An Integrin and Rho GTPase-Dependent Pinocytic Vacuole Mechanism Controls Capillary Lumen Formation in Collagen and Fibrin Matrices

ABSTRACTLumen extension in intracellular tubes can occur by the directed fusion of vesicles with an invading apical membrane domain. Within the C. elegans excretory cell, which contains an intracellular tube, the exocyst vesicle-tethering complex is enriched at the lumenal membrane domain and is required for tube formation, suggesting that it targets vesicles needed for lumen extension. Here, we identify a polarity pathway that promotes intracellular tube formation by enriching the exocyst at the lumenal membrane. We show that the PAR polarity proteins PAR-6 and PKC-3/aPKC localize to the lumenal membrane domain and function within the excretory cell to promote lumen extension, similar to exocyst component SEC-5 and exocyst regulator RAL-1. Using acute protein depletion, we find that PAR-6 is required to recruit the exocyst to the lumenal membrane domain, whereas PAR-3, which functions as an exocyst receptor in mammalian cells, appears to be dispensable for exocyst localization and lumen extension. Finally, we show that the Rho GTPase CDC-42 and the RhoGEF EXC-5/FGD act as upstream regulators of lumen formation by recruiting PAR-6 and PKC-3 to the lumenal membrane. Our findings reveal a molecular pathway that connects Rho GTPase signaling, cell polarity, and vesicle-tethering proteins to promote lumen extension in intracellular tubes.

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THE ISLET VASCULARIZATION IN THE SPLEEN (A UNIQUE MECHANISM OF CAPILLARY LUMEN FORMATION AND A SEARCHED PARADIGM OF ADULT VASCULOGENESIS)

Transplantation Journal ◽

10.1097/00007890-200407271-01951 ◽

2004 ◽

Vol 78 ◽

pp. 710

Author(s):

M L.C. Madureira

Keyword(s):

Lumen Formation ◽

Capillary Lumen ◽

Unique Mechanism

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Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin-1☆

Microvascular Research ◽

10.1016/s0026-2862(03)00045-1 ◽

2003 ◽

Vol 66 (2) ◽

pp. 102-112 ◽

Cited By ~ 308

Author(s):

Martin N. Nakatsu ◽

Richard C.A. Sainson ◽

Jason N. Aoto ◽

Kevin L. Taylor ◽

Mark Aitkenhead ◽

...

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Lumen Formation ◽

Human Umbilical Vein ◽

Fibrin Gels ◽

Capillary Lumen ◽

Umbilical Vein Endothelial Cells ◽

Angiopoietin 1

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Endothelial Cell Apoptosis during Glomerular Capillary Lumen Formation In Vivo

Journal of the American Society of Nephrology ◽

10.1097/01.asn.0000061779.70530.06 ◽

2003 ◽

Vol 14 (5) ◽

pp. 1349-1354 ◽

Cited By ~ 47

Author(s):

W. Fierlbeck

Keyword(s):

Endothelial Cell ◽

Cell Apoptosis ◽

Endothelial Cell Apoptosis ◽

Lumen Formation ◽

Capillary Lumen ◽

Glomerular Capillary

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RhoJ is an endothelial cell-restricted Rho GTPase that mediates vascular morphogenesis and is regulated by the transcription factor ERG

Blood ◽

10.1182/blood-2010-10-315275 ◽

2011 ◽

Vol 118 (4) ◽

pp. 1145-1153 ◽

Cited By ~ 44

Author(s):

Lei Yuan ◽

Anastasia Sacharidou ◽

Amber N. Stratman ◽

Alexandra Le Bras ◽

Peter J. Zwiers ◽

...

Keyword(s):

Transcription Factor ◽

Rho Gtpases ◽

Rho Gtpase ◽

Proximal Promoter ◽

Collagen Matrices ◽

Lumen Formation ◽

Functional Sites ◽

Knock Down ◽

Downstream Effectors ◽

3D Collagen

Abstract ERG is a member of the ETS transcription factor family that is highly enriched in endothelial cells (ECs). To further define the role of ERG in regulating EC function, we evaluated the effect of ERG knock-down on EC lumen formation in 3D collagen matrices. Blockade of ERG using siRNA completely interferes with EC lumen formation. Quantitative PCR (QPCR) was used to identify potential downstream gene targets of ERG. In particular, we identified RhoJ as the Rho GTPase family member that is closely related to Cdc42 as a target of ERG. Knockdown of ERG expression in ECs led to a 75% reduction in the expression of RhoJ. Chromatin immunoprecipitation and transactivation studies demonstrated that ERG could bind to functional sites in the proximal promoter of the RhoJ gene. Knock-down of RhoJ similarly resulted in a marked reduction in the ability of ECs to form lumens. Suppression of either ERG or RhoJ during EC lumen formation was associated with a marked increase in RhoA activation and a decrease in Rac1 and Cdc42 activation and their downstream effectors. Finally, in contrast to other Rho GTPases, RhoJ exhibits a highly EC-restricted expression pattern in several different tissues, including the brain, heart, lung, and liver.

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