Structure of the yeast Pml1 splicing factor and its integration into the RES complex

The RES complex was previously identified in yeast as a splicing factor affecting nuclear pre-mRNA retention. This complex was shown to contain three subunits, namely Snu17, Bud13 and Pml1, but its mode of action remains ill-defined. To obtain insights into its function, we have performed a structural investigation of this factor. Production of a short N-terminal truncation of residues that are apparently disordered allowed us to determine the X-ray crystallographic structure of Pml1. This demonstrated that it consists mainly of a FHA domain, a fold which has been shown to mediate interactions with phosphothreonine-containing peptides. Using a new sensitive assay based on alternative splice-site choice, we show, however, that mutation of the putative phosphothreonine-binding pocket of Pml1 does not affect pre-mRNA splicing. We have also investigated how Pml1 integrates into the RES complex. Production of recombinant complexes, combined with serial truncation and mutagenesis of their subunits, indicated that Pml1 binds to Snu17, which itself contacts Bud13. This analysis allowed us to demarcate the binding sites involved in the formation of this assembly. We propose a model of the organization of the RES complex based on these results, and discuss the functional consequences of this architecture

Actin Polymerization Controls the Organization of WASH Domains at the Surface of Endosomes

Sorting of cargoes in endosomes occurs through their selective enrichment into sorting platforms, where transport intermediates are generated. The WASH complex, which directly binds to lipids, activates the Arp2/3 complex and hence actin polymerization onto such sorting platforms. Here, we analyzed the role of actin polymerization in the physiology of endosomal domains containing WASH using quantitative image analysis. Actin depolymerization is known to enlarge endosomes. Using a novel colocalization method that is insensitive to the heterogeneity of size and shape of endosomes, we further show that preventing the generation of branched actin networks induces endosomal accumulation of the WASH complex. Moreover, we found that actin depolymerization induces a dramatic decrease in the recovery of endosomal WASH after photobleaching. This result suggests a built-in turnover, where the actin network, i.e. the product of the WASH complex, contributes to the dynamic exchange of the WASH complex by promoting its detachment from endosomes. Our experiments also provide evidence for a role of actin polymerization in the lateral compartmentalization of endosomes: several WASH domains exist at the surface of enlarged endosomes, however, the WASH domains coalesce upon actin depolymerization or Arp2/3 depletion. Branched actin networks are thus involved in the regulation of the size of WASH domains. The potential role of this regulation in membrane scission are discussed

Talin dissociates from RIAM and associates to vinculin sequentially in response to the actomyosin force

International audienceCells reinforce adhesion strength and cytoskeleton anchoring in response to the actomyosin force. The mechanical stretching of talin, which exposes cryptic vinculin-binding sites, triggers this process. The binding of RIAM to talin could regulate this mechanism. However, the mechanosensitivity of the talin-RIAM complex has never been tested. It is also not known whether RIAM controls the mechanosensitivity of the talin-vinculin complex. To address these issues, we designed an in vitro microscopy assay with purified proteins in which the actomyosin force controls RIAM and vinculin-binding to talin. We demonstrate that actomyosin triggers RIAM dissociation from several talin domains. Actomyosin also provokes the sequential exchange of RIAM for vinculin on talin. The effect of RIAM on this forcedependent binding of vinculin to talin varies from one talin domain to another. This mechanism could allow talin to biochemically code a wide range of forces by selecting different combinations of partners

Interpretation of the piezometric fluctuations and precursors associated with the November 29, 2007, magnitude 7.4 earthquake in Martinique (Lesser Antilles) Interprétation des fluctuations piézométriques et des précurseurs associés au séisme de magnitude 7,4 du 29 novembre 2007 sur l'île de la Martinique (Petites Antilles)

International audiencen November 29, 2007, a 7.4 earthquake occurred near the volcanic island of Martinique (French West Indies). It was widely felt in the Caribbean. Piezometric level changes correlated with the earthquake were recorded by 12 of the 24 piezometers in the groundwater monitoring network. A methodology has been developed for the interpretation of long-duration piezometric anomalies. It enables us to demonstrate that the hydraulic conductivity increased at the scale of the whole aquifer, by an order of magnitude of 5 to 10%, as a consequence of the earthquake. With this methodology, it is possible to compute either the aquifer hydraulic conductivity increase during the earthquake or its hydrodynamic parameters: diffusivity and relative location of the piezometer along a flow line. It shows, for instance, that the amplitude of the piezometric change due to the earthquake is not directly related to its intensity, but rather to the structure and hydrodynamic properties of the aquifer and also to the location of the piezometer. It also proves that a piezometric increase due to an earthquake cannot be straightforwardly related to a decrease in the hydraulic conductivity of the aquifer. Consequently, in such an active geodynamical context, tectonic processes appear to be among the factors responsible of the magnitude of the hydraulic conductivity of shallow aquifers. Piezometric precursors of the earthquake were definitely observed, but the operational use of such signals is, as yet, far from obvious

