Multi-year dynamics of ranavirus, chytridiomycosis, and co-infections in a temperate host assemblage of amphibians.

Chytridiomycosis and ranavirosis are 2 emerging infectious diseases that have caused significant global amphibian decline. Although both have received much scrutiny, little is known about interactions between the 2 causative agents Batrachochytrium dendrobatidis (Bd) and ranavirus (Rv) at the individual host and population levels. We present the first longitudinal assessment of Bd, Rv, and co-infections of a temperate amphibian assemblage in North America. From 2012 to 2016, we assessed the temporal oscillations of Bd, Rv and co-infection dynamics in a sample of 729 animals representing 13 species. Bd, Rv, and co-infected amphibians were detected during all 5 yr. Bd, Rv, and co-infection prevalence all varied annually, with the lowest instances of each at 2.1% (2013), 7.9% (2016), and 0.6% (2016), respectively. The highest Bd, Rv, and co-infection prevalence were recorded in 2012 (26.8%), 2016 (38.3%), and 2015 (10.3%), respectively. There was no association between Bd or Rv infection prevalence and co-infection, either when assessing the entire amphibian assemblage as a whole (odds ratio 1.32, 95% CI: 0.83-2.1, p = 0.29) or within species for amphibians that were more numerically represented (n > 40, p > 0.05). This suggests neither Bd nor Rv facilitate host co-infections within the sampled host assemblage. Instead, the basis for co-infections is the spatiotemporal distribution of both pathogens. Despite lack of interplay between Bd and Rv in this population, our study highlights the importance of considering numerous pathogens that may be present within amphibian habitats in order to properly anticipate interactions that may have direct bearing on disease outcomes.

Vimentin Intermediate Filaments Template Microtubule Networks to Enhance Persistence in Cell Polarity and Directed Migration.

Increased expression of vimentin intermediate filaments (VIFs) enhances directed cell migration, but the mechanism behind VIFs' effect on motility is not understood. VIFs interact with microtubules, whose organization contributes to polarity maintenance in migrating cells. Here, we characterize the dynamic coordination of VIF and microtubule networks in wounded monolayers of retinal pigment epithelial cells. By genome editing, we fluorescently labeled endogenous vimentin and α-tubulin, and we developed computational image analysis to delineate architecture and interactions of the two networks. Our results show that VIFs assemble an ultrastructural copy of the previously polarized microtubule network. Because the VIF network is long-lived compared to the microtubule network, VIFs template future microtubule growth along previous microtubule tracks, thus providing a feedback mechanism that maintains cell polarity. VIF knockdown prevents cells from polarizing and migrating properly during wound healing. We suggest that VIFs' templating function establishes a memory in microtubule organization that enhances persistence in cell polarization in general and migration in particular.

Methods for Determining the Cellular Functions of Vimentin Intermediate Filaments.

The type III intermediate filament protein vimentin was once thought to function mainly as a static structural protein in the cytoskeleton of cells of mesenchymal origin. Now, however, vimentin is known to form a dynamic, flexible network that plays an important role in a number of signaling pathways. Here, we describe various methods that have been developed to investigate the cellular functions of the vimentin protein and intermediate filament network, including chemical disruption, photoactivation and photoconversion, biolayer interferometry, soluble bead binding assay, three-dimensional substrate experiments, collagen gel contraction, optical-tweezer active microrheology, and force spectrum microscopy. Using these techniques, the contributions of vimentin to essential cellular processes can be probed in ever further detail.

Abnormal intermediate filament organization alters mitochondrial motility in giant axonal neuropathy fibroblasts.

