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Vinculin binding angle in podosomes revealed by high resolution microscopy.

Walde M, Monypenny J, Heintzmann R, Jones GE, Cox S - PLoS ONE (2014)

Bottom Line: They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin.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.Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom ; Institute of Physical Chemistry, Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Jena, Germany.

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.

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Immunolocalisation analysis and comparison of clustered and individual podosomes.(A) Radial distributions of actin (black), paxillin (red), talin (green) and vinculin (blue). The normalized average intensity of the immunolocalisation is plotted with respect to the radial distance from the centre of the actin core. (B+C) Comparison of actin (B) and vinculin (C) distributions in clustered (blue) and individual cells (red).
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pone-0088251-g005: Immunolocalisation analysis and comparison of clustered and individual podosomes.(A) Radial distributions of actin (black), paxillin (red), talin (green) and vinculin (blue). The normalized average intensity of the immunolocalisation is plotted with respect to the radial distance from the centre of the actin core. (B+C) Comparison of actin (B) and vinculin (C) distributions in clustered (blue) and individual cells (red).

Mentions: In order to quantify the immunolocalisation of the ring proteins with respect to the actin core, podosomes were automatically identified in Matlab with a recently published algorithm [30], which separates podosomes in samples stained with phalloidin (for F-actin) based on intensity, size and shape. This algorithm has previously been applied to wide-field and confocal images of human dendritic cells [30]. After segmentation, the distribution of ring proteins can be measured. Here, we calculated the average normalized intensity in a podosome with respect to the distance in nanometres from the centre of its actin core. Ring masks of 25 nm thickness and increasing radius around the core were analysed. Protein distributions around the actin cores were evaluated in 480 podosomes stained for actin, 339 for paxillin, 401 for talin and 141 for vinculin. The resulting normalized intensity distributions are shown in Figure 5 and results are summarized in Table 1. Paxillin and vinculin form distinct rings with peaks at distances dpax≈275 nm and dvin≈225 nm. The paxillin ring is broader (FHWMpax≈385 nm) than the vinculin ring (FHWMvin≈320 nm). In the histogram, talin appeared almost homogeneously distributed, because the calculated average distribution contains a mixture of the different observed configurations and possibly intermediate situations.


Vinculin binding angle in podosomes revealed by high resolution microscopy.

Walde M, Monypenny J, Heintzmann R, Jones GE, Cox S - PLoS ONE (2014)

Immunolocalisation analysis and comparison of clustered and individual podosomes.(A) Radial distributions of actin (black), paxillin (red), talin (green) and vinculin (blue). The normalized average intensity of the immunolocalisation is plotted with respect to the radial distance from the centre of the actin core. (B+C) Comparison of actin (B) and vinculin (C) distributions in clustered (blue) and individual cells (red).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3921150&req=5

pone-0088251-g005: Immunolocalisation analysis and comparison of clustered and individual podosomes.(A) Radial distributions of actin (black), paxillin (red), talin (green) and vinculin (blue). The normalized average intensity of the immunolocalisation is plotted with respect to the radial distance from the centre of the actin core. (B+C) Comparison of actin (B) and vinculin (C) distributions in clustered (blue) and individual cells (red).
Mentions: In order to quantify the immunolocalisation of the ring proteins with respect to the actin core, podosomes were automatically identified in Matlab with a recently published algorithm [30], which separates podosomes in samples stained with phalloidin (for F-actin) based on intensity, size and shape. This algorithm has previously been applied to wide-field and confocal images of human dendritic cells [30]. After segmentation, the distribution of ring proteins can be measured. Here, we calculated the average normalized intensity in a podosome with respect to the distance in nanometres from the centre of its actin core. Ring masks of 25 nm thickness and increasing radius around the core were analysed. Protein distributions around the actin cores were evaluated in 480 podosomes stained for actin, 339 for paxillin, 401 for talin and 141 for vinculin. The resulting normalized intensity distributions are shown in Figure 5 and results are summarized in Table 1. Paxillin and vinculin form distinct rings with peaks at distances dpax≈275 nm and dvin≈225 nm. The paxillin ring is broader (FHWMpax≈385 nm) than the vinculin ring (FHWMvin≈320 nm). In the histogram, talin appeared almost homogeneously distributed, because the calculated average distribution contains a mixture of the different observed configurations and possibly intermediate situations.

Bottom Line: They consist of a core of F-actin and actin-regulating proteins, surrounded by a ring of adhesion-associated proteins such as vinculin.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.Furthermore, adjacent vinculin strands are observed nucleating at the corners of the podosomes, suggesting a mechanism for podosome growth.

View Article: PubMed Central - PubMed

Affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom ; Institute of Physical Chemistry, Abbe Center of Photonics, Friedrich-Schiller-Universität Jena, Jena, Germany.

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.

Show MeSH