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SUMMARY Neural crest (NC) cells undergo extensive migrations during development. Here, we couple in vivo live imaging at high resolution with custom software tools to reveal dynamic migratory behavior in chick embryos. Trunk NC cells migrate as individuals with both stochastic and biased features as they move dorsoventrally to form peripheral ganglia. Their leading edge displays a prominent fan-shaped lamellipodium that reorients upon cell-cell contact. Computational analysis reveals that when the lamellipodium of one cell touches the body of another, the two cells undergo “contact attraction,” often moving together and then separating via a pulling force exerted by lamellipodium. Targeted optical manipulation shows that cell interactions coupled with cell density generate a long-range biased random walk behavior, such that cells move from high to low density. In contrast to chain migration noted at other axial levels, the results show that individual trunk NC cells navigate the complex environment without tight coordination between neighbors., Graphical Abstract, In Brief Li et al. combine quantitative imaging with perturbation analysis to define the cellular dynamics driving trunk neural crest migration. Unlike chain migration at other axial levels, trunk neural crest cells move as individuals driven by the combined effect of lamellipodia mediated directionality, together with cell-cell contact and cell density.

Summary: Chirality is a property of asymmetry between an object and its mirror image. Most biomolecules and many cell types are chiral. In the left-right organizer (LRO), cilia-driven flows transfer such chirality to the body scale. However, the existence of cellular chirality within tissues remains unknown. Here, we investigate this question in Kupffer’s vesicle (KV), the zebrafish LRO. Quantitative live imaging reveals that cilia populating the KV display asymmetric orientation between the right and left sides, resulting in a chiral structure, which is different from the chiral cilia rotation. This KV chirality establishment is dynamic and depends on planar cell polarity. While its impact on left-right (LR) symmetry breaking remains unclear, we show that this asymmetry does not depend on the LR signaling pathway or flow. This work identifies a different type of tissue asymmetry and sheds light on chirality genesis in developing tissues. : During left-right axis specification, motile cilia are required for breaking the axis of symmetry. In this context, Ferreira et al. show that cilia orientation is chiral, defining an alternative type of asymmetry in the embryo. Keywords: Danio rerio, trilobite/Vangl2, lrdr1, Dnaaf1/Lrrc50, spaw, nodal, tissue asymmetry, PKD2/PC2/TRPP2/polycistin-2, blebbistatin, Rock2B

Convergent extension is an evolutionarily conserved collective cell movement that elongates the body axis of almost all animals and is required for the morphogenesis of several organ systems. Decades of study have revealed two distinct mechanisms of cell movement during CE, one based on cell crawling and the other on junction contraction. How these two behaviors collaborate during CE is not understood. Here, using quantitative live cell imaging we show that these two modes act both independently and in concert during CE, but that cell movement is more effective when the two modes are integrated via mechano-reciprocity. Based on these findings, we developed a novel computational model that for the first time treats crawling and contraction independently. This model not only confirmed the biomechanical efficacy of integrating the two modes, but also revealed for the first time how the two modes -and their integration- are influenced by cell adhesion. Finally, we use these new insights to further understand the complex CE phenotype resulting from loss of the C-cadherin interacting catenin Arvcf. These data are significant for providing new biomechanical and cell biological insights into a fundamental morphogenetic process that is implicated in human neural tube defects and skeletal dysplasias.

