Faculty Bibliography

Phase-separated biomolecular condensates of proteins and nucleic acids form functional membrane-less organelles (e.g. stress granules and P-bodies) in the mammalian cell cytoplasm and nucleus. In contrast to the long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA) associated with the endoplasmic reticulum (ER) and Golgi apparatus, we report that MxA formed membrane-less metastable (shape-changing) condensates in the cytoplasm. In our studies we used the same cell lines and methods as used by previous investigators but concluded that wt MxA formed variably-sized spherical or irregular bodies, filaments and even a reticulum distinct from ER/Golgi membranes. Moreover, in Huh7 cells, MxA structures associated with a novel cytoplasmic reticular meshwork of intermediate filaments. In live-cell assays, 1,6-hexanediol treatment led to rapid disassembly of GFP-MxA structures; FRAP revealed a relative stiffness with a mobile fraction of 0.24±0.02 within condensates, consistent with a higher-order MxA network structure. Remarkably, in intact cells, GFP-MxA condensates reversibly disassembled/reassembled within minutes of sequential decrease/increase respectively in tonicity of extracellular medium, even in low-salt buffers adjusted only with sucrose. Condensates formed from IFN-α-induced endogenous MxA also displayed tonicity-driven disassembly/reassembly. In vesicular stomatitis virus (VSV)-infected Huh7 cells, the nucleocapsid (N) protein, which participates in forming phase-separated viral structures, associated with spherical GFP-MxA condensates in cells showing an antiviral effect. These observations prompt comparisons with the extensive literature on interactions between viruses and stress granules/P-bodies. Overall, the new data correct a long-standing misinterpretation in the MxA literature, and provide evidence for membrane-less MxA biomolecular condensates in the uninfected cell cytoplasm.IMPORTANCE There is a long-standing belief that interferon (IFN)-inducible human "myxovirus resistance protein A" (MxA), which displays antiviral activity against several RNA and DNA viruses, associates with the endoplasmic reticulum (ER) and Golgi apparatus. We provide data to correct this misinterpretation, and further report that MxA forms membrane-less metastable (shape-changing) condensates in the cytoplasm consisting of variably-sized spherical or irregular bodies, filaments and even a reticulum. Remarkably, MxA condensates showed the unique property of rapid (within 1-3 min) reversible disassembly and reassembly in intact cells exposed sequentially to hypotonic and isotonic conditions. Moreover, GFP-MxA condensates included the VSV nucleocapsid (N) protein, a protein previously shown to form liquid-like condensates. Since intracellular edema and ionic changes are hallmarks of cytopathic effects of a viral infection, the tonicity-driven regulation of MxA condensates may reflect a mechanism for modulation of MxA function during viral infection.

The apical surface of the terminally differentiated mammalian urothelial umbrella cell is mechanically stable and highly impermeable, in part due its coverage by urothelial plaques consisting of 2D-crystals of uroplakin particles. The mechanism for regulating the uroplakin/plaque level is unclear. We found that genetic ablation of the highly tissue-specific sorting nexin Snx31, which localizes to plaques lining the multivesicular bodies (MVBs) in urothelial umbrella cells, abolishes MVBs suggesting that Snx31 plays a role in stabilizing the MVB-associated plaques by allowing them to achieve a greater curvature. Strikingly, Snx31 ablation also induces a massive accumulation of uroplakin-containing mitochondria-derived lipid droplets (LDs), which mediate uroplakin degradation via autophagy/lipophagy, leading to the loss of apical and fusiform vesicle plaques. These results suggest that MVBs play an active role in suppressing the excessive/wasteful endocytic degradation of uroplakins. Failure of this suppression mechanism triggers the formation of mitochondrial LDs so that excessive uroplakin membranes can be sequestered and degraded. Since mitochondrial LD formation, which occurs at a low level in normal urothelium, can also be induced by disturbance in uroplakin polymerization due to individual uroplakin-knockout and by arsenite, a bladder carcinogen, this pathway may represent an inducible, versatile urothelial detoxification mechanism. [Media: see text] [Media: see text] [Media: see text].