Giant axonal neuropathy (GAN) is a rare disease caused by mutations in the GAN gene, which encodes gigaxonin, an E3 ligase adapter that targets intermediate filament (IF) proteins for degradation in numerous cell types, including neurons and fibroblasts. The cellular hallmark of GAN pathology is the formation of large aggregates and bundles of IFs. In this study, we show that both the distribution and motility of mitochondria are altered in GAN fibroblasts and this is attributable to their association with vimentin IF aggregates and bundles. Transient expression of wild-type gigaxonin in GAN fibroblasts reduces the number of IF aggregates and bundles, restoring mitochondrial motility. Conversely, silencing the expression of gigaxonin in control fibroblasts leads to changes in IF organization similar to that of GAN patient fibroblasts and a coincident loss of mitochondrial motility. The inhibition of mitochondrial motility in GAN fibroblasts is not due to a global inhibition of organelle translocation, as lysosome motility is normal. Our findings demonstrate that it is the pathological changes in IF organization that cause the loss of mitochondrial motility.

Intermediate Filaments Play a Pivotal Role in Regulating Cell Architecture and Function.

Intermediate filaments (IFs) are composed of one or more members of a large family of cytoskeletal proteins, whose expression is cell- and tissue type-specific. Their importance in regulating the physiological properties of cells is becoming widely recognized in functions ranging from cell motility to signal transduction. IF proteins assemble into nanoscale biopolymers with unique strain-hardening properties that are related to their roles in regulating the mechanical integrity of cells. Furthermore, mutations in the genes encoding IF proteins cause a wide range of human diseases. Due to the number of different types of IF proteins, we have limited this short review to cover structure and function topics mainly related to the simpler homopolymeric IF networks composed of vimentin, and specifically for diseases, the related muscle-specific desmin IF networks.

The Sec7 guanine nucleotide exchange factor GBF1 regulates membrane recruitment of BIG1 and BIG2 guanine nucleotide exchange factors to the trans-Golgi network (TGN).

Three Sec7 guanine nucleotide exchange factors (GEFs) activate ADP-ribosylation factors (ARFs) to facilitate coating of transport vesicles within the secretory and endosomal pathways. GBF1 recruits COPI to pre-Golgi and Golgi compartments, whereas BIG1 and BIG2 recruit AP1 and GGA clathrin adaptors to the trans-Golgi network (TGN) and endosomes. Here, we report a functional cascade between these GEFs by showing that GBF1-activated ARFs (ARF4 and ARF5, but not ARF3) facilitate BIG1 and BIG2 recruitment to the TGN. We localize GBF1 ultrastructurally to the pre-Golgi, the Golgi, and also the TGN. Our findings suggest a model in which GBF1 localized within pre-Golgi and Golgi compartments mediates ARF activation to facilitate recruitment of COPI to membranes, whereas GBF1 localized at the TGN mediates ARF activation that leads to the recruitment of BIG1 and BIG2 to the TGN. Membrane-associated BIG1/2 then activates ARFs that recruit clathrin adaptors. In this cascade, an early acting GEF (GBF1) activates ARFs that mediate recruitment of late acting GEFs (BIG1/2) to coordinate coating events within the pre-Golgi/Golgi/TGN continuum. Such coordination may optimize the efficiency and/or selectivity of cargo trafficking through the compartments of the secretory pathway.

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation.

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ∼200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.

Transient expression of epitope-tagged proteins in mammalian cells.

Before the advent of molecular methods to tag proteins, the visualization of proteins within cells by immunoelectron microscopy required the use of highly specific antibodies directed against the protein of interest. Thus, only proteins for which antibodies were available could be visualized. Current technologies allow the detection of proteins for which specific antibodies are not available. This procedure involves the generation of DNA constructs that express the protein of interest tagged with an epitope that is recognized by a well-characterized commercially available antibody. Proteins can be tagged with a wide variety of epitopes, small and large, using commercially available vectors that allow expression in mammalian cells. Epitope-tagged proteins are easily transfected into many mammalian cell lines and, in most cases, tightly mimic the distribution of the endogenous protein. Prior to immunoelectron microscopy, expression and localization of tagged proteins can be assessed by Western blotting and immunofluorescence. Furthermore, specialized fluorescent tags, such as the green fluorescent protein (GFP), can be used to rapidly screen for transfection efficiency and localization. The use of epitope-tagged protein expression has increased the versatility of immunoelectron microscopy to explore the function of uncharacterized proteins for which highly specific antibodies are not available.