assembly of nuclear membranes and concomitantly with chromatin decondensation. In interphase cytosolic extract, ATP-dependent decondensation of prometaphase chromosomes is blocked by PP1-specific inhibitors. In contrast, a recombinant PNUTS(309-691) fragment accelerates chromosome decondensation. This decondensation-promoting activity requires the consensus RVXF PP1-binding motif of PNUTS, whereas a secondary, inhibitory PP1-binding site is dispensable. In a defined buffer system, PNUTS(309-691) also elicits decondensation in an exogenous PP1-dependent manner and, as in the cytosolic extract, a W401A (Thr401-->Ala) mutation that destroys PP1 binding abolishes this activity. The results illustrate an involvement of the PNUTS:PP1 holoenzyme in chromosome decondensation in vitro and argue that PNUTS functions as a PP1-targeting subunit in this process. We hypothesize that targeting of PNUTS to reforming nuclei in telophase may be a part of a signalling event promoting chromatin decondensation as cells re-enter interphase.

Cell Nucleus↗

PubMed2005

Long-term in vitro, cell-type-specific genome-wide reprogramming of gene expression.

We demonstrate a cell extract-based, genome-wide and heritable reprogramming of gene expression in vitro. Kidney epithelial 293T cells have previously been shown to take on T cell properties following a brief treatment with an extract of Jurkat T cells. We show here that 293T cells exposed for 1 h to a Jurkat cell extract undergo genome-wide, target cell-type-specific and long-lasting transcriptional changes. Microarray analyses indicate that on any given week after extract treatment, approximately 2500 genes are upregulated >3-fold, of which approximately 900 are also expressed in Jurkat cells. Concomitantly, approximately 1500 genes are downregulated or repressed, of which approximately 500 are also downregulated in Jurkat cells. Gene expression changes persist for over 30 passages ( approximately 80 population doublings) in culture. Target cell-type specificity of these changes is shown by the lack of activation or repression of Jurkat-specific genes by extracts of 293T cells or carcinoma cells. Quantitative RT-PCR analysis confirms the long-term transcriptional activation of genes involved in key T cell functions. Additionally, growth of cells in suspended aggregates, expression of CD3 and CD28 T cell surface markers, and interleukin-2 secretion by 293T cells treated with extract of adult peripheral blood T cells illustrate a functional nuclear reprogramming. Therefore, target cell-type-specific and heritable changes in gene expression, and alterations in cell function, can be promoted by extracts derived from transformed cells as well as from adult primary cells.

Adult↗

PubMed2005

Isolation and transcription profiling of purified uncultured human stromal stem cells: alteration of gene expression after in vitro cell culture.

Stromal stem cells proliferate in vitro and may be differentiated along several lineages. Freshly isolated, these cells have been too few or insufficiently pure to be thoroughly characterized. Here, we have isolated two populations of CD45-CD34+CD105+ cells from human adipose tissue which could be separated based on expression of CD31. Compared with CD31+ cells, CD31- cells overexpressed transcripts associated with cell cycle quiescence and stemness, and transcripts involved in the biology of cartilage, bone, fat, muscle, and neural tissues. In contrast, CD31+ cells overexpressed transcripts associated with endothelium and the major histocompatibility complex class II complex. Clones of CD31- cells could be expanded in vitro and differentiated into cells with characteristics of bone, fat, and neural-like tissue. On culture, transcripts associated with cell cycle quiescence, stemness, certain cytokines and organ specific genes were down-regulated, whereas transcripts associated with signal transduction, cell adhesion, and cytoskeletal +CD105+CD31- cells from human adipose tissue have stromal stem cell properties which may make them useful for tissue engineering.

Adipose Tissue↗

PubMed2004

Transient alteration of cell fate using a nuclear and cytoplasmic extract of an insulinoma cell line.

We report a transient modulation of cell fate in fibroblasts briefly exposed to an extract derived from the rat insulin-producing beta cell line, INS-1E. Primary fetal rat fibroblasts were reversibly permeabilized with Streptolysin O, incubated for 1h in a 15,000g INS-1E nuclear and cytoplasmic extract, resealed, and cultured. A first marker of change in cell fate was a reduction of cell and nuclear size within days of exposure to extract such that in some instances the fibroblasts resembled INS-1E cells. Second, two beta cell transcripts, Pdx-1 and insulin, were detected in the fibroblasts for up to 4 weeks. Third, (pro)insulin labeling was detected in 5-30% of the cells for a period of 8-14 days after incubation in extract. These phenotypes were absent from fibroblasts exposed to heat-treated INS-1E extracts, a human fibroblast cell line-derived extract or buffer. The results indicate that the extract of an insulinoma-derived cell line can promote at least a transient modification of cell fate towards a beta cell phenotype in non-beta cells. Because they are easily accessible, cell extracts may represent a practical source of material for investigating the mechanisms of alteration of a nuclear and cellular program.