BACKGROUND:Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. METHODS:We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO") 14 days post-tamoxifen (post-TAM) injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS:homeostasis. Similarly, PKC inhibition normalized spark frequency at comparable SR load levels. CONCLUSIONS:handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

PURPOSE/OBJECTIVE:To identify and characterize amyloid-like substance (ALS) in human and mouse oocytes and preimplantation embryos. METHODS:An experimental prospective pilot study. A total of 252 mouse oocytes and preimplantation embryos and 50 immature and in vitro matured human oocytes and parthenogenetic human embryos, from 11 consenting fertility patients, ages 18-45. Fluorescence intensity from immunofluorescent staining and data from confocal microscopy were quantified. Data were compared by one-way analysis of variance, with the least square-MEANS post-test, Pearson correlation coefficients (r), and bivariate analyses (t tests). ALS morphology was verified using transmission electron microscopy. RESULTS:Immunostaining for ALS appears throughout the zona pellucida, as well as in the cytoplasm and nucleus of mouse and human oocytes, polar bodies, and parthenogenetic embryos, and mouse preimplantation embryos. In mouse, 2-cell embryos exhibited the highest level of ALS (69000187.4 ± 6733098.07). Electron microscopy confirmed the presence of ALS. In humans, fresh germinal vesicle stage oocytes exhibited the highest level of ALS (4164.74088 ± 1573.46) followed by metaphase I and II stages (p = 0.008). There was a significant negative association between levels of ALS and patient body mass index, number of days of ovarian stimulation, dose of gonadotropin used, time between retrieval and fixation, and time after the hCG trigger. Significantly higher levels of ALS were found in patients with AMH between 1 and 3 ng/ml compared to < 1 ng/ml. CONCLUSION/CONCLUSIONS:We demonstrate for the first time the presence, distribution, and change in ALS throughout some stages of mouse and human oocyte maturation and embryonic development. We also determine associations between ALS in human oocytes with clinical characteristics.

Tissue microstructure modeling of diffusion MRI signal is an active research area striving to bridge the gap between macroscopic MRI resolution and cellular-level tissue architecture. Such modeling in neuronal tissue relies on a number of assumptions about the microstructural features of axonal fiber bundles, such as the axonal shape (e.g., perfect cylinders) and the fiber orientation dispersion. However, these assumptions have not yet been validated by sufficiently high-resolution 3-dimensional histology. Here, we reconstructed sequential scanning electron microscopy images in mouse brain corpus callosum, and introduced a random-walker (RaW)-based algorithm to rapidly segment individual intra-axonal spaces and myelin sheaths of myelinated axons. Confirmed by a segmentation based on human annotations initiated with conventional machine-learning-based carving, our semi-automatic algorithm is reliable and less time-consuming. Based on the segmentation, we calculated MRI-relevant estimates of size-related parameters (inner axonal diameter, its distribution, along-axon variation, and myelin g-ratio), and orientation-related parameters (fiber orientation distribution and its rotational invariants; dispersion angle). The reported dispersion angle is consistent with previous 2-dimensional histology studies and diffusion MRI measurements, while the reported diameter exceeds those in other mouse brain studies. Furthermore, we calculated how these quantities would evolve in actual diffusion MRI experiments as a function of diffusion time, thereby providing a coarse-graining window on the microstructure, and showed that the orientation-related metrics have negligible diffusion time-dependence over clinical and pre-clinical diffusion time ranges. However, the MRI-measured inner axonal diameters, dominated by the widest cross sections, effectively decrease with diffusion time by ~ 17% due to the coarse-graining over axonal caliber variations. Furthermore, our 3d measurement showed that there is significant variation of the diameter along the axon. Hence, fiber orientation dispersion estimated from MRI should be relatively stable, while the "apparent" inner axonal diameters are sensitive to experimental settings, and cannot be modeled by perfectly cylindrical axons.

