Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1.

Synaptotagmin-1 (Syt1) is a major calcium sensor for rapid neurotransmitter release in neurons and hormone release in many neuroendocrine cells. It possesses two tandem cytosolic C2 domains that bind calcium, negatively charged phospholipids, and the neuronal SNARE complex. Calcium binding to Syt1 triggers exocytosis, but how this occurs is not well understood.

CALHM2 is a mitochondrial protein import channel that regulates fatty acid metabolism.

For mitochondrial metabolism to occur in the matrix, multiple proteins must be imported across the two (inner and outer) mitochondrial membranes. Classically, two protein import channels, TIM/TOM, are known to perform this function, but whether other protein import channels exist is not known. Here, using super-resolution microscopy, proteomics, and electrophysiological techniques, we identify CALHM2 as the import channel for the ECHA subunit of the mitochondrial trifunctional protein (mTFP), which catalyzes β-oxidation of fatty acids in the mitochondrial matrix.

Unraveling cellular complexity with transient adapters in highly multiplexed super-resolution imaging.

Mapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput.

An integrated platform for high-throughput nanoscopy.

Single-molecule localization microscopy enables three-dimensional fluorescence imaging at tens-of-nanometer resolution, but requires many camera frames to reconstruct a super-resolved image. This limits the typical throughput to tens of cells per day. While frame rates can now be increased by over an order of magnitude, the large data volumes become limiting in existing workflows.

Use of Ecto-Tagged Integrins to Monitor Integrin Exocytosis and Endocytosis.

Controlled exocytosis and endocytosis of integrin adhesion receptors is required for normal cell adhesion, migration, and signaling. In this chapter, we describe the design of functional β1 integrins carrying extracellular fluorescent or chemically traceable tags (ecto-tag) and methods for their use to image β1 integrin trafficking in cells. We provide approaches to generate cells in which endogenous β1 integrins are replaced by ecto-tagged integrins containing a pH-sensitive fluorophore pHluorin or a HaloTag and describe strategies using photobleaching, selective extracellular/intracellular labeling, and chase, quenching, and blocking to reveal β1 integrin exocytosis, endocytosis, and recycling by live total internal reflection fluorescence (TIRF) microscopy.

Endothelial β-arrestins regulate mechanotransduction by the type II bone morphogenetic protein receptor in primary cilia.

Modulation of endothelial cell behavior and phenotype by hemodynamic forces involves many signaling components, including cell surface receptors, intracellular signaling intermediaries, transcription factors, and epigenetic elements. Many of the signaling mechanisms that underlie mechanotransduction by endothelial cells are inadequately defined. Here we sought to better understand how β-arrestins, intracellular proteins that regulate agonist-mediated desensitization and integration of signaling by transmembrane receptors, may be involved in the endothelial cell response to shear stress.

Fibroblasts secrete fibronectin under lamellipodia in a microtubule- and myosin II-dependent fashion.

Fibronectin (FN) is an essential structural and regulatory component of the extracellular matrix (ECM), and its binding to integrin receptors supports cell adhesion, migration, and signaling. Here, using live-cell microscopy of fibroblasts expressing FN tagged with a pH-sensitive fluorophore, we show that FN is secreted predominantly at the ventral surface of cells in an integrin-independent manner. Locally secreted FN then undergoes β1 integrin-dependent fibrillogenesis.

Syndecan-2 selectively regulates VEGF-induced vascular permeability.

Vascular endothelial growth factor (VEGF)- driven increase in vascular permeability is a key feature of many disease states associated with inflammation and ischemic injury, contributing significantly to morbidity and mortality in these settings. Despite its importance, no specific regulators that preferentially control VEGF-dependent increase in permeability versus its other biological activities, have been identified. Here we report that a proteoglycan Syndecan-2 (Sdc2) regulates the interaction between a transmembrane phosphatase DEP1 and VEGFR2 by controlling cell surface levels of DEP1.

Liquid-liquid phase separation facilitates the biogenesis of secretory storage granules.

Insulin is synthesized by pancreatic β-cells and stored into secretory granules (SGs). SGs fuse with the plasma membrane in response to a stimulus and deliver insulin to the bloodstream. The mechanism of how proinsulin and its processing enzymes are sorted and targeted from the trans-Golgi network (TGN) to SGs remains mysterious.

Multimodal imaging of synaptic vesicles with a single probe.

A complete understanding of synaptic-vesicle recycling requires the use of multiple microscopy methods to obtain complementary information. However, many currently available probes are limited to a specific microscopy modality, which necessitates the use of multiple probes and labeling paradigms. Given the complexity of vesicle populations and recycling pathways, having new single-vesicle probes that could be used for multiple microscopy techniques would complement existing sets of tools for studying vesicle function.

Exocyst complex mediates recycling of internal cilia.

Primary cilia are slender, cellular antennae that sense extracellular stimuli, and their absence or dysfunction plays a role in numerous human diseases. Prior work has indicated a role of the exocyst tethering complex in cilia biogenesis and maintenance, with the underlying paradigm that the exocyst targets vesicles to the ciliary base to deliver ciliary cargoes. However, the role of the exocyst vis-à-vis to primary cilia in living cells and during stimulation is unknown.

An active tethering mechanism controls the fate of vesicles.

