BioPixel Reveals How Podosomes and Microtubule +TIPs Regulate Intracellular Transport

Abstract

Podosomes are dynamic, actin-rich adhesive structures found in monocyte-derived cells such as macrophages, dendritic cells, and osteoclasts. These specialized cellular domains coordinate cellular motility, extracellular matrix remodeling, and intracellular trafficking by integrating cytoskeletal elements with membrane dynamics. Structurally, podosomes consist of a dense F-actin core surrounded by an adhesion ring containing integrins and cytoskeletal linker proteins. While their actin dynamics have been extensively studied, the mechanisms integrating podosome activity with microtubule-dependent transport and microtubule plus-end tracking proteins (+TIPs) remain incompletely understood.

This work introduces BioPixel, a computational platform combining deep learning-based podosome segmentation with spatial analysis of protein interactions, providing a robust alternative to existing tools like ImageJ, Poji, and proprietary software. BioPixel was developed to overcome limitations of conventional analysis methods, which rely on manual thresholding and lack 3D quantification capabilities. The platform offers dual functionality: precise quantification of protein localization patterns relative to podosome architecture, and statistical mapping of proximity ligation assay (PLA) signals with single-molecule resolution.

When applied to macrophage podosomes, this integrated approach generated comprehensive localization profiles for actin regulators including drebrin, resolving distinct subdomain targeting patterns. Structure-function analysis revealed that the coiled-coil (CC), helical (Hel), and C-terminal regions of drebrin are collectively required for cap localization, while the ADFH and proline-rich (PP) domains are dispensable. The platform's PLA analysis module identified preferential formation of drebrin-EB3-clathrin complexes at podosome peripheries, functionally linking actin remodeling to microtubule-based transport through interaction with the +TIP protein EB3. The spatial analysis provided by BioPixel suggests these molecular complexes may play a key role in podosome functions related to matrix interactions and cytoskeletal coordination.

This work provides evidence for podosomes as potential integrated hubs for cytoskeletal crosstalk and offers new insights into the spatial organization of molecular interactions within these structures. The BioPixel platform combines the flexibility of open-source tools with the precision typically associated with proprietary software, eliminating the need for multiple specialized programs. While initially developed for podosome research, its modular architecture is designed for expansion to diverse biological questions. This unified approach provides quantitative 3D mapping of protein organization and interactions at subcellular resolution, advancing studies of molecular architecture in both physiological and pathological contexts.