12
downstream kinase signaling necessary to regulate vascular tube morphogenesis
[83]. Cdc42 and Par3 have also been shown to control lumen formation in epithelial
cells, and a recent study shows that Par3 and β1 integrins co-regulate arteriolar
lumen formation in vivo using a conditional β1 integrin subunit knockout mouse
system [110]. Thus, this latter work again confirms our prior conclusions obtained
in vitro showing that β1 integrins, Cdc42, and polarity proteins control the lumen
and tube formation process in 3D matrix environments [30, 36, 66, 67, 79, 83, 95].
1.3.6 Key Role for Polarized Subapical Microtubule
Modifications to Direct the Trafficking and Fusion
of Pinocytic Vacuoles to Regulate Apical Membrane
Development During EC Lumen Formation
Recent studies indicate that an important step in EC lumen and tube assembly is for
the increased accumulation of posttranslationally modified tubulins (i.e., acetylated
and detyrosinated tubulin) [63] (Fig. 1.2). Both of these modifications, which are
associated with increased tubulin polymer stability, are upregulation during EC
lumen formation and correlate with the lumen formation process. We demonstrated
that the microtubule tip complex proteins, EB1, p150 glued, and Clasp1, play a key
role in regulating lumen formation as well as the accumulation of both acetylated
and detyrosinated tubulin [63]. In addition, we identified HDAC6 and sirtuin2 as
negative regulators of lumen formation due to their ability to reduce tubulin acetylation via their activity as tubulin deacetylases. siRNA suppression of these deacetylases increased lumen formation, while increased expression of them decreased it
[63]. Further support for this conclusion is that addition of the HDAC6 inhibitor,
tubacin, leads to increased EC lumen and tube formation [79]. Of great interest here
is that these modified tubulins accumulate in a polarized manner subapically during
EC lumen and tube assembly, while in contrast, filamentous actin (F-actin) accumulates in a distinct basal location [64, 79]. This subapical polarized region where
acetylated and detyrosinated tubulin accumulates is precisely the location where
vacuole and vesicle fusion events occur, to control the development of the apical
membrane surface during this process. Interestingly, Cdc42 shows strong colocalization with acetylated tubulin in these regions of vacuole/vesicle fusion [79].
Thus, one of the central features of the EC lumen formation process is the creation
of cytoskeletal asymmetry and tracks where vesicles can be trafficked to this subapical domain, to polarize the lumen formation process. Furthermore, our data suggests that this subapical membrane accumulation of modified tubulins is necessary
to stabilize the apical membrane and maintain a stable tube structure [63, 64, 79].
We demonstrated in past studies that disruption of microtubules led to rapid collapse of tube networks [10].
G. E. Davis
downstream kinase signaling necessary to regulate vascular tube morphogenesis
[83]. Cdc42 and Par3 have also been shown to control lumen formation in epithelial
cells, and a recent study shows that Par3 and β1 integrins co-regulate arteriolar
lumen formation in vivo using a conditional β1 integrin subunit knockout mouse
system [110]. Thus, this latter work again confirms our prior conclusions obtained
in vitro showing that β1 integrins, Cdc42, and polarity proteins control the lumen
and tube formation process in 3D matrix environments [30, 36, 66, 67, 79, 83, 95].
1.3.6 Key Role for Polarized Subapical Microtubule
Modifications to Direct the Trafficking and Fusion
of Pinocytic Vacuoles to Regulate Apical Membrane
Development During EC Lumen Formation
Recent studies indicate that an important step in EC lumen and tube assembly is for
the increased accumulation of posttranslationally modified tubulins (i.e., acetylated
and detyrosinated tubulin) [63] (Fig. 1.2). Both of these modifications, which are
associated with increased tubulin polymer stability, are upregulation during EC
lumen formation and correlate with the lumen formation process. We demonstrated
that the microtubule tip complex proteins, EB1, p150 glued, and Clasp1, play a key
role in regulating lumen formation as well as the accumulation of both acetylated
and detyrosinated tubulin [63]. In addition, we identified HDAC6 and sirtuin2 as
negative regulators of lumen formation due to their ability to reduce tubulin acetylation via their activity as tubulin deacetylases. siRNA suppression of these deacetylases increased lumen formation, while increased expression of them decreased it
[63]. Further support for this conclusion is that addition of the HDAC6 inhibitor,
tubacin, leads to increased EC lumen and tube formation [79]. Of great interest here
is that these modified tubulins accumulate in a polarized manner subapically during
EC lumen and tube assembly, while in contrast, filamentous actin (F-actin) accumulates in a distinct basal location [64, 79]. This subapical polarized region where
acetylated and detyrosinated tubulin accumulates is precisely the location where
vacuole and vesicle fusion events occur, to control the development of the apical
membrane surface during this process. Interestingly, Cdc42 shows strong colocalization with acetylated tubulin in these regions of vacuole/vesicle fusion [79].
Thus, one of the central features of the EC lumen formation process is the creation
of cytoskeletal asymmetry and tracks where vesicles can be trafficked to this subapical domain, to polarize the lumen formation process. Furthermore, our data suggests that this subapical membrane accumulation of modified tubulins is necessary
to stabilize the apical membrane and maintain a stable tube structure [63, 64, 79].
We demonstrated in past studies that disruption of microtubules led to rapid collapse of tube networks [10].
G. E. Davis