Polyploidy and Mitotic Cell Death are Two Distinct HIV-1 Vpr-Driven Outcomes in Renal Tubule Epithelial Cells

Given the emerging epidemic of renal disease in HIV+ patients and the fact that HIV DNA and RNA persist in the kidneys of HIV+ patients despite therapy, it is necessary to understand the role of direct HIV-1 infection of the kidney. HIV-associated kidney disease pathogenesis is attributed in large part to viral proteins. Expression of Vpr in renal tubule epithelial cells (RTECs) induces G2 arrest, apoptosis and polyploidy. The ability of a subset of cells to overcome the G2/M block and progress to polyploidy is not well understood. Polyploidy frequently associates with a bypass of cell death and disease pathogenesis. Given the ability of the kidney to serve as a unique compartment for HIV-1 infection, and the observed occurrence of polyploid cells in HIV+ renal cells, it is critical to understand the mechanisms and consequences of Vpr-induced polyploidy.

Here I determined effects of HIV-1 Vpr expression in renal cells using highly efficient transduction with VSV.G pseudotyped lentiviral vectors expressing Vpr in the HK2 human tubule epithelial cell line. Using FACS, fluorescence microscopy, and live cell imaging I show that G2 escape immediately precedes a critical junction between two distinct outcomes in Vpr+ RTECs: mitotic cell death and polyploidy. Vpr+ cells that evade aberrant mitosis and become polyploid have a substantially higher survival rate than those that undergo complete mitosis, and this survival correlates with enrichment for polyploidy in cell culture over time. Further, I identify a novel role for ATM kinase in promoting G2 arrest escape and polyploidy in this context. In summary, my work identifies ATM-dependent override of Vpr-mediated G2/M arrest as a critical determinant of cell fate Vpr+ RTECs. Further, our work highlights how a poorly understood HIV mechanism, ploidy increase, may offer insight into key processes of reservoir establishment and disease pathogenesis in HIV+ kidneys.

UNC-6 (netrin) stabilizes oscillatory clustering of the UNC-40 (DCC) receptor to orient polarity.

The receptor deleted in colorectal cancer (DCC) directs dynamic polarizing activities in animals toward its extracellular ligand netrin. How DCC polarizes toward netrin is poorly understood. By performing live-cell imaging of the DCC orthologue UNC-40 during anchor cell invasion in Caenorhabditis elegans, we have found that UNC-40 clusters, recruits F-actin effectors, and generates F-actin in the absence of UNC-6 (netrin). Time-lapse analyses revealed that UNC-40 clusters assemble, disassemble, and reform at periodic intervals in different regions of the cell membrane. This oscillatory behavior indicates that UNC-40 clusters through a mechanism involving interlinked positive (formation) and negative (disassembly) feedback. We show that endogenous UNC-6 and ectopically provided UNC-6 orient and stabilize UNC-40 clustering. Furthermore, the UNC-40-binding protein MADD-2 (a TRIM family protein) promotes ligand-independent clustering and robust UNC-40 polarization toward UNC-6. Together, our data suggest that UNC-6 (netrin) directs polarized responses by stabilizing UNC-40 clustering. We propose that ligand-independent UNC-40 clustering provides a robust and adaptable mechanism to polarize toward netrin.

Opening Basement Membrane Gaps During C. elegans Uterine-Vulval Attachment

The basement membrane (BM) is a highly conserved form of extracellular matrix that underlies or surrounds and supports most animal tissues. BMs are crossed by cells during various remodeling events in development, immune surveillance, or during cancer metastasis. Because BMs are dense and not easily penetrable, most of these cells must open a gap in order to facilitate their migration. The mechanisms by which cells execute these changes are poorly understood. A developmental event that requires the opening of a BM gap is C. elegans uterine-vulval connection. The anchor cell (AC), a specialized uterine cell, creates a de novo BM gap. Subsequent widening of the BM gap involves the underlying vulval precursor cells (VPCs) and the π cells, uterine neighbors of the AC through non-proteolytic BM sliding. Using forward and reverse genetic screening, transcriptome profiling, and live-cell imaging, I investigated how the cells in these tissues accomplish BM gap formation. In Chapter 2, I identify two potentially novel regulators of BM breaching, isolated through a large-scale forward genetic screen and characterize the invasion defect in these mutants. In Chapter 3, I describe single-cell transcriptome sequencing of the invasive AC. In Chapter 4, I describe the role of the π cells in opening the nascent BM gap. A complete developmental pathway for this process has been elucidated: the AC induces the π fate through Notch signaling, after which the π cells upregulate the Sec14 family protein CTG-1, which in turn restricts the trafficking of DGN-1 (dystroglycan), a laminin receptor, allowing the BM to slide. Chapter 5 outlines the implications of these discoveries.

