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A comprehensive understanding of the SARS-CoV-2 infection dynamics and the ensuing host immune responses is needed to explain the pathogenesis as it relates to viral transmission. Here, we report results from an ongoing, longitudinal, workplace clinical surveillance study that address knowledge gaps surrounding SARS-CoV-2 in vivo kinetics, particularly in the earliest stages after exposure. Nine study participants who developed COVID-19 between November, 2020 and March, 2021 were monitored at high temporal resolution for three months in terms of viral loads as well as associated inflammatory biomarker and antibody responses. Analysis of the resulting datasets, supported by Bayesian modeling, allowed the underlying kinetic processes to be described, yielding a number of unexpected findings. Early viral replication is more rapid (median doubling time, 3.1 hours) than previously thought, shrinking the window between exposure and viral shedding. Results from our small study give a rare insight into the life-cycle of COVID-19 infection and hold a number of important biological, clinical, and public health implications.

Global efforts aimed at preventing human immunodeficiency virus type one (HIV-1) infection in vulnerable populations appear to be stalling, limiting our ability to control the epidemic. Long-acting, controlled drug administration from subdermal implants holds significant potential by reducing the compliance burden associated with frequent dosing. We, and others, are exploring the development of complementary subdermal implant technologies delivering the potent prodrug, tenofovir alafenamide (TAF). The current report addresses knowledge gaps in the preclinical pharmacology of long-acting, subdermal TAF delivery using several mouse models. Systemic drug disposition during TAF implant dosing was explained by a multi-compartment pharmacokinetic (PK) model. Imaging mass spectrometry was employed to characterize the spatial distribution of TAF and its principal five metabolites in local tissues surrounding the implant. Humanized mouse studies determined the effective TAF dose for preventing vaginal and rectal HIV-1 acquisition. Our results represent an important step in the development of a safe and effective TAF implant for HIV-1 prevention.

Age-related macular degeneration (AMD) is a progressive eye disease and the most common cause of blindness among the elderly. AMD is characterized by early atrophy of the choriocapillaris and retinal pigment epithelium (RPE). Although AMD is a multifactorial disease with many environmental and genetic risk factors, a hallmark of the disease is the origination of extracellular deposits, or drusen, between the RPE and Bruch membrane. Human retinal G-protein-coupled receptor (RGR) gene generates an exon-skipping splice variant of RGR-opsin (RGR-d; NP_001012740) that is a persistent component of small and large drusen. Herein, the findings show that abnormal RGR proteins, including RGR-d, are pathogenic in an animal retina with degeneration of the choriocapillaris, RPE, and photoreceptors. A frameshift truncating mutation resulted in severe retinal degeneration with a continuous band of basal deposits along the Bruch membrane. RGR-d produced less severe disease with choriocapillaris and RPE atrophy, including focal accumulation of abnormal RGR-d protein at the basal boundary of the RPE. Degeneration of the choriocapillaris was marked by a decrease in endothelial CD31 protein and choriocapillaris breakdown at the ultrastructural level. Fundus lesions with patchy depigmentation were characteristic of old RGR-d mice. RGR-d was mislocalized in cultured cells and caused a strong cell growth defect. These results uphold the notion of a potential hidden link between AMD and a high-frequency RGR allele.

Neurons use multiple modes of endocytosis, including clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE), during mild and intense neuronal activity, respectively, to maintain stable neurotransmission. While molecular players modulating CME are well characterized, factors regulating ADBE and mechanisms coordinating CME and ADBE activations remain poorly understood. Here we report that Minibrain/DYRK1A (Mnb), a kinase mutated in autism and up-regulated in Down’s syndrome, plays a novel role in suppressing ADBE. We demonstrate that Mnb, together with calcineurin, delicately coordinates CME and ADBE by controlling the phosphoinositol phosphatase activity of synaptojanin (Synj) during varying synaptic demands. Functional domain analyses reveal that Synj’s 5′-phosphoinositol phosphatase activity suppresses ADBE, while SAC1 activity is required for efficient ADBE. Consequently, Parkinson’s disease mutation in Synj’s SAC1 domain impairs ADBE. These data identify Mnb and Synj as novel regulators of ADBE and further indicate that CME and ADBE are differentially governed by Synj’s dual phosphatase domains.

Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. We have investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions where Drosophila larvae are terminally arrested, we have characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). While wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites, and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales.