SUMMARY Drug development is hampered by poor target selection. Phenotypic screens using neurons differentiated from patient stem cells offer the possibility to validate known and discover novel disease targets in an unbiased fashion. To identify targets for managing hyperexcitability, a pathological feature of amyotrophic lateral sclerosis (ALS), we design a multi-step screening funnel using patient-derived motor neurons. High-content live cell imaging is used to evaluate neuronal excitability, and from a screen against a chemogenomic library of 2,899 target-annotated compounds, 67 reduce the hyperexcitability of ALS motor neurons carrying the SOD1(A4V) mutation, without cytotoxicity. Bioinformatic deconvolution identifies 13 targets that modulate motor neuron excitability, including two known ALS excitability modulators, AMPA receptors and Kv7.2/3 ion channels, constituting target validation. We also identify D2 dopamine receptors as modulators of ALS motor neuron excitability. This screen demonstrates the power of human disease cell-based phenotypic screens for identifying clinically relevant targets for neurological disorders., Graphical abstract, In brief Motor neuron hyperexcitability is observed in both ALS patients and their iPSC-derived neurons. Combining a high-content live imaging excitability phenotypic assay, high-throughput screening against a cross-annotated chemogenomic library, and bioinformatic enrichment analysis, Huang et al. identify targets modulating the hyperexcitability of ALS patient-derived motor neurons in an unbiased manner.

Summary: How do stem cells respond to signals to initiate differentiation? Here, we show that, despite uniform exposure to differentiation-inducing extracellular signals, individual human embryonic stem cells (hESCs) respond heterogeneously. To track how hESCs incipiently exit pluripotency, we established a system to differentiate hESCs as single cells and conducted live imaging to track their very first cell division. We followed the fate of their earliest daughters as they remained undifferentiated or differentiated toward the primitive streak (the earliest descendants of pluripotent cells). About 30%–50% of the time, hESCs divided to yield one primitive streak and one undifferentiated daughter. The undifferentiated daughter cell was innately resistant to WNT signaling and could not respond to this primitive-streak-specifying differentiation signal. Hence, the first division of differentiating hESCs sometimes yields daughters with diverging fates, with implications for the efficiency of directed differentiation protocols and the underlying rules of lineage commitment. : Brown et al. show that 30%–50% of the time, the first division of differentiating human embryonic stem cells generates one differentiated and one undifferentiated daughter cell. These asymmetric fate outcomes have implications for the efficiency of stem cell differentiation protocols and the mechanisms underlying lineage commitment. Keywords: embryonic stem cell, differentiation, cell division, primitive streak, live imaging

Summary Cell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations., Graphical Abstract, Highlights • Endogenous mRuby-PCNA as an all-in-one cell cycle reporter • Automated image analysis pipeline to segment, track, and classify based on PCNA • Quantitative analysis of cyclin oscillations during a complete cell cycle • Cyclin D1 maintains G1 phase and prevents the transition into quiescence, Zerjatke et al. present endogenously tagged PCNA as an all-in-one cell cycle reporter for living cells to classify all cell cycle phases and quiescence using a single fluorescent channel. Visualizing endogenous cyclin D1 in living cells, they show that cyclin D1 maintains G1 phase and prevents the transition into quiescence.

Mammalian cells have two fundamentally different states, proliferative and quiescent, but our understanding of how and why cells switch between these states is limited. We previously showed that actively proliferating populations contain a subpopulation that enters quiescence (G0) in an apparently stochastic manner. Using single-cell time-lapse imaging of CDK2 activity and DNA damage, we now show that unresolved endogenous replication stress in the previous (mother) cell cycle prompts p21-dependent entry of daughter cells into quiescence immediately after mitosis. Furthermore, the amount of time daughter cells spend in quiescence is correlated with the extent of inherited damage. Our study thus links replication errors in one cell cycle to the fate of daughter cells in the subsequent cell cycle. More broadly, this work reveals that entry into quiescence is not purely stochastic but has a strong deterministic component arising from a memory of events that occurred in the previous generation(s).

