A5011932661- Cellular transport and secretion
- Advanced Fluorescence Microscopy Techniques
- Lipid Membrane Structure and Behavior
- Advanced Electron Microscopy Techniques and Applications
- Advanced biosensing and bioanalysis techniques
- Cell Image Analysis Techniques
- Genetic and Kidney Cyst Diseases
- Cell Adhesion Molecules Research
- RNA Interference and Gene Delivery
- Retinal Development and Disorders
- Cellular Mechanics and Interactions
- Biochemical and Structural Characterization
- Photoreceptor and optogenetics research
- Angiogenesis and VEGF in Cancer
- Endoplasmic Reticulum Stress and Disease
- Plant Reproductive Biology
- Erythrocyte Function and Pathophysiology
- Signaling Pathways in Disease
- Fungal and yeast genetics research
- Pancreatic function and diabetes
- Renal and related cancers
- Protist diversity and phylogeny
- Lymphatic System and Diseases
- Near-Field Optical Microscopy
- Calcium signaling and nucleotide metabolism
Yale University
2015-2024
ETH Zurich
2016
University of Puerto Rico, Medical Sciences Campus
2008
University of Puerto Rico System
2005
Fluorescence nanoscopy, or super-resolution microscopy, has become an important tool in cell biological research. However, because of its usually inferior resolution the depth direction (50–80 nm) and rapidly deteriorating thick samples, practical application been effectively limited to two dimensions thin samples. Here, we present development whole-cell 4Pi single-molecule switching nanoscopy (W-4PiSMSN), optical nanoscope that allows imaging three-dimensional (3D) structures at 10- 20-nm...
Neuropilin 1 (NRP1) plays an important but ill-defined role in VEGF-A signaling and vascular morphogenesis. We show that mice with a knockin mutation ablates the NRP1 cytoplasmic tail (Nrp1cyto) have normal angiogenesis impaired developmental adult arteriogenesis. The arteriogenic defect was traced to absence of PDZ-dependent interaction between VEGF receptor 2 (VEGFR2) complex synectin, which delayed trafficking endocytosed VEGFR2 from Rab5+ EAA1+ endosomes. This led increased PTPN1...
Membrane traffic along the endocytic and exocytic pathways relies on appropriate localization activation of a series different Rab GTPases. Rabs are activated by specific guanine nucleotide exchange factors (GEFs) inactivated GTPase-activating proteins (GAPs). GEF cascades, in which one its GTP-bound form recruits that activates next pathway, can account for sequential Rabs, but it does not explain how first is after has been activated. We present evidence counter-current GAP cascade serves...
Abstract We report a lipid‐based strategy to visualize Golgi structure and dynamics at super‐resolution in live cells. The method is based on two novel reagents: trans ‐cyclooctene‐containing ceramide lipid (Cer‐TCO) highly reactive, tetrazine‐tagged near‐IR dye (SiR‐Tz). These reagents assemble via an extremely rapid “tetrazine‐click” reaction into Cer‐SiR, photostable “vital dye” that enables prolonged live‐cell imaging of the apparatus by 3D confocal STED microscopy. Cer‐SiR nontoxic...
Significance The biochemical reactions that drive cellular life are housed in distinct membrane enclosed compartments known as organelles. Whereas proteins targeting to different organelles well developed, little is regarding how lipids sorted We engineered a protein from marine organism into fluorescent “biosensor” of sphingomyelin (SM), sphingolipid produced the Golgi apparatus but major component plasma membrane. By monitoring SM dynamics live cells, we discovered transported its site...
Insulin is synthesized by pancreatic β-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus deliver insulin bloodstream. The mechanism of how proinsulin its processing enzymes are sorted targeted from trans-Golgi network (TGN) remains mysterious. No cargo receptor for has been identified. Here, we show that chromogranin (CG) proteins undergo liquid–liquid phase separation (LLPS) at mildly acidic pH lumen TGN, recruit clients like...
Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of small GTPase ARF1 in mediating transport steps at Golgi. Besides its well-established generating COPI vesicles, find that is also involved formation long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde retrograde carriers are both largely free classical Golgi coat proteins coatomer (COPI) clathrin. Instead, they contain along their entire length a density estimated to...
Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves extracorporeal exposure white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in context ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, therapeutic activity has been attributed selective cytotoxicity on circulating CTCL cells. However, only fraction WBCs exposed ECP, and 8-MOP inactive absence UVA light, implying that other...
Platelet-monocyte “adhesion synapse” initiates physiologic biogenesis of cross-presenting dendritic cells.
Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide–sensitive fusion attachment protein receptors (SNAREs), spatiotemporal dynamics tethers relative to are poorly characterized. The most extensively studied tethering complex is exocyst, which spatially targets vesicles sites on plasma membrane. By using a mammalian genetic replacement strategy, we were able assemble fluorescently tagged Sec8 into exocyst...
Integrins are abundant heterodimeric cell-surface adhesion receptors essential in multicellular organisms. Integrin function is dynamically modulated by endo-exocytic trafficking, however, major mysteries remain about where, when, and how this occurs living cells. To address this, here we report the generation of functional recombinant β1 integrins with traceable tags inserted an extracellular loop. We demonstrate that these 'ecto-tagged' expressed, localize to adhesions, exhibit normal...
As a master regulator of endothelial cell function, vascular growth factor receptor-2 (VEGFR2) activates multiple downstream signaling pathways that are critical for development and normal vessel function. VEGFR2 trafficking through various endosomal compartments modulates its output. Accordingly, proteins regulate the speed direction by which traffics endosomes have been demonstrated to be particularly important arteriogenesis. However, little is known about how these control implications...
Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes plasma membrane probe DiI-TCO photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within to generate HIDE DiI-SiR. Using DiI-SiR, visualized filopodia dynamics in HeLa 25 min at 0.5 s resolution, dynamic contact-mediated repulsion events...
Nearly all cells have a primary cilia on their surface, which functions as cellular antennae. Primary assembly begins intracellularly and eventually emerges extracellularly. However, current ciliogenesis assays, detect length number, do not monitor ciliary stages. We developed new assay that detects antibody access to fluorescently tagged transmembrane protein, revealed three states: classified 'inside,' 'outside,' or 'partial' cilia. Strikingly, most in RPE only partially emerged many...
Abstract Performing multi-color nanoscopy for extended times is challenging due to the rapid photobleaching rate of most fluorophores. Here we describe a new fluorophore (Yale-595) and bio-orthogonal labeling strategy that enables two-color super-resolution (STED) 3D confocal imaging two organelles simultaneously using high-density environmentally sensitive (HIDE) probes. Because HIDE probes are small, cell-permeant molecules, they can visualize dual organelle dynamics in hard-to-transfect...
Abstract Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles their target membranes. However, interplay between tethering and has remained enigmatic. Here, through optogenetic control either a natural tether—the exocyst complex—or an artificial tether, we report that regulates mode fusion. We find mainly undergo kiss-and-run instead full in absence functional exocyst. Full is rescued optogenetically restoring function, manner likely dependent...