Primary tumor cells continue to evolve novel modes of metastatic escape into distant tissues. These escape strategies require migratory plasticity, and are characterized by interconvertability between mesenchymal and amoeboid cancer cell motility programs. Morphological plasticity relies upon localized disassembly/reassembly of cytoskeletal structures that either drive and/or support the traction and protrusion cycles. Central to this process are Rho family GTP-binding proteins and their downstream effecters, formins. Here we demonstrate a functional role for mDia2 and its binding partner DIP in the interconversion of mesenchymal and amoeboid motility modes in cancer cells in 2D and 3D matrices. Co-recruitment of DIP and mDia2 to the plasma membrane is co-incident with bleb extrusion and blebbing is dependent upon DIP expression and mDia2 function. Our data reveal a physiological trigger for DIP-directed mDia2-dependent blebbing in mesenchymal breast cancer cells- CXCL12- which stimulates blebbing both in 2D and 3D matrices, while driving the assembly of the DIP:mDia2 complex to blebs.
The migratory plasticity in both 2D and 3D was dependent upon mDia2 and DIP expression/activation. As the Rho-dependent release of autoinhibited mDia2 is required for DIP:mDia2 interaction 
, these data suggest an important interplay between Rho activation and the DIP-driven mDia2-dependent amoeboid transition in both 2D and 3D matrices. This contrasts with 2D studies suggesting that DIP, through association with p190RhoGAP and VAV2, suppresses RhoA activity to negatively impact stress fiber formation and cellular migration 
. Forced DIP overexpression in COS cells induced a transient decrease in GTP-RhoA upon EGF stimulation that was recoverable within 20 min. Rac-GTP levels were simultaneous elevated, consistent with lamellipodia formation, although DIP expression alone was not sufficient to activate Rac in another study 
. In our study, in 2D and 3D matrices, EGF (Figure S1
, data not shown) or CXCL12 induced an amoeboid transition in MDA-MB-231 cells through 60 min and DIP depletion in MDA-MB-435S cells induced a mesenchymal transition. These data suggest that Rho activity predominates the cellular signaling milieu to induce the formation of the mDia2:DIP signaling axis, thus maintaining an amoeboid morphology. However, it must be considered that spatial and temporal localization of DIP with its binding partners (e.g
., mDia2, N-WASp, palladin, p53 IRES or Src 
) may control the assembly of DIP-directed complexes that functionally regulate actin dynamics. Importantly, our studies examine the contribution of DIP upon membrane dynamics in the context of 3D matrices, where the activity of Rho GTPases controlling migratory plasticity and the nature of actin-rich protrusion formed can be very distinct from those observed in 2D.
DIP and mDia2 predominantly co-associate at membranes associated with blebs in M2, MDA-MB-231 and MDA-MB-435S cells. These data point to a potential mechanism for blebbing and amoeboid transitions (). In this model, localized GTP-Rho binds to and releases the autoinhibited mDia2 (Step 1), allowing it to complex with DIP at the cell cortex (Step 2). The consequence is disruption of F-actin assembly at the membrane and localized weakening of the actin cortex to induce bleb extrusion. Our data demonstrate distinct localization of mDia2 alone to the rim of extruded blebs (), revealing a second potential function for mDia2 in the re-assembly of the F-actin cortex preceding bleb retraction 
(Step 3, 4a). RhoA localized with F-actin to the rims of extruded and retracting blebs 
, suggesting a role for RhoA effecter proteins like mDia2 in maintaining blebs. However, mDia2 interacts with multiple Rho GTPases, including RhoA–C (reviewed in 
) and Cdc42 
. While it is uncertain if specific mDia2-interacting Rho GTPases drive the blebbing process, FHOD-mediated blebbing was dependent upon interaction with Rac 
, while RhoA was required for SH4-domain-induced blebbing 
. Yet, GTP-Rac was shown to negatively regulate pMLC2, diminishing actinomyosin contractility and inhibiting the amoeboid motility program; Rho/ROCK pro-amoeboid signaling conversely directed Rac inhibition, via activation of the Rac GAP ARH-GAP22 
, indicating a tightly controlled balance in Rho/Rac signaling in driving morphological transitions. Finally, we observed punctate DIP and mDia2 co-localization to the bleb rims and propose that this interaction induces a secondary break in the re-establishing F-actin cortex to mediate formation of compound blebs, or lobopodia 
mDia2:DIP-driven mechanism for bleb and lobopodia formation: bulbous structures driving amoeboid motility in 3D matrices.