Errors in protein synthesis have historically been assumed to be detrimental to the cell. While there are many reports that translational errors are consequential, there is a growing body of evidence that some mistranslation events may be tolerated or even beneficial. Using two models of mistranslation, we compare the direct phenotypic effects of these events in Escherichia coli. This work provides insight into the threshold for tolerance of specific mistranslation events that were previously predicted to be broadly neutral to proteome integrity. Furthermore, these data reveal the effects of mistranslation beyond the general unfolded stress response, leading to global translational reprogramming., Mechanisms have evolved to prevent errors in replication, transcription, and translation of genetic material, with translational errors occurring most frequently. Errors in protein synthesis can occur at two steps, during tRNA aminoacylation and ribosome decoding. Recent advances in protein mass spectrometry have indicated that previous reports of translational errors have potentially underestimated the frequency of these events, but also that the majority of translational errors occur during ribosomal decoding, suggesting that aminoacylation errors are evolutionarily less tolerated. Despite that interpretation, there is evidence that some aminoacylation errors may be regulated, and thus provide a benefit to the cell, while others are clearly detrimental. Here, we show that while it has been suggested that regulated Thr-to-Ser substitutions may be beneficial, there is a threshold beyond which these errors are detrimental. In contrast, we show that errors mediated by alanyl-tRNA synthetase (AlaRS) are not well tolerated and induce a global stress response that leads to gross perturbation of the Escherichia coli proteome, with potentially catastrophic effects on fitness and viability. Tolerance for Ala mistranslation appears to be much lower than with other translational errors, consistent with previous reports of multiple proofreading mechanisms targeting mischarged tRNAAla. These results demonstrate the essential role of aminoacyl-tRNA proofreading in optimizing cellular fitness and suggest that any potentially beneficial effects of mistranslation may be confined to specific amino acid substitutions.

Synapses exhibit an astonishing degree of adaptive plasticity in healthy and disease states. We have investigated whether synapses also adjust to life stages imposed by novel developmental programs for which they were never molded by evolution. Under conditions in which Drosophila larvae are terminally arrested, we have characterized synaptic growth, structure and function at the neuromuscular junction (NMJ). Although wild-type larvae transition to pupae after 5 days, arrested third instar (ATI) larvae persist for 35 days, during which time NMJs exhibit extensive overgrowth in muscle size, presynaptic release sites and postsynaptic glutamate receptors. Remarkably, despite this exuberant growth, stable neurotransmission is maintained throughout the ATI lifespan through a potent homeostatic reduction in presynaptic neurotransmitter release. Arrest of the larval stage in stathmin mutants also reveals a degree of progressive instability and neurodegeneration that was not apparent during the typical larval period. Hence, an adaptive form of presynaptic depression stabilizes neurotransmission during an extended developmental period of unconstrained synaptic growth. More generally, the ATI manipulation provides a powerful system for studying neurodegeneration and plasticity across prolonged developmental timescales.

Research on neuropeptide function has advanced rapidly, yet there is still no spatio-temporally resolved method to measure the release of neuropeptides in vivo. Here we introduce Neuropeptide Release Reporters (NPRRs): novel genetically-encoded sensors with high temporal resolution and genetic specificity. Using the Drosophila larval neuromuscular junction (NMJ) as a model, we provide evidence that NPRRs recapitulate the trafficking and packaging of native neuropeptides, and report stimulation-evoked neuropeptide release events as real-time changes in fluorescence intensity, with sub-second temporal resolution.

The ubiquitin ligase Highwire restrains synaptic growth and promotes evoked neurotransmission at NMJ synapses in Drosophila . Highwire regulates synaptic morphology by downregulating the MAP3K Wallenda, but excess Wallenda signaling does not account for the decreased presynaptic release observed in highwire mutants. Hence, Highwire likely has a second substrate that inhibits neurotransmission. Highwire targets the NAD + biosynthetic and axoprotective enzyme dNmnat to regulate axonal injury responses. dNmnat localizes to synapses and interacts with the active zone protein Bruchpilot, leading us to hypothesize that Highwire promotes evoked release by downregulating dNmnat. Here, we show that excess dNmnat is necessary in highwire mutants and sufficient in wild‐type larvae to reduce quantal content, likely via disruption of active zone ultrastructure. Catalytically active dNmnat is required to drive defects in evoked release, and depletion of a second NAD + synthesizing enzyme is sufficient to suppress these defects in highwire mutants, suggesting that excess NAD + biosynthesis is the mechanism inhibiting neurotransmission. Thus, Highwire downregulates dNmnat to promote evoked synaptic release, suggesting that Highwire balances the axoprotective and synapse‐inhibitory functions of dNmnat.

