The polarity protein Scrib is essential for directed endothelial cell migration.
BACKGROUND
Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells.


OBJECTIVE
We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis.


RESULTS
Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP)(s843) transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels.


CONCLUSIONS
Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis.
Institut für Kardiovaskuläre Physiologie, Goethe-Universität, Theodor-Stern-Kai 7, Frankfurt am Main, Germany. michaelis@vrc.uni-frankfurt.de
U Ruth
UR
Michaelis
Emmanouil
E
Chavakis
Christoph
C
Kruse
Benno
B
Jungblut
David
D
Kaluza
Katalin
K
Wandzioch
Yosif
Y
Manavski
Heinrich
H
Heide
Marie-Josée
MJ
Santoni
Michael
M
Potente
Johannes A
JA
Eble
Jean-Paul
JP
Borg
Ralf P
RP
Brandes
D000818:Animals; D054443:Cell Migration Assays; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D016764:Cell Polarity / Q000502:physiology*; D042783:Endothelial Cells / Q000502:physiology; D061307:Human Umbilical Vein Endothelial Cells / Q000502:physiology*; D006801:Humans; D039482:Integrin alpha5 / Q000378:metabolism*; D039564:Integrin alphaV / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D051379:Mice; D018919:Neovascularization, Physiologic / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*
D039482:Integrin alpha5; D039564:Integrin alphaV; D008565:Membrane Proteins; D034741:RNA, Small Interfering; C480591:SCRIB protein, human; D025521:Tumor Suppressor Proteins