Chromatin moves with subdiffusive and spatially constrained dynamics within the cell nucleus. Here, we use single-locus tracking by time-lapse fluorescence microscopy to uncover information regarding the forces that influence chromatin movement following the induction of a persistent DNA double-strand break (DSB). Using improved time-lapse imaging regimens, we monitor trajectories of tagged DNA loci at a high temporal resolution, which allows us to extract biophysical parameters through robust statistical analysis. Polymer modeling based on these parameters predicts chromatin domain expansion near a DSB and damage extrusion from the domain. Both phenomena are confirmed by live imaging in budding yeast. Calculation of the anomalous exponent of locus movement allows us to differentiate forces imposed on the nucleus through the actin cytoskeleton from those that arise from INO80 remodeler-dependent changes in nucleosome organization. Our analytical approach can be applied to high-density single-locus trajectories obtained in any cell type.

The retina of lower vertebrates grows continuously by integrating new neurons generated from progenitors in the ciliary margin zone (CMZ). Whether the mammalian CMZ provides the neural retina with retinal cells is controversial. Live imaging of embryonic retina expressing eGFP in the CMZ shows that cells migrate laterally from the CMZ to the neural retina where differentiated retinal ganglion cells (RGCs) reside. Because Cyclin D2, a cell-cycle regulator, is enriched in ventral CMZ, we analyzed Cyclin D2-/- mice to test whether the CMZ is a source of retinal cells. Neurogenesis is diminished in Cyclin D2 mutants, leading to a reduction of RGCs in the ventral retina. In line with these findings, in the albino retina, the decreased production of ipsilateral RGCs is correlated with fewer Cyclin D2+ cells. Together, these results implicate the mammalian CMZ as a neurogenic site that produces RGCs and whose proper generation depends on Cyclin D2 activity.

Summary: Emerging evidence suggests that crosstalk between glioma cells and the brain microenvironment may influence brain tumor growth. To date, known reciprocal interactions among these cells have been limited to the release of paracrine factors. Combining a genetic strategy with longitudinal live imaging, we find that individual gliomas communicate with distinct sets of non-glioma cells, including glial cells, neurons, and vascular cells. Transfer of genetic material is achieved mainly through extracellular vesicles (EVs), although cell fusion also plays a minor role. We further demonstrate that EV-mediated communication leads to the increase of synaptic activity in neurons. Blocking EV release causes a reduction of glioma growth in vivo. Our findings indicate that EV-mediated interaction between glioma cells and non-glioma brain cells alters the tumor microenvironment and contributes to glioma development. : Non-glioma cells form a unique tumor microenvironment and are critical for glioma progression. Gao et al. find that individual gliomas communicate with distinct sets of non-glioma cells, including glial cells, neurons, and vascular cells. Transfer of genetic material is achieved mainly through extracellular vesicles (EVs). Keywords: glioma, glia, astrocyte, interaction, extracellular vesicle, tumor microenvironment, cell fusion, neuron, exosome

Summary: Budding yeast cells undergo a limited number of divisions before they enter senescence and die. Despite recent mechanistic advances, whether and how molecular events are temporally and causally linked during the transition to senescence remain elusive. Here, using real-time observation of the accumulation of extrachromosomal rDNA circles (ERCs) in single cells, we provide evidence that ERCs build up rapidly with exponential kinetics well before any physiological decline. We then show that ERCs fuel a massive increase in ribosomal RNA (rRNA) levels in the nucleolus, which do not mature into functional ribosomes. This breakdown in nucleolar coordination is followed by a loss of nuclear homeostasis, thus defining a chronology of causally related events leading to cell death. A computational analysis supports a model in which a series of age-independent processes lead to an age-dependent increase in cell mortality, hence explaining the emergence of aging in budding yeast. : The accumulation of extrachromosomal rDNA circles (ERCs) is a hallmark of aging in budding yeast. Morlot et al. show that ERCs accumulate ahead of senescence onset and fuel an excessive rDNA transcription, ultimately leading to impaired nuclear homeostasis and an irreversible cell cycle slowdown. Keywords: live cell imaging, microfluidics, yeast replicative aging, ribosome biogenesis, nucleolus, rDNA, ERCs

