Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Jun 5;22(11):1005-12.
doi: 10.1016/j.cub.2012.03.064. Epub 2012 May 17.

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Jennifer Block  1 , Dennis Breitsprecher, Sonja Kühn, Moritz Winterhoff, Frieda Kage, Robert Geffers, Patrick Duwe, Jennifer L Rohn, Buzz Baum, Cord Brakebusch, Matthias Geyer, Theresia E B Stradal, Jan Faix, Klemens Rottner
Affiliations

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Jennifer Block et al. Curr Biol. .

Abstract

Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.

PubMed Disclaimer

Figures

Figure 1
Figure 1
FMNL2 Is Widely Expressed and Accumulates at the Tips of Lamellipodia and Filopodia (A) Microarray analyses indicating high levels of FMNL2 gene transcription in various murine and human cell lines. (B) Confirmation of FMNL2 expression by western blotting in cell lines as in (A). α-tubulin was used as loading control; FMNL2 migrates at an apparent molecular weight of 150 kDa plus ∼30 kDa for the EGFP-tagged variant. The additional band most prominent in murine cell lines corresponded to FMNL3 (Figure S3G), as indicated. (C) Domain structure and splice variants of FMNL2 are illustrated. DID, Diaphanous inhibitory domain; DD, dimerization domain; FH1/2, formin homology 1/2; WH2, WASP homology 2; DAD, Diaphanous autoregulatory domain. FMNL2 is spliced into at least two isoforms, designated FMNL2A (Uniprot identifier Q96PY5-1) and FMNL2B (Uniprot identifier Q96PY5-3), which differ at their C-terminal ends as depicted. (D) Immunolabeling showing that endogenous FMNL2/FMNL3 are concentrated in F-actin-rich lamellipodia (white arrows, zoom-in: Figure S1A) of B16-F1 melanoma cells. Scale bar represents 30 μm. (E) EGFP-tagged, constitutively active FMNL2 (ΔDAD) targets to the tips of protruding filopodia (white arrows) and lamellipodia (white arrowheads) but is displaced in the course of retraction (asterisk; see also Figures S1B and S1C). Time is in s; scale bars represent 2 μm (left panel) and 10 μm (right panel).
Figure 2
Figure 2
FMNL2 Is Regulated by Cdc42 and Myristoylation (A–C) ITC measurements of the interactions between triphosphate-loaded Rho-GTPases and purified FMNL2 N terminus (FMNL2N, aa 2–379). (A) Binding of Cdc42⋅GppNHp to FMNL2N revealed a dissociation constant (KD) of 3.13 ± 0.38 μM. ΔH and TΔS corresponded to −5.33 ± 0.16 kcal/mol and 2.19 kcal/mol, respectively, with a molar ratio of 0.51. No binding was detected with either diphosphate-loaded Cdc42 (B) or Rac1⋅GppNHp (C). (D) Subcellular targeting of FMNL2 is affected by constitutively active Cdc42, but not Rac1. EGFP-FMNL2 expressed in B16-F1 cells is entirely cytosolic (top) but targeted to the cell periphery upon coexpression of active Cdc42 (middle). Constitutively active Rac1 (bottom) lacked this effect, consistent with ITC measurements (C) and despite significant lamellipodia stimulation. Scale bar represents 10 μm. (E) FMNL2 and FMNL3 (but not FMNL1) harbor the canonical myristoylation consensus sequence MGXXXS at their N termini. (F) FMNL2B capable of myristoylation due to C-terminal tagging with EGFP (myr-FMNL2-EGFP) targets to protruding lamellipodia in B16-F1 cells (left), but an analogous construct harboring a point mutation prohibiting myristoylation (G2A-FMNL2-EGFP) does not (middle). Targeting of G2A-FMNL2-EGFP is restored by coexpression with constitutively active Cdc42 (right). Scale bars represent 10 μm. (G) Myristoylation and Cdc42 both mediate activation of FMNL2, but not its subcellular targeting. Summary of subcellular localization of constructs as depicted in B16-F1 cells or fibroblasts lacking Cdc42 (Cdc42−/−) and their parental controls (Cdc42+/−). For images see Figures S3A and S3B. Specific accumulation in the lamellipodium was categorized as follows: +++, very strong localization; ++, clear, specific enrichment; +, weak enrichment; (+), weak localization in some but not all cells examined; (−), no enrichment; n.d., not determined. Note that nonmyristoylatable, active FMNL2 (G2A-FMNL2ΔDAD-EGFP) localizes to lamellipodia even in Cdc42−/− cells.
Figure 3
Figure 3
