Zooming in on the molecular mechanisms of endocytic budding by time-resolved electron microscopy

Endocytic budding implies the remodeling of a plasma membrane portion from a flat sheet to a closed vesicle. Clathrin- and actin-mediated endocytosis in yeast has proven a very powerful model to study this process, with more than 60 evolutionarily conserved proteins involved in fashioning primary endocytic vesicles. Major progress in the field has been made during the last decades by defining the sequential recruitment of the endocytic machinery at the cell cortex using live-cell fluorescence microscopy. Higher spatial resolution has been recently achieved by developing time-resolved electron microscopy methods, allowing for the first time the visualization of changes in the plasma membrane shape, coupled to the dynamics of the endocytic machinery. Here, we highlight these advances and review recent findings from yeast and mammals that have increased our understanding of where and how endocytic proteins may apply force to remodel the plasma membrane during different stages of the process.     

Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination

Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.     

Mutations in the RNA exosome component gene EXOSC3 cause pontocerebellar hypoplasia and spinal motor neuron degeneration

RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration.     

Rediscovered population of Mexican Plateau spotted whiptail lizard, Aspidoscelis septemvittata , from México, D.F.

Multiple surveys carried out by herpetologists in México, D.F., during the 1980s failed to find specimens of Aspidoscelis septemvitatta (Squamata: Teiidae). However, 10 specimens were recently collected to the east of México, D.F., inside a protected area, Sierra de Santa Catarina. The Sierra de Santa Catarina has been heavily modified by human activity and habitation. Morphological and natural history information about the specimens are presented, as well as notes on locality.     

Comparative life history for populations of the Sceloporus grammicus complex (Squamata: Phrynosomatidae)

A Teotihuacán population of the Sceloporus grammicus complex was compared with other previously studied populations (Parque Nacional Zoquiapan [PNZ], Monte Alegre Ajusco [MAA], Pedregal San Angel [PSA], Cantimplora [CA], Capulín, Laguna, Paredon, Michilia, and south Texas) for variations in several life history characteristics (SVL at sexual maturity, reproductive period, ovulation and gestation time, and litter size). Mean body size at sexual maturity of females from Teotihuacán was larger than PNZ, MAA, CA, and Capulín. Reproductive period (vitellogenesis, ovulation, gestation, and birth) for the Teotihuacán population was the shortest of all populations. In the Teotihuacán population, gestation time was similar to the Capulínand MAA populations but was shorter than all other populations except the Michilia population. Embryonic development at ovulation varied among populations, with Teotihuacán and Capulín showing earlier stages (stages 1) at ovulation than all other populations. Teotihuacán, PSA, PNZ, and Texas all showed similar litter size, which were larger than Laguna, Paredon, MAA, Capulín, and CA populations. Differences in reproductive characteristics of these populations could indicate phylogenetically constrained, reproductively isolated populations, or they may be explained as merely responses to different environments.     

A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition

Mutations affecting ciliary components cause ciliopathies. As described here, we investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel and Joubert syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2 and Cc2d2a. Components of this complex co-localize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes Joubert syndrome. Thus, a transition zone complex of Meckel and Joubert syndrome proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.     

The sheddase activity of ADAM17/TACE is regulated by the tetraspanin CD9

ADAM17/TACE is a metalloproteinase responsible for the shedding of the proinflammatory cytokine TNF-α and many other cell surface proteins involved in development, cell adhesion, migration, differentiation, and proliferation. Despite the important biological function of ADAM17, the mechanisms of regulation of its metalloproteinase activity remain largely unknown. We report here that the tetraspanin CD9 and ADAM17 partially co-localize on the surface of endothelial and monocytic cells. In situ proximity ligation, co-immunoprecipitation, crosslinking, and pull-down experiments collectively demonstrate a direct association between these molecules. Functional studies reveal that treatment with CD9-specific antibodies or neoexpression of CD9 exert negative regulatory effects on ADAM17 sheddase activity. Conversely, CD9 silencing increased the activity of ADAM17 against its substrates TNF-α and ICAM-1. Taken together, our results show that CD9 associates with ADAM17 and, through this interaction, negatively regulates the sheddase activity of ADAM17.