DTC migration is indispensible for gonadogenesis in C. elegans
hermaphrodites. Multiple factors are involved in facilitating this process by driving motility and/or regulating pathfinding. One of the key events during DTC migration is a change in direction by cell turning. Wild-type DTCs execute a turn away from the ventral basement membrane toward dorsal, and then they turn on the dorsal basement membrane toward the midbody (Cram et al., 2006
; Hedgecock et al., 1987
; Nishiwaki, 1999
). In this report, we have characterized mig-38
, a gene that is required for execution of the turn toward the midbody. mig-38
encodes a novel protein that acts in a DTC-autonomous manner to promote this turn on both the dorsal (as in wild-type) and ventral (as in the unc-6
) ventralized mutant) surfaces. Our results link this protein to cell adhesion and motility. First, the turning defects are exacerbated with double knockdown of MIG-38 and either INA-1/α integrin or MIG-15/NIK, which binds to PAT-3/β integrin cytoplasmic domain (Poinat et al., 2002
). Second, knockdown of mig-38
expression restores DTC motility that is lost with talin RNAi. Since talin also interacts with integrin receptors (Pfaff et al., 1998
; Tadokoro et al., 2003
; Wegener et al., 2007
), the suppressive effect when both genes are knocked down suggests that MIG-38 acts on DTC motility through a pathway that does not include talin.
The integrin heterodimer INA-1/PAT-3 has a major role in driving DTC motility (Baum and Garriga, 1997
; Meighan and Schwarzbauer, 2007
). This integrin is also involved in turning since simultaneous knockdown of mig-38
expression by RNAi significantly enhanced the penetrance of a no-turn, extended linear gonad arm phenotype. Turning defects were also observed with mig-38
RNAi treatment of ina-1
) hypomorphs, but the percentage of defects was not increased over RNAi treatment of wild type nematodes (unpublished observations). ina-1
) has a missense mutation in the extracellular domain of the integrin that does not affect ligand binding, and the distinctive phenotype of this ina-1
allele may result from interactions with two different extracellular ligands (Baum and Garriga, 1997
). Proteins that act on the cytoplasmic tails of the integrins, as proposed for MIG-38, may not be able to modulate the effects of this extracellular mutation. Reduction of mig-15/
NIK gene function produced the same turning defect as mig-38
RNAi, albeit with lower penetrance, and knockdown of both genes together significantly increased the number of turning defective gonad arms. Therefore, mig-38
genetically interacts with ina-1
and the associated kinase gene mig-15
to regulate turning. We propose that MIG-38 acts through MIG-15 to affect the function of the INA-1/PAT-3 heterodimer in this process ().
Fig. 6 Model for the role of MIG-38 in DTC turning. The integrin heterodimer INA-1/PAT-3 is required for motility and turning of the DTCs and its activity is regulated by two associated proteins, talin and MIG-15. (A) Downregulation of MIG-38 with either MIG-15 (more ...)
Talin promotes integrin activity by binding to the β integrin cytoplasmic domain (Pfaff et al., 1998
; Shattil et al., 2010
; Tadokoro et al., 2003
; Wegener et al., 2007
) and it also links integrins to the actin cytoskeleton (Critchley, 2009
). Loss of talin had a similar effect on DTC turning as loss of MIG-38 (this report and (Cram et al., 2006
), but talin is also required for DTC movement since reduction of its expression gave a significant proportion of short gonad arms. These effects on turning and motility suggest that talin binding to PAT-3/β integrin has a dual role in DTC migration. It is needed for forward motility so that when it is knocked down, the DTCs stop prematurely. For those cells that continue forward movement, talin is needed to change direction and, in its absence, the cells do not turn back toward the midbody region. Since talin binds directly to the β integrin cytoplasmic domain and stimulates integrin activity, the reduction-of-talin effects could result from reduced levels of integrin activity during movement and/or at the turn. Knockdown of talin expression will also change the connection between integrins and the actin cytoskeleton (Critchley, 2009
; Shattil et al., 2010
), which suggests that perturbation of cell shape changes may also contribute to the defects in motility and turning.
Our data show that reduction of talin expression causes a motility defect that is suppressed by MIG-38 depletion. This suggests that mig-38
affects a motility-promoting pathway, but one that is separate from talin (). Knockdown of MIG-15 or NCK-1 counteracted the suppressive effects of MIG-38 leading to an increase in short gonad arms, linking these molecules to cell motility. Vertebrate studies implicate MIG-15/NIK in regulating changes in cell morphology through phosphorylation of actin-modulating ERM proteins resulting in extension of lamellipodia (Baumgartner et al., 2006
). Also, mammalian NIK regulates adhesion through promoting integrin activation downstream of Eph receptors (Becker et al., 2000
). MIG-15/NIK is involved in cell movement and pathfinding of migrating neurons and neuroblasts in C. elegans
(Chapman et al., 2008
; Poinat et al., 2002
; Shakir et al., 2006
). This kinase also regulates development and maintenance of cell polarity in Q neuroblasts (Chapman et al., 2008
). In a loss-of-function mig-15
) mutant, more than one-third of the Q cells elaborated cell extensions in the wrong direction or failed to polarize at all (Chapman et al., 2008
). Similar to these findings in neurons, our data point to a dual role for MIG-15/NIK in controlling motility and directionality of DTCs through integrins ().
The binding partner of MIG-15/NIK, NCK-1/Nck adapter protein, serves as a link between cell surface receptors, including integrins, and the actin cytoskeleton (Buday et al., 2002
; Li et al., 2001
). Numerous signaling molecules bind to Nck in different cellular systems, and assessing the physiological importance of these interactions is a subject of active investigation (Buday et al., 2002
; Funasaka et al., 2010
; Lettau et al., 2009
). Nck has been implicated in axon guidance and cell migration in flies (Buday et al., 2002
), and our data link NCK-1 to cell motility since its loss increased the penetrance of the short gonad arm phenotype in the absence of talin. Therefore, we suggest that the MIG-15/NCK-1 complex is involved in promoting DTC motility ().
Can we reconcile a synergistic role for MIG-38 with one integrin-associated protein, MIG-15, and an opposing role with another, talin? We propose that both MIG-15/NCK and talin contribute to motility through their interactions with PAT-3/β integrin (). Our data suggest that mig-38 negatively impacts the function of the MIG-15/NCK-1 complex such that the reduction of both MIG-38 and talin allows this complex to substitute for talin and promote motility. The proposed inhibitory effect of a MIG-38 pathway on MIG-15/NCK-1 may promote dissociation of the complex, thus allowing MIG-15 to interact with other factors and participate in integrin-mediated turning (). In this model, MIG-38 positively affects MIG-15 and its interaction with INA-1/PAT-3 integrin to promote a change in the direction of DTC migration (). Given its apparent localization to both the cytoplasm and nucleus, MIG-38 could act directly on this pathway or indirectly perhaps through effects on gene expression. Thus when MIG-38 is depleted, stimulation of a turning pathway is reduced, but MIG-15 could still couple with NCK-1 and promote DTC motility.
Mechanistically, MIG-38 might function in turning through control of cell polarity. MIG-15/NIK has previously been shown to control the development of cell polarity (Chapman et al., 2008
) and our findings show a synergistic effect between MIG-38 and MIG-15/NIK in turning raising the possibility that MIG-38 acts as a switch from forward movement to turning by invoking MIG-15 function in repolarization.