Single-cell functional analysis of parathyroid adenomas reveals distinct classes of calcium sensing behaviour in primary hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder caused by a failure of calcium sensing secondary to tumour development in one or more of the parathyroid glands. Parathyroid adenomas are comprised of distinct cellular subpopulations of variable clonal status that exhibit differing degrees of calcium responsiveness. To gain a clearer understanding of the relationship among cellular identity, tumour composition and clinical biochemistry in PHPT, we developed a novel single cell platform for quantitative evaluation of calcium sensing behaviour in freshly resected human parathyroid tumour cells. Live-cell intracellular calcium flux was visualized through Fluo-4-AM epifluorescence, followed by in situ immunofluorescence detection of the calcium sensing receptor (CASR), a central component in the extracellular calcium signalling pathway. The reactivity of individual parathyroid tumour cells to extracellular calcium stimulus was highly variable, with discrete kinetic response patterns observed both between and among parathyroid tumour samples. CASR abundance was not an obligate determinant of calcium responsiveness. Calcium EC50 values from a series of parathyroid adenomas revealed that the tumours segregated into two distinct categories. One group manifested a mean EC50 of 2.40 mM (95% CI: 2.37-2.41), closely aligned to the established normal range. The second group was less responsive to calcium stimulus, with a mean EC50 of 3.61 mM (95% CI: 3.45-3.95). This binary distribution indicates the existence of a previously unappreciated biochemical sub-classification of PHPT tumours, possibly reflecting distinct etiological mechanisms. Recognition of quantitative differences in calcium sensing could have important implications for the clinical management of PHPT.

Live volumetric (4D) visualization and guidance of in vivo human ophthalmic surgery with intraoperative optical coherence tomography.

Minimally-invasive microsurgery has resulted in improved outcomes for patients. However, operating through a microscope limits depth perception and fixes the visual perspective, which result in a steep learning curve to achieve microsurgical proficiency. We introduce a surgical imaging system employing four-dimensional (live volumetric imaging through time) microscope-integrated optical coherence tomography (4D MIOCT) capable of imaging at up to 10 volumes per second to visualize human microsurgery. A custom stereoscopic heads-up display provides real-time interactive volumetric feedback to the surgeon. We report that 4D MIOCT enhanced suturing accuracy and control of instrument positioning in mock surgical trials involving 17 ophthalmic surgeons. Additionally, 4D MIOCT imaging was performed in 48 human eye surgeries and was demonstrated to successfully visualize the pathology of interest in concordance with preoperative diagnosis in 93% of retinal surgeries and the surgical site of interest in 100% of anterior segment surgeries. In vivo 4D MIOCT imaging revealed sub-surface pathologic structures and instrument-induced lesions that were invisible through the operating microscope during standard surgical maneuvers. In select cases, 4D MIOCT guidance was necessary to resolve such lesions and prevent post-operative complications. Our novel surgical visualization platform achieves surgeon-interactive 4D visualization of live surgery which could expand the surgeon's capabilities.

Magnetic resonance imaging of graded skeletal muscle injury in live rats.

INTRODUCTION: Increasing number of stretch-shortening contractions (SSCs) results in increased muscle injury. METHODS: Fischer Hybrid rats were acutely exposed to an increasing number of SSCs in vivo using a custom-designed dynamometer. Magnetic resonance imaging (MRI) imaging was conducted 72 hours after exposure when rats were infused with Prohance and imaged using a 7T rodent MRI system (GE Epic 12.0). Images were acquired in the transverse plane with typically 60 total slices acquired covering the entire length of the hind legs. Rats were euthanized after MRI, the lower limbs removed, and tibialis anterior muscles were prepared for histology and quantified stereology. RESULTS: Stereological analyses showed myofiber degeneration, and cellular infiltrates significantly increased following 70 and 150 SSC exposure compared to controls. MRI images revealed that the percent affected area significantly increased with exposure in all SSC groups in a graded fashion. Signal intensity also significantly increased with increasing SSC repetitions. DISCUSSION: These results suggest that contrast-enhanced MRI has the sensitivity to differentiate specific degrees of skeletal muscle strain injury, and imaging data are specifically representative of cellular histopathology quantified via stereological analyses.

Live Imaging of Host-Parasite Interactions in a Zebrafish Infection Model Reveals Cryptococcal Determinants of Virulence and Central Nervous System Invasion.

