Pattern formation during organogenesis requires precise cytoskeletal alterations in response to a variety of morphogenic stimuli. Our data show that the modular Shh effector MIM directly remodels the actin cytoskeleton by bundling actin. MIM activity is inducible and can be controlled by regulating expression via Shh signaling or by modulating activation domain interactions with RPTPδ.
The data reported here point out a crucial role for the coiled-coil domain in MIM-dependent bundling activity. In vitro, the dimerization domain aligns two actin filament binding domains to allow bundling to occur, just as it does in α-actinin and other bundling proteins. The biochemical and genetic data presented in this work with full-length MIM, in conjunction with previous biochemical data using the MIM NH2
terminus (Yamagishi et al., 2004
), suggest that there is a specific interaction between the MIM dimer and actin filaments, although the exact stoichiometry, affinity, and orientation of binding of the protein on the filament will require more careful biophysical studies. However, the importance of this domain is illustrated by the observation that having the activation domain without the coiled-coil domain (MIMΔN399 or MIMΔN159) is not sufficient for membrane association, strong bundling, or RPTP relocalization. This suggests that in the cell, recognition of MIM by RPTPs at the membrane requires a three-dimensional surface provided by the alignment of the dimerization domain. Interestingly, a search of GenBank sequences reveals two other proteins that have related dimerization sequences, the recently identified ABBA (Yamagishi et al., 2004
) and IRSp53 (Miki et al., 2000
; Nakagawa et al., 2003
), which share 90% and 25% identity, respectively. The similarity between MIM and these dimerization domains suggests that MIM may form heterodimers with other family members, much like members of the plakin or ezrin/radixin/moesin subfamilies of cytoskeletal regulators. Preliminary data suggest that MIM can form heterodimers (unpublished data) with ABBA, which points to additional diversity in the ability to generate cytoplasmic projections.
Our data suggest that MIM belongs to a growing family of cytoskeletal regulators that have transcriptional effects. Previously reported data indicate that MIM forms a cytoplasmic complex with Suppressor of Fused and the transcription factor Gli to regulate transcription (Callahan et al., 2004
). This nuclear effect is in direct contrast to the cytoplasmic and membrane effects of actin bundling shown here. Because of recent data suggesting a role for actin binding in transcription (Olave et al., 2002
), we considered the possibility that transcription was dependent on the MIM bundling domain. However, we observed that MIM potentiates transcription even without the self-association or WH2 domains that are required for actin bundling or monomeric actin binding. This supports the idea that actin bundling and transcriptional potentiation are mediated through distinct domains. Other proteins have been identified and suggested to regulate the cytoskeleton and transcription, including the Wnt pathway regulators β-catenin and plakoglobin (Moon et al., 2002
; Maeda et al., 2004
). Interestingly, the identification of separable domains differs from other regulators such as β-catenin that use the same domain (armadillo repeats 3–8) to bind to either adherens junctions or to TCF transcription factors (Rubinfeld et al., 1993
; Su et al., 1993
; Hulsken et al., 1994
; Sadot et al., 1998
Another aspect of the modular nature of MIM is the identification of distinct sequences outside the actin bundling domain that regulate bundling activity at sites of cytoplasmic projections. Colocalization studies, together with binding and cell biological experiments with a blocking polypeptide, support an important interaction domain between the RPTP D2 domain and MIM amino acids 408–538 (–). RPTPs are known to assemble into large complexes of proteins that regulate the subjacent cytoskeleton during retinal and motor neuron axon pathfinding (for review see Johnson and Van Vactor, 2003
). Recent data indicate that some associated proteins function to localize RPTPs to focal adhesions and neuronal synapses. For example, liprin binds to the D2 domain of another type IIa RPTP, LAR, and is required for LAR function at the synapse, in part by localizing LAR to the synapse (Serra-Pages et al., 1995
; Kaufmann et al., 2002
). Our data suggest a similar function for the activation domain of MIM on RPTPδ to assemble both at the membrane into specialized membrane domains. Future experiments will address whether liprin and MIM are part of the same complex and direct the RPTPs to similar or different compartments at the membrane.
The activation domain of MIM greatly enhances MIM cytoskeletal remodeling in vivo through interaction with RPTPδ. Because the cross-linking activity of many bundling proteins is activated by dephosphorylation (Zhai et al., 2001
), it is tempting to speculate that MIM activity could be controlled via a competition between tyrosine phosphatases and tyrosine kinases, such as Abl or Src. This is consistent with the known association of Abl kinase with Type IIa RPTPs (Wills et al., 1999
). Supporting this idea is the strong effect of phosphatase inhibitors on MIM localization and cytoskeletal activity. However, the fact that MIM408-GAP43 rescues much of the cytoskeletal phenotype by localizing MIM to focal adhesions () suggests that RPTP may be playing a localizing, rather than a catalytic, role with MIM. This is supported by the ability of MIM to localize a catalytically dead RPTP to the membrane () and our observation that the apparent size of MIM protein does not change in vanadate-treated cells (unpublished data). Similar results have been seen with the fly LAR protein, in which catalytically inactive LAR can rescue LAR-null animals (Krueger et al., 2003
). We speculate that modification of non-RPTP accessory proteins may be required to activate MIM-dependent actin bundling activity at the membrane.
Our data provide a framework for how actin bundling proteins like MIM may coordinate effects of both global and local signaling pathways on the cytoskeleton during development. Morphogens such as Shh induce cytoskeletal regulators such as MIM and then rely on MIM's interaction with RPTPs to localize actin bundles. Interestingly, in the neural tube, MIM localizes to Shh-dependent and Islet-1–positive motor neurons, which have been shown to express RPTPδ in rats (Sommer et al., 1997
). This suggests that Shh signaling and RPTP may cooperate to control motor neuron morphogenesis through MIM during spinal cord development. Future studies to examine how the activation domain of MIM regulates precise cytoskeletal changes in vivo will enhance our understanding of how morphogens such as Shh control organogenesis.