SUMMARY The epidermis regenerates continually to maintain a protective barrier at the body’s surface composed of differentiating keratinocytes. Maturation of this stratified tissue requires that keratinocytes undergo wholesale organelle degradation upon reaching the outermost tissue layers to form compacted, anucleate cells. Through live imaging of organotypic cultures of human epidermis, we find that regulated breakdown of mitochondria is critical for epidermal development. Keratinocytes in the upper layers initiate mitochondrial fragmentation, depolarization, and acidification upon upregulating the mitochondrion-tethered autophagy receptor NIX. Depleting NIX compromises epidermal maturation and impairs mitochondrial elimination, whereas ectopic NIX expression accelerates keratinocyte differentiation and induces premature mitochondrial fragmentation via the guanosine triphosphatase (GTPase) DRP1. We further demonstrate that inhibiting DRP1 blocks NIX-mediated mitochondrial breakdown and disrupts epidermal development. Our findings establish mitochondrial degradation as a key step in terminal keratinocyte differentiation and define a pathway operating via the mitophagy receptor NIX in concert with DRP1 to drive epidermal morphogenesis., In Brief Using live microscopy of human organotypic epidermis, Simpson et al. demonstrate how keratinocytes degrade their mitochondria in the upper tissue layers during their final stage of differentiation. By upregulating expression of the mitophagy receptor NIX, keratinocytes initiate DRP1- dependent mitochondrial fragmentation, a process critical for epidermal tissue maturation., Graphical Abstract

SUMMARY In many tissues, stem cell (SC) proliferation is dynamically adjusted to regenerative needs. How SCs adapt their metabolism to meet the demands of proliferation and how changes in such adaptive mechanisms contribute to age-related dysfunction remain poorly understood. Here, we identify mitochondrial Ca2+ uptake as a central coordinator of SC metabolism. Live imaging of genetically encoded metabolite sensors in intestinal SCs (ISCs) of Drosophila reveals that mitochondrial Ca2+ uptake transiently adapts electron transport chain flux to match energetic demand upon proliferative activation. This tight metabolic adaptation is lost in ISCs of old flies, as declines in mitochondrial Ca2+ uptake promote a “Warburg-like” metabolic reprogramming toward aerobic glycolysis. This switch mimics metabolic reprogramming by the oncogene RasV12 and enhances ISC hyperplasia. Our data identify a critical mechanism for metabolic adaptation of tissue SCs and reveal how its decline sets aging SCs on a metabolic trajectory reminiscent of that seen upon oncogenic transformation., Graphical Abstract, In Brief Morris et al. uncover a Ca2+-mediated adjustment of mitochondrial electron transport chain flux that links increased ATP production to proliferation in Drosophila intestinal stem cells. Declines in mitochondrial Ca2+ uptake in aging stem cells promote a Warburg-like metabolic reprogramming reminiscent of that observed upon oncogenic transformation.

SummaryErrors during cell division in oocytes and early embryos are linked to birth defects in mammals. Bipolar spindle assembly in early mouse embryos is unique in that three or more acentriolar microtubule-organizing centers (MTOCs) are initially formed and are then clustered into two spindle poles. Using a knockout mouse and live imaging of spindles in embryos, we demonstrate that MTOC clustering during the blastocyst stage requires augmin, a critical complex for MT-dependent MT nucleation within the spindle. Functional analyses in cultured cells with artificially increased numbers of centrosomes indicate that the lack of intra-spindle MT nucleation, but not loss of augmin per se or overall reduction of spindle MTs, is the cause of clustering failure. These data suggest that onset of mitosis with three or more MTOCs is turned into a typical bipolar division through augmin-dependent intra-spindle MT assembly.

