Tau forms synaptic nano-biomolecular condensates controlling the dynamic clustering of recycling synaptic vesicles.

Neuronal communication relies on the release of neurotransmitters from various populations of synaptic vesicles. Despite displaying vastly different release probabilities and mobilities, the reserve and recycling pool of vesicles co-exist within a single cluster suggesting that small synaptic biomolecular condensates could regulate their nanoscale distribution. Here, we performed a large-scale activity-dependent phosphoproteome analysis of hippocampal neurons in vitro and identified Tau as a highly phosphorylated and disordered candidate protein. Single-molecule super-resolution microscopy revealed that Tau undergoes liquid-liquid phase separation to generate presynaptic nanoclusters whose density and number are regulated by activity. This activity-dependent diffusion process allows Tau to translocate into the presynapse where it forms biomolecular condensates, to selectively control the mobility of recycling vesicles. Tau, therefore, forms presynaptic nano-biomolecular condensates that regulate the nanoscale organization of synaptic vesicles in an activity-dependent manner.

Presynaptic targeting of botulinum neurotoxin type A requires a tripartite PSG-Syt1-SV2 plasma membrane nanocluster for synaptic vesicle entry.

The unique nerve terminal targeting of botulinum neurotoxin type A (BoNT/A) is due to its capacity to bind two receptors on the neuronal plasma membrane: polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Whether and how PSGs and SV2 may coordinate other proteins for BoNT/A recruitment and internalization remains unknown. Here, we demonstrate that the targeted endocytosis of BoNT/A into synaptic vesicles (SVs) requires a tripartite surface nanocluster. Live-cell super-resolution imaging and electron microscopy of catalytically inactivated BoNT/A wildtype and receptor-binding-deficient mutants in cultured hippocampal neurons demonstrated that BoNT/A must bind coincidentally to a PSG and SV2 to target synaptic vesicles. We reveal that BoNT/A simultaneously interacts with a preassembled PSG-synaptotagmin-1 (Syt1) complex and SV2 on the neuronal plasma membrane, facilitating Syt1-SV2 nanoclustering that controls endocytic sorting of the toxin into synaptic vesicles. Syt1 CRISPRi knockdown suppressed BoNT/A- and BoNT/E-induced neurointoxication as quantified by SNAP-25 cleavage, suggesting that this tripartite nanocluster may be a unifying entry point for selected botulinum neurotoxins that hijack this for synaptic vesicle targeting.

Fyn nanoclustering requires switching to an open conformation and is enhanced by FTLD-Tau biomolecular condensates.

Fyn is a Src kinase that controls critical signalling cascades and has been implicated in learning and memory. Postsynaptic enrichment of Fyn underpins synaptotoxicity in dementias such as Alzheimer's disease and frontotemporal lobar degeneration with Tau pathology (FTLD-Tau). The FLTD P301L mutant Tau is associated with a higher propensity to undergo liquid-liquid phase separation (LLPS) and form biomolecular condensates. Expression of P301L mutant Tau promotes aberrant trapping of Fyn in nanoclusters within hippocampal dendrites by an unknown mechanism. Here, we used single-particle tracking photoactivated localisation microscopy to demonstrate that the opening of Fyn into its primed conformation promotes its nanoclustering in dendrites leading to increased Fyn/ERK/S6 downstream signalling. Preventing the auto-inhibitory closed conformation of Fyn through phospho-inhibition or through perturbation of its SH3 domain increased Fyn's nanoscale trapping, whereas inhibition of the catalytic domain had no impact. By combining pharmacological and genetic approaches, we demonstrate that P301L Tau enhanced both Fyn nanoclustering and Fyn/ERK/S6 signalling via its ability to form biomolecular condensates. Together, our findings demonstrate that Fyn alternates between a closed and an open conformation, the latter being enzymatically active and clustered. Furthermore, pathogenic immobilisation of Fyn relies on the ability of P301L Tau to form biomolecular condensates, thus highlighting the critical importance of LLPS in controlling nanoclustering and downstream intracellular signalling events.

