RefilinB (FAM101B) targets filamin A to organize perinuclear actin networks and regulates nuclear shape.

The intracellular localization and shape of the nucleus plays a central role in cellular and developmental processes. In fibroblasts, nuclear movement and shape are controlled by a specific perinuclear actin network made of contractile actin filament bundles called transmembrane actin-associated nuclear (TAN) lines that form a structure called the actin cap. The identification of regulatory proteins associated with this specific actin cytoskeletal dynamic is a priority for understanding actin-based changes in nuclear shape and position in normal and pathological situations. Here, we first identify a unique family of actin regulators, the refilin proteins (RefilinA and RefilinB), that stabilize specifically perinuclear actin filament bundles. We next identify the actin-binding filamin A (FLNA) protein as the downstream effector of refilins. Refilins act as molecular switches to convert FLNA from an actin branching protein into one that bundles. In NIH 3T3 fibroblasts, the RefilinB/FLNA complex organizes the perinuclear actin filament bundles forming the actin cap. Finally, we demonstrate that in epithelial normal murine mammary gland (NmuMG) cells, the RefilinB/FLNA complex controls formation of a new perinuclear actin network that accompanies nuclear shape changes during the epithelial-mesenchymal transition (EMT). Our studies open perspectives for further functional analyses of this unique actin-based network and shed light on FLNA function during development and in human syndromes associated with FLNA mutations.

S100P is a novel interaction partner and regulator of IQGAP1.

Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 µM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.

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 Ca(2+)-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.

IQGAP1 protein specifies amplifying cancer cells in glioblastoma multiforme.

The accurate identification and thorough characterization of tumorigenic cells in glioblastomas are essential to enhance our understanding of their malignant behavior and for the design of strategies that target this important cell population. We report here that, in rat brain, the scaffolding protein IQGAP1 is a marker of brain nestin+ amplifying neural progenitor cells. In a rat model of glioma, IQGAP1 also characterizes a subpopulation of nestin+ amplifying tumor cells in glioblastoma-like tumors but not in tumors with oligodendroglioma features. We next confirmed that IQGAP1 represents a new marker that may help to discriminate human glioblastoma from oligodendrogliomas. In human glioblastoma exclusively, IQGAP1 specifies a subpopulation of amplifying nestin+ cancer cells. Neoplastic IQGAP1+ cells from glioblastoma can be expanded in culture and possess all the characteristics of cancer stem-like progenitors. The similarities between amplifying neural progenitors and glioblastoma amplifying cancer cells may have significant implications for understanding the biology of glioblastoma.

Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics.

Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling.

Distinct outcomes of CRL-Nedd8 pathway inhibition reveal cancer cell plasticity.

Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/ß-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR-ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR-ERG interaction.

Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates.

Sumoylation during genotoxic stress regulates the composition of DNA repair complexes. The yeast metalloprotease Wss1 clears chromatin-bound sumoylated proteins. Wss1 and its mammalian analog, DVC1/Spartan, belong to minigluzincins family of proteases. Wss1 proteolytic activity is regulated by a cysteine switch mechanism activated by chemical stress and/or DNA binding. Wss1 is required for cell survival following UV irradiation, the smt3-331 mutation and Camptothecin-induced formation of covalent topoisomerase 1 complexes (Top1cc). Wss1 forms a SUMO-specific ternary complex with the AAA ATPase Cdc48 and an adaptor, Doa1. Upon DNA damage Wss1/Cdc48/Doa1 is recruited to sumoylated targets and catalyzes SUMO chain extension through a newly recognized SUMO ligase activity. Activation of Wss1 results in metalloprotease self-cleavage and proteolysis of associated proteins. In cells lacking Tdp1, clearance of topoisomerase covalent complexes becomes SUMO and Wss1-dependent. Upon genotoxic stress, Wss1 is vacuolar, suggesting a link between genotoxic stress and autophagy involving the Doa1 adapter.

ATAD3B is a human embryonic stem cell specific mitochondrial protein, re-expressed in cancer cells, that functions as dominant negative for the ubiquitous ATAD3A.

Here we report on the identification of a human pluripotent embryonic stem cell (hESC) specific mitochondrial protein that is re-expressed in cancer cells, ATAD3B. ATAD3B belongs to the AAA+ ATPase ATAD3 protein family of mitochondrial proteins specific to multicellular eukaryotes. Using loss- and gain-of-function approaches, we show that ATAD3B associates with the ubiquitous ATAD3A species, negatively regulates the interaction of ATAD3A with matrix nucleoid complexes and contributes to a mitochondria fragmentation phenotype. We conclude that ATAD3B is a negative regulator of ATAD3A and may function as an adaptor of mitochondrial homeostasis and metabolism in hESCs and cancer cells.

The AAA+ ATPase ATAD3A controls mitochondrial dynamics at the interface of the inner and outer membranes.

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.

The calcium-dependent interaction between S100B and the mitochondrial AAA ATPase ATAD3A and the role of this complex in the cytoplasmic processing of ATAD3A.

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.

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 beta-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.

Identification of an AHNAK binding motif specific for the Annexin2/S100A10 tetramer.

The Annexin2 tetramer (A2t), which consists of two Annexin2 molecules bound to a S100A10 dimer, is implicated in membrane-trafficking events. Here, we showed using a yeast triple-hybrid experiment and in vitro binding assay that Annexin2 is required for strong binding of S100A10 to the C-terminal domain of the protein Ahnak. We also revealed that this effect involves only the Annexin2 N-terminal tail, which is implicated in S100A10/Annexin2 tetramerization. The minimal A2t binding motif (A2tBP1) in Ahnak was mapped to a 20-amino acid peptide, and this peptide is highly specific for A2t. We also identified a second A2t binding motif (A2tBP2) present in the N-terminal domain of Ahnak, which binds to A2t, albeit with less affinity. When overexpressed as an EGFP fusion protein in MDCK cells, A2tBPs cofractionate in a calcium-dependent manner and co-immunoprecipitate with S100A10 and Annexin2. In living cells, A2tBPs target EGFP to the cytoplasm as does Annexin2. In response to oxidative and mechanical stress, EGFP-A2tBPs relocalize within minutes to the plasma membrane; a behavior shared with Annexin2-GFP. These results suggest that the A2t complex exists within the cytoplasm of resting living cells and that its localization at the plasma membrane relies on cellular signaling. Together, our data demonstrate that A2tBP1 is a specific A2t complex binding domain and may be a powerful tool to help elucidate A2t structure and cellular functions.