Introduction: Noncoding small RNAs in the retina regulate gene expression by targeting and repressing mRNA. Small RNAs are secreted in extracellular vesicles (EVs). Analysis of EVs released from developing retinal tissue is an essential step in elucidating the role of EV molecular cargo and signalling during retinogenesis. A number of canonical genes are associated with retinal cell fate determination during development, but EV-mediated gene regulation in the retinal microenvironment remains undefined. In this study, we characterize the microRNA, tRNA, and piRNA composition of EVs secreted from human induced pluripotent stem cell (hiPSCs) - derived 3D retinas at three developmental time points that correlate with hallmarks of retinal cell differentiation and lamination in vivo.
Method(s): Retinal organoids were generated from hiPSCs. We selected three developmental time points (day 42, 63 and 90) that represent distinctive stages during normal retinal cell fate specification and lamination. We analysed the release rate, concentration, morphology and content (miRNA, tRNA and pi- RNA) of EVs released from human hiPSCs-derived 3D retinas.
Result(s): The genetic signalling, developmental time course and morphogenesis of these retinal organoids were comparable to those of developing human retinas in vivo. According to Gene Ontology analysis, miRNA targets at the earliest stage of development were more relevant to early differentiation and cell morphogenesis, whereas miRNA targets at the later stages were more relevant to cell proliferation, cell differentiation, and cell migration. Summary/Conclusion: For the first time, this work demonstrates the rate of release and concentration of EVs from developing hiPSC-derived 3D retinal tissue. We report a large variety of small RNAs in EVs from hiPSC-derived 3D retinas, including miRNAs, tRNAs and piRNAs. The full range of small RNAs detected in our EVs may act as regulatory elements to modulate gene activity and may serve as biomarkers of normal development. This work represents the first sequencing analysis of small RNA species contained in hiPSCderived 3D retinas and their released EVs

Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique 2D-crystals of 16-nm particles ("urothelial plaques") covering the apical urothelial surface. Although uroplakins are highly expressed only in mouse urothelium and are often referred to as being urothelium-specific, they are also expressed in several nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms and ovary/oocytes. In oocytes, uroplakins co-localize with CD9 on cell surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine-phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs' fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require 2D-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form 2D- crystalline plaques during mammalian divergence enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, in order to protect/modify the apical surface of the modern-day mammalian urothelium.

Germ granules are non-membranous ribonucleoprotein granules deemed the hubs for post-transcriptional gene regulation and functionally linked to germ cell fate across species. Little is known about the physical properties of germ granules and how these relate to germ cell function. Here we study two types of germ granules in the Drosophila embryo: cytoplasmic germ granules that instruct primordial germ cells (PGCs) formation and nuclear germ granules within early PGCs with unknown function. We show that cytoplasmic and nuclear germ granules are phase transitioned condensates nucleated by Oskar protein that display liquid as well as hydrogel-like properties. Focusing on nuclear granules, we find that Oskar drives their formation in heterologous cell systems. Multiple, independent Oskar protein domains synergize to promote granule phase separation. Deletion of Oskar's nuclear localization sequence specifically ablates nuclear granules in cell systems. In the embryo, nuclear germ granules promote germ cell divisions thereby increasing PGC number for the next generation.

Aim of the study/UNASSIGNED:Interferon (IFN)-α is now established as a treatment modality in various human cancers. The IFN-α-inducible human "myxovirus resistance protein A" (MxA) is a cytoplasmic dynamin-family large GTPase primarily characterized for its broad-spectrum antiviral activity and, more recently, for its anti-tumor and anti-metastasis effects. We characterized the association of IFN-α-induced MxA with cytoplasmic structures in human Huh7 cancer cells and in primary endothelial cells. Material and methods/UNASSIGNED:We re-evaluated the long-standing inference that MxA associated with the smooth ER using double-label immunofluorescence techniques and the ER structural protein RTN4 as a marker for smooth ER in IFN-α-treated cells. We also evaluated the relationship of exogenously expressed HA-MxA and GFP-MxA with mitochondria, and characterized cytoplasmic GFP-MxA structures using correlated light and electron microscopy (CLEM). Results and conclusions/UNASSIGNED:We discovered that IFN-α-induced endogenous MxA associated with variably-sized endosome-like and reticular cytoplasmic structures which were distinct from the ER. Thin-section EM studies of GFP-MxA expressing Huh7 cells showed that GFP-MxA formed variably-sized clusters of vesiculotubular elements to form endosome-like "MxA bodies". Many of these clusters stretched out alongside cytoskeletal elements to give the appearance of a cytoplasmic "MxA reticulum". This MxA meshwork was distinct from but adjacent to mitochondria. GFP-MxA expressing Huh7 cells showed reduced MitoTracker uptake and swollen mitochondria by thin-section EM. The new data identify cytoplasmic MxA structures as novel organelles, and suggest cross-talk between MxA structures and mitochondria that might account for the increased anti-tumoral efficacy of IFN-α combined with ligands that activate other pattern-sensing receptor pathways.