Collectively, the results presented in this study suggest that ITSN2 is involved in the activation of Cdc42 to regulate spindle orientation during mitosis. To perform this function, ITSN2 localizes to the centrosomes, both in interphase cells and in cells in mitosis. The EH domains of ITSN2 mediate the localization of ITSN2 at the centrosomes. Thus, ITSN2 operates as a spatial regulator for Cdc42 activity, which is also associated to the spindle poles during cell division. Then, Cdc42 is required somehow for the normal function of the spindle machinery, which is involved in regulating the orientation of the mitotic spindle at the centrosomes and for the normal formation of the lumen in epithelial morphogenesis.
The mechanisms and external factors that regulate spindle orientation in epithelial cells are poorly understood. The majority of these studies have been performed using asymmetric division models (for review see Siller and Doe, 2009
). However, it has been postulated that the main players of this process in mammalian epithelial cells may be the same, including the dynein–dynactin complex, Gα proteins, LGN, NuMA (nuclear mitotic apparatus protein), the Par complex, and the Rho GTPase Cdc42 (for review see Siller and Doe, 2009
). The molecular mechanism associated with the function of Rho GTPases is generally related to its subcellular localization (Iden and Collard, 2008
). We have shown that Cdc42 localizes to the mitotic spindle in the centrosomal region, where it can activate downstream effectors that control the mitotic spindle machinery. An essential question is how Cdc42 associates with the spindle poles. We found that ITSN2 is localized in centrosomes, through the EH domains of ITSN2. Therefore, ITSN2 could recruit and activate Cdc42 at the spindle poles. In fact, silencing of ITSN2 significantly reduces the amount of active Cdc42 and that of the Cdc42 associated with the spindle poles. In addition, ITSN2 seems to mediate protein–protein interactions with other centrosomal proteins through the SH3 domains, and thus, it might scaffold signaling platforms for Cdc42 at this location. Therefore, a potential mechanism for Cdc42 function in the orientation of the mitotic spindles might be mediated by ITSN2, which would be responsible not only for activating Cdc42 in the centrosomal region but also for scaffolding proteins involved in downstream signaling.
In addition to the data presented in this study for the role of Cdc42 by controlling mitotic spindle orientation in the MDCK cells, Cdc42 activity was also recently described to regulate epithelial morphogenesis and spindle orientation in Caco-2 cells, a mammalian intestinal model of epithelial morphogenesis (Jaffe et al., 2008
). However, the link between Cdc42, spindle orientation, and the formation of the lumen described previously was strictly correlative (Jaffe et al., 2008
). To clarify this issue, we have shown that disruption of LGN, a known essential component of the spindle orientation machinery, also caused a defect lumen formation similar to the defect caused by the silencing of ITSN2 and Cdc42, which suggests a direct relationship between these two processes.
The mechanism of spindle rotation may involve centrosome movement directed by interactions between the astral microtubules and the cell cortex. Therefore, the connection of astral microtubules to centrosomes and the cell cortex must be fully coordinated. A role for the actin cytoskeleton and motors such as the dynein–dynactin complex has been proposed in this process (Schuyler and Pellman, 2001
; Kunda and Baum, 2009
). The actin cytoskeleton is one of the main downstream effects of Rho GTPase function. To stabilize the interaction between the cell cortex, centrosomes, and the astral microtubules, Cdc42 regulates actin remodeling via different effectors such as Pak (p21-activated kinase), N-WASP, and formins in mammalian cells (Narumiya and Yasuda, 2006
). Moreover, in yeast, a Cdc42 GEF complex (Bem1–cdc24p, functionally similar to ITSN) interacts with PAK and regulates the polarization of the cell division machinery (Heil-Chapdelaine et al., 1999
). A previous study has elucidated a route in which Cdc42 may regulate the activation of Pak2 at the centrosomes, which in turn regulates the spindle assembly machinery, including Aurora A, dynein–dynactin, and NuMA (Mitsushima et al., 2009
). In other series of experiments, Cdc42 and its effector mammalian Dia3 have been shown to regulate the biorientation of the chromosomes, which involved attachment of the plus ends of microtubules to kinetochores, to ensure alignment of chromosomes during metaphase and their correct segregation during anaphase (Yasuda et al., 2004
). Therefore, although the function of Cdc42 in spindle positioning is clear, the effectors involved and the molecular mechanism acting downstream of Cdc42 remain to be identified.
Different epithelial tissues undergo events of massive membrane trafficking in the initial steps of morphogenesis (Bryant and Mostov, 2008
). Previous data have demonstrated a role for Cdc42 in vesicle trafficking in polarized epithelial cells (Müsch et al., 2001
). We have observed a function for Cdc42 in the exocytosis of gp135-containing vesicles and the remodeling of tight junctions in calcium switch experiments. In addition, we have found a greater effect of silencing Cdc42 on lumen formation and mitotic spindle orientation than silencing ITSN2. Our present data suggest the existence of another GEF as regulator for Cdc42 during this process. Interestingly, we have detected ITSN2 at intracellular locations in MDCK cells, which might be endosomal membranes. Multiple studies have demonstrated an important function for ITSN2 during endocytosis (Pucharcos et al., 2000
; McGavin et al., 2001
). Recently, it has been shown that endocytosis can act to promote cell polarity in different models (Georgiou et al., 2008
, Harris and Tepass, 2008
; Leibfried et al., 2008
). Even more, some proteins associated with endocytosis have been described to be required for spindle positioning in mitosis (Royle et al., 2005
; Liu and Zheng, 2009
). The possibility that ITSN2 regulates cell polarity through endocytosis remains to be investigated.
In summary, we have identified ITSN2 as an activator of Cdc42 at the centrosomes to regulate spindle orientation during mitosis. It has been recently described that the apical membrane and the lumen originate from the place where the midbody is formed during cytokinesis (Schlüter et al., 2009
). Therefore, the regulation of the orientation of the mitotic spindle, which determines the location of the midbody, would be essential to ensure the formation of a single lumen.