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Chromatin regulatory processes physically take place in the environment of the cell nucleus, which is filled with the chromosomes and a plethora of smaller biomolecules. The nucleus contains macromolecular assemblies of different sizes, from nanometer-sized protein complexes to micrometer-sized biomolecular condensates, chromosome territories, and nuclear bodies. This multiscale organization impacts the transport processes within the nuclear interior, the global mechanical properties of the nucleus, and the way the nucleus senses and reacts to mechanical stimuli. Here, we discuss recent work on these aspects, including microrheology and micromanipulation experiments assessing the material properties of the nucleus and its subcomponents. We summarize how the properties of multiscale media depend on the time and length scales probed in the experiment, and we reconcile seemingly contradictory observations made on different scales. We also revisit the concept of liquid-like and solid-like material properties for complex media such as the nucleus. We propose that the nucleus can be considered a multiscale viscoelastic medium composed of three major components with distinct properties: the lamina, the chromatin network, and the nucleoplasmic fluid. This multicomponent organization enables the nucleus to serve its different functions as a reaction medium on the nanoscale and as a mechanosensor and structural scaffold on the microscale.

Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo . However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro , enhancing IRSp53-dependent membrane extensions in vivo . Cdc42 binds to and controls the cellular distribution of the IRSp53–Eps8 complex, supporting the existence of a Cdc42–IRSp53–Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53–Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.

Actin capping and cross-linking proteins regulate the dynamics and architectures of different cellular protrusions. Eps8 is the founding member of a unique family of capping proteins capable of side-binding and bundling actin filaments. However, the structural basis through which Eps8 exerts these functions remains elusive. Here, we combined biochemical, molecular, and genetic approaches with electron microscopy and image analysis to dissect the molecular mechanism responsible for the distinct activities of Eps8. We propose that bundling activity of Eps8 is mainly mediated by a compact four helix bundle, which is contacting three actin subunits along the filament. The capping activity is mainly mediated by a amphipathic helix that binds within the hydrophobic pocket at the barbed ends of actin blocking further addition of actin monomers. Single-point mutagenesis validated these modes of binding, permitting us to dissect Eps8 capping from bundling activity in vitro. We further showed that the capping and bundling activities of Eps8 can be fully dissected in vivo, demonstrating the physiological relevance of the identified Eps8 structural/functional modules. Eps8 controls actin-based motility through its capping activity, while, as a bundler, is essential for proper intestinal morphogenesis of developing Caenorhabditis elegans.

Caveolae are specialized domains of the plasma membrane, which play key roles in signaling, endocytosis and mechanosensing. Using total internal reflection fluorescent microscopy (TIRF-M), we observe that the exocyst subunit Exo70 forms punctuate structures at the plasma membrane and partially localizes with caveolin-1, the main component of caveolae. Upon cell detachment, we found that Exo70 accumulates with caveolin-1-positive vesicular structures. Upon cell re-adhesion, caveolin-1 traffics back to the plasma membrane in a multistep process involving microtubules and actin cytoskeleton. In addition, silencing of Exo70 redirects caveolin-1 to focal adhesions identified by markers such as α5 integrin or vinculin. Based on these findings, we conclude that Exo70 is involved in caveolin-1 recycling to the plasma membrane during re-adhesion of the cells to the substratum.

The regulation of filopodia plays a crucial role during neuronal development and synaptogenesis. Axonal filopodia, which are known to originate presynaptic specializations, are regulated in response to neurotrophic factors. The structural components of filopodia are actin filaments, whose dynamics and organization are controlled by ensembles of actin-binding proteins. How neurotrophic factors regulate these latter proteins remains, however, poorly defined. Here, using a combination of mouse genetic, biochemical, and cell biological assays, we show that genetic removal of Eps8, an actin-binding and regulatory protein enriched in the growth cones and developing processes of neurons, significantly augments the number and density of vasodilator-stimulated phosphoprotein (VASP)-dependent axonal filopodia. The reintroduction of Eps8 wild type (WT), but not an Eps8 capping-defective mutant, into primary hippocampal neurons restored axonal filopodia to WT levels. We further show that the actin barbed-end capping activity of Eps8 is inhibited by brain-derived neurotrophic factor (BDNF) treatment through MAPK-dependent phosphorylation of Eps8 residues S624 and T628. Additionally, an Eps8 mutant, impaired in the MAPK target sites (S624A/T628A), displays increased association to actin-rich structures, is resistant to BDNF-mediated release from microfilaments, and inhibits BDNF-induced filopodia. The opposite is observed for a phosphomimetic Eps8 (S624E/T628E) mutant. Thus, collectively, our data identify Eps8 as a critical capping protein in the regulation of axonal filopodia and delineate a molecular pathway by which BDNF, through MAPK-dependent phosphorylation of Eps8, stimulates axonal filopodia formation, a process with crucial impacts on neuronal development and synapse formation.

