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Multi-scale imaging of a minimal eukaryotic pathogen

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Microsporidia, an early-diverging group of fungi, are tiny, single-celled parasites that infect a wide range of animal species, from worms and honey bees to humans. In humans, these opportunistic pathogens can cause life-threatening infections in immunocompromised individuals. To initiate an infection, microsporidia harness a specialized harpoon-like invasion apparatus called the polar tube (PT) to gain entry into host cells. The PT is tightly coiled within the transmissible extracellular spore, and is about 20 times the length of the spore. Once triggered, the PT is rapidly ejected, within milliseconds, and is thought to penetrate the host cell, acting as a conduit for the transfer of infectious cargo into the host, to initiate infection. Once inside host cells, microsporidia create a niche which is permissive to their development. We combine optical microscopy, Volume electron microscopy and structural cell biology to decipher the 3-dimensional organization, dynamics, and mechanism of the polar tube, parasite development, and host-parasite interactions

Autofluorescence lifetime imaging of immune cell metabolism in situ

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In recent years it became apparent that to understand immune cell regulation and function, immunologists must study metabolism. Studies rely on in vitro and ex vivo approaches and bulk measurements, that fall short of capturing the context- and time-dependent nature of immune cells in vivo. Using zebrafish models of injury, we show that fluorescence lifetime imaging microscopy of endogenous metabolic coenzymes, NAD(P)H and FAD, can measure dynamic changes in the intracellular metabolism of macrophages within the native wound microenvironment. This single cell-based approach can also resolve heterogeneity within a responding cell population, making it a valuable tool in immunometabolism to study immune response during inflammation and infection

Miniscopes: Visualizing the brain in motion

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Miniscope is a series of inexpensive, open-source head-mounted microscopes that use wide-field fluorescence imaging to record neural activity in awake, freely moving mice. There is also a wire-free version that enables us to track hundreds-to-thousands of neurons across days to weeks to months and longer in a freely behaving animal. Hundreds of labs worldwide are using and building on this powerful technology.

Mapping astrocytic networks through tissue clearing

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Astrocytes are non-neuronal glial cells in the central nervous system with myriad functions: they maintain the blood brain barrier, recycle neurotransmitters, and are even thought to be a component of every neuronal synapse. We have found that astrocytes also form gap junctional networks that help neurons survive degeneration. Brain regions connected by astrocytes support one another metabolically – but these connections also make linked regions collectively vulnerable to degenerative stress. Astrocyte networks have never been mapped, a critical need considering that these networks may influence the course of neurodegeneration. Here, we use a combination of novel biomolecular tools, clearing, and light sheet imaging to reveal for the first time the shape, extent, and potential plasticity of astrocytic networks in intact brains

Visualizing melanocyte transit across the basement membrane

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Metastasis accounts for the vast majority of cancer deaths, yet the very process by which it begins – invasion across a basement membrane (BM) – is poorly understood. The BM is a dense, ultrathin extracellular matrix that lines the organs and vasculature, where it serves as a selective filter and protective barrier. During metastasis, cancer cells must cross this barrier to escape the resident tissue and colonize distant organs; an example of this being metastatic melanoma, where transformed melanocytes must first migrate across the dermal-epidermal junction (DEJ) BM to escape to the deeper dermal layer. Late-stage metastatic melanoma cells share a common gene signature with embryonic melanocytes, called melanoblasts, which also cross the DEJ BM as part of their normal development. Critically, it is difficult to predict which cancers will breach a BM, because the process by which cells cross this otherwise impenetrable barrier is not well understood under both normal and pathogenic conditions. The key bottleneck in our understanding has been the ability to visualize this process live in mammals, with most studies being undertaken in cell culture or invertebrates. We have developed the mTurquoise2-Col4a1 mouse model in which all BMs are fluorescently labelled, together with methods to perform long-term live imaging in mouse embryos. These tools allow us to visualize the process of mammalian BM crossing, live, in vivo for the first time, ultimately enabling us to begin describing the fundamental cellular and molecular mechanisms underpinning BM breach

