11
Interestingly, previous collaborative studies revealed the role of another small
GTPase, RhoJ, which also plays a role during these events [79, 105]. One of key
remaining questions is to define the specific role for each of these individual
GTPases. Together they appear to control membrane pinocytosis leading to intracellular vacuole formation, trafficking, and fusion of these vacuoles (and possibly
other vesicular structures such as Weibel-Palade bodies) to contribute membrane to
the developing apical surface. Interestingly, they appear to also regulate key cytoskeletal modifications such acetylation and detyrosination of tubulin which accumulates subapically to surround and regulate vacuole/vesicle transport (and thus
direct new membrane) to the apical domain [79]. In addition, they may stimulate
vesicle fusion and exocytic events to create this new polarized apical membrane.
Clearly, more work is needed here to define the role of these GTPases and their
downstream effectors at the different stages of this process. Of interest to this point
is our finding that Rasip1, a Ras and Rap effector, strongly targets apically during
the lumen formation process [79]. Apical targeting of Rac1, k-Ras, and Rap1b has
also been observed, and interestingly, Cdc42 appears to accumulate predominantly
subapically and can interestingly co-localize with acetylated tubulin in this subapical domain [79]. This co-localized region is where vacuole to vacuole fusion events
occur to create and expand the apical membrane surface.
1.3.5 Cdc42 Coupling to Cell Polarity Pathways Controls EC
Lumen and Tube Formation
A major function of Cdc42 is its ability to affect cell polarity signaling, by interfacing with the polarity proteins Par6, Par3, and atypical PKC isoforms [40, 73]. Cell
polarity signaling controls directional cell motility that involves Cdc42 [40]. In fact,
active Cdc42 (i.e., Cdc42-GTP) binds directly to Par6 which then couples to Par3, a
scaffold protein that also interacts with atypical protein kinase C isoforms, such as
PKCζ [73]. We reported that Cdc42-dependent EC lumen and tube formation was
dependent on Par6b, Par3, and PKCζ [66] (Fig. 1.3). Thus, this work reveals a fundamental role for Cdc42-dependent polarity signaling in EC tubulogenesis. Par3 is
known to interact with a number of other cell surface proteins including members
of the junction adhesion molecule (Jam) family (i.e., Jam-A, Jam-B, and Jam-C)
[38, 39]. Our most recent work reveals that Jam-B and Jam-C associate with Par3 in
ECs to control EC lumen formation in 3D collagen matrices [83]. Furthermore,
these Jam proteins co-assemble into a defined EC lumen signaling complex consisting of α2β1, MT1-MMP, Jam-C, Jam-B, Par3, Par6b, and Cdc42-GTP that is
responsible for the ability of ECs to form tubes in 3D collagen matrices [83] (see
later on). Disruption of any member of this complex markedly interferes with the
ability of ECs to form tubes [83]. These lumen signaling complexes are also directly
coupled to the kinase cascade discussed earlier including PKCε, Src, Pak, Raf, and
Erk1/Erk2, since blockade of these complexes completely interferes with the
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…
Interestingly, previous collaborative studies revealed the role of another small
GTPase, RhoJ, which also plays a role during these events [79, 105]. One of key
remaining questions is to define the specific role for each of these individual
GTPases. Together they appear to control membrane pinocytosis leading to intracellular vacuole formation, trafficking, and fusion of these vacuoles (and possibly
other vesicular structures such as Weibel-Palade bodies) to contribute membrane to
the developing apical surface. Interestingly, they appear to also regulate key cytoskeletal modifications such acetylation and detyrosination of tubulin which accumulates subapically to surround and regulate vacuole/vesicle transport (and thus
direct new membrane) to the apical domain [79]. In addition, they may stimulate
vesicle fusion and exocytic events to create this new polarized apical membrane.
Clearly, more work is needed here to define the role of these GTPases and their
downstream effectors at the different stages of this process. Of interest to this point
is our finding that Rasip1, a Ras and Rap effector, strongly targets apically during
the lumen formation process [79]. Apical targeting of Rac1, k-Ras, and Rap1b has
also been observed, and interestingly, Cdc42 appears to accumulate predominantly
subapically and can interestingly co-localize with acetylated tubulin in this subapical domain [79]. This co-localized region is where vacuole to vacuole fusion events
occur to create and expand the apical membrane surface.
1.3.5 Cdc42 Coupling to Cell Polarity Pathways Controls EC
Lumen and Tube Formation
A major function of Cdc42 is its ability to affect cell polarity signaling, by interfacing with the polarity proteins Par6, Par3, and atypical PKC isoforms [40, 73]. Cell
polarity signaling controls directional cell motility that involves Cdc42 [40]. In fact,
active Cdc42 (i.e., Cdc42-GTP) binds directly to Par6 which then couples to Par3, a
scaffold protein that also interacts with atypical protein kinase C isoforms, such as
PKCζ [73]. We reported that Cdc42-dependent EC lumen and tube formation was
dependent on Par6b, Par3, and PKCζ [66] (Fig. 1.3). Thus, this work reveals a fundamental role for Cdc42-dependent polarity signaling in EC tubulogenesis. Par3 is
known to interact with a number of other cell surface proteins including members
of the junction adhesion molecule (Jam) family (i.e., Jam-A, Jam-B, and Jam-C)
[38, 39]. Our most recent work reveals that Jam-B and Jam-C associate with Par3 in
ECs to control EC lumen formation in 3D collagen matrices [83]. Furthermore,
these Jam proteins co-assemble into a defined EC lumen signaling complex consisting of α2β1, MT1-MMP, Jam-C, Jam-B, Par3, Par6b, and Cdc42-GTP that is
responsible for the ability of ECs to form tubes in 3D collagen matrices [83] (see
later on). Disruption of any member of this complex markedly interferes with the
ability of ECs to form tubes [83]. These lumen signaling complexes are also directly
coupled to the kinase cascade discussed earlier including PKCε, Src, Pak, Raf, and
Erk1/Erk2, since blockade of these complexes completely interferes with the
1 Molecular Control of Capillary Tube Morphogenesis and Maturation Through…