Early, nonlethal ploidy and genome size quantification using confocal microscopy in zebrafish embryos.

Ploidy transitions through whole genome duplication have shaped evolution by allowing the sub- and neo-functionalization of redundant copies of highly conserved genes to express novel traits. The nuclear:cytoplasmic (n:c) ratio is maintained in polyploid vertebrates resulting in larger cells, but body size is maintained by a concomitant reduction in cell number. Ploidy can be manipulated easily in most teleosts, and the zebrafish, already well established as a model system for biomedical research, is therefore an excellent system in which to study the effects of increased cell size and reduced cell numbers in polyploids on development and physiology. Here we describe a novel technique using confocal microscopy to measure genome size and determine ploidy non-lethally at 48 h post-fertilization (hpf) in transgenic zebrafish expressing fluorescent histones. Volumetric analysis of myofiber nuclei using open-source software can reliably distinguish diploids and triploids from a mixed-ploidy pool of embryos for subsequent experimentation. We present an example of this by comparing heart rate between confirmed diploid and triploid embryos at 54 hpf.

Spatiotemporal evolution of COVID-19 infection and detection within night light networks: comparative analysis of USA and China.

The spatial distribution of population affects disease transmission, especially when shelter in place orders restrict mobility for a large fraction of the population. The spatial network structure of settlements therefore imposes a fundamental constraint on the spatial distribution of the population through which a communicable disease can spread. In this analysis we use the spatial network structure of lighted development as a proxy for the distribution of ambient population to compare the spatiotemporal evolution of COVID-19 confirmed cases in the USA and China. The Visible Infrared Imaging Radiometer Suite (VIIRS) Day/Night Band sensor on the NASA/NOAA Suomi satellite has been imaging night light at ~ 700 m resolution globally since 2012. Comparisons with sub-kilometer resolution census observations in different countries across different levels of development indicate that night light luminance scales with population density over ~ 3 orders of magnitude. However, VIIRS' constant ~ 700 m resolution can provide a more detailed representation of population distribution in peri-urban and rural areas where aggregated census blocks lack comparable spatial detail. By varying the low luminance threshold of VIIRS-derived night light, we depict spatial networks of lighted development of varying degrees of connectivity within which populations are distributed. The resulting size distributions of spatial network components (connected clusters of nodes) vary with degree of connectivity, but maintain consistent scaling over a wide range (5 × to 10 × in area & number) of network sizes. At continental scales, spatial network rank-size distributions obtained from VIIRS night light brightness are well-described by power laws with exponents near -2 (slopes near -1) for a wide range of low luminance thresholds. The largest components (104 to 105 km2) represent spatially contiguous agglomerations of urban, suburban and periurban development, while the smallest components represent isolated rural settlements. Projecting county and city-level numbers of confirmed cases of COVID-19 for the USA and China (respectively) onto the corresponding spatial networks of lighted development allows the spatiotemporal evolution of the epidemic (infection and detection) to be quantified as propagation within networks of varying connectivity. Results for China show rapid nucleation and diffusion in January 2020 followed by rapid decreases in new cases in February. While most of the largest cities in China showed new confirmed cases approaching zero before the end of February, most of these cities also showed distinct second waves of cases in March or April. Whereas new cases in Wuhan did not approach zero until mid-March, as of December 2020 it has not yet experienced a second wave of cases. In contrast, the results for the USA show a wide range of trajectories, with an abrupt transition from slow increases in confirmed cases in a small number of network components in January and February, to rapid geographic dispersion to a larger number of components shortly before mobility reductions occurred in March. Results indicate that while most of the upper tail of the network had been exposed by the end of March, the lower tail of the component size distribution has only shown steep increases since mid-June.

Combining Single Molecule Super-Resolution Imaging Techniques to Unravel the Nanoscale Organization of the Presynapse.

