Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies.

Spatially controlled cell adhesion on electron microscopy supports remains a bottleneck in specimen preparation for cellular cryo-electron tomography. Here, we describe contactless and mask-free photo-micropatterning of electron microscopy grids for site-specific deposition of extracellular matrix-related proteins. We attained refined cell positioning for micromachining by cryo-focused ion beam milling. Complex micropatterns generated predictable intracellular organization, allowing direct correlation between cell architecture and in-cell three-dimensional structural characterization of the underlying molecular machinery.

Spatial integration of mechanical forces by α-actinin establishes actin network symmetry.

Cell and tissue morphogenesis depend on the production and spatial organization of tensional forces in the actin cytoskeleton. Actin network architecture is made of distinct modules characterized by specific filament organizations. The assembly of these modules are well described, but their integration in a cellular network is less understood. Here, we investigated the mechanism regulating the interplay between network architecture and the geometry of the extracellular environment of the cell. We found that α-actinin, a filament crosslinker, is essential for network symmetry to be consistent with extracellular microenvironment symmetry. It is required for the interconnection of transverse arcs with radial fibres to ensure an appropriate balance between forces at cell adhesions and across the actin network. Furthermore, this connectivity appeared necessary for the ability of the cell to integrate and to adapt to complex patterns of extracellular cues as they migrate. Our study has unveiled a role of actin filament crosslinking in the spatial integration of mechanical forces that ensures the adaptation of intracellular symmetry axes in accordance with the geometry of extracellular cues.This article has an associated First Person interview with the first author of the paper.

Variation in traction forces during cell cycle progression.

BACKGROUND INFORMATION: Tissue morphogenesis results from the interplay between cell growth and mechanical forces. While the impact of geometrical confinement and mechanical forces on cell proliferation has been fairly well characterised, the inverse relationship is much less understood. Here, we investigated how traction forces vary during cell cycle progression. RESULTS: Cell shape was constrained on micropatterned substrates in order to distinguish variations in cell contractility from cell size increase. We performed traction force measurements of asynchronously dividing cells expressing a cell-cycle reporter, to obtain measurements of contractile forces generated during cell division. We found that forces tend to increase as cells progress through G1, before reaching a plateau in S phase, and then decline during G2. CONCLUSIONS: While cell size increases regularly during cell cycle progression, traction forces follow a biphasic behaviour based on specific and opposite regulation of cell contractility during early and late growth phases. SIGNIFICANCE: These results highlight the key role of cellular signalling in the regulation of cell contractility, independently of cell size and shape. Non-monotonous variations of cell contractility during cell cycle progression are likely to impact the mechanical regulation of tissue homoeostasis in a complex and non-linear manner.

Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.

Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

A radiation hybrid map of the European sea bass (Dicentrarchus labrax) based on 1581 markers: Synteny analysis with model fish genomes.

The selective breeding of fish for aquaculture purposes requires the understanding of the genetic basis of traits such as growth, behaviour, resistance to pathogens and sex determinism. Access to well-developed genomic resources is a prerequisite to improve the knowledge of these traits. Having this aim in mind, a radiation hybrid (RH) panel of European sea bass (Dicentrarchus labrax) was constructed from splenocytes irradiated at 3000 rad, allowing the construction of a 1581 marker RH map. A total of 1440 gene markers providing ~4400 anchors with the genomes of three-spined stickleback, medaka, pufferfish and zebrafish, helped establish synteny relationships with these model species. The identification of Conserved Segments Ordered (CSO) between sea bass and model species allows the anticipation of the position of any sea bass gene from its location in model genomes. Synteny relationships between sea bass and gilthead seabream were addressed by mapping 37 orthologous markers. The sea bass genetic linkage map was integrated in the RH map through the mapping of 141 microsatellites. We are thus able to present the first complete gene map of sea bass. It will facilitate linkage studies and the identification of candidate genes and Quantitative Trait Loci (QTL). The RH map further positions sea bass as a genetic and evolutionary model of Perciformes and supports their ongoing aquaculture expansion.

The biochemical composition of the actomyosin network sets the magnitude of cellular traction forces.

The regulation of cellular force production relies on the complex interplay between a well-conserved set of proteins of the cytoskeleton: actin, myosin, and α-actinin. Despite our deep knowledge of the role of these proteins in force production at the molecular scale, our understanding of the biochemical regulation of the magnitude of traction forces generated at the entire-cell level has been limited, notably by the technical challenge of measuring traction forces and the endogenous biochemical composition in the same cell. In this study, we developed an alternative Traction-Force Microscopy (TFM) assay, which used a combination of hydrogel micropatterning to define cell adhesion and shape and an intermediate fixation/immunolabeling step to characterize strain energies and the endogenous protein contents in single epithelial cells. Our results demonstrated that both the signal intensity and the area of the Focal Adhesion (FA)-associated protein vinculin showed a strong positive correlation with strain energy in mature FAs. Individual contents from actin filament and phospho-myosin displayed broader deviation in their linear relationship to strain energies. Instead, our quantitative analyzes demonstrated that their relative amount exhibited an optimum ratio of phospho-myosin to actin, allowing maximum force production by cells. By contrast, although no correlation was identified between individual α-actinin content and strain energy, the ratio of α-actinin to actin filaments was inversely related to strain energy. Hence, our results suggest that, in the cellular model studied, traction-force magnitude is dictated by the relative numbers of molecular motors and cross-linkers per actin filament, rather than the amounts of an individual component in the cytoskeletal network. This assay offers new perspectives to study in more detail the complex interplay between the endogenous biochemical composition of individual cells and the force they produce.

