Biology subjects

Wester, M. J.

Publications and source records attributed to Wester, M. J..

6 recordsLinked to original sources

bioRxiv2023

Antigen Geometry Tunes Mast Cell Signaling Through Distinct FcϵRI Aggregation andStructural Changes

Fc receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs) are critical components of the innate immune system that bridge adaptive antibody recognition to cellular effector responses. In allergic responses, the high-affinity IgE receptor, Fc{varepsilon}RI, is activated when multivalent antigens crosslink receptor-bound IgE, yet the molecular mechanisms linking antigen structure to signaling output remain incompletely understood. Here, we compare two antigens presenting identical IgE-binding haptens but differing in geometry: the high-valency, heterogeneous DNP-BSA and the defined trivalent antigen DF3. We find that these ligands elicit distinct patterns of degranulation and Fc{varepsilon}RI {gamma}-chain phosphorylation, correlating with differences in the recruitment of the inhibitory lipid phosphatase SHIP1. Monte Carlo simulations predicted that each antigen generates receptor aggregates with distinct size, complexity, and inter-receptor spacing. Using direct stochastic optical reconstruction microscopy (dSTORM) and Bayesian Grouping of Localizations (BaGoL) analysis, we directly visualized the nanoscale aggregate geometry and found that DF3 induced smaller, more linear aggregates with tighter receptor spacing than DNP-BSA. Together, our results show that antigen properties, including size, valency, and epitope spacing, modulate Fc{varepsilon}RI aggregate architecture and tune the balance of positive and negative signaling to ultimately shape mast cell outcomes. Statement of SignificanceAllergic immune responses are initiated when multivalent antigens aggregate IgE-bound Fc{varepsilon}RI on mast cells, yet the relationship between antigen structure and signaling strength remains unclear. This study combines biochemical assays, Monte Carlo modeling, and super-resolution imaging to show that allergen properties, specifically valency and nanoscale epitope spacing, govern the geometry of Fc{varepsilon}RI aggregates and the balance of activating and inhibitory signals. These findings establish a direct mechanistic link between antigen structure and immune receptor output, providing new insight into how physical antigen features encode mast cell responses.

biophysics↗

bioRxiv2021

Robust, Fiducial-Free Drift Correction for Super-resolution Imaging

We describe a robust, fiducial-free method of drift correction for use in single molecule localization-based super-resolution methods. The method combines periodic 3D registration of the sample using brightfield images with a fast post-processing algorithm that corrects residual registration errors and drift between registration events. The method is robust to low numbers of collected localizations, requires no specialized hardware, and provides stability and drift correction for an indefinite time period.

biophysics↗

bioRxiv2020

Comparing Lifeact and Phalloidin for Super-resolution Imaging of Actin in Fixed Cells

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide lifeact. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a (d)STORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

cell biology↗

bioRxiv2019

Sub-Nanometer Precision using Bayesian Grouping of Localizations

Single-molecule localization microscopy super-resolution methods such as DNA-PAINT and (d)STORM can generate multiple observed localizations over the time course of data acquisition from each dye or binding site that are not a priori assigned to those specific dyes or binding sites. We describe a Bayesian method of grouping and combining localizations from multiple blinking/binding events that can improve localization precision to better than one nanometer. The known statistical distribution of the number of binding/blinking events per dye/docking strand along with the precision of each localization event are used to estimate the true number and location of emitters in closely-spaced clusters.

biophysics↗

bioRxiv2019

Bayesian Multiple Emitter Fitting using Reversible Jump Markov Chain Monte Carlo

In single molecule localization-based super-resolution imaging, high labeling density or the desire for greater data collection speed can lead to clusters of overlapping emitter images in the raw super-resolution image data. We describe a Bayesian inference approach to multiple-emitter fitting that uses Reversible Jump Markov Chain Monte Carlo to identify and localize the emitters in dense regions of data. This formalism can take advantage of any prior information, such as emitter intensity and density. The output is both a posterior probability distribution of emitter locations that includes uncertainty in the number of emitters and the background structure, and a set of coordinates and uncertainties from the most probable model.

biophysics↗

bioRxiv2017

Mannan molecular sub-structures control nanoscale glucan exposure in Candida

N-linked mannans (N-mannans) in the cell wall of Candida albicans are thought to mask {beta}-(1,3)-glucan from recognition by Dectin-1, contributing to innate immune evasion. Lateral cell wall exposures of glucan on Candida albicans are predominantly single receptor-ligand interaction sites and are restricted to nanoscale geometries. Candida species exhibit a range of basal glucan exposures and their mannans also vary in size and complexity at the molecular level. We used super resolution fluorescence imaging and a series of protein mannosylation mutants in C. albicans and C. glabrata to investigate the role of specific N-mannan features in regulating the nanoscale geometry of glucan exposure. Decreasing acid labile mannan abundance and -(1,6)-mannan backbone length correlated most strongly with increased density and nanoscopic size of glucan exposures in C. albicans and C. glabrata, respectively. Additionally, a C. albicans clinical isolate with high glucan exposure produced similarly perturbed N-mannan structures and exhibited similar changes to nanoscopic glucan exposure geometry. We conclude that acid labile N-mannan controls glucan exposure geometry at the nanoscale. Furthermore, variations in glucan nanoexposure characteristics are clinically relevant and are likely to impact the nature of the pathogenic surface presented to innate immunocytes at dimensions relevant to receptor engagement, aggregation and signaling.

microbiology↗