Neural Wiskott–Aldrich syndrome protein (N-WASP) and WAVE are members of a family of proteins that use the Arp2/3 complex to stimulate actin assembly in actin-based motile processes. By entering into distinct macromolecular complexes, they act as convergent nodes of different signalling pathways. The role of WAVE in generating lamellipodial protrusion during cell migration is well established. Conversely, the precise cellular functions of N-WASP have remained elusive. Here, we report that Abi1, an essential component of the WAVE protein complex, also has a critical role in regulating N-WASP-dependent function. Consistently, Abi1 binds to N-WASP with nanomolar affinity and, cooperating with Cdc42, potently induces N-WASP activity in vitro . Molecular genetic approaches demonstrate that Abi1 and WAVE, but not N-WASP, are essential for Rac-dependent membrane protrusion and macropinocytosis. Conversely, Abi1 and N-WASP, but not WAVE, regulate actin-based vesicular transport, epidermal growth factor receptor (EGFR) endocytosis, and EGFR and transferrin receptor (TfR) cell-surface distribution. Thus, Abi1 is a dual regulator of WAVE and N-WASP activities in specific processes that are dependent on actin dynamics.

Chromatin regulatory processes physically take place in the environment of the cell nucleus, which is filled with the chromosomes and a plethora of smaller biomolecules. The nucleus contains macromolecular assemblies of different sizes, from nanometer-sized protein complexes to micrometer-sized biomolecular condensates, chromosome territories, and nuclear bodies. This multiscale organization impacts the transport processes within the nuclear interior, the global mechanical properties of the nucleus, and the way the nucleus senses and reacts to mechanical stimuli. Here, we discuss recent work on these aspects, including microrheology and micromanipulation experiments assessing the material properties of the nucleus and its subcomponents. We summarize how the properties of multiscale media depend on the time and length scales probed in the experiment, and we reconcile seemingly contradictory observations made on different scales. We also revisit the concept of liquid-like and solid-like material properties for complex media such as the nucleus. We propose that the nucleus can be considered a multiscale viscoelastic medium composed of three major components with distinct properties: the lamina, the chromatin network, and the nucleoplasmic fluid. This multicomponent organization enables the nucleus to serve its different functions as a reaction medium on the nanoscale and as a mechanosensor and structural scaffold on the microscale.

β‐Thymosin (βT) and WH2 domains are widespread, intrinsically disordered actin‐binding peptides that display significant sequence variability and different regulations of actin self‐assembly in motile and morphogenetic processes. Here, we reveal the structural mechanisms by which, in their 1:1 stoichiometric complexes with actin, they either inhibit assembly by sequestering actin monomers like Thymosin‐β4, or enhance motility by directing polarized filament assembly like Ciboulot βT. We combined mutational, functional or structural analysis by X‐ray crystallography, SAXS (small angle X‐ray scattering) and NMR on Thymosin‐β4, Ciboulot, TetraThymosinβ and the long WH2 domain of WASP‐interacting protein. The latter sequesters G‐actin with the same molecular mechanisms as Thymosin‐β4. Functionally different βT/WH2 domains differ by distinct dynamics of their C‐terminal half interactions with G‐actin pointed face. These C‐terminal interaction dynamics are controlled by the strength of electrostatic interactions with G‐actin. At physiological ionic strength, a single salt bridge with actin located next to their central LKKT/V motif induces G‐actin sequestration in both isolated long βT and WH2 domains. The results open perspectives for elucidating the functions of βT/WH2 domains in other modular proteins. β‐Thymosin/WH2 domain‐containing proteins can either sequester actin monomers or promote filament assembly. These opposing functions are defined a single residue on their actin interaction interface, which determines the dynamics of actin binding.

Maud Hertzog is also affiliated with Laboratoire de Microbiologie et Génétique Moléculaire, LMGM-CNRS UMR 5100, Université Paul Sabatier, Toulouse, France, and Gaëlle Le Dez is also affiliated with CNRS, UMR 6061, Institut Génétique et Développement de Rennes, Université Rennes 1, UEB, IFR 140, Rennes, France. Citation: Hertzog M, Monteiro P, Le Dez G, Chavrier P (2013) Correction: Exo70 Subunit of the Exocyst Complex Is Involved in Adhesion-Dependent Trafficking of Caveolin-1.