A gene-based radiation hybrid map of the gilthead sea bream Sparus aurata refines and exploits conserved synteny with Tetraodon nigroviridis.

BACKGROUND: Comparative teleost studies are of great interest since they are important in aquaculture and in evolutionary issues. Comparing genomes of fully sequenced model fish species with those of farmed fish species through comparative mapping offers shortcuts for quantitative trait loci (QTL) detections and for studying genome evolution through the identification of regions of conserved synteny in teleosts. Here a comparative mapping study is presented by radiation hybrid (RH) mapping genes of the gilthead sea bream Sparus aurata, a non-model teleost fish of commercial and evolutionary interest, as it represents the worldwide distributed species-rich family of Sparidae. RESULTS: An additional 74 microsatellite markers and 428 gene-based markers appropriate for comparative mapping studies were mapped on the existing RH map of Sparus aurata. The anchoring of the RH map to the genetic linkage map resulted in 24 groups matching the karyotype of Sparus aurata. Homologous sequences to Tetraodon were identified for 301 of the gene-based markers positioned on the RH map of Sparus aurata. Comparison between Sparus aurata RH groups and Tetraodon chromosomes (karyotype of Tetraodon consists of 21 chromosomes) in this study reveals an unambiguous one-to-one relationship suggesting that three Tetraodon chromosomes correspond to six Sparus aurata radiation hybrid groups. The exploitation of this conserved synteny relationship is furthermore demonstrated by in silico mapping of gilthead sea bream expressed sequence tags (EST) that give a significant similarity hit to Tetraodon. CONCLUSION: The addition of primarily gene-based markers increased substantially the density of the existing RH map and facilitated comparative analysis. The anchoring of this gene-based radiation hybrid map to the genome maps of model species broadened the pool of candidate genes that mainly control growth, disease resistance, sex determination and reversal, reproduction as well as environmental tolerance in this species, all traits of great importance for QTL mapping and marker assisted selection. Furthermore this comparative mapping approach will facilitate to give insights into chromosome evolution and into the genetic make up of the gilthead sea bream.

Dissipation of contractile forces: the missing piece in cell mechanics.

Mechanical forces are key regulators of cell and tissue physiology. The basic molecular mechanism of fiber contraction by the sliding of actin filament upon myosin leading to conformational change has been known for decades. The regulation of force generation at the level of the cell, however, is still far from elucidated. Indeed, the magnitude of cell traction forces on the underlying extracellular matrix in culture is almost impossible to predict or experimentally control. The considerable variability in measurements of cell-traction forces indicates that they may not be the optimal readout to properly characterize cell contractile state and that a significant part of the contractile energy is not transferred to cell anchorage but instead is involved in actin network dynamics. Here we discuss the experimental, numerical, and biological parameters that may be responsible for the variability in traction force production. We argue that limiting these sources of variability and investigating the dissipation of mechanical work that occurs with structural rearrangements and the disengagement of force transmission is key for further understanding of cell mechanics.

Microtubule stabilization drives 3D centrosome migration to initiate primary ciliogenesis.

Primary cilia are sensory organelles located at the cell surface. Their assembly is primed by centrosome migration to the apical surface, yet surprisingly little is known about this initiating step. To gain insight into the mechanisms driving centrosome migration, we exploited the reproducibility of cell architecture on adhesive micropatterns to investigate the cytoskeletal remodeling supporting it. Microtubule network densification and bundling, with the transient formation of an array of cold-stable microtubules, and actin cytoskeleton asymmetrical contraction participate in concert to drive apical centrosome migration. The distal appendage protein Cep164 appears to be a key actor involved in the cytoskeleton remodeling and centrosome migration, whereas intraflagellar transport 88's role seems to be restricted to axoneme elongation. Together, our data elucidate the hitherto unexplored mechanism of centrosome migration and show that it is driven by the increase and clustering of mechanical forces to push the centrosome toward the cell apical pole.

Kank2 activates talin, reduces force transduction across integrins and induces central adhesion formation.

Integrin-based adhesions play critical roles in cell migration. Talin activates integrins and flexibly connects integrins to the actomyosin cytoskeleton, thereby serving as a 'molecular clutch' that transmits forces to the extracellular matrix to drive cell migration. Here we identify the evolutionarily conserved Kank protein family as novel components of focal adhesions (FAs). Kank proteins accumulate at the lateral border of FAs, which we term the FA belt, and in central sliding adhesions, where they directly bind the talin rod domain through the Kank amino-terminal (KN) motif and induce talin and integrin activation. In addition, Kank proteins diminish the talin-actomyosin linkage, which curbs force transmission across integrins, leading to reduced integrin-ligand bond strength, slippage between integrin and ligand, central adhesion formation and sliding, and reduced cell migration speed. Our data identify Kank proteins as talin activators that decrease the grip between the integrin-talin complex and actomyosin to regulate cell migration velocity.

