Jacques Baudier

S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage

During the postnatal development, astrocytic cells in the neocortex progressively lose their neural stem cell (NSC) potential, whereas this peculiar attribute is preserved in the adult subventricular zone (SVZ). To understand this fundamental difference, many reports suggest that adult subventricular GFAP‐expressing cells might be maintained in immature developmental stage. Here, we show that S100B, a marker of glial cells, is absent from GFAP‐expressing cells of the SVZ and that its onset of expression characterizes a terminal maturation stage of cortical astrocytic cells. Nevertheless, when cultured in vitro, SVZ astrocytic cells developed as S100B expressing cells, as do cortical astrocytic cells, suggesting that SVZ microenvironment represses S100B expression. Using transgenic s100b‐EGFP cells, we then demonstrated that S100B expression coincides with the loss of neurosphere forming abilities of GFAP expressing cells. By doing grafting experiments with cells derived from β‐actin‐GFP mice, we next found that S100B expression in astrocytic cells is repressed in the SVZ, but not in the striatal parenchyma. Furthermore, we showed that treatment with epidermal growth factor represses S100B expression in GFAP‐expressing cells in vitro as well as in vivo. Altogether, our results indicate that the S100B expression defines a late developmental stage after which GFAP‐expressing cells lose their NSC potential and suggest that S100B expression is repressed by adult SVZ microenvironment.

Effect of S-100 proteins and calmodulin on Ca2+-induced disassembly of brain microtubule proteins in vitro

The brain-specific S-100 protein, discovered in 1965 [l] is a mixture of two very similar proteins, the S-100a and S-100b protein. These proteins are dimers of highly homologous subunits: S-1OOa (a/I) and S-100b (/3/3) [2,3]. Both proteins are small (M, 20 000) very acidic and water soluble. It is assumed, that they are mainly located in the cytosol of glial cells [4] but they have also been found bound to membranes [5,6]. The biological activity of the S-100 proteins remains unknown. However, these proteins share two typical amino acid sequences in their primary structure, associated with the calcium-binding domain [7] which indicates that they also belong to the calcium-binding protein family, such as among others, calmodulin, troponin C and parvalbumin. Therefore, it has been proposed that a calciumsensitizing factor or factors should regulate the microtubule disassembly in vivo [ 10,111. Calmodulin has often been suggested [9,12,13] to have this role as it potentiates the disassembly effect of Ca2+ [ 121 and is found to be localized at the ends of the mitotic spindle [ 131. We now report the effect of S100 proteins on the Ca2+-induced disassembly of microtubule proteins in comparison with the effect of calmodulin. We found that S-100 protein induced disassembly of microtubules with a higher efficiency than calmodulin at mM Ca2 + levels.

AHNAK interaction with the annexin 2/S100A10 complex regulates cell membrane cytoarchitecture.

Remodelling of the plasma membrane cytoarchitecture is crucial for the regulation of epithelial cell adhesion and permeability. In Madin-Darby canine kidney cells, the protein AHNAK relocates from the cytosol to the cytosolic surface of the plasma membrane during the formation of cell–cell contacts and the development of epithelial polarity. This targeting is reversible and regulated by Ca2+-dependent cell–cell adhesion. At the plasma membrane, AHNAK associates as a multimeric complex with actin and the annexin 2/S100A10 complex. The S100A10 subunit serves to mediate the interaction between annexin 2 and the COOH-terminal regulatory domain of AHNAK. Down-regulation of both annexin 2 and S100A10 using an annexin 2–specific small interfering RNA inhibits the association of AHNAK with plasma membrane. In Madin-Darby canine kidney cells, down-regulation of AHNAK using AHNAK-specific small interfering RNA prevents cortical actin cytoskeleton reorganization required to support cell height. We propose that the interaction of AHNAK with the annexin 2/S100A10 regulates cortical actin cytoskeleton organization and cell membrane cytoarchitecture.

AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration

Mutations in dysferlin cause limb girdle muscular dystrophy 2B, Miyoshi myopathy and distal anterior compartment myopathy. Dysferlin is proposed to play a role in muscle membrane repair. To gain functional insight into the molecular mechanisms of dysferlin, we have searched for dysferlin‐interacting proteins in skeletal muscle. By coimmunoprecipitation coupled with mass spectrometry, we demonstrate that AHNAK interacts with dysferlin. We defined the binding sites in dysferlin and AHNAK as the C2A domain in dysferlin and the carboxyterminal domain of AHNAK by glutathione S‐transferase (GST)‐pull down assays. As expected, the N‐terminal domain of myoferlin also interacts with the carboxyterminal domain of AHNAK. In normal skeletal muscle, dysferlin and AHNAK colo‐calize at the sarcolemmal membrane and T‐tubules. In dysferlinopathies, reduction or absence of dysferlin correlates with a secondary muscle‐specific loss of AHNAK. Moreover, in regenerating rat muscle, dysfer‐lin and AHNAK showed a marked increase and cyto‐plasmic localization, consistent with the direct interaction between them. Our data suggest that dysferlin participates in the recruitment and stabilization of AHNAK to the sarcolemma and that AHNAK plays a role in dysferlin membrane repair process. It may also have significant implications for understanding the biology of AHNAK‐containing exocytotic vesicles, “en‐largosomes, ” in plasma membrane remodeling and repair.—Huang Y., Laval S. H., van Remoortere A., Baudier J., Benaud C., Anderson L. V. B., Straub V., Deelder A., Frants R. R., den Dunnen J. T., Bushby K., van der Maarel S. M. AHNAK, a novel component of the dysferlin protein complex, redistributes to the cytoplasm with dysferlin during skeletal muscle regeneration. FASEB J. 21, 732–742 (2007)

