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The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.
© 2018, Fenix et al.

Thallium (Tl+) flux assays enable imaging of potassium (K+) channel activity in cells and tissues by exploiting the permeability of K+ channels to Tl+ coupled with a fluorescent Tl+ sensitive dye. Common Tl+ sensing dyes utilize fluorescein as the fluorophore though fluorescein exhibits certain undesirable properties in these assays including short excitation wavelengths and pH sensitivity. To overcome these drawbacks, the replacement of fluorescein with rhodols was investigated. A library of 13 rhodol-based Tl+ sensors was synthesized and their properties and performance in Tl+ flux assays evaluated. The dimethyl rhodol Tl+ sensor emerged as the best of the series and performed comparably to fluorescein-based sensors while demonstrating greater pH tolerance in the physiological range and excitation and emission spectra 30 nm red-shifted from fluorescein.

This study reports the synthesis and testing of a family of rhodamine pro-fluorophores and an enzyme capable of converting pro-fluorophores to Rhodamine 110. We prepared a library of simple N,N'-diacyl rhodamines and investigated porcine liver esterase (PLE) as an enzyme to activate rhodamine-based pro-fluorophores. A PLE-expressing cell line generated an increase in fluorescence rapidly upon pro-fluorophore addition demonstrating the rhodamine pro-fluorophores are readily taken up and fluorescent upon PLE-mediated release. Rhodamine pro-fluorophore amides trifluoroacetamide (TFAm) and proponamide (PAm) appeared to be the best substrates using a cell-based assay using PLE expressing HEK293. Our pro-fluorophore series showed diffusion into live cells and resisted endogenous hydrolysis. The use of our engineered cell line containing the exogenous enzyme PLE demonstrated the rigorousness of amide masking when compared to cells not containing PLE. This simple and selective pro-fluorophore rhodamine pair with PLE offers the potential to be used in vitro and in vivo fluorescence based assays.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purpose - Mammalian central nervous system axons fail to regenerate after injury. Contributing factors include limited intrinsic growth capacity and an inhibitory glial environment. Inflammation-induced optic nerve regeneration (IIR) is thought to boost retinal ganglion cell (RGC) intrinsic growth capacity through progrowth gene expression, but effects on the inhibitory glial environment of the optic nerve are unexplored. To investigate progrowth molecular changes associated with reactive gliosis during IIR, we developed an imaging mass spectrometry (IMS)-based approach that identifies discriminant molecular signals in and around optic nerve crush (ONC) sites.
Methods - ONC was performed in rats, and IIR was established by intravitreal injection of a yeast cell wall preparation. Optic nerves were collected at various postcrush intervals, and longitudinal sections were analyzed with matrix-assisted laser desorption/ionization (MALDI) IMS and data mining. Immunohistochemistry and confocal microscopy were used to compare discriminant molecular features with cellular features of reactive gliosis.
Results - IIR increased the area of the crush site that was occupied by a dense cellular infiltrate and mass spectral features consistent with lysosome-specific lipids. IIR also increased immunohistochemical labeling for microglia and macrophages. IIR enhanced clearance of lipid sulfatide myelin-associated inhibitors of axon growth and accumulation of simple GM3 gangliosides in a spatial distribution consistent with degradation of plasma membrane from degenerated axons.
Conclusions - IIR promotes a robust phagocytic response that improves clearance of myelin and axon debris. This growth-permissive molecular remodeling of the crush injury site extends our current understanding of IIR to include mechanisms extrinsic to the RGC.

To better understand the roles of microRNAs in glial function, we used a conditional deletion of Dicer1 (Dicer-CKO) in retinal Müller glia (MG). Dicer1 deletion from the MG leads to an abnormal migration of the cells as early as 1 month after the deletion. By 6 months after Dicer1 deletion, the MG form large aggregations and severely disrupt normal retinal architecture and function. The most highly upregulated gene in the Dicer-CKO MG is the proteoglycan Brevican (Bcan) and overexpression of Bcan results in similar aggregations of the MG in wild-type retina. One potential microRNA that regulates Bcan is miR-9, and overexpression of miR-9 can partly rescue the effects of Dicer1 deletion on the MG phenotype. We also find that MG from retinitis pigmentosa patients display an increase in Brevican immunoreactivity at sites of MG aggregation, linking the retinal remodeling that occurs in chronic disease with microRNAs.

