PMID:24236012

Citation	De Clercq, S, Boucherie, C, Vandekerckhove, J, Gettemans, J and Guillabert, A (2013) L-plastin nanobodies perturb matrix degradation, podosome formation, stability and lifetime in THP-1 macrophages. PLoS ONE 8:e78108
Abstract	Podosomes are cellular structures acting as degradation 'hot-spots' in monocytic cells. They appear as dot-like structures at the ventral cell surface, enriched in F-actin and actin regulators, including gelsolin and L-plastin. Gelsolin is an ubiquitous severing and capping protein, whereas L-plastin is a leukocyte-specific actin bundling protein. The presence of the capping protein CapG in podosomes has not yet been investigated. We used an innovative approach to investigate the role of these proteins in macrophage podosomes by means of nanobodies or Camelid single domain antibodies. Nanobodies directed against distinct domains of gelsolin, L-plastin or CapG were stably expressed in macrophage-like THP-1 cells. CapG was not enriched in podosomes. Gelsolin nanobodies had no effect on podosome formation or function but proved very effective in tracing distinct gelsolin populations. One gelsolin nanobody specifically targets actin-bound gelsolin and was effectively enriched in podosomes. A gelsolin nanobody that blocks gelsolin-G-actin interaction was not enriched in podosomes demonstrating that the calcium-activated and actin-bound conformation of gelsolin is a constituent of podosomes. THP-1 cells expressing inhibitory L-plastin nanobodies were hampered in their ability to form stable podosomes. Nanobodies did not perturb Ser5 phosphorylation of L-plastin although phosphorylated L-plastin was highly enriched in podosomes. Furthermore, nanobody-induced inhibition of L-plastin function gave rise to an irregular and unstable actin turnover of podosomes, resulting in diminished degradation of the underlying matrix. Altogether these results indicate that L-plastin is indispensable for podosome formation and function in macrophages.
Links	PubMed PMC3827245 Online version:10.1371/journal.pone.0078108
Keywords	Actin Cytoskeleton/metabolism; Cell Line, Tumor; Extracellular Matrix/metabolism; Gelatin/metabolism; Gelsolin/metabolism; Humans; Macrophages/drug effects; Macrophages/metabolism; Macrophages/ultrastructure; Matrix Metalloproteinases, Secreted/metabolism; Membrane Glycoproteins/immunology; Membrane Glycoproteins/metabolism; Microfilament Proteins/immunology; Microfilament Proteins/metabolism; Organelles/metabolism; Phosphoproteins/immunology; Phosphoproteins/metabolism; Protein Transport; Proteolysis; Single-Domain Antibodies/pharmacology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
HUMAN:GELS	part_of	GO:0002102: podosome	ECO:0000314: direct assay evidence used in manual assertion		C	part_of:(CL:0000576)	Seeded From UniProt	complete
HUMAN:GELS	enables	GO:0003779: actin binding	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:GELS	involved_in	GO:0071801: regulation of podosome assembly	ECO:0000315: mutant phenotype evidence used in manual assertion		P	occurs_in:(CL:0000235)	Seeded From UniProt	complete
HUMAN:GELS	part_of	GO:0030864: cortical actin cytoskeleton	ECO:0000314: direct assay evidence used in manual assertion		C	part_of:(CL:0000235)	Seeded From UniProt	complete
HUMAN:GELS	NOT|involved_in	GO:0022617: extracellular matrix disassembly	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
HUMAN:MMP9	part_of	GO:0005615: extracellular space	ECO:0000314: direct assay evidence used in manual assertion		C	part_of:(CL:0000235)	Seeded From UniProt	complete
HUMAN:MMP9	enables	GO:0008237: metallopeptidase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete
HUMAN:MMP2	part_of	GO:0005615: extracellular space	ECO:0000314: direct assay evidence used in manual assertion		C	part_of:(CL:0000235)	Seeded From UniProt	complete
HUMAN:MMP2	enables	GO:0008237: metallopeptidase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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