Nuclear expression of S100B in oligodendrocyte progenitor cells correlates with differentiation toward the oligodendroglial lineage and modulates oligodendrocytes maturation

The S100B protein belongs to the S100 family of EF-hand calcium binding proteins implicated in cell growth and differentiation. Here, we show that in the developing and the adult mouse brain, S100B is expressed in oligodendroglial progenitor cells (OPC) committed to differentiate into the oligodendrocyte (OL) lineage. Nuclear S100B accumulation in OPC correlates with the transition from the fast dividing multipotent stage to the morphological differentiated, slow proliferating, pro-OL differentiation stage. In the adult, S100B expression is down-regulated in mature OLs that have established contacts with their axonal targets, suggesting a nuclear S100B function during oligodendroglial cells maturation. In vitro, the morphological transformation and maturation of pro-OL cells are delayed in the absence of S100B. Moreover, mice lacking S100B show an apparent delay in OPC maturation in response to demyelinating insult. We propose that nuclear S100B participates in the regulation of oligodendroglial cell maturation.

Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis.

ABSTRACT In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5°C). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha-ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5°C, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G1 arrest in S100B-expressing REF (S100B-REF) cells at 37.5°C that is phenotypically indistinguishable from p53-mediated G1arrest at the permissive temperature (32°C). S100B-REF cells proceeding from G1 underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis.

The Giant Protein AHNAK Is a Specific Target for the Calcium- and Zinc-binding S100B Protein POTENTIAL IMPLICATIONS FOR Ca2+ HOMEOSTASIS REGULATION BY S100B

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val135 is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca2+-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca2+-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca2+- and Zn2+-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn2+ equivalents/mol S100B enhances Ca2+-dependent S100B-AHNAK interaction and that the effect of Zn2+ relies on Zn2+-dependent regulation of S100B affinity for Ca2+. Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca2+ homeostasis.

The AAA+ ATPase ATAD3A Controls Mitochondrial Dynamics at the Interface of the Inner and Outer Membranes

ABSTRACT Dynamic interactions between components of the outer (OM) and inner (IM) membranes control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA+ ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central transmembrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA+ ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using dominant-negative mutants, including a defective ATP-binding mutant and a truncated 50-amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms.

Concerted Regulation of Wild-Type p53 Nuclear Accumulation and Activation by S100B and Calcium-Dependent Protein Kinase C

ABSTRACT The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G1 checkpoint control in S100B-expressing mouse embryo fibroblasts and rat embryo fibroblasts (REF cells) which express the temperature-sensitive p53Val135 mutant (C. Scotto, J. C. Deloulme, D. Rousseau, E. Chambaz, and J. Baudier, Mol. Cell. Biol. 18:4272–4281, 1998). We investigated in this study the contributions of S100B and calcium-dependent PKC (cPKC) signalling pathways to the activation of wild-type p53. We first confirmed that S100B expression in mouse embryo fibroblasts enhanced specific nuclear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Mutation of the five putative cPKC phosphorylation sites on murine p53 into alanine or aspartic residues had no significant effect on p53 nuclear localization, suggesting that the cPKC effect on p53 nuclear translocation is indirect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear translocation and activation was confirmed with REF cells expressing S100B (S100B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. Stimulation of S100B-REF cells with the PKC activator phorbol ester phorbol myristate acetate (PMA) promoted specific nuclear translocation of the wild-type p53Val135 species in cells positioned in early G1 phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G1 arrest at the nonpermissive temperature (37.5°C). PMA-dependent growth arrest was linked to the cell apoptosis response to UV irradiation. In contrast, growth arrest mediated by a temperature shift to 32°C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S100B to promote wild-type p53 nuclear translocation in early G1 phase and activation of a p53-dependent G1checkpoint control.

Calcium-dependent Interaction of S100B with the C-terminal Domain of the Tumor Suppressor p53

In vitro, the S100B protein interacts with baculovirus recombinant p53 protein and protects p53 from thermal denaturation. This effect is isoform-specific and is not observed with S100A1, S100A6, or calmodulin. Using truncated p53 proteins in the N-terminal (p531–320) and C-terminal (p5373–393) domains, we localized the S100B-binding region to the C-terminal region of p53. We have confirmed a calcium-dependent interaction of the S100B with a synthetic peptide corresponding to the C-terminal region of p53 (residues 319–393 in human p53) using plasmon resonance experiments on a BIAcore system. In the presence of calcium, the equilibrium affinity of the S100B for the C-terminal region of p53 immobilized on the sensor chip was 24 ± 10 nm. To narrow down the region within p53 involved in S100B binding, two synthetic peptides, O1357–381 (residues 357–381 in mouse p53) and YF-O2320–346 (residues 320–346 in mouse p53), covering the C-terminal region of p53 were compared for their interaction with purified S100B. Only YF-O2 peptide interacts with S100B with high affinity. The YF-O2 motif is a critical determinant for the thermostability of p53 and also corresponds to a domain responsible for cytoplasmic sequestration of p53. Our results may explain the rescue of nuclear wild type p53 activities by S100B in fibroblast cell lines expressing the temperature-sensitive p53val135 mutant at the nonpermissive temperature.