Cunningham and Littleton preview work from Goel et al. that describes a mechanism by which neurons regulate synaptic output after alterations in synapse size or active zone number., How neurons stabilize their overall synaptic strength following conditions that alter synaptic morphology or function is a key question in neuronal homeostasis. In this issue, Goel et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201807165) find that neurons stabilize synaptic output despite disruptions in synapse size, active zone number, or postsynaptic function by controlling the delivery of active zone material and active zone size.

Summary High-pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high-pressure frozen, freeze-substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around –60°C.

Summary In this study, we quantitatively investigate the role of the M97 protein for viral morphogenesis in murine cytomegalovirus (MCMV)-infected fibroblast cells. For this purpose, a statistical analysis is performed for the spatial distribution of nuclear B-capsids (devoid of DNA, containing the scaffold) and C-capsids (filled with DNA). Cell nuclei infected with either wild-type or an M97 deletion mutant were compared. Univariate and multivariate point process characteristics (like Ripley's K-function, the L-function and the nearest neighbour distance distribution function) are investigated in order to describe and quantify the effects that the deletion of M97 causes to the process of DNA packaging into nucleocapsids. The estimation of the function L(r) −r reveals that with respect to the wild type there is an increased frequency of point pairs at a very short distance (less than approximately 100 nm) for both the B-capsids as well as for the C-capsids. For the M97 deletion mutant type this is no longer true. Here only the C-capsids show such a clustering behaviour, whereas for B-capsids it is almost nonexistant. Estimations of functionals such as the nearest neighbour distance distribution function confirmed these results. Thereby, a quantification is provided for the effect that the deletion of M97 leads to a loss of typical nucleocapsid clustering in MCMV-infected nuclei.

We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis.

Defining the ultrastructure of endocytic sites and localization of endocytic proteins in Saccharomyces cerevisiae by immunoelectron microscopy is central in understanding the mechanisms of membrane deformation and scission during endocytosis. We show that an improved sample preparation protocol based on high-pressure freezing, freeze substitution, and low-temperature embedding allows us to maintain the cellular fine structure and to immunolabel green fluorescent protein–tagged endocytic proteins or actin in the same sections. Using this technique we analyzed the stepwise deformation of endocytic membranes and immunolocalized the endocytic proteins Abp1p, Sla1p, Rvs167p, and actin, and were able to draw a clear ultrastructural distinction between endocytic sites and eisosomes by immunolocalizing Pil1p. In addition to defining the geometry and the fine structure of budding yeast endocytic sites, we observed associated actin filaments forming a cage-like meshwork around the endocytic membrane.

During endocytic vesicle formation, distinct subdomains along the membrane invagination are specified by different proteins, which bend the membrane and drive scission. Bin-Amphiphysin-Rvs (BAR) and Fer-CIP4 homology-BAR (F-BAR) proteins can induce membrane curvature and have been suggested to facilitate membrane invagination and scission. Two F-BAR proteins, Syp1 and Bzz1, are found at budding yeast endocytic sites. Syp1 arrives early but departs from the endocytic site before formation of deep membrane invaginations and scission. Using genetic, spatiotemporal, and ultrastructural analyses, we demonstrate that Bzz1, the heterodimeric BAR domain protein Rvs161/167, actin polymerization, and the lipid phosphatase Sjl2 cooperate, each through a distinct mechanism, to induce membrane scission in yeast. Additionally, actin assembly and Rvs161/167 cooperate to drive formation of deep invaginations. Finally, we find that Bzz1, acting at the invagination base, stabilizes endocytic sites and functions with Rvs161/167, localized along the tubule, to achieve proper endocytic membrane geometry necessary for efficient scission. Together, our results reveal that dynamic interplay between a lipid phosphatase, actin assembly, and membrane-sculpting proteins leads to proper membrane shaping, tubule stabilization, and scission.

Correlative light and electron microscopy represents the ultimate goal for the visualization of cell biological processes. In theory, it is possible to combine the strengths of both methods, that is, the live-cell imaging of the movement of GFP-tagged proteins captured by fluorescence microscopy with an image of the fine structural context surrounding the tagged protein imaged and localized by immunoelectron microscopy. In practice, inherent technical limitations of the two individual methods and their combination make the technique very complex to handle. Here, we present a high-pressure freezing and freeze-substitution protocol which fulfills the key criterion for correlative microscopy, namely, the ability to achieve excellent visibility of fine structures without disrupting the antigens recognized by the immunolabeling protocol. This is achieved by a fixative-free freeze-substitution and low-temperature embedding.