Interpretation for the role of actin on WASH dynamics on and off endosomes.

<p>To explain how actin depolymerization increases the steady state amount of WASH on endosomes and decreases the exchange of endosomal WASH for cytosolic WASH, we propose that actin branched networks promote the detachment of WASH from endosomes. For example, endosomal WASH may bind to the active Arp2/3 complex at branched junctions of the actin network, and detach from the endosome as the branched junction moves backward because of filament elongation.</p

Quantification of the coalescence of WASH domains upon actin depolymerization.

<p>(A) Image processing workflow (see also Methods). Confocal slices were acquired for GFP-WASH and mCherry-Rab5Q79L channels and processed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039774#pone-0039774-g002" target="_blank">Fig. 2</a> B, except that the wavelet filter was substituted by an ‘Adaptative blind’ deconvolution step. (B) Image stacks were segmented in 3D for both channels (23 cells, 2179 endosomes for control; 22 cells, 1221 endosomes for LatA). The average number of WASH domains (± s.e.m.) and their average apparent surface (± s.e.m.) were plotted as a function of endosome volume. The average number of WASH domains increases with endosome volume (p<0.001 Kruskal-Wallis one way analysis of variance on ranks). Moreover, upon actin depolymerization, the number of WASH domains in large endosomes decreases (*: p<0.05 compared with control within the same volume bin, Kruskal-Wallis test followed by a Dunn pairwise comparison). Concomitantly, the surface occupied by WASH domains increases (* p<0.001 compared with control within the same volume bin, two way ANOVA followed by a Tukey pairewise comparison). Altogether, these data suggest that WASH domains coalesce upon actin depolymerization.</p

Depletion of the Arp2/3 complex increases the amount of endosomal WASH.

<p>(A) Stable 3T3 cells expressing GFP-WASH were depleted from the Arp2/3 complex using siRNAs and analyzed by Western Blot. (B) siRNA-transfected cells were treated with 10 µM nocodazole for 1 h, then processed for immunofluorescence using antibodies recognizing EEA1 and the p16Arc subunit of the Arp2/3 complex, and observed by spinning disk confocal microscopy (single planes). Scale bar: 10 µm. Arp2/3 depletion increases endosomal WASH staining. (C–D) Image stacks (36 cells, 6046 endosomes for Ctrl siRNA; 31 cells, 3889 endosomes for p34Arc #1 siRNA; 26 cells, 3470 endosomes for p34Arc #2 siRNA) were processed and presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039774#pone-0039774-g002" target="_blank">Fig. 2</a>D–E after normalization of endosomes volumes. Upon Arp2/3 complex depletion, the intensity and ‘apparent concentration’ of GFP-WASH domains increases but does not depend on endosome volume. *: p<0.001 compared with control within the same volume bin, two way ANOVA using treatments and volume bins as parameters, followed by pairwise comparisons using Tukey test.</p

Actin depolymerization increases the amount of endosomal WASH.

<p>(A) 3T3 cells were loaded with fluorescent Transferrin (Tf) until equilibrium, then treated with 0.2 µM Latrunculin A (LatA) for 10 min, 1 µM CytochalasinD (CytoD) for 30 min, or carrier in the continuous presence of Tf. Cells were processed for immunofluorescence using WASH antibody and observed by spinning disk confocal microscopy. A single plane is displayed. Scale bar: 10 µm (1 µm in inserts). LatA and CytoD treatments increase the fluorescence signal of WASH. (B) Since endosomes are clustered in the perinuclear region, the increase of WASH intensity was quantified on isolated endosomes from the cell periphery (see Methods). Both LatA and CytoD treatments induce a statistically significant increase of the WASH fluorescence signal (n refers to the number of endosomes, *: p<0.001, one way ANOVA followed by a Tukey pairwise comparison).</p