In this work, using RNAi, we have examined the involvement of three mDia isoforms in cytokinesis and identified mDia2 as an essential mDia isoform required for cytokinesis in NIH 3T3 cells and C2C12 cells ( and Figure S3). We have also analyzed localization of the mDia isoforms and found that mDia2 localizes specifically in the cleavage furrow during cytokinesis. We have found that depletion of mDia2 substantially reduced the amount of F-actin accumulating in the cleavage furrow but did not affect accumulation of other contractile ring components such as RhoA, myosin, anillin, and pERM in the equatorial region. However, without mDia2, these components could not be properly integrated into the contractile ring, but apparently formed incomplete complexes, which were shifted away from the equatorial region and induced contraction at aberrant site of a dividing cell.
Our findings summarized above can thus not only suggest functions of mDia2 in cytokinesis but also address to some of the major questions regarding cytokinesis. First, given that mDia molecules can stabilize and align microtubules (Ishizaki et al., 2001
; Palazzo et al., 2001
; Yasuda et al., 2004
) and that the cleavage plane is suggested to be specified by spindle microtubules that are stabilized in the equatorial region (Canman et al., 2003
), it is possible that mDia isoforms are involved in determination of the cleavage plane. However, the above observation that depletion of mDia2 does not interfere with RhoA accumulation in the equator argues against this idea and rather suggests that RhoA accumulates first in the cleavage plane and recruit mDia2 there. This is consistent with the property of mDia2 to bind to members of the Rho GTPases such as RhoA, Rac1, and Cdc42 (Alberts et al., 1998
; Yasuda et al., 2004
). However, binding to RhoA cannot explain selective localization of mDia2 to the cleavage furrow, because other mDia isoforms can bind to RhoA as well. Further analysis is therefore required to elucidate a mechanism for selective localization of mDia2 to the cleavage furrow.
Second, it is intriguing that mDia2 localizes in the cleavage furrow and that its depletion reduced the F-actin amount there. Previously, it was argued whether F-actin is formed in the cleavage furrow or formed elsewhere and transported to the furrow (Wang, 2005
; Eggert et al., 2006
). Given that mDia molecules are capable of catalyzing actin nucleation and polymerization, our results strongly suggest that the majority of F-actin is produced in situ in the cleavage furrow by the action of mDia2 and accumulate there. Then, what functions do actin filaments induced by mDia2 exert in cytokinesis? Abnormal contraction at aberrant sites apparently by the contractile ring components including RhoA, myosin, anillin, and pERM in mDia2-depleted cells suggests that with mDia2 depletion and/or with depletion of mDia2-induced F-actin, the contractile ring is not properly organized and is not maintained at the prospective site of the cleavage furrow, which indicates that mDia2 and mDia2-induced F-actin link these components together to form the contractile ring and stabilize its position in the equatorial region. Formins such as mDia2 can produce long, straight actin filaments. The structure of the contractile ring was studied by electron microscopy in fission yeast and newt eggs, and these studies revealed that it consists of anti-parallel bundles of straight actin filaments that are bound to the plasma membrane through barbed ends (Mabuchi et al., 1988
; Kamasaki et al., 2007
). Although it is argued whether such structure is applied also to the contractile ring in mammalian cells (Eggert et al., 2006
), it is tempting to speculate that mDia2 induces straight actin filaments of opposite directionality in the prospective site of the cleavage furrow, which provide an actin-based scaffold encircling the equatorial region of dividing cells and facilitate formation of the contractile ring complex by incorporating other components of the ring such as myosin, anillin, and pERM to this actin scaffold. By such actions, mDia2 may restrict the movement of the contractile ring and stabilize its position. mDia2 may also function in anchoring the actin filaments of the contractile ring to the plasma membrane, because it accumulates in the equatorial cell cortex and its binds to the barbed end of actin filaments. Our results are thus consistent with and have substantially extended the findings by Dean et al. (2005)
, who examined localization of myosin in dividing Drosophila
S2 cells subjected to RNAi for diaphanous and showed that diaphanous is required for maintenance of myosin II to the cleavage furrow. It has to be mentioned, however, that construction of the contractile ring may not be governed solely by mDia2 but by interdependent actions of the contractile ring components including mDia2. Recently, anillin has been reported to bind myosin, and RNAi of anillin induces a phenotype similar to that we have found in mDia2-depleted cells (Straight et al., 2005
Finally, in this study, we also noted that the mitotic cells depleted of mDia2 exhibited mild oscillatory contractions during prometaphase and metaphase and that the cortical localization of myosin was not uniform as typically seen in control cells (Movies S4, S7, and S8). Mitotic cell rounding is the process in which a flat interphase cell becomes spherical and is associated with rearrangement of the actin cytoskeleton, de-adhesion, and an increase in cortical rigidity (Maddox and Burridge, 2003
; Thery and Bornens, 2006
). Maddox and Burridge (2003)
reported that mitotic cell rounding requires activation of RhoA. In this study, we observed that mDia2 localized to the cell margin in rounding NIH 3T3 cells and mDia2-depleted cells exhibit impaired mitotic rounding ( and B). The oscillatory contractions of mDia2-depleted cells mentioned above may be caused by impaired rigidity of mitotic cells in the absence of mDia2. Eisenmann et al. (2007)
showed that expression of Dip, which they claimed as an inhibitory binding protein for mDia2, induced nonapoptotic blebbing in HeLa cells, which is thought to be caused by breaks in cortical rigidity. These results suggest that, in addition to its action in cytokinesis, mDia2 also function in maintenance of cortical rigidity and rounding of mitotic cells. Given that cytokinesis is now recognized as a consequence of many events occurring globally in the cell cortex through cell division (Maddox and Burridge, 2003
; Wang, 2005
; Mukhina et al., 2007
), these results may suggest that mDia2 functions not only by inducing F-actin in the cleavage furrow but also by regulating the cortical rigidity globally. It may shift the balance of the contractility of mitotic cells by shifting its accumulation in the cell dependent on the phase of cell division. Elucidation of a mechanism how functions of mDia2 in different phases of cell division is regulated may unravel how cells execute mitosis and cytokinesis properly through adjusting cell morphogenesis with chromosome separation.