A Computational Approach to Detect Gap Junction Plaques and Associate Them with Cells in Fluorescent Images

Abstract

Fundamental to understanding how tissues function is understanding how the cells that compose them communicate. In most tissues and organs, cells do not exist as homotypic monolayers but rather coexist as multiple different cell types in close proximity to one another. In some cases, these relationships are random and have no functional significance; others are symbiotic, where the presence and proximity of a neighboring cell are vital to another cell's quiescence, specification, maturation, function, survival, and so on. Although biochemical, genetic, genomic, and proteomic methods have revealed a wealth of molecules known to participate in cell signaling pathways, many such signaling networks have been established using in vitro systems, and thus it is still unclear how cells within a threedimensional (3D), functional tissue environment use these pathways.This question and the increasing importance of 3D microenvironments in cancer (Håkanson et al. 2011) and stem cell biology (Doetsch et al. 1997;Shen et al. 2008;Tavazoie et al. 2008;Kazanis et al. 2010) SummaryIntercellular signaling is a fundamental requirement for complex biological system function and survival. Communication between adjoining cells is largely achieved via gap junction channels made up of multiple subunits of connexin proteins, each with unique selectivity and regulatory properties. Intercellular communication via gap junction channels facilitates transmission of an array of cellular signals, including ions, macromolecules, and metabolites that coordinate physiological processes throughout tissues and entire organisms. Although current methods used to quantify connexin expression rely on number or area density measurements in a field of view, they lack cellular assignment, distance measurement capabilities (both within the cell and to extracellular structures), and complete automation. We devised an automated computational approach built on a contour expansion algorithm platform that allows connexin protein detection and assignment to specific cells within complex tissues. In addition, parallel implementation of the contour expansion algorithm allows for high-throughput analysis as the complexity of the biological sample increases. This method does not depend specifically on connexin identification and can be applied more widely to the analysis of numerous immunocytochemical markers as well as to identify particles within tissues such as nanoparticles, gene delivery vehicles, or even cellular fragments such as exosomes or microparticles. (J Histochem Cytochem 61:283-293, 2013)

Connexin 43-mediated neurovascular interactions regulate neurogenesis in the adult brain subventricular zone

“…To evaluate the expression of Cx proteins in NSCs in the murine SVZ, we first labeled the NSCs using a BrdU-labeling protocol in which label-retaining cells (LRCs) represent qNSCs. 34 We found that a percentage of SVZ LRC-NSCs express multiple Cx proteins, and a higher percentage of LRC-NSCs express Cx43 ( Figure 2B ), like ECs. To determine whether Cx43 is present between NSCs and ECs in the SVZ in vivo , we performed immunohistochemistry and high-resolution confocal imaging of SVZ coronal sections.…”

“…Quantification of connexin protein colocalization with LRCs or ECs was performed on confocal z stack images by a computational approach using FARSIGHT for nuclear segmentation and MATLAB. 34 …”

Connexin 43-mediated Neurovascular Interactions Regulate Neurogenesis in the Adult Brain Subventricular Zone

“…Using immunohistochemistry and antibodies against different Cx proteins and CD31, which is expressed by all ECs, we found a low percentage of ECs expressing Cx26 and Cx31, while a high percentage of ECs express Cx43 ( Figure 2a ). To evaluate the expression of Cx proteins in NSCs in the murine SVZ, we first labeled the NSCs using a BrdU-labeling protocol in which label-retaining cells (LRC) represent quiescent NSCs 33 . We found that a percentage of SVZ LRC-NSCs express multiple Cx proteins, and a higher percentage of LRC-NSCs express Cx43 ( Figure 2b ), similar to ECs.…”

“…Briefly, wild-type mice received BrdU (7.5mg/ml) i.p injections twice daily for 5 days and sacrificed 4 wk after the last injection. Quantification of connexin protein colocalization with LRCs or ECs was performed on confocal z-stack images by a computational approach using FARSIGHT for nuclear segmentation and MATLAB programs as previously published in the lab 33 .…”

“…Fastq-files of raw reads were generated with bcl2fastq2_v2.19.0 and then uploaded to the usegalaxy.org platform 70 for quality control and trimming (Galaxy ToolShed v 1.0.2; FASTQ/A short-reads preprocessing tools: http://hannonlab.cshl.edu/fastx_toolkit/) 71,72 . Sequences were aligned to the hg38genome using Kallisto 73 colocalization with LRCs or ECs was performed on confocal z-stack images by a computational approach using FARSIGHT for nuclear segmentation and MATLAB programs as previously published in the lab 33 .…”