SummaryThe local synthesis of transmembrane proteins underlies functional specialization of dendritic microdomains in neuronal plasticity. It is unclear whether these proteins have access to the complete machinery of the secretory pathway following local synthesis. In this study, we describe a probe called pGolt that allows visualization of Golgi-related organelles for live imaging in neurons. We show that pGolt labels a widespread microsecretory Golgi satellite (GS) system that is, in contrast to Golgi outposts, present throughout basal and apical dendrites of all pyramidal neurons. The GS system contains glycosylation machinery and is localized between ERGIC and retromer. Synaptic activity restrains lateral movement of ERGIC, GS, and retromer close to one another, allowing confined processing of secretory cargo. Several synaptic transmembrane proteins pass through and recycle back to the GS system. Thus, the presence of an ER-ERGIC-GS-retromer microsecretory system in all neuronal dendrites enables autonomous local control of transmembrane protein synthesis and processing.

Neurulation is a critical period in all vertebrates and results in the formation of the neural tube, which gives rise to the CNS. Apical constriction is one of the fundamental morphogenetic movements that drives neural tube closure. Using live imaging, we show that apical constriction during the neurulation is a stepwise process driven by cell-autonomous and asynchronous contraction pulses followed by stabilization steps. Our data suggest that contraction events are triggered by cell-autonomous Ca(2+) flashes and are driven by a transient contractile apical pool of actin. In addition, we provide evidence that the cell autonomy and asynchrony of contraction are required for the correct spatial distribution of constriction and, as a result, are critical for tissue morphogenesis. Finally, we identify Calpain2 as a regulator of apical constriction and show that it is required for the stabilization step, but is dispensable during contraction. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved. 13 2189 2202 Cited By :6

Numerous mammalian cells contain abundant Zn(2+) in their secretory granules, yet available Zn(2+) sensors lack the desired specificity and sensitivity for imaging granular Zn(2+). We developed a fluorescent zinc granule indicator, ZIGIR, that possesses numerous desired properties for live cell imaging, including >100-fold fluorescence enhancement, membrane permeability, and selective enrichment to acidic granules. The combined advantages endow ZIGIR with superior sensitivity and specificity for imaging granular Zn(2+). ZIGIR enables separation of heterogenous β cells based on their insulin content and sorting of mouse islets into pure α cells and β cells. In human islets, ZIGIR facilitates sorting of endocrine cells into highly enriched α cells and β cells, reveals unexpectedly high Zn(2+) activity in the somatostatin granule of some δ cells, and uncovers variation in the glucagon content among human α cells. We expect broad applications of ZIGIR for studying Zn(2+) biology and Zn(2+)-rich secretory granules and for engineering β cells with high insulin content for treating diabetes.

Summary: An intriguing question in developmental biology is how do developmental processes achieve high reproducibility among individuals? An in-depth analysis of information contained in phenotypic variability provides an important perspective to address this question. In this work, we present a quantitative and functional analysis of cell position variability during Caenorhabditis elegans embryogenesis. We find that cell position variability is highly deterministic and regulated by intrinsic and extrinsic mechanisms. Positional variability is determined by cell lineage identity and is coupled to diverse developmental properties of cells, including embryonic localization, cell contact, and left-right symmetry. Temporal dynamics of cell position variability are highly concordant, and fate specification contributes to a systems-wide reduction of variability that could provide a buffering strategy. Positional variability is stringently regulated throughout embryogenesis and cell-cell junctions function to restrict variability. Our results provide insight into systems properties and spatiotemporal control of cellular variability during development. : How do developmental processes achieve high reproducibility? Using live imaging and single-cell analyses, Li et al. show that the variability of cell position during C. elegans embryogenesis is highly deterministic and temporally dynamic, regulated by intrinsic and extrinsic mechanisms. Fate specification and cell-cell junctions contribute to the reduction of variability. Keywords: cellular variability, cell position, embryogenesis, live imaging, cell lineage tracing, single-cell analysis, C. elegans

