Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy.

Robert P J Nieuwenhuizen,Sjoerd Stallinga,Bernd Rieger,Kees Jalink,Leila Nahidiazar,Erik M M Manders

doi:10.1371/journal.pone.0131756

Abstract

Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of molecules and orientational alignment of the structures on which they reside as co-orientation. Because the orientation varies with the length scale at which it is evaluated, we consider this scale as a separate informative dimension in the analysis. Additionally we introduce a data driven method for testing the statistical significance of the co-orientation and provide a method for visualizing the local co-orientation strength in images. We demonstrate our methods on simulated localization microscopy data of filamentous structures, as well as experimental images of similar structures acquired with localization microscopy in different color channels. We also show that in cultured primary HUVEC endothelial cells, filaments of the intermediate filament vimentin run close to and parallel with microtubuli. In contrast, no co-orientation was found between keratin and actin filaments. Co-orientation between vimentin and tubulin was also observed in an endothelial cell line, albeit to a lesser extent, but not in 3T3 fibroblasts. These data therefore suggest that microtubuli functionally interact with the vimentin network in a cell-type specific manner.

Highlights

Cytoskeletal protein networks serve a number of crucial roles in living cells
We demonstrate our methods on simulated localization microscopy data of filament structures, as well as experimental images of filamentous structures acquired with localization microscopy in different color channels
In order to detect which parts of a region of interest exhibit the strongest co-orientation, we developed a scheme for visualizing the local co-orientation strength

Summary

Introduction

Cytoskeletal protein networks serve a number of crucial roles in living cells. Traditionally, three types of cytoskeletal networks are discriminated [1]. Over 60 different proteins such as keratins, vimentin and lamins have been identified, most of which have a strict cell type-specific distribution Whereas each of these filament systems, their subunits and methods of polymerization have been the subject of many thousands of studies, remarkably little is known on how the three principal filament systems may interact and collaborate to keep the cell alive and functioning. Proposed measures include: the pair-correlation function between coordinates in two color channels [11]; a hypothetical potential energy function that is estimated from the distances from each localization to the nearest neighbor in the other color channel [12]; and the rank correlation between the distances from a localization to its neighbors in the same color channel on the one hand and distances to its neighbors in the other channel on the other hand [13] All these analyses only consider the spatial proximity of molecules in different color channels. Software for our co-orientation analysis is freely available in the form of Matlab code at http://www.diplib.org/add-ons/

Materials and Methods

Results

Discussion