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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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SIM images show polygonal structure of podosomes.(A) Immunolocalisation of actin (red) and vinculin (blue) in podosomes. SIM image of podosomes recorded with 3 different grating directions and 5 grating phase positions showing the actin core surrounded by an vinculin-enriched protein ring. (B) SIM images of vinculin rings of podosomes, imaged with 5 different grating directions. The podosome rings appear to have a polygonal structure with both 3 and 5 grating directions. (C, D) SIM images of vinculin rings. Vinculin strands on the corners of podosomes are visible. (E, F) Immunolocalisation of actin (red) and paxillin (blue) in podosomes. SIM images were recorded with 5 grating directions and 5 grating phase positions. Paxillin rings appear polygonal, too, but are more punctate than the vinculin rings. All scale bars are 2 m.
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pone-0088251-g002: SIM images show polygonal structure of podosomes.(A) Immunolocalisation of actin (red) and vinculin (blue) in podosomes. SIM image of podosomes recorded with 3 different grating directions and 5 grating phase positions showing the actin core surrounded by an vinculin-enriched protein ring. (B) SIM images of vinculin rings of podosomes, imaged with 5 different grating directions. The podosome rings appear to have a polygonal structure with both 3 and 5 grating directions. (C, D) SIM images of vinculin rings. Vinculin strands on the corners of podosomes are visible. (E, F) Immunolocalisation of actin (red) and paxillin (blue) in podosomes. SIM images were recorded with 5 grating directions and 5 grating phase positions. Paxillin rings appear polygonal, too, but are more punctate than the vinculin rings. All scale bars are 2 m.

Mentions: Both SIM and STED were used to image the vinculin ring of podosomes, with the SIM measurements also imaging actin (Figure 2A). Podosomes were found to be widely distributed over the substratum-attached side of the cell. It was clearly visible that each podosome consisted of an actin core surrounded by a vinculin-rich ring, as expected from the current podosome model. However, the vinculin rings appeared to be polygonal rather than round (Figure 2B–D). For SIM, images were taken with both three and five grating directions, with the observed structures remaining the same.


Vinculin binding angle in podosomes revealed by high resolution microscopy.

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

SIM images show polygonal structure of podosomes.(A) Immunolocalisation of actin (red) and vinculin (blue) in podosomes. SIM image of podosomes recorded with 3 different grating directions and 5 grating phase positions showing the actin core surrounded by an vinculin-enriched protein ring. (B) SIM images of vinculin rings of podosomes, imaged with 5 different grating directions. The podosome rings appear to have a polygonal structure with both 3 and 5 grating directions. (C, D) SIM images of vinculin rings. Vinculin strands on the corners of podosomes are visible. (E, F) Immunolocalisation of actin (red) and paxillin (blue) in podosomes. SIM images were recorded with 5 grating directions and 5 grating phase positions. Paxillin rings appear polygonal, too, but are more punctate than the vinculin rings. All scale bars are 2 m.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0088251-g002: SIM images show polygonal structure of podosomes.(A) Immunolocalisation of actin (red) and vinculin (blue) in podosomes. SIM image of podosomes recorded with 3 different grating directions and 5 grating phase positions showing the actin core surrounded by an vinculin-enriched protein ring. (B) SIM images of vinculin rings of podosomes, imaged with 5 different grating directions. The podosome rings appear to have a polygonal structure with both 3 and 5 grating directions. (C, D) SIM images of vinculin rings. Vinculin strands on the corners of podosomes are visible. (E, F) Immunolocalisation of actin (red) and paxillin (blue) in podosomes. SIM images were recorded with 5 grating directions and 5 grating phase positions. Paxillin rings appear polygonal, too, but are more punctate than the vinculin rings. All scale bars are 2 m.
Mentions: Both SIM and STED were used to image the vinculin ring of podosomes, with the SIM measurements also imaging actin (Figure 2A). Podosomes were found to be widely distributed over the substratum-attached side of the cell. It was clearly visible that each podosome consisted of an actin core surrounded by a vinculin-rich ring, as expected from the current podosome model. However, the vinculin rings appeared to be polygonal rather than round (Figure 2B–D). For SIM, images were taken with both three and five grating directions, with the observed structures remaining the same.

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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