RESUMO
The three-dimensional arrangement of chromatin encodes regulatory traits important for nuclear processes such as transcription and replication. Chromatin topology is in part mediated by the architectural protein CCCTC-binding factor (CTCF) that binds to the boundaries of topologically associating domains. Whereas sites of CTCF interactions are well characterized, little is known on how long CTCF binds to chromatin and how binding evolves during the cell cycle. We monitored CTCF-chromatin interactions by live cell single molecule tracking in different phases of the cell cycle. In G1-, S-, and G2-phases, a majority of CTCF molecules was bound transiently (â¼0.2 s) to chromatin, whereas minor fractions were bound dynamically (â¼4 s) or stably (>15 min). During mitosis, CTCF was mostly excluded from chromatin. Our data suggest that CTCF scans DNA in search for two different subsets of specific target sites and provide information on the timescales over which topologically associating domains might be restructured. During S-phase, dynamic and stable interactions decreased considerably compared to G1-phase, but were resumed in G2-phase, indicating that specific interactions need to be dissolved for replication to proceed.
Assuntos
Ciclo Celular/fisiologia , Cromatina/metabolismo , Proteínas Repressoras/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular , Humanos , Cinética , Imagem Molecular , Ligação ProteicaRESUMO
Rho GTPases are central regulators of the cytoskeleton and, in humans, are controlled by 145 multidomain guanine nucleotide exchange factors (RhoGEFs) and GTPase-activating proteins (RhoGAPs). How Rho signalling patterns are established in dynamic cell spaces to control cellular morphogenesis is unclear. Through a family-wide characterization of substrate specificities, interactomes and localization, we reveal at the systems level how RhoGEFs and RhoGAPs contextualize and spatiotemporally control Rho signalling. These proteins are widely autoinhibited to allow local regulation, form complexes to jointly coordinate their networks and provide positional information for signalling. RhoGAPs are more promiscuous than RhoGEFs to confine Rho activity gradients. Our resource enabled us to uncover a multi-RhoGEF complex downstream of G-protein-coupled receptors controlling CDC42-RHOA crosstalk. Moreover, we show that integrin adhesions spatially segregate GEFs and GAPs to shape RAC1 activity zones in response to mechanical cues. This mechanism controls the protrusion and contraction dynamics fundamental to cell motility. Our systems analysis of Rho regulators is key to revealing emergent organization principles of Rho signalling.