The fusion of synaptic vesicles with the plasma membrane underpins neurotransmission. A number of presynaptic proteins play a critical role in overcoming the energy barrier inherent to the fusion of the negatively charged vesicular and plasma membranes. Emerging concepts suggest that this process is hierarchical and dependent on rapid and transient reorganization of proteins in and out of small nanoclusters located in the active zones of nerve terminals. Examining the nanoscale organization of presynaptic molecules requires super-resolution microscopy to overcome the limits of conventional light microscopy. In this chapter, we describe three super-resolution techniques that allow for the examination of the nanoscale organization of proteins within live hippocampal nerve terminals. We used (1) single-particle tracking photoactivated localization microscopy (sptPALM) to resolve the mobility and clustering of syntaxin1A (STX1A), (2) universal Point Accumulation Imaging in Nanoscale Topography (uPAINT) to study the mobility of a pool of vesicular-associated membrane protein 2 (VAMP2) transiting on the plasma membrane, and (3) subdiffractional Tracking of Internalized Molecules (sdTIM) to track VAMP2-positive recycling synaptic vesicles in conjunction with Cholera Toxin subunit B (CTB), which has recently been shown to be trafficked retrogradely from the presynapse to the cell body via signaling endosomes.

An Epilepsy-Associated SV2A Mutation Disrupts Synaptotagmin-1 Expression and Activity-Dependent Trafficking.

The epilepsy-linked gene SV2A, has a number of potential roles in the synaptic vesicle (SV) life cycle. However, how loss of SV2A function translates into presynaptic dysfunction and ultimately seizure activity is still undetermined. In this study, we examined whether the first SV2A mutation identified in human disease (R383Q) could provide information regarding which SV2A-dependent events are critical in the translation to epilepsy. We utilized a molecular replacement strategy in which exogenous SV2A was expressed in mouse neuronal cultures of either sex, which had been depleted of endogenous SV2A to mimic the homozygous human condition. We found that the R383Q mutation resulted in a mislocalization of SV2A from SVs to the plasma membrane, but had no effect on its activity-dependent trafficking. This SV2A mutant displayed reduced mobility when stranded on the plasma membrane and reduced binding to its interaction partner synaptotagmin-1 (Syt1). Furthermore, the R383Q mutant failed to rescue reduced expression and dysfunctional activity-dependent trafficking of Syt1 in the absence of endogenous SV2A. This suggests that the inability to control Syt1 expression and trafficking at the presynapse may be key in the transition from loss of SV2A function to seizure activity.SIGNIFICANCE STATEMENT SV2A is a synaptic vesicle (SV) protein, the absence or dysfunction of which is linked to epilepsy. However, the series of molecular events that result in this neurological disorder is still undetermined. We demonstrate here that the first human mutation in SV2A identified in an individual with epilepsy displays reduced binding to synaptotagmin-1 (Syt1), an SV protein essential for synchronous neurotransmitter release. Furthermore, this mutant cannot correct alterations in both Syt1 expression and trafficking when expressed in the absence of endogenous SV2A (to mimic the homozygous human condition). This suggests that the inability to control Syt1 expression and trafficking may be key in the transition from loss of SV2A function to seizure activity.

Matrix Metalloproteinase 13 Activity is Required for Normal and Hypoxia-Induced Precocious Hatching in Zebrafish Embryos.

Hypoxia induces precocious hatching in zebrafish, but we do not have a clear understanding of the molecular mechanisms regulating the activation of the hatching enzyme or how these mechanisms trigger precocious hatching under unfavorable environmental conditions. Using immunohistochemistry, pharmacological inhibition of matrix metalloproteinase 13 (Mmp13), and in vivo zymography, we show that Mmp13a is present in the hatching gland just as embryos become hatching competent and that Mmp13a activity is required for both normal hatching and hypoxia-induced precocious hatching. We conclude that Mmp13a likely functions in activating the hatching enzyme zymogen and that Mmp13a activity is necessary but not sufficient for hatching in zebrafish. This study highlights the broad nature of MMP function in development and provides a non-mammalian example of extra-embryonic processes mediated by MMP activity.

Kappa chain maturation helps drive rapid development of an infant HIV-1 broadly neutralizing antibody lineage.

