Molecular Basis for the Dual Function of Eps8 on Actin Dynamics: Bundling and Capping

The unusual dual functions of the actin-binding protein EPS8 as an actin capping and actin bundling factor are mapped to distinct structural features of the protein and to distinct physiological activities in vivo

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae

Abstract RecA-mediated Homologous Recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae ( Sp RecA), in comparison to the Escherichia coli RecA ( Ec RecA) paradigm. Ec RecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block Ec RecA nucleofilament growth. We report that neither of the two paralogous pneumococcal SSBs could assist Sp RecA polymerization on ssDNA. Instead, we found that the conserved RadA helicase promotes this Sp RecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native Sp RecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the Ec RecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that Sp RecA and Ec RecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes

Eps8 Regulates Axonal Filopodia in Hippocampal Neurons in Response to Brain-Derived Neurotrophic Factor (BDNF)

A novel signaling cascade controlling actin polymerization in response to extracellular signals regulates filopodia formation and likely also neuronal synapse formation

Exo70 Subunit of the Exocyst Complex Is Involved in Adhesion-Dependent Trafficking of Caveolin-1

<div><p>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.</p> </div

Exo70 is required for Cav1 transport to the plasma membrane.

<p>(<b>A, B</b>) Hela cells expressing Cav1-mRFP and α5-integrin-GFP either mock-treated (A) or treated with a specific siRNA to silencing Exo70 (B) were maintained in suspension for 1 h and replated on fibronectin for 3 h, and visualized using time-lapse spinning disk microscopy. Scale bars, 5 µm. (<b>A′, B′</b>) Intensity profile of Cav1 (red) and α5-integrin (green) along the white lines shown in panel A and B. (<b>C</b>) Co-localization analysis of Cav1-mRFP and α5-integrin-GFP in cells as in panels A and B in a 20-pixel width region along the cell periphery of mock- or Exo70 siRNA-depleted cells using two specific siRNAs (siRNA 7c and 7d). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052627#s2" target="_blank">Results</a> are the average of mean percentage of co-localization ± s.e.m. of three experiments. ** <i>P</i><0.05. Experiment has been repeated three times; with number of cell analysed: n = 19 for mock cells, n = 17 for siRNA 7c, and n = 17 for siRNA 7d.</p

Silencing of Exo70 leads to Cav1 accumulation in focal adhesions.

<p>Mock-treated (<b>A</b>) or Exo70-depleted cells (<b>B</b>) were detached for 1 h and replated on fibronectin-coated substrate for 3 h, and then fixed and stained for endogenous vinculin and Cav1. Scale bars, 5 µm. Co-localization between Cav1 and vinculin was quantified in a 20-pixel width region from the cell periphery and compared in Exo70-depleted with two independent siRNAs <i>vs</i>. mock-treated cells (<b>C</b>). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052627#s2" target="_blank">Results</a> in panel C are the average of mean percentage of co-localization ± s.e.m. of three experiments. ** <i>P</i><0.05. Experiment has been repeated three times with number of cells analysed: n = 25 mock cells, n = 9 for cells treated with Exo70 siRNA 7c, and n = 23 for siRNA 7d.</p