Wright, M. D. | bioRxiv Science

bioRxiv2022

Flexible and open-source programs for quantitative image analysis in microbial ecology

Epifluorescence microscopy is an essential tool for obtaining reliable estimates of the abundance of marine microorganisms including viruses. However, computational analysis is required to gain consistent and quantitative data from digital microscopy images. Many imaging programs are proprietary and cost-prohibitive. The currently available free imaging programs are often platform specific and/or lack the flexibility to analyze microscopy images from natural samples, such as the planktonic environment, which can contain challenges such as debris and high background signals. Here we describe two MATLAB-based open-source image analysis programs that work across computer platforms and provide the tools to analyze a range of image types and cell sizes with a user-friendly interface. The Microbial Image Analysis (MiA) program aims to provide flexibility for the selection, identification, and quantification of cells that vary in size and fluorescence intensity within natural microbial communities. The Viral Image Analysis (ViA) program aims to provide an effective means for quantifying viral abundances from epifluorescence images as well as enumerating the intensity of a primary and secondary stain. In this paper, we provide an overview of the functionality of the MiA and ViA programs and highlight specific program features through several microbial image case studies.

microbiology↗

bioRxiv2019

Dynamic regulation of CD45 by tetraspanin CD53

T cells are central to the adaptive immune response, playing a role in both the direct and indirect killing of pathogens and transformed cells. The activation of T cells is the result of a complex signaling cascade, initiated at the T cell receptor (TCR), and ending with the induction of proliferation. CD45, a member of the protein tyrosine phosphatase family, is one of the most abundant membrane proteins on T cells and functions by regulating activation directly downstream of the TCR. As a result of alternative splicing, CD45 can be expressed in multiple isoforms, naive T cells express the CD45RA isoform, while activated T cells gain expression of CD45RO, which has been proposed to increase signaling. Though the importance of CD45 in TCR signaling, proliferation and cytokine production is well established, little is known about the regulation of CD45 activity. We discovered that the immune-specific tetraspanin CD53 directly affects the stability and function of CD45RO in T cells. We have identified CD53 as a T cell co-stimulatory molecule in primary human and murine cells. Furthermore, we have shown that the absence of CD53 leads to an altered CD45 isoform expression as a result of decreased CD45RO stability on the cell surface. This instability was accompanied by increased mobility as measured by FRAP. Together, this indicates that CD53 functions as a stabilizer of CD45RO, and therefore as a positive regulator of TCR signaling at the T cell surface. Our data provides novel insight into the role of tetraspanins in the regulation of immune signaling and may provide a new avenue for the regulation of T cell signaling.

immunology↗