HIV-infected infants develop broadly neutralizing plasma responses with more rapid kinetics than adults, suggesting the ontogeny of infant responses could better inform a path to achievable vaccine targets. Here we reconstruct the developmental lineage of BF520.1, an infant-derived HIV-specific broadly neutralizing antibody (bnAb), using computational methods developed specifically for this purpose. We find that the BF520.1 inferred naive precursor binds HIV Env. We also show that heterologous cross-clade neutralizing activity evolved in the infant within six months of infection and that, ultimately, only 2% SHM is needed to achieve the full breadth of the mature antibody. Mutagenesis and structural analyses reveal that, for this infant bnAb, substitutions in the kappa chain were critical for activity, particularly in CDRL1. Overall, the developmental pathway of this infant antibody includes features distinct from adult antibodies, including several that may be amenable to better vaccine responses.

Multisensor Analysis of Spectral Dimensionality and Soil Diversity in the Great Central Valley of California.

Planned hyperspectral satellite missions and the decreased revisit time of multispectral imaging offer the potential for data fusion to leverage both the spectral resolution of hyperspectral sensors and the temporal resolution of multispectral constellations. Hyperspectral imagery can also be used to better understand fundamental properties of multispectral data. In this analysis, we use five flight lines from the Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) archive with coincident Landsat 8 acquisitions over a spectrally diverse region of California to address the following questions: (1) How much of the spectral dimensionality of hyperspectral data is captured in multispectral data?; (2) Is the characteristic pyramidal structure of the multispectral feature space also present in the low order dimensions of the hyperspectral feature space at comparable spatial scales?; (3) How much variability in rock and soil substrate endmembers (EMs) present in hyperspectral data is captured by multispectral sensors? We find nearly identical partitions of variance, low-order feature space topologies, and EM spectra for hyperspectral and multispectral image composites. The resulting feature spaces and EMs are also very similar to those from previous global multispectral analyses, implying that the fundamental structure of the global feature space is present in our relatively small spatial subset of California. Finally, we find that the multispectral dataset well represents the substrate EM variability present in the study area - despite its inability to resolve narrow band absorptions. We observe a tentative but consistent physical relationship between the gradation of substrate reflectance in the feature space and the gradation of sand versus clay content in the soil classification system.

Quantitative drinking water arsenic concentrations in field environments using mobile phone photometry of field kits.

Arsenic (As) groundwater contamination is common yet spatially heterogeneous within most environments. It is therefore necessary to measure As concentrations to determine whether a water source is safe to drink. Measurement of As in the field involves using a test strip that changes color in the presence of As. These tests are relatively inexpensive, but results are subjective and provide binned categorical data rather than exact determinations of As concentration. The goal of this work was to determine if photos of field kit test strips taken on mobile phone cameras could be used to extract more precise, continuous As concentrations. As concentrations for 376 wells sampled from Araihazar, Bangladesh were analyzed using ICP-MS, field kit and the new mobile phone photo method. Results from the field and lab indicate that normalized RGB color data extracted from images were able to accurately predict As concentrations as measured by ICP-MS, achieving detection limits of 9.2µg/L, and 21.9µg/L for the lab and field respectively. Data analysis is most consistent in the laboratory, but can successfully be carried out offline following image analysis, or on the mobile phone using basic image analysis software. The accuracy of the field method was limited by variability in image saturation, and variation in the illumination spectrum (lighting) and camera response. This work indicates that mobile phone cameras can be used as an analytical tool for quantitative measures of As and could change how water samples are analyzed in the field more widely, and that modest improvements in the consistency of photographic image collection and processing could yield measurements that are both accurate and precise.

Matrix metalloproteinases in neural development: a phylogenetically diverse perspective.

The matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases originally characterized as secreted proteases responsible for degrading extracellular matrix proteins. Their canonical role in matrix remodelling is of significant importance in neural development and regeneration, but emerging roles for MMPs, especially in signal transduction pathways, are also of obvious importance in a neural context. Misregulation of MMP activity is a hallmark of many neuropathologies, and members of every branch of the MMP family have been implicated in aspects of neural development and disease. However, while extraordinary research efforts have been made to elucidate the molecular mechanisms involving MMPs, methodological constraints and complexities of the research models have impeded progress. Here we discuss the current state of our understanding of the roles of MMPs in neural development using recent examples and advocate a phylogenetically diverse approach to MMP research as a means to both circumvent the challenges associated with specific model organisms, and to provide a broader evolutionary context from which to synthesize an understanding of the underlying biology.

