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The crosstalk between growth factor and adhesion receptors is key for cell growth and migration. In pathological settings, these receptors are drivers of cancer. Yet, how growth and adhesion signals are spatially organized and integrated is poorly understood. Here we use quantitative fluorescence and electron microscopy to reveal a mechanism where flat clathrin lattices partition and activate growth factor signals via a coordinated response that involves crosstalk between epidermal growth factor receptor (EGFR) and the adhesion receptor β5-integrin. We show that ligand-activated EGFR, Grb2, Src, and β5-integrin are captured by clathrin coated-structures at the plasma membrane. Clathrin structures dramatically grow in response to EGF into large flat plaques and provide a signaling platform that link EGFR and β5-integrin through Src-mediated phosphorylation. Disrupting this EGFR/Src/β5-integrin axis prevents both clathrin plaque growth and dampens receptor signaling. Our study reveals a reciprocal regulation between clathrin lattices and two different receptor systems to coordinate and enhance signaling. These findings have broad implications for the regulation of growth factor signaling, adhesion, and endocytosis. The integration and organization of growth factor signaling, adhesion, and endocytosis is poorly understood. Here the authors use light and electron microscopy to shed light on the role of flat clathrin lattices and cell adhesion in growth factor signaling.

Clathrin assemblies on the cell membrane are critical for endocytosis and signal transduction in cells. Specifically, the Ω-shaped clathrin assembly functions as the coat of endocytic vesicles, while the flat clathrin assembly, also known as the flat clathrin lattice (FCL), serves as a signaling hub for various pathways. Multiple flat clathrin lattices exist on the cell membrane, and these lattices grow after epidermal growth factor stimulation (EGF) and then return to baseline. In this work, we used a particle-based model to simulate the assembly and disassembly of flat clathrin lattices to capture these dynamics. We found that the formation of flat clathrin lattices is highly dynamic, that is, cluster number, size and dwelling time often change even in the absence of any stimulus. Moreover, these key features are affected by adaptor protein 2 (AP-2) number, clathrin-clathrin binding rate, and clathrin diffusion coefficient. Specifically, an increase in AP-2 number leads to the transition from no cluster, short-lived multiple small clusters, to a long-lasting single giant cluster. An increased clathrin-clathrin binding rate or decreased clathrin diffusion coefficient both result in an increased cluster number, reduced cluster size, and shortened dwelling time. Furthermore, we also predicted that under EGF stimulation, simultaneous changes in the AP-2 number, the clathrin-clathrin binding rate, and the clathrin diffusion coefficient can reproduce the experimentally observed trend of FCLs: an increase in cluster number and size in the first 30 minutes, followed by a decrease after 30 minutes. These findings reveal kinetic mechanisms underlying the formation of multiple FCLs and how EGF regulates FCL dynamics.

Cryo-electron tomography (cryoET) provides sub-nanometer protein structure within the dense cellular environment. Existing sample preparation methods are insufficient at accessing the plasma membrane and its associated proteins. Here, we present a correlative cryo-electron tomography pipeline optimally suited to image large ultra-thin areas of isolated basal and apical plasma membranes. The pipeline allows for angstrom-scale structure determination with subtomogram averaging and employs a genetically encodable rapid chemically-induced electron microscopy visible tag for marking specific proteins within the complex cellular environment. The pipeline provides efficient, distributable, low-cost sample preparation and enables targeted structural studies of identified proteins at the plasma membrane of mammalian cells. Cryo-electron tomography requires thin samples. Few cell-thinning techniques have been standardized. Here, the authors provide and validate a method to prepare, label, and image proteins at mammalian cell plasma membranes for sub-nanometer structural analysis.