Contrary to our previous biochemical studies 
and the above mechanism, recent studies in S. pombe
revealed that the yeast DIP orthologue, dip1p, failed to inhibit assembly of actin cable at the cell tips 
and did not act as a formin regulator. However, the for3p FH2 domain shares less than 40% identity with the FH2 domain of its murine orthologue, mDia2, and subtle differences between mDia FH2 domains influence DIP’s ability to negatively regulate FH2-mediated F-actin assembly; despite their similarity, DIP binds both the mDia1 and mDia2 FH2 domains, yet only negatively regulates the latter 
. As with mDia1, the for3p FH2 domain may preclude dip3p from acting as a negative regulator, whereas other S. pombe
FH2 domain-containing formins may be potent targets for dip1p-mediated inhibition. Currently, the exact nature of subtleties in the primary sequence or secondary structures of FH2 domains conferring both the ability to bind and be regulated by DIP are unclear. Interestingly, while both the mDia1 and mDia2 FH2 domains polymerize F-actin, only mDia2 bundles F-actin filaments 
. Further studies would be interesting to determine the parameters FH2 domains must conform to in order to be regulated by DIP.
It is uncertain if DIP-directed blebs in invading amoeboid cells are polarized towards a chemotactic gradient to drive protrusion and directional migration. Invading MDA-MB-231 cells formed copious blebs that polarized to the contracting cell rear proximal to the uropod, and migration speed and uropod formation were mDia dependent 
, suggesting that blebbing may drive polarized contraction as opposed to forward membrane extension. However, Marshall and colleagues revealed the presence of a rigid, ezrin-enriched uropod at the rear of amoeboid, invasive A375 melanoma cells 
; the presence of the rearward ROCK-dependent uropod inhibited proximal bleb formation, essentially establishing rear polarity through bleb inhibition. Our data show that MDA-MB-231 cells invading towards CXCL12 through collagen matrices have polarized compound blebs extending towards the gradient (), indicating a potential role for CXCL12 in directing bleb and/or cellular polarity. In fact, during zebrafish germline migration, CXCL12 gradients drive localized increases in Ca2+
to establish and stabilize protrusions that become the leading edge of the cell 
. Experiments directed at understanding contributions of chemokine gradients to uropod formation and/or bleb polarization at the front/rear edge of migrating cancer cells are indeed warranted.
Finally, our data reveal a novel role for CXCL12 in inducing amoeboid transitions in MDA-MB-231 cells, and in recruiting the mDia2:DIP complex to blebs. CXCL12 and its receptor CXCR4 are implicated in the metastasis of several cancers including breast cancer metastasis to lung and lymph node 
. CXCL12 is highly expressed in tissues that are prevalent sites of distant breast cancer metastasis 
. CXCR4 knockdown decreased MDA-MB-231 cell invasion 
. Therefore, amoeboid transitions in migrating breast cancer cells induced via exposure to CXCL12 may play a role in one or multiple steps in metastasis. Indeed, Rho degradation via the ubiquitin ligase Smurf1 locally impaired Rho/ROCK signaling and promoted mesenchymal motility, while Smurf1 inhibition induced amoeboid motility in tumor cells in subcutaneous xenografts 
. This transition was accompanied by dramatic increases in the number of tumor cells in and proximal to blood vessels, suggesting a linkage between amoeboid motility and intravasation. Ultimately, dynamic transition between mesenchymal and amoeboid motility may function during distinct stages of the metastatic process (e.g.,
localized invasion) when the microenvironment requires morphological adaptation of tumor cells. Together, these studies and data presented herein demonstrate an important role for DIP-directed mDia2-dependent F-actin dynamics in regulating morphological plasticity in motile cancer cells, and reveal a novel role for CXCL12 in inducing amoeboid transitions within 3D matrices. These studies underscore the importance of understanding amoeboid motility in promoting mechanistic steps driving tumor metastasis.