High-pressure freezing (HPF) has been around since the mid-1980s as a cryopreparation technique for biological electron microscopy. It has taken quite some time to "catch on" but with the recent interest in cellular tomography and electron microscopy of vitreous cryosections it has been used more frequently. While HPF is relatively easy to do, there are a number of steps, such as loading the sample into the specimen carrier correctly, that are critical to the success of this method. In this chapter we discuss some of the "little" things that can make the difference between successful or unsuccessful freezing. We cover all aspects of HPF, from specimen loading to removing your sample from the carriers in polymerized resin. Our goal is to make it easier and more reliable for HPF users to get well-frozen samples for their research.

The yeast Saccharomyces cerevisiae is a model organism widely used to study cell biological processes because of its easy genomic manipulation and its close relatedness to higher eukaryotes. For electron microscopy, the good freezing properties and the small size of yeast cells make it a nearly ideal specimen for the development of cryopreparation techniques. Here we report on the development of a method to correlate yeast cells by live-fluorescence and electron microscopy with the potential to achieve sub-second correlation times. This is possible by plunge-freezing of an optically transparent sample sandwich, so that the temporal resolution is only determined by the transfer speed from the fluorescence microscope to the freezing device. While direct correlation was not yet achieved, the system already offers the possibility to verify the state of the identical population of cells by fluorescence microscopy immediately before freezing and processing for transmission electron microscopy.

Correlative light and electron microscopy represents the ultimate goal for the visualization of cell biological processes. In theory, it is possible to combine the strengths of both methods, that is, the live-cell imaging of the movement of GFP-tagged proteins captured by fluorescence microscopy with an image of the fine structural context surrounding the tagged protein imaged and localized by immunoelectron microscopy. In practice, inherent technical limitations of the two individual methods and their combination make the technique very complex to handle. Here, we present a high-pressure freezing and freeze-substitution protocol which fulfills the key criterion for correlative microscopy, namely, the ability to achieve excellent visibility of fine structures without disrupting the antigens recognized by the immunolabeling protocol. This is achieved by a fixative-free freeze-substitution and low-temperature embedding.

High-pressure freezing (HPF) has been around since the mid-1980s as a cryopreparation technique for biological electron microscopy. It has taken quite some time to "catch on" but with the recent interest in cellular tomography and electron microscopy of vitreous cryosections it has been used more frequently. While HPF is relatively easy to do, there are a number of steps, such as loading the sample into the specimen carrier correctly, that are critical to the success of this method. In this chapter we discuss some of the "little" things that can make the difference between successful or unsuccessful freezing. We cover all aspects of HPF, from specimen loading to removing your sample from the carriers in polymerized resin. Our goal is to make it easier and more reliable for HPF users to get well-frozen samples for their research.

The tegument protein pp65 of human cytomegalovirus (HCMV) represents the major component of mature virus particles. Nevertheless, deletion of pp65 has been shown to have no effects on virus replication and morphogenesis in fibroblasts in vitro. We have studied the HCMV virion composition in the absence of pp65 and viral growth of a pp65 stop mutant in different cell types, including monocyte-derived macrophages. Two stop codons at amino acids 11 and 12 of pp65 were introduced by bacterial artificial chromosome mutagenesis into the endotheliotropic strain TB40/E. Clear changes of the tegument composition could be observed in purified mutant virus particles, where the amount of tegument protein pUL25 was drastically reduced. In addition, pUL69 and the virally encoded protein kinase UL97 were undetectable in the pp65 stop mutant. Expression of pUL69 in infected cells was unaltered while pUL25 accumulated in the absence of pp65, thus demonstrating that only incorporation into virus particles is dependent on pp65. Coimmunoprecipitation experiments using lysates of infected cells revealed an interaction between pUL69 and pp65. This interaction was verified in pull-down experiments using transfected cells, which showed that pp65 and pUL69 do not require the presence of other viral proteins for their interaction. We conclude that pp65 is required for the incorporation of other viral proteins into the virus particle and thus is involved in the protein-protein interaction network leading to normal tegument formation. When studying growth kinetics of the pp65 stop mutant in different cell types, we found a severe impairment of viral growth in monocyte-derived macrophages, showing for the first time a strong cell-specific role of pp65 in viral growth.

