Biology subjects

Pereira, P. M.

Publications and source records attributed to Pereira, P. M..

5 recordsLinked to original sources

bioRxiv2018

Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation

Single-molecule localization microscopy (SMLM) techniques allow near molecular scale resolution (~ 20nm) as well as precise and robust analysis of protein organization at different scales. SMLM hardware, analytics and probes have been the focus of a variety of studies and are now commonly used in laboratories across the world. Protocol reliability and artefact identification are increasingly seen as important aspects of super-resolution microscopy. The reliability of these approaches thus requires in-depth evaluation so that biological findings are based on solid foundations. Here we explore how different fixation approaches that disrupt or preserve the actin cytoskeleton affect membrane protein organization. Using CD4 as a model, we show that fixation-mediated disruption of the actin cytoskeleton correlates with changes in CD4 membrane organization. We highlight how these artefacts are easy to overlook and how careful sample preparation is essential for extracting meaningful results from super-resolution microscopy.

cell biology↗

bioRxiv2018

NanoJ: a high-performance open-source super-resolution microscopy toolbox

Super-resolution microscopy has become essential for the study of nanoscale biological processes. This type of imaging often requires the use of specialised image analysis tools to process a large volume of recorded data and extract quantitative information. In recent years, our team has built an open-source image analysis framework for super-resolution microscopy designed to combine high performance and ease of use. We named it NanoJ - a reference to the popular ImageJ software it was de-veloped for. In this paper, we highlight the current capabilities of NanoJ for several essential processing steps: spatio-temporal alignment of raw data (NanoJ-Core), super-resolution image re-construction (NanoJ-SRRF), image quality assessment (NanoJ-SQUIRREL), structural modelling (NanoJ-VirusMapper) and control of the sample environment (NanoJ-Fluidics). We expect to expand NanoJ in the future through the development of new tools designed to improve quantitative data analysis and measure the reliability of fluorescent microscopy studies.

bioinformatics↗

bioRxiv2018

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

NK cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here, we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at the nanometer scale with the activating receptor NKG2D upon immune synapse (IS) formation. NKG2D and Ly49A signal integration and colocalization was studied using NKG2D-GFP and Ly49A-RFP-expressing primary NK cells, forming ISs with NIH3T3 target cells, with or without expression of single chain trimer (SCT) H2-Dd and an extended form of SCT H2-Dd-CD4 MHC-I molecules. Nanoscale colocalization was assessed by Forster resonance energy transfer (FRET) between NKG2D-GFP and Ly49A-RFP and measured for each synapse. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at a nanometer scale leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D consequently impairing Ly49A-mediated inhibition. Thus, our data shows NK cell signal integration is critically dependent on the dimensions of NK cell ligand-receptor pairs by affecting their relative nanometer-scale colocalization at the IS. Together, our results suggest the balance of NK cell signals, and NK cell responses, are determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse.

immunology↗

bioRxiv2018

TMEM16F activation by Ca2+ triggers plasmalemma expansion and directs PD-1 trafficking

TMEM16F, an ion channel gated by high cytoplasmic Ca2+, is required for cell surface phosphatidylserine exposure during platelet aggregation and T cell activation. Here we demonstrate in Jurkat T cells and HEK293 cells that TMEM16F activation triggers large-scale surface membrane expansion in parallel with lipid scrambling. Following TMEM16F mediated scrambling and surface expansion, cells undergo extensive membrane shedding. The membrane compartment that expands the cell surface does not involve endoplasmic reticulum or acidified lysosomes. Surprisingly, T cells lacking TMEM16F expression not only fail to expand surface membrane, but instead rapidly internalize membrane via massive endocytosis (MEND). The T cell co-receptor PD-1 is selectively shed when TMEM16F triggers membrane expansion, while it is selectively internalized in the absence of TMEM16F. Its participation in this trafficking is determined by its single transmembrane domain. Thus, we establish a fundamental role for TMEM16F as a regulator of Ca2+-activated membrane trafficking.

cell biology↗

bioRxiv2017

NanoJ-SQUIRREL: quantitative mapping and minimisation of super-resolution optical imaging artefacts

Most super-resolution microscopy methods depend on steps that contribute to the formation of image artefacts. Here we present NanoJ-SQUIRREL, an ImageJ-based analytical approach providing a quantitative assessment of super-resolution image quality. By comparing diffraction-limited images and super-resolution equivalents of the same focal volume, this approach generates a quantitative map of super-resolution defects, as well as methods for their correction. To illustrate its broad applicability to super-resolution approaches we apply our method to Localization Microscopy, STED and SIM images of a variety of in-cell structures including microtubules, poxviruses, neuronal actin rings and clathrin coated pits. We particularly focus on single-molecule localisation microscopy, where super-resolution reconstructions often feature imperfections not present in the original data. By showing the quantitative evolution of data quality over these varied sample preparation, acquisition and super-resolution methods we display the potential of NanoJ-SQUIRREL to guide optimization of superresolution imaging parameters.

biophysics↗