Abstract
At the plasma membrane, transmembrane receptors are at the interface between cells and their environment. They allow sensing and transduction of chemical and mechanical extracellular signals. The spatial distribution of receptors and the specific recruitment of receptor subunits to the cell membrane is crucial for the regulation of signaling and cell behavior. However, it is challenging to define what regulates such spatial patterns for receptor localization, as cell shapes are extremely diverse when cells are maintained in standard culture conditions. Bone morphogenetic protein receptors (BMPRs) are serine-threonine kinases, which build heteromeric complexes of BMPRI and II. These are especially interesting targets for receptor distribution studies, since the signaling pathways triggered by BMPR-complexes depends on their dimerization mode. They might exist as preformed complexes, or assemble upon binding of BMP, triggering cell signaling which leads to differentiation or migration. In this work we analyzed BMPR receptor distributions in single cells grown on micropatterns, which allow not only to control cell shape, but also the distribution of intracellular organelles and protein assemblies. We developed a script called ComRed (Center Of Mass Receptor Distribution), which uses center of mass calculations to analyze the shift and spread of receptor distributions according to the different cell shapes. ComRed was tested by simulating changes in experimental data showing that shift and spread of distributions can be reliably detected. Our ComRed-based analysis of BMPR-complexes indicates that receptor distribution depends on cell polarization. The absence of a coordinated internalization after addition of BMP suggests that a rapid and continual recycling of BMPRs might occur. Receptor complexes formation and localization in cells induced by BMP might yield insights into the local regulation of different signaling pathways.
Highlights
Bone morphogenetic proteins (BMPs) are growth factors involved in a plethora of cellular processes, like cell differentiation [1], adhesion, and migration [2,3]
BMPRIa and Alk2 are only expressed slightly [12,16]. This simplifies the analysis of spatial distribution in human umbilical cord vein endothelial cells (HUVECs), as we expect no significant competition between BMPRIa and BMPRIb for complexing with BMPRII
In this study the spatial distribution of Bone morphogenetic protein receptors (BMPRs) subunits Ib and II in fluorescence microscopy images was studied by using Center Of Mass Receptor Distribution (ComRed), a script which analyzes center of mass differences
Summary
Bone morphogenetic proteins (BMPs) are growth factors involved in a plethora of cellular processes, like cell differentiation [1], adhesion, and migration [2,3]. Image correlation spectroscopy was used to analyze the receptor distributions of BMPRIa and BMPRII in COS7 and A431 cells. Upon BMP induction, a significantly different distribution after inhibition of clathrin-mediated endocytosis could be observed [8] These studies did not analyze spatial distributions within whole cells. ComRed analyzes the whole cell distributions of cellular receptors in fluorescence microscopy images, acquired after cell immunostaining It uses data from upstream programs, for example Ilastik [26,27], that segment images automatically to compute the center of mass of receptor clusters. BMPRIa and Alk are only expressed slightly [12,16] This simplifies the analysis of spatial distribution in HUVECs, as we expect no significant competition between BMPRIa and BMPRIb for complexing with BMPRII. ComRed is not restricted to the analysis of BMPRs and in combination with micropatterning techniques it might be a useful tool to analyze the spatial distributions of any receptor of interest
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