Histopathology and microcystin distribution in Lymnaea stagnalis (Gastropoda) following toxic cyanobacterial or dissolved microcystin-LR exposure.

The accumulation of hepatotoxic microcystins (MCs) in gastropods has been demonstrated to be higher following grazing of toxic cyanobacteria than from MCs dissolved in ambient water. Previous studies, however, did not adequately consider MCs covalently bound to protein phosphatases, which may represent a considerably part of the MC body burden. Thus, using an immunohistochemical method, we examined and compared the histopathology and organ distribution of covalently bound MCs in Lymnaea stagnalis following a 5-week exposure to (i) dmMC-LR, dmMC-RR, and MC-YR-producing Planktothrix agardhii (5 microg MC-LReqL(-1)) and (ii) dissolved MC-LR (33 and 100 microgL(-1)). A subsequent 3-week depuration investigated potential MC elimination and tissue regeneration. Following both exposures, bound MCs were primarily observed in the digestive gland and tract of L. stagnalis. Snails exposed to toxic cyanobacteria showed severe and widespread necrotic changes in the digestive gland co-occurring with a pronounced cytoplasmic presence of MCs in digestive cells and in the lumen of digestive lobules. Snails exposed to dissolved MC-LR showed moderate and negligible pathological changes of the digestive gland co-occurring with a restrained presence of MCs in the apical membrane of digestive cells and in the lumen of digestive lobules. These results confirm lower uptake of dissolved MC-LR and correspondingly lower cytotoxicity in the digestive gland of L. stagnalis. In contrast, after ingestion of MC-containing cyanobacterial filaments, the most likely longer residual time within the digestive gland and/or the MC variant involved (e.g., MC-YR) allowed for increased MC uptake, consequently a higher MC burden in situ and thus a more pronounced ensuing pathology. While no pathological changes were observed in kidney, foot and the genital gland, MCs were detected in spermatozoids and oocytes of all exposed snails, most likely involving a hemolymph transport from the digestive system to the genital gland. The latter results indicate the potential for adverse impact of MCs on gastropod health and reproduction as well as the possible transfer of MCs to higher trophic levels of the food web.

Functional analysis of a subset of canine olfactory receptor genes.

In this paper, we explored the level complexity of the combinatorial olfactory code that allows mammals with a repertoire of about thousand putatively active olfactory receptors encoded in their genomes to recognize and identify a much larger repertoire of odorant molecules. To that end, we cloned 38 canine OR genes belonging to the same OR gene family and transiently expressed them in a subclone of embryonic human kidney cells (HEK293) permanently expressing the G(olf) subunit. Using a Ca(2+) imaging approach, we established for example that as many as 26 out of the 38 cloned OR elicited a Ca(2+) response when exposed to octanal, whereas 10 responded to nonanal, other aldehydes providing intermediate responses. Altogether, these results demonstrated that the combinatorial code is quite complex in support to the highly developed sense of olfaction demonstrated by dogs.

The first radiation hybrid map of a perch-like fish: the gilthead seabream (Sparus aurata L).

Among Teleosts, Perciformes are the largest order of fishes and include numerous species of commercial importance. Perciformes also comprise species of primary interest for evolutionary studies and analysis of the sex determination systems and sex chromosome plasticity. Unfortunately, genomics tools and resources for Perciformes remain to be developed. Here, we report the production of a seabream whole-genome radiation hybrid (RH) panel in which quality was ascertained by the construction of a 2-Mb-resolution RH map. The map encompasses 440 markers (288 microsatellites, 82 gene-based markers, and 70 STS) suitable for linkage analysis and comparative mapping studies. Achievement of a RH panel and a whole-genome RH map should contribute to establishing seabream as a fish model among the Perciformes and should be of importance in aquaculture for marker-assisted selection, improvement of growth performance, and disease management. Development of RH maps in a cost-effective manner for other fishes with the described methodology will offer a powerful approach in aquaculture and will provide extended capabilities for comparing vertebrate genome evolution.

Facilitating genome navigation: survey sequencing and dense radiation-hybrid gene mapping.

Accurate and comprehensive sequence coverage for large genomes has been restricted to only a few species of specific interest. Lower sequence coverage (survey sequencing) of related species can yield a wealth of information about gene content and putative regulatory elements. But survey sequences lack long-range continuity and provide only a fragmented view of a genome. Here we show the usefulness of combining survey sequencing with dense radiation-hybrid (RH) maps for extracting maximum comparative genome information from model organisms. Based on results from the canine system, we propose that from now on all low-pass sequencing projects should be accompanied by a dense, gene-based RH map-construction effort to extract maximum information from the genome with a marginal extra cost.