Animals↗

PubMed2004

Differentiation of human adipose tissue stem cells using extracts of rat cardiomyocytes.

We report the differentiation of human adipose tissue stem cells (ATSCs) to take on cardiomyocyte properties following transient exposure to a rat cardiomyocyte extract. Reversibly permeabilized ATSCs were incubated for 1h in a nuclear and cytoplasmic extract of rat cardiomyocytes, resealed with CaCl(2), and cultured. Three weeks after exposure to extract, ATSCs expressed several cardiomyocyte markers including sarcomeric alpha-actinin, desmin, and cardiac troponin I, and displayed targeted expression of the gap junction protein connexin 43. Formation of binucleated and striated cells, and spontaneous beating in culture were also observed. A low proportion of intact ATSCs exposed to the extract also showed signs of alpha-actinin and connexin 43 expression. Additional evidence of differentiation was provided by induction of expression of nuclear lamin A/C, a marker of terminally differentiated cells, and a remarkable increase in cell cycle length. Together with our previous data, this study suggests that alteration of cell fate using cellular extracts may be applied to multiple cell types. Cell extracts may also prove useful for investigating the molecular mechanisms of stem cell differentiation.

Actinin↗

PubMed2004

Signalling to the nucleus via A-kinase anchoring proteins.

The cell nucleus is a highly dynamic organelle whose function and structure during the cell cycle is tightly controlled. A number of signals triggered by external stimuli or intracellular clocks are relayed to the nucleus by protein kinases and phosphatases. Specificity of action of kinases and phosphatases can be achieved by their recruitment into multiprotein complexes targeted to discrete subcellular or subnuclear loci. One class of molecules targeting signalling units within single complexes are A-kinase anchoring proteins or AKAPs. AKAPs not only target enzymes to their substrate but may also regulate enzyme activity. This chapter highlights the role of nuclear AKAPs in relaying and modulating protein kinase and phosphatase signals to the nucleus or chromosomes.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Cloned calves from chromatin remodeled in vitro.

We have developed a novel system for remodeling mammalian somatic nuclei in vitro prior to cloning by nuclear transplantation. The system involves permeabilization of the donor cell and chromatin condensation in a mitotic cell extract to promote removal of nuclear factors solubilized during chromosome condensation. The condensed chromosomes are transferred into enucleated oocytes prior to activation. Unlike nuclei of nuclear transplant embryos, nuclei of chromatin transplant embryos exhibit a pattern of markers closely resembling that of normal embryos. Healthy calves were produced by chromatin transfer. Compared with nuclear transfer, chromatin transfer shows a trend toward greater survival of cloned calves up to at least 1 mo after birth. This is the first successful demonstration of a method for directly manipulating the somatic donor chromatin prior to transplantation. This procedure should be useful for investigating mechanisms of nuclear reprogramming and for making improvements in the efficiency of mammalian cloning.

Animals↗

PubMed2003

In vitro modulation of the interaction between HA95 and LAP2beta by cAMP signaling.

The nuclear envelope mediates key functions by interacting with chromatin. We recently reported an interaction between the chromatin- and nuclear matrix-associated protein HA95 and the inner nuclear membrane integral protein LAP2beta, implicated in initiation of DNA replication (Martins et al. (2003) J. Cell Biol. 160, 177-188). Here, we show that in vitro, interaction between HA95 and LAP2beta is modulated by cAMP signaling via PKA. Exposure of an anti-HA95 immune precipitate from interphase HeLa cells to a mitotic extract promotes ATP-dependent release of LAP2beta from the HA95 complex. This coincides with Ser and Thr phosphorylation of HA95 and LAP2beta. Inhibition of PKA with PKI abolishes phosphorylation of HA95 and dissociation of LAP2beta from HA95, although LAPbeta remains phosphorylated. Antagonizing cAMP signaling in mitotic extract also abolishes the release of LAP2beta from HA95; however, disrupting PKA anchoring to A-kinase anchoring proteins has no effect. Inhibition of CDK activity in the extract greatly reduces LAP2beta phosphorylation but does not prevent LAP2beta release from HA95. Inhibition of PKC, MAP kinase, or CaM kinase II does not affect mitotic extract-induced dissociation of LAP2beta from HA95. PKA phosphorylates HA95 but not LAP2beta in vitro and elicits a release of LAP2beta from HA95. CDK1 or PKC phosphorylates LAP2beta within the HA95 complex, but neither kinase induces LAP2beta release. Our results indicate that in vitro, the interaction between HA95 and LAP2beta is influenced by a PKA-mediated phosphorylation of HA95 rather than by CDK1- or PKC-mediated phosphorylation of LAP2beta. This suggests an additional level of regulation of a chromatin-nuclear envelope interaction in dividing cells.

Animals↗