Project description:Brain postnatal development is characterized by critical periods of experience dependent remodeling. Maturation of local circuits inhibitory neurons terminate this period of enhanced plasticity. Astroglial cells are known to influence excitatory and inhibitory synaptic transmission as well as network activity through active signaling mechanisms. Although these can be developmentally regulated, the role of astrocytes in the timing of post-natal critical period is unknown. Here we show in the visual cortex that astrocytes con-trol the maturation of inhibitory neurons and thereby closure of the critical period. We uncover a novel underlying pathway involving regulation of the extracellular matrix that allows interneurons maturation via astroglial connexin signaling. We find that timing of the critical period closure is controlled by a marked upregulation of the astroglial protein connexin 30 that inhibits expression of the matrix degrading enzyme MMP9 through the RhoA-GTPase pathway. Our results thus demonstrate that astrocytes not only influ-ence neuronal activity but are also key elements in the experience–dependent wiring of brain circuits. Therefore, astrocytes represent a new cellular partner to consider in our understanding of the post-natal shaping of neuronal activities, hence providing a new target to alleviate malfunctions associated to im-paired closure of the critical period and settling of synaptic circuits.
Project description:In this study we performed 2 interactomes. To identify interactants of RNF111 that are dependent of the RING domain, we performed qualitative interactome comparison of HEK-293 cells transfected with GFP, GFP-RNF111-wt or GFP-RNF11-C933A mutated in its RING domain. This led to the identification of UBXN7 as a RING-dependent partner for RNF111. To identify UBXN7 UAS dependant partners, we performed quantitative interactome comparison of HEK-293 cells transfected with GFP, GFP-UBXN7-UAS.
Project description:This study focuses on the impact of ECM (extra-cellular matrix) over cell processes such as proliferation, differentiation or mineralization which is more specifically related to our dental pulp cells. We cultured these dental pulp stem cells (DPSC) in mineralization growth or normal growth conditions. After 21 days, cells were incubated in decellularized solution until no intact cells are seen. After ECM was washed, we studied the mineralization associated to these two different ECM. Matrisome proteins were identified through proteomics. DPSC matrisome is composed of 225 individual different proteins. We classified them according to different categories, the 3 core matrisome categories: glycoproteins, collagens, proteoglycans and the 3 matrisome associated proteins categories: the regulators, affiliated and secreted. When comparing the proteins in the N-ECM and OM-ECM, most of the core matrisome proteins are downregulated in OM conditions, except 3 glycoproteins, as well as regulators and secreted factors. However, annexins were found to be upregulated in OM condition. Cell adhesion, tensile strength and growth factor binding are over-represented in NM ECM. The collagen group and glycoproteins were higher in N-ECM than OM-ECM Thereafter, gingival stem cells (GSC), the less inherit osteogenic potency cells, were seeded on these N-ECM and OM-ECM. When GSCs were seeded on DPSC-derived ECM, the OM-ECM dramatically promoted mineral deposition compared with N-ECM. We hypothesize that annexins could participate in the osteogenic inductive properties. ECM plays a pivotal role in many physiological processes, it regulates cells behavior and can orient cell differentiation. Dental pulp and oral mucosa share embryological origins but differ in their reactions to insults. Dental pulp can mineralize while oral mucosa heals ad integrum. We hypothesize that ECM participate in these characteristics.
Project description:Cell adhesion to the extracellular matrix occurs through integrin-mediated focal adhesions, which sense the mechanical properties of the substrate and impact cellular functions such as cell migration. Mechanotransduction at focal adhesions affects the actomyosin network and contributes to cell migration. Despite being key players in cell adhesion and migration, the role of microtubules in mechanotransduction has been overlooked. Here, we show that substrate rigidity increases microtubule acetylation through β1 integrin signalling in primary rat astrocytes. Moreover, αTAT1, the enzyme responsible for microtubule acetylation, interacts with a major mechanosensing focal adhesion protein, Talin, and is able to tune the distribution of focal adhesions depending on the matrix rigidity. αTAT1 also reorganises the actomyosin network, increases traction force generation and cell migration speed on stiff substrates. Mechanistically, acetylation of microtubules promotes the release of microtubule-associated RhoGEF, GEF-H1 into the cytoplasm, which then leads to RhoA activation and high actomyosin contractility. Thus, we propose a novel feedback loop involving a crosstalk between microtubules and actin in mechanotransduction at focal adhesions whereby, cells sense the rigidity of the substrate through integrin-mediated adhesions, modulate their levels of microtubule acetylation, which then controls the actomyosin cytoskeleton and force transmission on the substrate to promote mechanosensitive cell migration.