Background To unravel the evolutionary history of a complex group, a comprehensive reconstruction of its phylogenetic relationships is crucial. This requires meticulous taxon sampling and careful consideration of multiple characters to ensure a complete and accurate reconstruction. The phylogenetic position of the Orestias genus has been estimated partly on unavailable or incomplete information. As a consequence, it was assigned to the family Cyprindontidae, relating this Andean fish to other geographically distant genera distributed in the Mediterranean, Middle East and North and Central America. In this study, using complete genome sequencing, we aim to clarify the phylogenetic position of Orestias within the Cyprinodontiformes order. Results We sequenced the genome of three Orestias species from the Andean Altiplano. Our analysis revealed that the small genome size in this genus (~ 0.7 Gb) was caused by a contraction in transposable element (TE) content, particularly in DNA elements and short interspersed nuclear elements (SINEs). Using predicted gene sequences, we generated a phylogenetic tree of Cyprinodontiformes using 902 orthologs extracted from all 32 available genomes as well as three outgroup species. We complemented this analysis with a phylogenetic reconstruction and time calibration considering 12 molecular markers (eight nuclear and four mitochondrial genes) and a stratified taxon sampling to consider 198 species of nearly all families and genera of this order. Overall, our results show that phylogenetic closeness is directly related to geographical distance. Importantly, we found that Orestias is not part of the Cyprinodontidae family, and that it is more closely related to the South American fish fauna, being the Fluviphylacidae the closest sister group. Conclusions The evolutionary history of the Orestias genus is linked to the South American ichthyofauna and it should no longer be considered a member of the Cyprinodontidae family. Instead, we submit that Orestias belongs to the Orestiidae family, as suggested by Freyhof et al. (2017), and that it is the sister group of the Fluviphylacidae family, distributed in the Amazonian and Orinoco basins. These two groups likely diverged during the Late Eocene concomitant with hydrogeological changes in the South American landscape.

Climatic and oceanographic events occurring during the last glacial cycle in the Humboldt Current System (HCS) have left genetic footprints in marine invertebrate populations. The objective of this study was to evaluate the effect of the glacial period on Octopus mimus populations found throughout the HCS. This species lays a large number of small eggs which hatch into planktonic paralarvae with the potential to undergo wide dispersal. We sequenced the COIII gene to perform phylogeographic analyses of 197 octopuses sampled from seven localities. The genetic diversity of Octopus mimus was low and decreased towards the southern end of the distribution range, which comprises a single population. The haplotype genealogy and Bayesian Skyride plot suggest that O. mimus underwent a demographic expansion after the last glacial maximum (LGM). This would imply a contraction of the range of this organism toward northern latitudes during the LGM followed by southward expansion and recolonization once the contemporary interglacial period began.

Clarifying the taxonomic status and distribution of endangered species is crucial to their conservation. In this study, we contrasted different lines of evidence (morphology, mtDNA, and nucDNA: microsatellites and SNP) to clarify the taxonomic status of endangered Telmatobius water frog species and unidentified populations that inhabit the Salt Puna in Chile. We studied population differentiation and species divergence by performing morphometric, population genetic and species delimitation analyses. The results confirmed the species status of Telmatobius fronteriensis and T. philippii, as they exhibited morphometric, mitochondrial and genomic SNP divergence. Although Bayes factor delimitation analysis indicated that the Telmatobius populations of Ascotán and Carcote could represent a new species, their few mitochondrial differences and similar morphology with respect to T. philippii suggested otherwise. Instead, they can be considered an evolutionarily significant unit of T. philippii that has differentiated from the type locality. These results extend the geographic distribution of T. philippii, which is categorized as critically endangered by the IUCN.

Species delimitation in minute freshwater snails is often difficult to perform using solely shell morphology. The problem intensifies when invasive species spread within the distribution range of morphologically similar native species. In Chile, the Truncatelloidean snails are represented by the native genera Heleobia and Potamolithu s plus the invasive mudsnail Potamopyrgus antipodarum , which can easily be confused. Using an integrative approach, we performed molecular phylogenetic analysis and studied reproductive and morphological features to identify superficially similar forms inhabiting the central area of the country. Truncatelloidean snails were identified in 40 of 51 localities sampled, 10 containing Potamopyrgus antipodarum , 23 Heleobia and 7 Potamolithus . Based on these results and previously published data, the known distribution of the mudsnail in Chile encompasses 6 hydrological basins, including 18 freshwater ecosystems. The finding of the mudsnails in several type localities of native species/subspecies of “ Heleobia ” that were not find in situ suggests species replacement or significant extinction of native fauna, a hypothesis supported by the restudy of type material that shows that endemic forms belong to the genus Potamolithu s. This study shows the usefulness of integrative taxonomy not only resolving complex taxa with cryptic morphology but also measuring the extent of an ongoing invasion.