Non-Human Primates Harbor Diverse Mammalian and Avian Astroviruses Including Those Associated with Human Infections.

Astroviruses (AstVs) are positive sense, single-stranded RNA viruses transmitted to a wide range of hosts via the fecal-oral route. The number of AstV-infected animal hosts has rapidly expanded in recent years with many more likely to be discovered because of the advances in viral surveillance and next generation sequencing. Yet no study to date has identified human AstV genotypes in animals, although diverse AstV genotypes similar to animal-origin viruses have been found in children with diarrhea and in one instance of encephalitis. Here we provide important new evidence that non-human primates (NHP) can harbor a wide variety of mammalian and avian AstV genotypes, including those only associated with human infection. Serological analyses confirmed that >25% of the NHP tested had antibodies to human AstVs. Further, we identified a recombinant AstV with parental relationships to known human AstVs. Phylogenetic analysis suggests AstVs in NHP are on average evolutionarily much closer to AstVs from other animals than are AstVs from bats, a frequently proposed reservoir. Our studies not only demonstrate that human astroviruses can be detected in NHP but also suggest that NHP are unique in their ability to support diverse AstV genotypes, further challenging the paradigm that astrovirus infection is species-specific.

Sensitivity of crop cover to climate variability: insights from two Indian agro-ecoregions.

Crop productivity in India varies greatly with inter-annual climate variability and is highly dependent on monsoon rainfall and temperature. The sensitivity of yields to future climate variability varies with crop type, access to irrigation and other biophysical and socio-economic factors. To better understand sensitivities to future climate, this study focuses on agro-ecological subregions in Central and Western India that span a range of crops, irrigation, biophysical conditions and socioeconomic characteristics. Climate variability is derived from remotely-sensed data products, Tropical Rainfall Measuring Mission (TRMM - precipitation) and Moderate Resolution Imaging Spectroradiometer (MODIS - temperature). We examined green-leaf phenologies as proxy for crop productivity using the MODIS Enhanced Vegetation Index (EVI) from 2000 to 2012. Using both monsoon and winter growing seasons, we assessed phenological sensitivity to inter-annual variability in precipitation and temperature patterns. Inter-annual EVI phenology anomalies ranged from -25% to 25%, with some highly anomalous values up to 200%. Monsoon crop phenology in the Central India site is highly sensitive to climate, especially the timing of the start and end of the monsoon and intensity of precipitation. In the Western India site, monsoon crop phenology is less sensitive to precipitation variability, yet shows considerable fluctuations in monsoon crop productivity across the years. Temperature is critically important for winter productivity across a range of crop and management types, such that irrigation might not provide a sufficient buffer against projected temperature increases. Better access to weather information and usage of climate-resilient crop types would play pivotal role in maintaining future productivity. Effective strategies to adapt to projected climate changes in the coming decades would also need to be tailored to regional biophysical and socio-economic conditions.

A novel Bayesian method for detection of APOBEC3-mediated hypermutation and its application to zoonotic transmission of simian foamy viruses.

Simian Foamy Virus (SFV) can be transmitted from non-human primates (NHP) to humans. However, there are no documented cases of human to human transmission, and significant differences exist between infection in NHP and human hosts. The mechanism for these between-host differences is not completely understood. In this paper we develop a new Bayesian approach to the detection of APOBEC3-mediated hypermutation, and use it to compare SFV sequences from human and NHP hosts living in close proximity in Bangladesh. We find that human APOBEC3G can induce genetic changes that may prevent SFV replication in infected humans in vivo.

Simian foamy virus infection of rhesus macaques in Bangladesh: relationship of latent proviruses and transcriptionally active viruses.

