Abstract
The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.
Highlights
The vascular system is an intricate, well defined structure that forms in every fetus with striking accuracy and has the ability to remodel itself to ensure proper delivery of oxygen and nutrients in every corner of the body
The following four fractions were analyzed: Invasiveϩ was defined as the fraction of VEGFA-stimulated embryoid body (EB) cultures that contained blood vessels and cells migrating out from the EB core
A similar microarray experiment where tube-forming endothelial cells harvested from three-dimensional collagen cultures were compared with proliferating endothelial cells cultured on fibronectin, recently identified tie1, vegfr1, and notch4 as differentiation-regulated genes [20]
Summary
The vascular system is an intricate, well defined structure that forms in every fetus with striking accuracy and has the ability to remodel itself to ensure proper delivery of oxygen and nutrients in every corner of the body. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. We show that endothelial cells in developing blood vessels express elevated levels of exoc3l2, and that the EXOC3L2 protein associates with components of the exocyst complex.
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