Making endothelial cells move : a study of Angiomotin and binding partners

Angiogenesis is a process crucial for tumor growth and metastasis formation and
encompasses the control of endothelial directional migration, anastomosis and lumen
formation. We have previously shown that Angiomotin (Amot), a membrane-associated
scaffold protein, plays an essential role in controlling endothelial cell migration and
cell shape. Amot is critical for normal development as more than 75% of amot deficient
mouse embryos die in utero due to vascular defects.



In this thesis, the vascular system of the mice that survive gestation are studied.
These mice are apparently normal in regards of body weight, kidney function and
reproduction. Further, the vascular system did not exhibit any changes in perfusion or
vascular density. However, the growth of Lewis Lung Carcinoma tumor xenograft was
significantly impaired in amot deficient mice as compared to sibling wt mice. Analysis
of the tumor vasculature revealed a marked change in vessel morphology and
decreased vascular density and perfusion. These data argue that Amot, besides its role
in development, plays a critical role in pathological angiogenesis. Gene expression
analysis shows that the loss of amot expression leads to indirect effects, which activates
NF-κB regulated genes and results in an inflammatory phenotype in vitro and in vivo.



As the thesis title implies, the function of Amot in endothelial cell migration has
been investigated by identifying and studying its binding partners and their functions.
The polarity proteins Patj and Mupp1 (plus Pals1) are presented as binding partners to
the Amot PDZ-binding motif. This is of interest since establishing cell polarity is a key
step in directional endothelial migration. Migration also depends on correct actin
polymerization, driven by Rho GTPases. We show that Amot is essential for the
localization of RhoA activity at the leading edge of a migrating cell and that the Rho
GEF (activator of RhoA) Syx1 also associates to Patj/Mupp1. Finally, we show that the
interaction of the PDZ-binding motif to Patj/Mupp1/Syx1 is essential for vessel
migration in developing zebrafish embryos.



Furthermore, tight junction-associated tumor suppressor Merlin (nf2) is identified as
a binder to the Amot coiled-coil domain. Rich1, a negative regulator of Rac1 (also a
RhoGTPase), has been shown to bind to Amot through the same domain. We show that
Amot regulates Rac1 activity and subsequent MAPK pathway activation through Merlin
and Rich1 and that Merlin and Rich1 compete for the same binding site on Amot,
resulting in Rac1 regulation. We hypothesize that Rich1 is inhibited by its binding to
Amot, leaving Rac1 in its active state, able to polymerize actin. Upon binding to
Merlin, Amot releases Rich1 and Rac1 is inhibited. Finally, we show that Schwann cell
tumors, caused by loss of nf2 expression, exhibit a decreased growth rate upon loss of
amot expression, leading to prolonged mouse survival. This further emphasizes the
potential for Amot as a drug target and indicates that anti-Amot therapy could be used
to treat Schwanoma patients.



Amot localizes to the lamellipodia of migrating cells, a region of the cell membrane
with specific lipid composition. The Amot coiled-coil domain has been shown to be a
conserved lipid-binding domain. I propose a model where migratory cues trigger the
change in lipid composition of the cell membrane, concentrating lipids to which Amot
binds to the lamellipodia. Amot brings with it the complex of Patj/Mupp1:Syx localizing RhoA activity and thus actin polymerization at the leading edge