The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ.

The endoplasmic reticulum's (ER's) structure is directly linked to the many functions of the ER, but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects the local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule, increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4.

The nanoscale organization of reticulon 4 shapes local endoplasmic reticulum structure in situ.

The endoplasmic reticulum’s (ER) structure is directly linked to the many functions of the ER but its formation is not fully understood. We investigate how the ER-membrane curving protein reticulon 4 (Rtn4) localizes to and organizes in the membrane and how that affects local ER structure. We show a strong correlation between the local Rtn4 density and the local ER membrane curvature. Our data further reveal that the typical ER tubule possesses an elliptical cross-section with Rtn4 enriched at either end of the major axis. Rtn4 oligomers are linear-shaped, contain about five copies of the protein, and preferentially orient parallel to the tubule axis. Our observations support a mechanism in which oligomerization leads to an increase of the local Rtn4 concentration with each molecule increasing membrane curvature through a hairpin wedging mechanism. This quantitative analysis of Rtn4 and its effects on the ER membrane result in a new model of tubule shape as it relates to Rtn4. Summary: Rtn4 forms linear-shaped oligomers that contain an average of five Rtn4 proteins, localize to the sides of elliptical tubules, prefer orientations near parallel to the tubule axis, and increase local curvature of the ER membrane by increasing local Rtn4 density.

Bioorthogonal, Fluorogenic Targeting of Voltage-Sensitive Fluorophores for Visualizing Membrane Potential Dynamics in Cellular Organelles.

Electrical potential differences across lipid bilayers play foundational roles in cellular physiology. Plasma membrane voltage is the most widely studied; however, the bilayers of organelles like mitochondria, lysosomes, nuclei, and the endoplasmic reticulum (ER) also provide opportunities for ionic compartmentalization and the generation of transmembrane potentials. Unlike plasma membranes, organellar bilayers, cloistered within the cell, remain recalcitrant to traditional approaches like patch-clamp electrophysiology. To address the challenge of monitoring changes in organelle membrane potential, we describe the design, synthesis, and application of the LUnAR RhoVR (Ligation Unquenched for Activation and Redistribution Rhodamine-based Voltage Reporter) for optically monitoring membrane potential changes in the ER of living cells. We pair a tetrazine-quenched RhoVR for voltage sensing with a transcyclooctene (TCO)-conjugated ceramide (Cer-TCO) for targeting to the ER. Bright fluorescence is observed only at the coincidence of the LUnAR RhoVR and TCO in the ER, minimizing non-specific, off-target fluorescence. We show that the product of the LUnAR RhoVR and Cer-TCO is voltage-sensitive and that the LUnAR RhoVR can be targeted to an intact ER in living cells. Using the LUnAR RhoVR, we use two-color, ER-localized, fast voltage imaging coupled with cytosolic Ca2+ imaging to validate the electroneutrality of Ca2+ release from internal stores. Finally, we use the LUnAR RhoVR to directly visualize functional coupling between the plasma-ER membranes in patch clamped cell lines, providing the first direct evidence of the sign of the ER potential response to plasma membrane potential changes. We envision that the LUnAR RhoVR, along with other existing organelle-targeting TCO probes, could be applied widely for exploring organelle physiology.

Extremely Bright, Near-IR Emitting Spontaneously Blinking Fluorophores Enable Ratiometric Multicolor Nanoscopy in Live Cells.

New bright, photostable, emission-orthogonal fluorophores that blink without toxic additives are needed to enable multicolor, live-cell, single-molecule localization microscopy (SMLM). Here we report the design, synthesis, and biological evaluation of Yale676sb, a photostable, near-IR-emitting fluorophore that achieves these goals in the context of an exceptional quantum yield (0.59). When used alongside HMSiR, Yale676sb enables simultaneous, live-cell, two-color SMLM of two intracellular organelles (ER + mitochondria) with only a single laser and no chemical additives.

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. The extended time domain possible using these tools reveals dynamic nanoscale targeting between different organelles.

Effectiveness of participatory ergonomic interventions on health outcomes: a systematic review.

The objective of this study was to conduct a systematic review of the literature on the effectiveness of participatory ergonomic (PE) interventions for improving workers' health. The search strategy targeted six electronic databases and identified 442 potential articles. Each article was examined by pairs of reviewers for relevance (assessed a participative ergonomic workplace intervention, with at least one health outcome, published in English in peer reviewed literature). Twenty-three articles met relevance criteria and were then appraised for methodological strength. Using a best evidence synthesis approach, 12 studies that were rated as 'medium' or higher provided partial to moderate evidence that PE interventions have a positive impact on: musculoskeletal symptoms, reducing injuries and workers' compensation claims, and a reduction in lost days from work or sickness absence. However, the magnitude of the effect requires more precise definition.