The dynamic TRPV2 ion channel proximity proteome reveals functional links of calcium flux with cellular adhesion factors NCAM and L1CAM in neurite outgrowth.

TRPV2 voltage-insensitive, calcium-permeable ion channels play important roles in cancer progression, immune response, and neuronal development. Despite TRPV2's physiological impact, underlying endogenous proteins mediating TRPV2 responses and affected signaling pathways remain elusive. Using quantitative peroxidase-catalyzed (APEX2) proximity proteomics we uncover dynamic changes in the TRPV2-proximal proteome and identify calcium signaling and cell adhesion factors recruited to the molecular channel neighborhood in response to activation. Quantitative TRPV2 proximity proteomics further revealed activation-induced enrichment of protein clusters with biological functions in neural and cellular projection. We demonstrate a functional connection between TRPV2 and the neural immunoglobulin cell adhesion molecules NCAM and L1CAM. NCAM and L1CAM stimulation robustly induces TRPV2 [Ca2+]I flux in neuronal PC12 cells and this TRPV2-specific [Ca2+]I flux requires activation of the protein kinase PKCα. TRPV2 expression directly impacts neurite lengths that are modulated by NCAM or L1CAM stimulation. Hence, TRPV2's calcium signaling plays a previously undescribed, yet vital role in cell adhesion, and TRPV2 calcium flux and neurite development are intricately linked via NCAM and L1CAM cell adhesion proteins.

Contribution of the Golgi apparatus in morphogenesis of a virus-induced cytopathic vacuolar system.

The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of a peroxidase-tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here, we propose a model for the conversion of the GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our results have implications for alphavirus-induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.

Coronavirus infection induces progressive restructuring of the endoplasmic reticulum involving the formation and degradation of double membrane vesicles.

Coronaviruses rearrange endoplasmic reticulum (ER) membranes to form a reticulovesicular network (RVN) comprised predominantly of double membrane vesicles (DMVs) involved in viral replication. While portions of the RVN have been analyzed by electron tomography (ET), the full extent of the RVN is not known, nor how RVN formation affects ER morphology. Additionally the precise mechanism of DMV formation has not been observed. In this work, we examined large volumes of coronavirus-infected cells at multiple timepoints during infection using serial-section ET. We provide a comprehensive 3D analysis of the ER and RVN which gives insight into the formation mechanism of DMVs as well as the first evidence for their lysosomal degradation. We also show that the RVN breaks down late in infection, concurrent with the ER becoming the main budding compartment for new virions. This work provides a broad view of the multifaceted involvement of ER membranes in coronavirus infection.

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells.

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

Morphological properties of collagen fibers in porcine lamina propria.

OBJECTIVES: Collagen influences the biomechanical properties of vocal folds. Altered collagen morphology has been implicated in dysphonia associated with aging and scarring. Documenting the morphological properties of native collagen in healthy vocal folds is essential to understand the structural and functional alterations to collagen with aging and disease. Our primary objective was to quantify the morphological properties of collagen in the vocal fold lamina propria. Our secondary exploratory objective was to investigate the effects of pepsin exposure on the morphological properties of collagen in the lamina propria. STUDY DESIGN: Experimental, in vitro study with porcine model. METHODS: Lamina propria was dissected from 26 vocal folds and imaged with atomic force microscopy (AFM). Morphological data on d-periodicity, diameter, and roughness of collagen fibers were obtained. To investigate the effects of pepsin exposure on collagen morphology, vocal fold surface was exposed to pepsin or sham challenge before lamina propria dissection and AFM imaging. RESULTS: The d-periodicity, diameter, and roughness values for native vocal fold collagen are consistent with literature reports of collagen fibers in other body tissues. Pepsin exposure on vocal fold surface did not appear to change the morphological properties of collagen fibers in the lamina propria. CONCLUSIONS: Quantitative data on collagen morphology were obtained at nanoscale resolution. Documenting collagen morphology in healthy vocal folds is critical for understanding the physiological changes to collagen with aging and scarring and for designing biomaterials that match the native topography of lamina propria.

Chemotherapeutic selectivity conferred by selenium: a role for p53-dependent DNA repair.

Selenium in various chemical forms has been the subject of cancer chemoprevention trials, but, more recently, selenium has been used in combination with DNA-damaging chemotherapeutics. Specifically, selenium protected tissues from dose-limiting toxicity and, in fact, allowed delivery of higher chemotherapeutic doses. At the same time, selenium did not protect cancer cells. Therefore, we seek to define the genetic basis for the observed selectivity of selenium in combination chemotherapeutics. The tumor suppressor p53 is mutated in the vast majority of cancers, but is by definition wild-type in nontarget tissues such as bone marrow and gut epithelium, tissues that are often dose-limiting due to DNA damage. We used primary, low-passage mouse embryonic fibroblasts that are wild-type or null for p53 genes to test differential effects of selenium. Seleno-l-methionine, nontoxic by itself, was used to pretreat cell cultures before exposure to UV radiation or UV-mimetic cancer chemotherapy drugs. Seleno-l-methionine pretreatment caused a DNA repair response, which protected from subsequent challenge with DNA-damaging agents. The observed DNA repair response and subsequent DNA damage protection were p53 dependent as neither was observed in p53-null cells. The data suggest that (a) p53 may be an important genetic determinant that distinguishes normal cells from cancer cells, and (b) combinatorial chemotherapeutics that act by p53-dependent mechanisms may enhance chemotherapeutic efficacy by increasing the chemotherapeutic window distinguishing cancer cells from normal cells.