Structural Correlates of Rotavirus Cell Entry

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex—for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼700 Å inner capsid particle (“double-layered particle”, DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration

Quantitative Proteomics of an Amphibian Pathogen, Batrachochytrium dendrobatidis, following Exposure to Thyroid Hormone

Batrachochytrium dendrobatidis (Bd), a chytrid fungus, has increasingly been implicated as a major factor in the worldwide decline of amphibian populations. The fungus causes chytridiomycosis in susceptible species leading to massive die-offs of adult amphibians. Although Bd infects the keratinized mouthparts of tadpoles and negatively affects foraging behavior, these infections are non-lethal. An important morphogen controlling amphibian metamorphosis is thyroid hormone (T3). Tadpoles may be infected with Bd and the fungus may be exposed to T3 during metamorphosis. We hypothesize that exposure of Bd to T3 may induce the expression of factors associated with host colonization and pathogenicity. We utilized a proteomics approach to better understand the dynamics of the Bd-T3 interaction. Using liquid chromatography-mass spectrometry (LC-MS), we generated a data set of a large number of cytoplasmic and membrane proteins following exposure of Bd to T3. From these data, we identified a total of 263 proteins whose expression was significantly changed following T3 exposure. We provide evidence for expression of an array of proteins that may play key roles in both genomic and non-genomic actions of T3 in Bd. Additionally, our proteomics study shows an increase in several proteins including proteases and a class of uncommon crinkler and crinkler-like effector proteins suggesting their importance in Bd pathogenicity as well as those involved in metabolism and energy transfer, protein fate, transport and stress responses. This approach provides insights into the mechanistic basis of the Bd-amphibian interaction following T3 exposure

Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

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Role of the clathrin adaptor PICALM in normal hematopoiesis and polycythemia vera pathophysiology

Clathrin-dependent endocytosis is an essential cellular process shared by all cell types. Despite this, precisely how endocytosis is regulated in a cell-type-specific manner and how this key pathway functions physiologically or pathophysiologically remain largely unknown. PICALM, which encodes the clathrin adaptor protein PICALM, was originally identified as a component of the CALM/AF10 leukemia oncogene. Here we show, by employing a series of conditional Picalm knockout mice, that PICALM critically regulates transferrin uptake in erythroid cells by functioning as a cell-type-specific regulator of transferrin receptor endocytosis. While transferrin receptor is essential for the development of all hematopoietic lineages, Picalm was dispensable for myeloid and B-lymphoid development. Furthermore, global Picalm inactivation in adult mice did not cause gross defects in mouse fitness, except for anemia and a coat color change. Freeze-etch electron microscopy of primary erythroblasts and live-cell imaging of murine embryonic fibroblasts revealed that Picalm function is required for efficient clathrin coat maturation. We showed that the PICALM PIP(2) binding domain is necessary for transferrin receptor endocytosis in erythroblasts and absolutely essential for erythroid development from mouse hematopoietic stem/progenitor cells in an erythroid culture system. We further showed that Picalm deletion entirely abrogated the disease phenotype in a Jak2(V617F) knock-in murine model of polycythemia vera. Our findings provide new insights into the regulation of cell-type-specific transferrin receptor endocytosis in vivo. They also suggest a new strategy to block cellular uptake of transferrin-bound iron, with therapeutic potential for disorders characterized by inappropriate red blood cell production, such as polycythemia vera

Immunoprotective properties of primary Sertoli cells in mice: potential functional pathways that confer immune privilege

Primary Sertoli cells isolated from mouse testes survive when transplanted across immunological barriers and protect cotransplanted allogeneic and xenogeneic cells from rejection in rodent models. In contrast, the mouse Sertoli cell line (MSC-1) lacks immunoprotective properties associated with primary Sertoli cells. In this study, enriched primary Sertoli cells or MSC-1 cells were transplanted as allografts into the renal subcapsular area of naive BALB/c mice, and their survival in graft sites was compared. While Sertoli cells were detected within the grafts with 100% graft survival throughout the 20-day study, MSC-1 cells were rejected between 11 and 14 days, with 0% graft survival at 20 days posttransplantation. Nonetheless, the mechanism for primary Sertoli cell survival and immunoprotection remains unresolved. To identify immune factors or functional pathways potentially responsible for immune privilege, gene expression profiles of enriched primary Sertoli cells were compared with those of MSC-1 cells. Microarray analysis identified 2369 genes in enriched primary Sertoli cells that were differentially expressed at ±4-fold or higher levels than in MSC-1 cells. Ontological analyses identified multiple immune pathways, which were used to generate a list of 340 immune-related genes. Three functions were identified in primary Sertoli cells as potentially important for establishing immune privilege: suppression of inflammation by specific cytokines and prostanoid molecules, slowing of leukocyte migration by controlled cell junctions and actin polymerization, and inhibition of complement activation and membrane-associated cell lysis. These results increase our understanding of testicular immune privilege and, in the long-term, could lead to improvements in transplantation success

List of proteins that showed more than 2-fold increase in <i>Bd</i> exposed to T₃.

<p>Statistically significant expression at P <0.05.</p><p>The values are given as T₃ normalized spectral count (N-SC) Log₂ relative expression.</p><p>To calculate the absolute fold change, the conversion is applied as 2^ (T₃ N-SC).</p><p>^aAs given according to the <a href="http://www.broadinstitute.org" target="_blank">www.broadinstitute.org</a>.</p><p>List of proteins that showed more than 2-fold increase in <i>Bd</i> exposed to T₃.</p

Association of productive (A) and non-productive (B) particles with AP-2 clathrin adaptor, dynamin, and Rab5.

<p>“Productive particles” are those that bind and uncoat as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004355#ppat-1004355-g003" target="_blank">Figure 3</a>; “non-productive particles”, those that fail to uncoat within 30 mins of binding. Of 62 productive particles followed in cells transfected with σ2-eGFP (<b>A</b>, left), 50 did not colocalize with σ2 at any time, while 12 colocalized with σ2 at an early time point. Likewise, of 62 productive particles followed in Rab5-eGFP transfected cells (<b>A</b>, right), only 11 appeared to colocalize with Rab5, while of 166 non-productive particles (<b>B</b>, right), a substantial majority ended up in Rab5 endosomes. Cells stably transfected with σ2-eGFP or Rab5-eGFP or transiently transfected with dynamin-eGFP.</p

Internalization.

<p><b>A</b>. Image of three particles that uncoat after addition of fluorescently labeled m159 antibody to the medium. (Virus pre-bound to cells, excess medium withdrawn, and antibody-containing medium then added.) Particles that uncoat do not bind antibody. White line represents edge of cell; particles on the coverslip all bind antibody. <b>B</b>. Time to initiation of uncoating (decrease in VP7 and VP4 label intensities) of particles that do not bind antibody m159. The antibody was added 5–7 minutes after addition of virus to cells, as indicated by the arrow; images were collected every 6 seconds. <b>C</b>. EDTA pulse. Top panel: doubly labeled virus bound to cell, before and after EDTA pulse. Middle panel: DIC images of the same cell; Bottom panel: particles bound to coverslip. <b>D</b>. Percent of virus resistant to EDTA uncoating, as a function of time between addition of virus and pulse of EDTA (black open circles) and cumulative representation of data in 2B (gray solid circles).</p

Schematic illustration for the proteomics study to profile total proteins in <i>Batrachochytrium dendrobatidis</i> following exposure to T₃.

<p>Red boxes show regions where each sample lane of the gel was cut into slices for peptide extraction.</p