title: Essential diurnal Rac1 activation during retinal phagocytosis requires αvβ5 integrin but not tyrosine kinases focal adhesion kinase or Mer tyrosine kinase.
abstract: Diurnal phagocytosis of shed photoreceptor outer-segment particles by retinal pigment epithelial (RPE) cells belongs to a group of conserved clearance mechanisms employing αv integrins upstream of tyrosine kinases and Rho GTPases. In this study, we tested the interdependence of the tyrosine kinases focal adhesion kinase (FAK) and Mer tyrosine kinase (MerTK) and Rho GTPases during engulfment. RPE cells activated and redistributed Rac1, but not RhoA or Cdc42, during phagocytosis. Toxin B, overexpression of dominant-negative Rac1, or decreasing Rac1 expression prevented particle engulfment. Fluorescence microscopy showed that Rac1 inhibition had no obvious effect on F-actin arrangement in resting RPE but prevented recruitment of F-actin to surface-bound phagocytic particles. Quantification of active GTP-Rac1 in wild-type and mutant RPE in culture and in vivo revealed that Rac1 activation during phagocytosis requires αvβ5 integrin and its ligand milk fat globule EGF factor-8 (MFG-E8) but not the receptor tyrosine kinase MerTK. Abolishing tyrosine kinase signaling downstream of αvβ5 toward MerTK by inhibiting FAK specifically or tyrosine kinases generally neither prevented Rac1 activation nor F-actin recruitment during phagocytosis. Likewise, inhibiting Rac1 had no effect on FAK or MerTK activation. We conclude that MerTK activation via FAK and F-actin recruitment via Rac1 both require MFG-E8-ligated αvβ5 integrin. Both pathways are independently activated and required for clearance phagocytosis.
authors.affiliation: Department of Biological Sciences, Fordham University, Bronx, NY 10458, USA.
authors.forename: Yingyu
authors.initials: Y
authors.lastname: Mao
authors.forename: Silvia C
authors.initials: SC
authors.lastname: Finnemann
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism; D039564:Integrin alphaV / Q000378:metabolism*; D039641:Integrin beta Chains / Q000378:metabolism*; D010587:Phagocytosis; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D055213:Retinal Pigment Epithelium / Q000166:cytology* / Q000378:metabolism; D000076202:c-Mer Tyrosine Kinase; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
chemical_list: D039564:Integrin alphaV; D039641:Integrin beta Chains; D011518:Proto-Oncogene Proteins; C490765:Mertk protein, rat; D020794:Receptor Protein-Tyrosine Kinases; D000076202:c-Mer Tyrosine Kinase; D051416:Focal Adhesion Protein-Tyrosine Kinases; C499134:Rac1 protein, rat; D020830:rac1 GTP-Binding Protein