Cytokinesis is the process by which a cell physically divides in two at the conclusion of a cell cycle. In animal and fungal cells, this process is mediated by a conserved set of proteins including actin, type II myosin, IQGAP proteins, F-BAR proteins, and the septins. To facilitate biochemical and ultrastructural analysis of cytokinesis, we have isolated and partially purified the Saccharomyces cerevisiae cytokinetic apparatus. The isolated apparatus contains all components of the actomyosin ring for which we tested—actin, myosin heavy and light chain, and IQGAP—as well as septins and the cytokinetic F-BAR protein, Hof1p. We also present evidence indicating that the actomyosin rings associated with isolated cytokinetic apparati may be contractile in vitro, and show preliminary electron microscopic imaging of the cytokinetic apparatus. This first successful isolation of the cytokinetic apparatus from a genetically tractable organism promises to make possible a deeper understanding of cytokinesis. © 2009 Wiley-Liss, Inc.

Inactivation of gene products by dominant-negative (DN) mutants is a powerful tool to assign functions to proteins. Here, we present a two-step procedure to establish a random screen for DN alleles, using the essential murine cytomegalovirus gene M50 as an example. First, loss-of-function mutants from a linker-scanning library were tested for inhibition of virus reconstitution with the help of FLP-mediated ectopic insertion of the mutants into the viral genome. Second, DN candidates were confirmed by conditional expression of the inhibitory proteins in the virus context. This allowed the quantification of the inhibitory effect, the identification of the morphogenesis block, and the construction of DN mutants with improved activity. Based on these observations a DN mutant of the homologous gene (UL50) in human cytomegalovirus was predicted and constructed. Our data suggest that a proline-rich sequence motif in the variable region of M50/UL50 represents a new functional site which is essential for nuclear egress of cytomegalovirus capsids.

The tegument proteins ppUL35 and ppUL82 (pp71) of human cytomegalovirus (HCMV) physically interact and cooperatively activate the major immediate-early transcription. While an HCMV mutant lacking UL82 displayed a multiplicity of infection (MOI)-dependent growth, the biological significance of ppUL35 has not been addressed so far. We generated a mutant virus with a deletion of the UL35 gene. Using an MOI of 0.1, the progeny virus yield of this mutant was reduced by a factor of 1,000; however, when infected at a low MOI (0.01), the gene was essential. Characterization of the replication cycle showed that the mutant virus had two defects: when virus inoculum was standardized by the amount of viral DNA, a reduced immediate-early gene expression was observed, leading to a strongly delayed expression of lytic genes. A second defect was apparent in the virus assembly, as fewer enveloped particles and no dense bodies were present in cells infected with the mutant virus. However, the particles produced by wild-type and mutant viruses did not show significant ultrastructural differences. These results suggest an important role for ppUL35 in immediate-early gene expression and virus assembly.

Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007

High-pressure freezing followed by freeze substitution and plastic embedding is becoming a more widely used method for TEM sample preparation. Here, we have investigated the influence of solvents, fixative concentrations and water content in the substitution medium on the sample quality of high-pressure frozen, freeze-substituted and plastic embedded mammalian cell culture monolayers. We found that the visibility of structural details was optimal with acetone and that extraction increased with both increasing and decreasing solvent polarity. Interestingly, the addition of water to polar solvents increased the sample quality, while being destructive when added to apolar solvents. The positive effect of water addition is saturable in acetone and ethanol at 5%(v/v), but even addition of up to 20% water has no negative effect on the sample structure. Therefore, a medium based on acetone containing fixatives and 5% water is most optimal for the substitution of mammalian cell cultures. In addition, our results suggest that the presence of water is critical for the retention of structure at temperatures around -60 degrees C.

Leber's hereditary optic neuropathy (LHON), a disease associated with a mitochondrial DNA mutation, is characterized by blindness due to degeneration of retinal ganglion cells (RGCs) and their axons, which form the optic nerve. We show that a sustained pathological autophagy and compartment-specific mitophagy activity affects LHON patient-derived cells and cybrids, as well as induced pluripotent-stem-cell-derived neurons. This is variably counterbalanced by compensatory mitobiogenesis. The aberrant quality control disrupts mitochondrial homeostasis as reflected by defective bioenergetics and excessive reactive oxygen species production, a stress phenotype that ultimately challenges cell viability by increasing the rate of apoptosis. We counteract this pathological mechanism by using autophagy regulators (clozapine and chloroquine) and redox modulators (idebenone), as well as genetically activating mitochondrial biogenesis (PGC1-α overexpression). This study substantially advances our understanding of LHON pathophysiology, providing an integrated paradigm for pathogenesis of mitochondrial diseases and druggable targets for therapy.