Biology subjects

Roy, A. R.

Publications and source records attributed to Roy, A. R..

4 recordsLinked to original sources

bioRxiv2023

Nanoscale cellular organization of viral RNA and proteins in SARS-CoV-2 replication organelles

The SARS-CoV-2 viral infection transforms host cells and produces special organelles in many ways, and we focus on the replication organelle where the replication of viral genomic RNA (vgRNA) occurs. To date, the precise cellular localization of key RNA molecules and replication intermediates has been elusive in electron microscopy studies. We use super-resolution fluorescence microscopy and specific labeling to reveal the nanoscopic organization of replication organelles that contain vgRNA clusters along with viral double-stranded RNA (dsRNA) clusters and the replication enzyme, encapsulated by membranes derived from the host endoplasmic reticulum (ER). We show that the replication organelles are organized differently at early and late stages of infection. Surprisingly, vgRNA accumulates into distinct globular clusters in the cytoplasmic perinuclear region, which grow and accommodate more vgRNA molecules as infection time increases. The localization of ER labels and nsp3 (a component of the double-membrane vesicle, DMV) at the periphery of the vgRNA clusters suggests that replication organelles are enclosed by DMVs at early infection stages which then merge into vesicle packets as infection progresses. Precise co-imaging of the nanoscale cellular organization of vgRNA, dsRNA, and viral proteins in replication organelles of SARS-CoV-2 may inform therapeutic approaches that target viral replication and associated processes.

microbiology↗

bioRxiv2023

Curved adhesions mediate cell attachment to soft matrix fibres in 3D

Mammalian cells adhere to the extracellular matrix (ECM) and sense mechanical cues through integrin-mediated adhesions1, 2. Focal adhesions and related structures are the primary architectures that transmit forces between the ECM and the actin cytoskeleton. Although focal adhesions are abundant when cells are cultured on rigid substrates, they are sparse in soft environments that cannot support high mechanical tensions3. Here, we report a new class of integrin-mediated adhesions, curved adhesions, whose formation is regulated by membrane curvature instead of mechanical tension. In soft matrices made of protein fibres, curved adhesions are induced by membrane curvatures imposed by the fibre geometry. Curved adhesions are mediated by integrin [a]V{beta}5 and are molecularly distinct from focal adhesions and clathrin lattices. The molecular mechanism involves a previously unknown interaction between integrin {beta}5 and a curvature-sensing protein FCHo2. We find that curved adhesions are prevalent in physiologically relevant environments. Disruption of curved adhesions by knocking down integrin {beta}5 or FCHo2 abolishes the migration of multiple cancer cell lines in 3D matrices. These findings provide a mechanism of cell anchorage to natural protein fibres that are too soft to support the formation of focal adhesions. Given their functional importance for 3D cell migration, curved adhesions may serve as a therapeutic target for future development.

cell biology↗

bioRxiv2021

Exploring cell surface-nanopillar interactions with 3D super-resolution microscopy

Plasma membrane topography has been shown to strongly influence the behavior of many cellular processes such as clathrin-mediated endocytosis, actin rearrangements, and others. Recent studies have used 3D nanostructures such as nanopillars to imprint well-defined membrane curvatures (the "nano-bio interface"). In these studies, proteins and their interactions were probed by 2D fluorescence microscopy. However, the low resolution and limited axial detail of such methods are not optimal to determine the relative spatial position and distribution of proteins along a 100 nm-diameter object, which is below the optical diffraction limit. Here, we introduce a general method to explore the nanoscale distribution of proteins at the nano-bio interface with 10-20 nm precision using 3D single-molecule super-resolution (SR) localization microscopy. This is achieved by combining a silicone oil immersion objective and 3D double-helix point-spread function microscopy. We carefully optimize the objective to minimize spherical aberrations between quartz nanopillars and the cell. To validate the 3D SR method, we imaged the 3D shape of surface-labeled nanopillars and compared the results with electron microscopy measurements. Turning to transmembrane-anchored labels in cells, the high quality 3D SR reconstructions reveal the membrane tightly wrapping around the nanopillars. Interestingly, the cytoplasmic protein AP-2 involved in clathrin-mediated endocytosis accumulates along the nanopillar above a specific threshold of 1/R membrane curvature. Finally, we observe that AP-2 and actin preferentially accumulate at positive Gaussian curvature near the pillar caps. Our results establish a general method to investigate the nanoscale distribution of proteins at the nano-bio interface using 3D SR microscopy.

biophysics↗

bioRxiv2020

Exposure to maternal high-fat diet induces extensive changes in the brain of adult offspring

Maternal environmental exposures, such as high-fat diets, diabetes and obesity, can induce long term effects in offspring. These effects include increased risk of neurodevelopmental disorders (NDDs) including autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), depression and anxiety. The mechanisms underlying these late-life neurologic effects are unknown. In this article, we measured changes in the offspring brain and determined which brain regions are sensitive to maternal metabolic milieu and therefore may mediate NDD risk. We showed that mice exposed to maternal high-fat diet display extensive brain changes in adulthood despite being switched to low-fat diet at weaning. Brain regions impacted by early-life diet include the extended amygdalar system, which plays an important role in reward-seeking behaviour. Genes preferentially expressed in these regions have functions related to feeding behavior, while also being implicated in human NDDs, such as autism. Our data demonstrated that exposure to maternal high-fat diet in early-life leads to brain alterations that persist into adulthood, even after dietary modifications.

neuroscience↗