UNLABELLED: The human fungal pathogen Cryptococcus neoformans is capable of infecting a broad range of hosts, from invertebrates like amoebas and nematodes to standard vertebrate models such as mice and rabbits. Here we have taken advantage of a zebrafish model to investigate host-pathogen interactions of Cryptococcus with the zebrafish innate immune system, which shares a highly conserved framework with that of mammals. Through live-imaging observations and genetic knockdown, we establish that macrophages are the primary immune cells responsible for responding to and containing acute cryptococcal infections. By interrogating survival and cryptococcal burden following infection with a panel of Cryptococcus mutants, we find that virulence factors initially identified as important in causing disease in mice are also necessary for pathogenesis in zebrafish larvae. Live imaging of the cranial blood vessels of infected larvae reveals that C. neoformans is able to penetrate the zebrafish brain following intravenous infection. By studying a C. neoformans FNX1 gene mutant, we find that blood-brain barrier invasion is dependent on a known cryptococcal invasion-promoting pathway previously identified in a murine model of central nervous system invasion. The zebrafish-C. neoformans platform provides a visually and genetically accessible vertebrate model system for cryptococcal pathogenesis with many of the advantages of small invertebrates. This model is well suited for higher-throughput screening of mutants, mechanistic dissection of cryptococcal pathogenesis in live animals, and use in the evaluation of therapeutic agents. IMPORTANCE: Cryptococcus neoformans is an important opportunistic pathogen that is estimated to be responsible for more than 600,000 deaths worldwide annually. Existing mammalian models of cryptococcal pathogenesis are costly, and the analysis of important pathogenic processes such as meningitis is laborious and remains a challenge to visualize. Conversely, although invertebrate models of cryptococcal infection allow high-throughput assays, they fail to replicate the anatomical complexity found in vertebrates and, specifically, cryptococcal stages of disease. Here we have utilized larval zebrafish as a platform that overcomes many of these limitations. We demonstrate that the pathogenesis of C. neoformans infection in zebrafish involves factors identical to those in mammalian and invertebrate infections. We then utilize the live-imaging capacity of zebrafish larvae to follow the progression of cryptococcal infection in real time and establish a relevant model of the critical central nervous system infection phase of disease in a nonmammalian model.

Direct In Vivo Manipulation and Imaging of Calcium Transients in Neutrophils Identify a Critical Role for Leading-Edge Calcium Flux.

Calcium signaling has long been associated with key events of immunity, including chemotaxis, phagocytosis, and activation. However, imaging and manipulation of calcium flux in motile immune cells in live animals remain challenging. Using light-sheet microscopy for in vivo calcium imaging in zebrafish, we observe characteristic patterns of calcium flux triggered by distinct events, including phagocytosis of pathogenic bacteria and migration of neutrophils toward inflammatory stimuli. In contrast to findings from ex vivo studies, we observe enriched calcium influx at the leading edge of migrating neutrophils. To directly manipulate calcium dynamics in vivo, we have developed transgenic lines with cell-specific expression of the mammalian TRPV1 channel, enabling ligand-gated, reversible, and spatiotemporal control of calcium influx. We find that controlled calcium influx can function to help define the neutrophil's leading edge. Cell-specific TRPV1 expression may have broad utility for precise control of calcium dynamics in other immune cell types and organisms.

Whole-cell-analysis of live cardiomyocytes using wide-field interferometric phase microscopy.

We apply wide-field interferometric microscopy techniques to acquire quantitative phase profiles of ventricular cardiomyocytes in vitro during their rapid contraction with high temporal and spatial resolution. The whole-cell phase profiles are analyzed to yield valuable quantitative parameters characterizing the cell dynamics, without the need to decouple thickness from refractive index differences. Our experimental results verify that these new parameters can be used with wide field interferometric microscopy to discriminate the modulation of cardiomyocyte contraction dynamics due to temperature variation. To demonstrate the necessity of the proposed numerical analysis for cardiomyocytes, we present confocal dual-fluorescence-channel microscopy results which show that the rapid motion of the cell organelles during contraction preclude assuming a homogenous refractive index over the entire cell contents, or using multiple-exposure or scanning microscopy.

Pulsating tandem microbubble for localized and directional single-cell membrane poration.

The interaction of laser-generated tandem microbubble (maximum diameter of about 50  μm) with single (rat mammary carcinoma) cells is investigated in a 25-μm liquid layer. Antiphase and coupled oscillation of the tandem microbubble leads to the formation of alternating, directional microjets (with max microstreaming velocity of 10  m/s) and vortices (max vorticity of 350 000  s{-1}) in opposite directions. Localized and directional membrane poration (200 nm to 2  μm in pore size) can be produced by the tandem microbubble in an orientation and proximity-dependent manner, which is absent from a single oscillating microbubble of comparable size and at the same stand-off distance.

Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.

Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.

Magnetic Resonance Imaging Biomarkers of Renal Structure and Function

The kidney's major role in filtration depends on its high blood flow, concentrating mechanisms, and biochemical activation. The kidney's greatest strengths also lead to vulnerability for drug-induced nephrotoxicity and other renal injuries. The current standard to diagnose renal injuries is with a percutaneous renal biopsy, which can be biased and insufficient. In one particular case, biopsy of a kidney with renal cell carcinoma can actually initiate metastasis. Tools that are sensitive and specific to detect renal disease early are essential, especially noninvasive diagnostic imaging. While other imaging modalities (ultrasound and x-ray/CT) have their unique advantages and disadvantages, MRI has superb soft tissue contrast without ionizing radiation. More importantly, there is a richness of contrast mechanisms in MRI that has yet to be explored and applied to study renal disease.

The focus of this work is to advance preclinical imaging tools to study the structure and function of the renal system. Studies were conducted in normal and disease models to understand general renal physiology as well as pathophysiology. This dissertation is separated into two parts--the first is the identification of renal architecture with ex vivo MRI; the second is the characterization of renal dynamics and function with in vivo MRI. High resolution ex vivo imaging provided several opportunities including: 1) identification of fine renal structures, 2) implementation of different contrast mechanisms with several pulse sequences and reconstruction methods, 3) development of image-processing tools to extract regions and structures, and 4) understanding of the nephron structures that create MR contrast and that are important for renal physiology. The ex vivo studies allowed for understanding and translation to in vivo studies. While the structure of this dissertation is organized by individual projects, the goal is singular: to develop magnetic resonance imaging biomarkers for renal system.

The work presented here includes three ex vivo studies and two in vivo studies:

1) Magnetic resonance histology of age-related nephropathy in sprague dawley.

2) Quantitative susceptibility mapping of kidney inflammation and fibrosis in type 1 angiotensin receptor-deficient mice.

3) Susceptibility tensor imaging of the kidney and its microstructural underpinnings.

4) 4D MRI of renal function in the developing mouse.

5) 4D MRI of polycystic kidneys in rapamycin treated Glis3-deficient mice.

Imaging Learned Song Representations in Populations of Sensorimotor Neurons Essential to Vocal Communication

Perceiving or producing complex vocalizations such as speech and birdsongs require the coordinated activity of neuronal populations, and these activity patterns can vary over space and time. How learned communication signals are represented by populations of sensorimotor neurons essential to vocal perception and production remains poorly understood. Using a combination of two-photon calcium imaging, intracellular electrophysiological recording and retrograde tracing methods in anesthetized adult male zebra finches (Taeniopygia guttata), I addressed how the bird's own song and its component syllables are represented by the spatiotemporal patterns of activity of two spatially intermingled populations of projection neurons (PNs) in HVC, a sensorimotor area required for song perception and production. These experiments revealed that neighboring PNs can respond at markedly different times to song playback and that different syllables activate spatially intermingled HVC PNs within a small region. Moreover, noise correlation analysis reveals enhanced functional connectivity between PNs that respond most strongly to the same syllable and also provides evidence of a spatial gradient of functional connectivity specific to PNs that project to song motor nucleus (i.e. HVC_RA cells). These findings support a model in which syllabic and temporal features of song are represented by spatially intermingled PNs functionally organized into cell- and syllable-type networks.

An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

The Macrophage-Specific Promoter mfap4 Allows Live, Long-Term Analysis of Macrophage Behavior during Mycobacterial Infection in Zebrafish.

Transgenic labeling of innate immune cell lineages within the larval zebrafish allows for real-time, in vivo analyses of microbial pathogenesis within a vertebrate host. To date, labeling of zebrafish macrophages has been relatively limited, with the most specific expression coming from the mpeg1 promoter. However, mpeg1 transcription at both endogenous and transgenic loci becomes attenuated in the presence of intracellular pathogens, including Salmonella typhimurium and Mycobacterium marinum. Here, we describe mfap4 as a macrophage-specific promoter capable of producing transgenic lines in which transgene expression within larval macrophages remains stable throughout several days of infection. Additionally, we have developed a novel macrophage-specific Cre transgenic line under the control of mfap4, enabling macrophage-specific expression using existing floxed transgenic lines. These tools enrich the repertoire of transgenic lines and promoters available for studying zebrafish macrophage dynamics during infection and inflammation and add flexibility to the design of future macrophage-specific transgenic lines.