Project description:In the Caenorhabditis elegans germline, thousands of mRNAs are concomitantly expressed with antisense 22G-RNAs, which are loaded into the Argonaute CSR-1. Despite their essential functions for animal fertility and embryonic development, how CSR-1 22G-RNAs are produced remains unknown. Here, we show that CSR-1 slicer activity is primarily involved in triggering the synthesis of small RNAs on the coding sequences of germline mRNAs and post-transcriptionally regulates a fraction of targets. CSR-1-cleaved mRNAs prime the RNA-dependent RNA polymerase, EGO-1, to synthesize 22G-RNAs in phase with ribosome translation in the cytoplasm, in contrast to other 22G-RNAs mostly synthesized in germ granules. Moreover, codon optimality and efficient translation antagonize CSR-1 slicing and 22G-RNAs biogenesis. We propose that codon usage differences encoded into mRNA sequences might be a conserved strategy in eukaryotes to regulate small RNA biogenesis and Argonaute targeting
Project description:Cardiovascular events are the leading cause of death in patients with JAK2V617F myeloproliferative neoplasms. Their mechanisms are poorly understood. To investigate the role of microvesicles in these events, we performed a proteomic analysis of microvesicles derived from red blood cells from mice with a myeloproliferative neoplasms (Jak2V617F Flex/WT ;VE-cadherin-Cre) vs. littermate controls.
Project description:RUF6 is a ncRNA gene family that is transcribed by RNA Polymerase III but actively regulates the Pol II-transcribed var virulence gene family. Understanding how RUF6 ncRNA connect to downstream effectors is lacking. We developed an RNA-directed proteomic discovery (ChIRP-MS) protocol to identify in vivo RUF6 ncRNA protein interactions. The RUF6 ncRNA interactome was purified with biotinylated antisense oligonucleotides. Quantitative label-free mass spectrometry identified several unique proteins linked to gene transcription including. Affinity purification of Pf-DDX5 identified proteins originally found by our RUF6-ChIRP protocol, validating the technique’s robustness for identifying ncRNA interactomes in P. falciparum. Our work identifies a RUF6 ncRNA protein complex that interacts with RNA Pol II to sustain var gene expression.
Project description:The var multigene family encodes clonally variant surface antigens that are key to immune evasion and pathogenesis in the human malaria parasite, Plasmodium falciparum. Epigenetics and nuclear organization regulate the var gene family in a system of mutually exclusive expression; however, few factors have been shown to play a direct role in these processes. Thus, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for identification of novel factors associated with var genes in their natural chromatin context. A tagged, catalytically inactive Cas9 (“dCas9”) was targeted to the promoters or introns of a subset of var genes and subjected to immunoprecipitation followed by label-free LC-MS/MS. A non-targeted dCas9 served as a control.
Project description:Cdc42 is a small GTPase protein whose role in polarity is widely studied. However, in vertebrates there exist 2 isoforms arising due to alternative splicing. The function and localization of these have not been studied with respect to cell polarity and cell migration. We therefore perform a mass spec screen to identify interactors of the isoforms to better understand their cellular functions.
Project description:In this present project, we aimed to physiologically dissect the contribution of glycolipid- and lectin-driven endocytosis mechanism that operate within the mice intestine. We therefore decided to first characterize the expression profile of galectins in this tissue and then to focus on the specific implication of galectin 3 in that context and identify a potential binding partner, that may use this endocytic process for its internalization.