Summary: We describe a simple and direct approach to measure the progression of single DNA replication forks in living cells by monitoring two fluorescently labeled loci downstream of an origin of replication. We employ this approach to investigate the roles of several leading and lagging strand factors in overall replisome function and show that fork progression is strongly dependent on proper maturation of Okazaki fragments. We also demonstrate how related cellular phenotypes, such as cell-cycle progression and the dynamics of sister chromatid cohesion, may be simultaneously monitored and correlated to DNA replication at the single-cell level. : Dovrat et al. develop a simple approach based on live-cell imaging to measure the progression rates of single DNA replication forks at specific loci while simultaneously examining cell-cycle progression and sister chromatid cohesion. The rate of DNA replication is shown to be strongly dependent on full maturation of Okazaki fragments. Keywords: DNA replication, live-cell imaging, FROS, Okazaki fragment maturation, sister chromatid cohesion

Transcription of developmental genes is controlled by multiple enhancers. Frequently, more than one enhancer can activate transcription from the same promoter in the same cells. How is regulatory information from multiple enhancers combined to determine the overall expression output? We measure nascent transcription driven by a pair of shadow enhancers, each enhancer of the pair separately, and each duplicated, using live imaging in Drosophila embryos. This set of constructs allows us to quantify the input-output function describing signal integration by two enhancers. We show that signal integration performed by these shadow enhancers and duplications varies across the expression pattern, implying that how their activities are combined depends on the transcriptional regulators bound to the enhancers in different parts of the embryo. Characterizing signal integration by multiple enhancers is a critical step in developing conceptual and computational models of gene expression at the locus level, where multiple enhancers control transcription together.

Summary: A major challenge in cancer research is the complexity of the tumor microenvironment, which includes the host immunological setting. Inspired by the emerging technology of organ-on-chip, we achieved 3D co-cultures in microfluidic devices (integrating four cell populations: cancer, immune, endothelial, and fibroblasts) to reconstitute ex vivo a human tumor ecosystem (HER2+ breast cancer). We visualized and quantified the complex dynamics of this tumor-on-chip, in the absence or in the presence of the drug trastuzumab (Herceptin), a targeted antibody therapy directed against the HER2 receptor. We uncovered the capacity of the drug trastuzumab to specifically promote long cancer-immune interactions (>50 min), recapitulating an anti-tumoral ADCC (antibody-dependent cell-mediated cytotoxicity) immune response. Cancer-associated fibroblasts (CAFs) antagonized the effects of trastuzumab. These observations constitute a proof of concept that tumors-on-chip are powerful platforms to study ex vivo immunocompetent tumor microenvironments, to characterize ecosystem-level drug responses, and to dissect the roles of stromal components. : Inspired by the emerging technology of tumor-on-chip, Nguyen et al. reconstituted ex vivo a human tumor microenvironment (HER2+ breast cancer), characterized the ecosystem-level responses to the drug trastuzumab (Herceptin), and dissected the roles of stromal components (immune cells and fibroblasts), demonstrating the power of immunocompetent tumors-on-chip for preclinical drug studies. Keywords: tumor microenvironment, organ-on-chip, tumor-on-chip, trastuzumab, HER2+ breast cancer, cancer-associated fibroblasts, live cell imaging, microfluidics, pre-clinical models, immunotherapy

Summary Reprogramming of metabolic pathways determines cell functions and fate. In our work, we have used organelle-targeted ATP biosensors to evaluate cellular metabolic settings with high resolution in real time. Our data indicate that mitochondria dynamically supply ATP for glucose phosphorylation in a variety of cancer cell types. This hexokinase-dependent process seems to be reversed upon the removal of glucose or other hexose sugars. Our data further verify that mitochondria in cancer cells have increased ATP consumption. Similar subcellular ATP fluxes occurred in young mouse embryonic fibroblasts (MEFs). However, pancreatic beta cells, senescent MEFs, and MEFs lacking mitofusin 2 displayed completely different mitochondrial ATP dynamics, indicative of increased oxidative phosphorylation. Our findings add perspective to the variability of the cellular bioenergetics and demonstrate that live cell imaging of mitochondrial ATP dynamics is a powerful tool to evaluate metabolic flexibility and heterogeneity at a single-cell level.

