Biology subjects

Diaz-Rohrer, B.

Publications and source records attributed to Diaz-Rohrer, B..

3 recordsLinked to original sources

bioRxiv2024

Live Cell Painting: image-based profiling in live cells using Acridine Orange

Image-based profiling has been used to analyze cell health, drug mechanism of action, CRISPR-edited cells, and overall cytotoxicity. Cell Painting is a broadly used image-based assay that uses morphological features to capture how cells respond to treatments. However, this method requires cell fixation for staining, which prevents examining live cells. To address this limitation, here we present Live Cell Painting (LCP), a high-content method based on Acridine orange, a metachromatic dye that labels different organelles and cellular structures. We began by showing that LCP can be applied to follow acidic vesicle redistribution of cells exposed to acidic vesicles inhibitors. Next, we show that LCP can identify subtle changes in cells exposed to silver nanoparticles that are not detected by techniques such as MTT assay. In drug treatments, LCP was helpful in assessing the dose-response relationship and creating profiles that allow clustering of drugs that cause liver injury. Here, we present an affordable and easy-to-use image-based assay capable of assessing overall cell health and showing promise for use in various applications such as assessing drugs and nanoparticles. We envisage the use of Live Cell Painting as an initial screening of overall cell health while providing insights into new biological questions.

cell biology↗

bioRxiv2024

A Postdoctoral Training Program in Bioimage Analysis

We herein describe a postdoctoral training program designed to train biologists with microscopy experience in bioimage analysis. We detail the rationale behind the program, the various components of the training program, and outcomes in terms of works produced and the career effects on past participants. We analyze the results of an anonymous survey distributed to past and present participants, indicating overall high value of all 12 rated aspects of the program, but significant heterogeneity in which aspects were most important to each participant. Finally, we propose this model as a template for other programs which may want to train experts in professional skill sets, and discuss the important considerations when running such a program. We believe that such programs can have extremely positive impact for both the trainees themselves and the broader scientific community.

cell biology↗

bioRxiv2023

Partitioning to ordered membrane domains regulates the kinetics of secretory traffic

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting Golgi [~]2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.

cell biology↗