Simian foamy viruses (SFV) are complex retroviruses that are ubiquitous in nonhuman primates (NHP) and are zoonotically transmitted to humans, presumably through NHP saliva, by licking, biting, and other behaviors. We have studied SFV in free-ranging rhesus macaques in Bangladesh. It has been previously shown that SFV in immunocompetent animals replicates to detectable levels only in superficial epithelial cells of the oral mucosa, although latent proviruses are found in most, if not all, tissues. In this study, we compare DNA sequences from latent SFV proviruses found in blood cells of 30 Bangladesh rhesus macaques to RNA sequences of transcriptionally active SFV from buccal swabs obtained from the same animals. Viral strains, defined by differences in SFV gag sequences, from buccal mucosal specimens overlapped with those from blood samples in 90% of animals. Thus, latent proviruses in peripheral blood mononuclear cells (PBMC) are, to a great extent, representative of viruses likely to be transmitted to other hosts. The level of SFV RNA in buccal swabs varied greatly between macaques, with increasing amounts of viral RNA in older animals. Evidence of APOBEC3-induced mutations was found in gag sequences derived from the blood and oral mucosa.

Induction of spermatogenic synchrony by retinoic acid in neonatal mice.

Retinoic acid (RA) is required for the successful differentiation and meiotic entry of germ cells in the murine testis. The availability of RA to undifferentiated germ cells begins in a variable, uneven pattern during the first few days after birth and establishes the asynchronous pattern of germ cell differentiation in adulthood. It has been shown that synchronous spermatogenesis can be induced in 2 d postpartum mice, but not in adult mice, by treating vitamin A sufficient males with RA. In this study, neonatal males were treated at different ages with a single dose of RA and spermatogenesis was examined after recovery to adulthood. The failure of exogenous RA to alter asynchrony correlates with the appearance of meiotic preleptotene spermatocytes within the seminiferous epithelium.

Turning a spermatogenic wave into a tsunami: synchronizing murine spermatogenesis using WIN 18,446.

The BDADs (bis-[dichloroacetyl]-diamines) are compounds that can inhibit spermatogenesis via blocking the metabolism of vitamin A. We utilized one specific BDAD, WIN 18,446, to manipulate the endogenous production of retinoic acid (RA) in the testis to further investigate the action of this compound on mammalian sperm production. Transient treatment of adult male mice with WIN 18,446 blocked spermatogonial differentiation and induced significant changes in the cycle of the seminiferous epithelium. WIN 18,446 treatment of neonatal mice also blocked spermatogonial differentiation and, followed by injection of RA, induced synchronous spermatogenesis in adulthood. The net result was pulsatile, rather than normal continuous, release of sperm from the seminiferous epithelium. This study describes a novel technique that can enrich for specific germ cell populations within the testis, representing a valuable new tool for studying spermatogenesis.

Population dynamics of rhesus macaques and associated foamy virus in Bangladesh.

Foamy viruses are complex retroviruses that have been shown to be transmitted from nonhuman primates to humans. In Bangladesh, infection with simian foamy virus (SFV) is ubiquitous among rhesus macaques, which come into contact with humans in diverse locations and contexts throughout the country. We analyzed microsatellite DNA from 126 macaques at six sites in Bangladesh in order to characterize geographic patterns of macaque population structure. We also included in this study 38 macaques owned by nomadic people who train them to perform for audiences. PCR was used to analyze a portion of the proviral gag gene from all SFV-positive macaques, and multiple clones were sequenced. Phylogenetic analysis was used to infer long-term patterns of viral transmission. Analyses of SFV gag gene sequences indicated that macaque populations from different areas harbor genetically distinct strains of SFV, suggesting that geographic features such as forest cover play a role in determining the dispersal of macaques and SFV. We also found evidence suggesting that humans traveling the region with performing macaques likely play a role in the translocation of macaques and SFV. Our studies found that individual animals can harbor more than one strain of SFV and that presence of more than one SFV strain is more common among older animals. Some macaques are infected with SFV that appears to be recombinant. These findings paint a more detailed picture of how geographic and sociocultural factors influence the spectrum of simian-borne retroviruses.