Vesicle tethers are thought to underpin the efficiency of intracellular fusion by bridging vesicles to their target membranes. However, the interplay between tethering and fusion has remained enigmatic. Here, through optogenetic control of either a natural tether-the exocyst complex-or an artificial tether, we report that tethering regulates the mode of fusion.

Two-color nanoscopy of organelles for extended times with HIDE probes.

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 a bio-orthogonal labeling strategy that enables two-color super-resolution (STED) and 3D confocal imaging of two organelles simultaneously for extended times 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 cell lines by super-resolution for over an order of magnitude longer than with tagged proteins.

Platelet P-selectin initiates cross-presentation and dendritic cell differentiation in blood monocytes.

Dendritic cells (DCs) are adept at cross-presentation and initiation of antigen-specific immunity. Clinically, however, DCs produced by in vitro differentiation of monocytes in the presence of exogenous cytokines have been met with limited success. We hypothesized that DCs produced in a physiological manner may be more effective and found that platelets activate a cross-presentation program in peripheral blood monocytes with rapid (18 hours) maturation into physiological DCs (phDCs).

Endosome motility defects revealed at super-resolution in live cells using HIDE probes.

We report new lipid-based, high-density, environmentally sensitive (HIDE) probes that accurately and selectively image endo-lysosomes and their dynamics at super-resolution for extended times. Treatment of live cells with the small molecules DiICTCO or DiICTCO followed by in situ tetrazine ligation reaction with the silicon-rhodamine dye SiR-Tz generates the HIDE probes DiIC-SiR and DiIC-SiR in the endo-lysosomal membrane. These new probes support the acquisition of super-resolution videos of organelle dynamics in primary cells for more than 7 min with no detectable change in endosome structure or function.

Extracorporeal photochemotherapy induces bona fide immunogenic cell death.

Extracorporeal photochemotherapy (ECP) is employed for the management of cutaneous T cell lymphoma (CTCL). ECP involves the extracorporeal exposure of white blood cells (WBCs) to a photosensitizer, 8-methoxypsoralen (8-MOP), in the context of ultraviolet A (UVA) radiation, followed by WBC reinfusion. Historically, the therapeutic activity of ECP has been attributed to selective cytotoxicity on circulating CTCL cells.

Seeing the long tail: A novel green fluorescent protein, SiriusGFP, for ultra long timelapse imaging.

Background: Fluorescent proteins (FPs) have widespread uses in cell biology. However, the practical applications of FPs are significantly limited due to their rapid photobleaching and misfolding when fused to target proteins.

New Method: Using a combination of novel and known mutations to eGFP, we developed a well folded and very photostable variant, SiriusGFP.

S-Palmitoylation Sorts Membrane Cargo for Anterograde Transport in the Golgi.

While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted, and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by S-palmitoylation.

The Rab-effector protein RABEP2 regulates endosomal trafficking to mediate vascular endothelial growth factor receptor-2 (VEGFR2)-dependent signaling.

As a master regulator of endothelial cell function, vascular endothelial growth factor receptor-2 (VEGFR2) activates multiple downstream signaling pathways that are critical for vascular development and normal vessel function. VEGFR2 trafficking through various endosomal compartments modulates its signaling output. Accordingly, proteins that regulate the speed and direction by which VEGFR2 traffics through endosomes have been demonstrated to be particularly important for arteriogenesis.

Novel ecto-tagged integrins reveal their trafficking in live cells.

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 in living cells. To address this, here we report the generation of functional recombinant β1 integrins with traceable tags inserted in an extracellular loop.

Long-Term Live-Cell STED Nanoscopy of Primary and Cultured Cells with the Plasma Membrane HIDE Probe DiI-SiR.

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 of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR.

Long time-lapse nanoscopy with spontaneously blinking membrane probes.

Imaging cellular structures and organelles in living cells by long time-lapse super-resolution microscopy is challenging, as it requires dense labeling, bright and highly photostable dyes, and non-toxic conditions. We introduce a set of high-density, environment-sensitive (HIDE) membrane probes, based on the membrane-permeable silicon-rhodamine dye HMSiR, that assemble in situ and enable long time-lapse, live-cell nanoscopy of discrete cellular structures and organelles with high spatiotemporal resolution. HIDE-enabled nanoscopy movies span tens of minutes, whereas movies obtained with labeled proteins span tens of seconds.

A novel physiological role for ARF1 in the formation of bidirectional tubules from the Golgi.

Capitalizing on CRISPR/Cas9 gene-editing techniques and super-resolution nanoscopy, we explore the role of the small GTPase ARF1 in mediating transport steps at the Golgi. Besides its well-established role in generating COPI vesicles, we find that ARF1 is also involved in the formation of long (∼3 µm), thin (∼110 nm diameter) tubular carriers. The anterograde and retrograde tubular carriers are both largely free of the classical Golgi coat proteins coatomer (COPI) and clathrin.

Leptin Is Produced by Parathyroid Glands and Stimulates Parathyroid Hormone Secretion.

Objective: We asked if leptin and its cognate receptor were present in normal and diseased parathyroid glands, and if so, whether they had any functional effects on parathyroid hormone (PTH) secretion in parathyroid neoplasms.

Background: The parathyroid glands acting through PTH play a critical role in the regulation of serum calcium. Based on leptin's recently discovered role in bone metabolism, we hypothesized these glands were the sites of a functional interaction between these 2 hormones.