Vinculin Binding Angle in Podosomes Revealed by High Resolution Microscopy

Marie Walde,Rainer Heintzmann,Gareth E Jones,James Monypenny,Susan Cox,Cara Gottardi

doi:10.1371/journal.pone.0088251

Abstract

Podosomes are highly dynamic actin-rich adhesive structures formed predominantly by cells of the monocytic lineage, which degrade the extracellular matrix. They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin. We have characterised the structure of podosomes in macrophages, particularly the structure of the ring, using three super-resolution fluorescence microscopy techniques: stimulated emission depletion microscopy, structured illumination microscopy and localisation microscopy. Rather than being round, as previously assumed, we found the vinculin ring to be created from relatively straight strands of vinculin, resulting in a distinctly polygonal shape. The strands bind preferentially at angles between 116° and 135°. Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.

Highlights

Podosomes and the related invadopodia are receiving increasing attention due to their potential involvement in physiological events, such as monocyte extravasation and tissue transmigration, and pathological conditions such as atherosclerosis [1], osteoporosis [2] and cancer metastasis [3]
Samples stained for paxillin and F-actin showed similar polygonal structures for paxillin to those seen for vinculin
Immunofluorescent staining of paxillin and vinculin shows rings surrounding the actin core, confirming the current podosome model

Summary

Introduction

Podosomes and the related invadopodia are receiving increasing attention due to their potential involvement in physiological events, such as monocyte extravasation and tissue transmigration, and pathological conditions such as atherosclerosis [1], osteoporosis [2] and cancer metastasis [3]. Each podosome consists of a core of actin filaments surrounded by a ring containing integrin-associated proteins such as talin, vinculin and paxillin, amongst others [6,7]. The integrin-associated proteins, such as vinculin, appear to form round ring-like structures surrounding the acting core when imaged using confocal fluorescence microscopy (Figure 1A). Since their size is close to the diffraction limit (about 200 nm for standard labels) they are prime candidates for super-resolution imaging, as few details of the structure can be seen by standard fluorescence imaging

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