Summary Mucus separates gut-luminal microbes from the tissue. It is unclear how pathogens like Salmonella Typhimurium (S.Tm) can overcome this obstacle. Using live microscopy, we monitored S.Tm interactions with native murine gut explants and studied how mucus affects the infection. A dense inner mucus layer covers the distal colon tissue, limiting direct tissue access. S.Tm performs near-surface swimming on this mucus layer, which allows probing for colon mucus heterogeneities, but can also entrap the bacterium in the dense inner colon mucus layer. In the cecum, dense mucus fills only the bottom of the intestinal crypts, leaving the epithelium between crypts unshielded and prone to access by motile and non-motile bacteria alike. This explains why the cecum is highly infection permissive and represents the primary site of S.Tm enterocolitis in the streptomycin mouse model. Our findings highlight the importance of mucus in intestinal defense and homeostasis.

Summary T cells engage in two modes of interaction with antigen-presenting surfaces: stable synapses and motile kinapses. Although it is surmised that durable interactions of T cells with antigen-presenting cells involve synapses, in situ 3D imaging cannot resolve the mode of interaction. We have established in vitro 2D platforms and quantitative metrics to determine cell-intrinsic modes of interaction when T cells are faced with spatially continuous or restricted stimulation. All major resting human T cell subsets, except memory CD8 T cells, spend more time in the kinapse mode on continuous stimulatory surfaces. Surprisingly, we did not observe any concordant relationship between the mode and durability of interaction on cell-sized stimulatory spots. Naive CD8 T cells maintain kinapses for more than 3 hr before leaving stimulatory spots, whereas their memory counterparts maintain synapses for only an hour before leaving. Thus, durable interactions do not require stable synapses.

Mutations in proline-rich transmembrane protein 2 (PRRT2) are associated with a range of paroxysmal neurological disorders. PRRT2 predominantly localizes to the pre-synaptic terminals and is believed to regulate neurotransmitter release. However, the mechanism of action is unclear. Here, we use reconstituted single vesicle and bulk fusion assays, combined with live cell imaging of single exocytotic events in PC12 cells and biophysical analysis, to delineate the physiological role of PRRT2. We report that PRRT2 selectively blocks the trans SNARE complex assembly and thus negatively regulates synaptic vesicle priming. This inhibition is actualized via weak interactions of the N-terminal proline-rich domain with the synaptic SNARE proteins. Furthermore, we demonstrate that paroxysmal dyskinesia-associated mutations in PRRT2 disrupt this SNARE-modulatory function and with efficiencies corresponding to the severity of the disease phenotype. Our findings provide insights into the molecular mechanisms through which loss-of-function mutations in PRRT2 result in paroxysmal neurological disorders.

Summary During sexual intercourse, HIV-1 crosses epithelial barriers composing the genital mucosa, a poorly understood feature that requires an HIV-1-infected cell vectoring efficient mucosal HIV-1 entry. Therefore, urethral mucosa comprising a polarized epithelium and a stroma composed of fibroblasts and macrophages were reconstructed in vitro. Using this system, we demonstrate by live imaging that efficient HIV-1 transmission to stromal macrophages depends on cell-mediated transfer of the virus through virological synapses formed between HIV-1-infected CD4+ T cells and the epithelial cell mucosal surface. We visualized HIV-1 translocation through mucosal epithelial cells via transcytosis in regions where virological synapses occurred. In turn, interleukin-13 is secreted and HIV-1 targets macrophages, which develop a latent state of infection reversed by lipopolysaccharide (LPS) activation. The live observation of virological synapse formation reported herein is key in the design of vaccines and antiretroviral therapies aimed at blocking HIV-1 access to cellular reservoirs in genital mucosa.