β-Arrestins are key regulators and signal transducers of G protein-coupled receptors (GPCRs). The interaction between receptors and β-arrestins is generally believed to require both receptor activity and phosphorylation by GPCR kinases. In this study, we investigated whether β-arrestins are able to bind second messenger kinase-phosphorylated, but inactive receptors as well. Because heterologous phosphorylation is a common phenomenon among GPCRs, this mode of β-arrestin activation may represent a novel mechanism of signal transduction and receptor cross-talk. Here we demonstrate that activation of protein kinase C (PKC) by phorbol myristate acetate, G-coupled GPCR, or epidermal growth factor receptor stimulation promotes β-arrestin2 recruitment to unliganded AT angiotensin receptor (ATR). We found that this interaction depends on the stability lock, a structure responsible for the sustained binding between GPCRs and β-arrestins, formed by phosphorylated serine-threonine clusters in the receptor's C terminus and two conserved phosphate-binding lysines in the β-arrestin2 N-domain. Using improved FlAsH-based serine-threonine clusters β-arrestin2 conformational biosensors, we also show that the stability lock not only stabilizes the receptor-β-arrestin interaction, but also governs the structural rearrangements within β-arrestins. Furthermore, we found that β-arrestin2 binds to PKC-phosphorylated ATR in a distinct active conformation, which triggers MAPK recruitment and receptor internalization. Our results provide new insights into the activation of β-arrestins and reveal their novel role in receptor cross-talk.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Epithelial wound healing is an evolutionarily conserved process that requires coordination across a field of cells. Studies in many organisms have shown that cytosolic calcium levels rise within a field of cells around the wound and spread to neighboring cells, within seconds of wounding. Although calcium is a known potent second messenger and master regulator of wound-healing programs, it is unknown what initiates the rise of cytosolic calcium across the wound field. Here we use laser ablation, a commonly used technique for the precision removal of cells or subcellular components, as a tool to investigate mechanisms of calcium entry upon wounding. Despite its precise ablation capabilities, we find that this technique damages cells outside the primary wound via a laser-induced cavitation bubble, which forms and collapses within microseconds of ablation. This cavitation bubble damages the plasma membranes of cells it contacts, tens of microns away from the wound, allowing direct calcium entry from extracellular fluid into damaged cells. Approximately 45 s after this rapid influx of calcium, we observe a second influx of calcium that spreads to neighboring cells beyond the footprint of cavitation. The occurrence of this second, delayed calcium expansion event is predicted by wound size, indicating that a separate mechanism of calcium entry exists, corresponding to cell loss at the primary wound. Our research demonstrates that the damage profile of laser ablation is more similar to a crush injury than the precision removal of individual cells. The generation of membrane microtears upon ablation is consistent with studies in the field of optoporation, which investigate ablation-induced cellular permeability. We conclude that multiple types of damage, including microtears and cell loss, result in multiple mechanisms of calcium influx around epithelial wounds.
Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

High-resolution microscopy has traditionally come at the expense of field of view, resulting in suboptimal interpretation of protein distribution throughout large or complex samples. Likewise, a low-resolution microscopic approach inhibits the ability of researchers to precisely localize proteins of interest at the subcellular level. Until recently, the ability to combine the strengths of these approaches was limited and technically impractical for most laboratories to implement. Continued advances in microscope automation, sophisticated software applications, and modern workstations have enabled expansion of such combinatorial approaches to researchers outside computationally focused fields. Through image stitching, researchers can acquire large field-of-view, multidimensional datasets, at the diffraction limit of high-numerical aperture objectives to effectively map protein distribution in large samples with high precision. Here, we outline a protocol for acquisition of such datasets with the purpose of introducing inexperienced researchers to the methodology of large image stitching using the widely available technology of laser point-scanning confocal microscopy in combination with basic microscope automation and freely available software for post-acquisition processing.

Purpose - Prominin-1 (Prom1) is a transmembrane glycoprotein, which is expressed in stem cell lineages, and has recently been implicated in cancer stem cell survival. Mutations in the Prom1 gene have been shown to disrupt photoreceptor disk morphogenesis and cause an autosomal dominant form of Stargardt-like macular dystrophy (STGD4). Despite the apparent structural role of Prom1 in photoreceptors, its role in other cells of the retina is unknown. The purpose of this study is to investigate the role of Prom1 in the highly metabolically active cells of the retinal pigment epithelium (RPE).
Methods - Lentiviral siRNA and the genome editing CRISPR/Cas9 system were used to knockout Prom1 in primary RPE and ARPE-19 cells, respectively. Western blotting, confocal microscopy, and flow sight imaging cytometry assays were used to quantify autophagy flux. Immunoprecipitation was used to detect Prom1 interacting proteins.
Results - Our studies demonstrate that Prom1 is primarily a cytosolic protein in the RPE. Stress signals and physiological aging robustly increase autophagy with concomitant upregulation of Prom1 expression. Knockout of Prom1 increased mTORC1 and mTORC2 signaling, decreased autophagosome trafficking to the lysosome, increased p62 accumulation, and inhibited autophagic puncta induced by activators of autophagy. Conversely, ectopic overexpression of Prom1 inhibited mTORC1 and mTORC2 activities, and potentiated autophagy flux. Through interactions with p62 and HDAC6, Prom1 regulates autophagosome maturation and trafficking, suggesting a new cytoplasmic role of Prom1 in RPE function.
Conclusions - Our results demonstrate that Prom1 plays a key role in the regulation of autophagy via upstream suppression of mTOR signaling and also acting as a component of a macromolecular scaffold involving p62 and HDAC6.

Translocator protein (TSPO) is a validated target for molecular imaging of a variety of human diseases and disorders. Given its involvement in cholesterol metabolism, TSPO expression is commonly elevated in solid tumors, including glioma, colorectal cancer, and breast cancer. TSPO ligands capable of detection by optical imaging are useful molecular tracers for a variety of purposes that range from quantitative biology to drug discovery. Leveraging our prior optimization of the pyrazolopyrimidine TSPO ligand scaffold for cancer imaging, we report herein a new generation of TSPO tracers with superior binding affinity and suitability for optical imaging and screening. In total, seven candidate TSPO tracers were synthesized and vetted in this study; the most promising tracer identified (29, K = 0.19 nM) was the result of conjugating a high-affinity TSPO ligand to a fluorophore used routinely in biological sciences (FITC) via a functional carbon linker of optimal length. Computational modeling suggested that an n-alkyl linker of eight carbons in length allows for positioning of the bulky fluorophore distal to the ligand binding domain and toward the solvent interface, minimizing potential ligand-protein interference. Probe 29 was found to be highly suitable for in vitro imaging of live TSPO-expressing cells and could be deployed as a ligand screening and discovery tool. Competitive inhibition of probe 29 quantified by fluorescence and H-PK11195 quantified by traditional radiometric detection resulted in equivalent affinity data for two previously reported TSPO ligands. This study introduces the utility of TSPO ligand 29 for in vitro imaging and screening and provides a structural basis for the development of future TSPO imaging ligands bearing bulky signaling moieties.