Morphometrics has been used on Triatomines, a well-known phenotypically variable insect, to understand the process of morphological plasticity and infer the changes of this phenomenon. The following research was carried out in two regions of the inter-Andean valleys and two Chaco regions of Chuquisaca-Bolivia. Triatoma infestans adults were collected from the peridomestic (pens and chicken coops) along a geographic gradient in order to evaluate the morphological differentiation between groups and their pattern of sexual shape dimorphism. Geometric morphometric methods were applied on the wings and heads of T. infestans. The main findings include that we proved sexual dimorphism in heads and wings, determined the impact of environmental factors on size and shape and validated the impact of nutrition on head shape variation. These results show that geometric morphometric procedures can be used to provide key insight into the biological adaptation of T. infestans on different biotic (nutrition) and abiotic (environment) conditions, which could serve in understanding and evaluating infestation processes and further vector control programs.

Background The diversity and population genetic structure of many species have been shaped by historical and contemporary climatic changes. For the species of the South American Altiplano, the historical climatic changes are mainly related to the wet events of great magnitude and regional influence that occurred during the Pleistocene climatic oscillations (PCOs). In contrast, contemporary climate changes are associated with events of lesser magnitude and local influence related to intensifications of the South American Summer Monsoon (SASM). Although multiple studies have analyzed the effect of PCOs on the genetic patterns of highland aquatic species, little is known about the impact of contemporary climate changes in recent evolutionary history. Therefore, in this study, we investigated the change in population structure and connectivity using nuclear and mitochondrial markers throughout the distribution range of Heleobia ascotanensis , a freshwater Cochliopidae endemic to the Ascotán Saltpan. In addition, using geometric morphometric analyses, we evaluated the concomitance of genetic divergence and morphological differentiation. Results The mitochondrial sequence analysis results revealed the presence of highly divergent co-distributed and geographically nested haplotypes. This pattern reflects an extension in the distribution of groups that previously would have differentiated allopatrically. These changes in distribution would have covered the entire saltpan and would be associated with the large-scale wet events of the PCOs. On the other hand, the microsatellite results defined five spatially isolated populations, separated primarily by geographic barriers. Contemporary gene flow analyses suggest that post-PCO, climatic events that would have connected all populations did not occur. The morphometric analyses results indicate that there is significant morphological differentiation in the populations that are more isolated and that present the greatest genetic divergence. Conclusions The contemporary population structure and morphological variation of H. ascotanensis mainly reflect the post-PCO climatic influence. Although both markers exhibit high genetic structuring, the microsatellite and morphology results show the preponderant influence of fragmentation in recent evolutionary history. The contemporary genetic pattern shows that in species that have limited dispersal capabilities, genetic discontinuities can appear rapidly, erasing signs of historical connectivity.

The morphological variations of four populations of geographically isolated Triatoma infestans located in the area of inter-Andean valleys and Chaco of Chuquisaca, Bolivia, were evaluated. Fifty-three females and sixty-one males were collected in the peri-domicile and analyzed with geometric morphometrics tools to study the patterns of the head and wing shape variation. The principal component analysis and canonical variate analysis revealed morphological variations between the populations studied, which were then confirmed by the permutation test of the differences between populations using Mahalanobis and Procustes distances. The multivariate regression analysis shows that the centroid size influences the shape of the heads and wings. T. infestans of the inter-Andean valleys are longer in the head and wings compared to the population of the Chaco. We propose that the geometric shape variation may be explained by geographical changes in climatic conditions, peri-domiciliar habitats, food source quality, and the use of insecticides.