Poji: a Fiji-based tool for analysis of podosomes and associated proteins
Podosomes are micron-sized cytoskeletal structures which enable the adhesion to and the degradation of extracellular matrix by a variety of cells, including macrophages. The structure of podosomes comprises a core of branched F-actin, a surrounding ring of integrins and adhesion plaque proteins (e.g. vinculin), a cap of regulators and nucleators on the top of the core, as well as acto-myosin fibers that both extend laterally along the core and between individual podosomes to connect them to superstructures. The size, amount and protein composition of podosomes can vary both in homeostasis (e.g. the size difference between successor and precursor podosomes) and in response to external cues like matrices of different stiffness or the treatment with inhibitors and chemical reagents. As macrophages regularly form >100 podosomes per cell, manual analysis of individual podosomes would be an insurmountable task. Thus, we developed a semi-automatic tool to quickly and reliably analyse individual podosomes, the combined podosome covered area as well as general properties of cells, both individually and in relation to each other. This tool is designed for the characterization of podosomes in fixed cell images (including z-stacks), but can, among others, also be used to detect invadopodia and to correlate them with the area of local matrix degradation.
While the analysis of cells is conducted in automated processes, the setting of predefined parameters is user-dependent. These parameters comprise the manual definition of the cell area (with an optional additional selection of podosome clusters) and settings for podosome detection and further processing. While the splitting of z slices and channels (automatable by using an additional, optional macro) has to be done prior to starting the macro, all other settings are defined in interfaces of the macro. The settings include defining the noise threshold for local maxima recognition, as well as smoothing of images, to better detect the geometrical centre of podosomes. For further advices on using the macro, please refer to the user guide that is deposited together with the codes and premade analysis tables (see bottom of the page).
Analysis of images by using Poji can yield up to 19 different sets of data, that describe properties of the entire cell and of podosomes (collectively and individually) as well as calculating ratios between them. Examples of these calculated results are shown below.
This tool was originally published in:
Herzog et al., “Poji: a Fiji-based tool for analysis of podosomes and associated proteins”, Journal of Cell Science, 2020.
The code, together with associated premade analysis tables and an extensive user guide are deposited online at: