Formin-like 2 drives amoeboid invasive cell motility downstream of RhoC

Abstract

Invasive cell migration is a key step for cancer metastasis and involves Rho GTPase-controlled reorganization of the actin cytoskeleton. Altered Rho GTPase expression is found in various malignancies. Particularly, the closely related GTPases RhoA and RhoC are upregulated in many aggressive tumours, but specific effectors that distinguish between these two GTPases to explain mechanistic differences have not been identified. The formins are by far the largest family of Rho GTPase effectors and are characterized by the actin-nucleating formin homology 2 domain. Using siRNA-based screening against all 15 human formins, we systematically analysed their functions in 3D cell motility using three different cancer cell lines. These results reveal distinct requirements for specific formins in amoeboid versus mesenchymal invasive cell migration. Importantly, by knocking down all Rho proteins, we identified formin-like 2 (FMNL2) as a specific RhoC effector, showing selective interaction of FMNL2 with active RhoC, but not RhoA or RhoB. Functional analysis shows that RhoC regulates autoinhibition of FMNL2, whereas suppression of FMNL2 inhibits RhoC-, but not RhoA-dependent, rounded invasive cell migration. Thus, our data uncover a novel regulatory and functional interaction between RhoC and FMNL2 for modulating cell shape and invasiveness and provide mechanistic insight into RhoC-specific signalling events.

The actin-associating protein Tm5NM1 blocks mesenchymal motility without transition to amoeboid motility

“…Importantly, amoeboid motility is highly dependant on extensive reorganization of the actin cytoskeleton (Eichinger et al, 1999;Sahai, 2007;Kitzing et al, 2010). We have shown that elevated Tm5NM1 causes reduced sensitivity to agents that reorganize the actin cytoskeleton (Bryce et al, 2003;Creed et al, 2008), therefore, Tm5NM1 may block the dynamic response of the cytoskeleton that is required to switch to amoeboid type motility.…”

Protein N-Myristoylation Is Required for Cellular Morphological Changes Induced by Two Formin Family Proteins, FMNL2 and FMNL3

“…29) FMNL2 is also autoinhibited and is activated by GTPase RhoC. 30,31) In contrast, FMNL3 has been found to be constitutively active in cells even though its DID and DAD bind to one another in vitro. 29) However, a recent study found that FMNL3, but not FMNL2, is a RhoC-specific target, and is involved in cancer cell invasion.…”

Differing and isoform-specific roles for the formin DIAPH3 in plasma membrane blebbing and filopodia formation

“…Similar to a previous screen for the role of formins in cancer cell invasion [28], silencing against all 21 actin nucleators known to be expressed in human non-muscle cells was achieved by transfection with SMARTpool siRNAs consisting of four distinct siRNAs against each target. 96 h post transfection, cells were replated and the frequency of cells with prominent PM blebs quantified.…”

“…qPCR was performed with 7500 System Software (Applied Biosystems) using Power SYBR Green PCR Master Mix (Applied Biosystems). Primers used for qPCR are summarized in Supplementary information, Figure S9 and were partially validated previously [28]. The DIAPH3 amplicon is between 3154-3255 bp of DIAPH3 Isof3 and is thus present within all putative isoforms except for Isof2.…”