Whole brain imaging for quantitative neural profiling

Recent advancements in tissue clearing and whole mount imaging have enabled high-content quantitative analysis of cellular complexity in large intact organs like adult mouse brain. Our group has continued to develop new versions of iDISCO-family tissue clearing protocols to enable more facile and accurate imaging of diverse genetically and molecularly defined patterns across whole mouse brains, as well as large human brain samples. Complete and well-preserved tissue morphology in 3D imaging also facilitates incorporation of automatic data registration and analysis pipelines to integrate different imaging modalities for brain-wide anatomical and patho-histological profiling across scales

Architecture and dynamics of erythrocyte ankyrin-1 complexes

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The stability and shape of the erythrocyte membrane is provided by the ankyrin-1 complex, but how it tethers the spectrin-actin cytoskeleton to the lipid bilayer and the nature of its association with the band 3 anion exchanger and the Rhesus glycoproteins remains unknown. Here we present structures of ankyrin-1 complexes purified from human erythrocytes, and sub-tomogram averages of the same complexes from native membrane vesicles. We reveal the architecture of a core complex of ankyrin-1, the Rhesus proteins RhAG and RhCE, the band 3 anion exchanger, protein 4.2 and glycophorin A. The distinct T-shaped conformation of membrane-bound ankyrin-1 facilitates recognition of RhCE and unexpectedly, the water channel aquaporin-1. Together, our results uncover the molecular details of ankyrin-1 association with the erythrocyte membrane, and illustrate the mechanism of ankyrin-mediated membrane protein clustering

Toward scalable and accessible image registration with BigWarp

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The registration of biological images is a crucial part of many analyses, and manual registration is often necessary when automatic methods are inadequate. This need motivated us to develop BigWarp: software for non-linear manual registration. BigWarp has played an important role for several projects involving different collaborations and imaging modalities. In this talk, I will present a series of case studies that demonstrate both BigWarp's common and niche registration workflows, features unique to BigWarp that have made it accessible to non-expert users and enabling of science, and how broader communities have shaped its growth and future directions

The rhythm of cell walking: pulses of actomyosin contraction drive pulsatile cell protrusion dynamics in collective cell migration

Cells migrate collectively to form organs, close wounds, and in the case of disease, metastasize. The motility of singly migrating cells is driven largely by an interplay between Rho GTPase signaling and the actin network. Whether cells migrating as collectives use the same machinery for motility in vivo is unclear. To address this question, we are using the zebrafish posterior lateral line primordium as a model for collective cell migration. The primordium is a tissue of about 140 cells that migrates directly under the skin of the zebrafish embryo from behind the ear to the tip of the tail. Using a spinning disk light microscopy, we imaged the highly dynamic protrusion behaviors of the primordium cells. We discovered that active RhoA clusters as puncta on the basal sides of the primordium cells while active Rac/Cdc42 localizes fairly uniform at the membranes of the cells without clustering. RhoA and actomyosin puncta pulse at a certain frequency at the base of the primordium cell protrusion. Combining drug inhibitors and tissue-specific expression of genes targeting RhoA, we determined that the pulses of active RhoA and actomyosin are required for primordium motility. The pulses of RhoA signaling stimulate actin polymerization at the tip of the protrusions, myosin II-dependent actin flow, and protrusion retraction at the base of the protrusions, and deform the basement membrane underneath the migrating primordium. These results suggest that RhoA-induced pulses of actomyosin not only constitute the motor that pulls the primordium forward but also set the rhythm that regulates cell protrusion dynamics, a scenario that likely underlies collective migration in other contexts

Sudden cardiac arrest in young athletes: Seeing molecular mechanisms through advanced microscopy

Our laboratory seeks to identify the causes of sudden cardiac death in young athletes, and to develop strategies to treat individuals at risk. Our main focus is on an inheritable disease called Arrhythmogenic Right Ventricular Cardiomyopathy, caused by pathogenic variants in the gene coding for the desmosomal protein Plakophilin-2 (PKP2).  For this presentation, I will focus on results recently obtained through our collaboration with scientists at the NYU Microscopy Core. Advanced methodologies that we have used include pre-embedded, volumetric immuno-electron microscopy of cardiac tissue, and cell expansion combined with structured illumination for sub-diffraction limit images of adult murine cardiac myocytes. These and other visualization strategies have given us unparalleled insight into the ultrastructural platform on which the cells transition from a single gene deficiency to total functional disruption and the sudden loss of an effective heartbeat.