FMNL2 Requires Profilin to Elongate Actin Filaments Derived from Spontaneous or Arp2/3 Complex-Mediated Nucleation (A) FMNL2 inhibits actin polymerization in the absence of profilin. A total of 3 μM G-actin (10% pyrene labeled) was polymerized in 1× KMEI buffer in the presence of FMNL2(8P)-C at concentrations as indicated. (B) FMNL2 processively assembles actin filaments. Spontaneous assembly of 1.3 μM actin (23% Alexa 488 labeled) in the presence of 20 nM FMNL2(8P)-C (top) or 5 nM FMNL2(8P)-C and 5 μM profilin (middle) or 10 nM FMNL2(21P)-C and 5 μM profilin (bottom) monitored by TIRFM. Circles mark barbed ends captured by FMNL2 molecules passively absorbed to the coverslip; arrows indicate dim, buckling filaments elongated by FMNL2. Time is in seconds. Scale bars represent 10 μm. See Movie S3. (C) Examples of individual, spontaneously growing, or FMNL2(21P)-C-assembled filaments increasing in length over time in the presence or absence of profilin. (D) Quantification of actin filament elongation rates as determined by TIRFM. (E) FMNL2 does not significantly enhance nucleation in the absence or presence of profilin. Filament numbers counted for actin alone or upon addition of profilin (5 μM) or FMNL2(21P)-C (10 nM), or both, as indicated. (F) FMNL2 elongates actin filaments nucleated by Arp2/3 complex. Spontaneous assembly of 1.3 μM actin (23% ATTO 488 labeled) in the presence of the proteins indicated. Arrowheads mark branch points generated by Arp2/3 complex, except for red ones that mark branch and/or filament end capped by FMNL2 in the absence of profilin. White circles mark barbed ends of spontaneously growing filaments, and green, blue, or white circles at the bottom mark dim filaments elongated by FMNL2. Scale bars represent 5 μm. See Movie S4. (G) Quantification of filaments elongated by FMNL2(21P)-C and profilin in the presence or absence of activated Arp2/3 complex at concentrations as in (F). (H) Quantification of actin filament elongation from experiments as shown in (G). Error bars in (D), (E), (G), and (H) represent mean ± SD from at least three independent measurements. See Figure S3.
Figure 4
Figure 4
FMNL2 Regulates Rates of Lamellipodia Protrusion and Cell Migration (A) Western blot using anti-FMNL2 antibody and extracts from B16-F1 cells transfected with mock construct or a vector mediating knockdown of FMNL2 (FMNL2 RNAi). α-tubulin was used as a loading control. (B) Analysis of protrusion rates of lamellipodia formed in mock or FMNL2 knockdown cells, with transfected cells identified by GFP expression. Insets show representative regions from images recorded by phase-contrast time-lapse microscopy and subjected to protrusion rate analyses as indicated. Time is in minutes and seconds; scale bars in overview panels represent 20 μm and in insets 5 μm. (C) Box and whisker plots showing results from quantification of lamellipodia protrusion velocities in mock (arithmetic mean, 1.26 μm/min; SD, 0.62 μm/min) and FMNL2 knockdown cells (arithmetic mean, 0.96 μm/min; SD, 0.62 μm/min). The line within the box indicates median, and the box boundaries contain 50% (25%–75%) and the whiskers 80% (10%–90%) of all measurements, whereas outlying points are shown as dots. Difference in protrusion velocity was confirmed to be statistically significant by Mann-Whitney rank sum test. (D) Migration efficiency of B16-F1 control (arithmetic mean, 0.36 μm/min; SD, 0.13 μm/min) versus FMNL2 knockdown cells (arithmetic mean, 0.298 μm/min; SD, 0.12 μm/min). Data were displayed and confirmed to be statistically significant as in (C). Data in (C) and (D) are derived from at least three independent experiments. (E) Model for FMNL2 function in lamellipodium protrusion is illustrated. Actin filaments are generated by nucleation through Arp2/3 complex or perhaps alternative mechanisms (actin nucleator). Engagement of FMNL2 is preceded by release from autoinhibition possibly mediated by a myristoyl switch (not shown) and by interaction with active Cdc42. FMNL2 dimers then capture preformed actin filament barbed ends and promote their elongation in a profilin/actin-dependent manner. Elongation by FMNL2 is proposed to accelerate lamellipodium protrusion in coordination with other factors, e.g., the Ena/VASP family member VASP. Cdc42 contributes to activation but not recruitment of FMNL2 to the lamellipodium tip, which is mediated by an unknown factor (X) remaining to be established. See Figure S3 and Movie S5.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Campellone K.G., Welch M.D. A nucleator arms race: cellular control of actin assembly. Nat. Rev. Mol. Cell Biol. 2010;11:237–251. - PMC - PubMed
    1. Ridley A.J. Life at the leading edge. Cell. 2011;145:1012–1022. - PubMed
    1. Schönichen A., Geyer M. Fifteen formins for an actin filament: a molecular view on the regulation of human formins. Biochim. Biophys. Acta. 2010;1803:152–163. - PubMed
    1. Chesarone M.A., DuPage A.G., Goode B.L. Unleashing formins to remodel the actin and microtubule cytoskeletons. Nat. Rev. Mol. Cell Biol. 2010;11:62–74. - PubMed
    1. Faix J., Rottner K. The making of filopodia. Curr. Opin. Cell Biol. 2006;18:18–25. - PubMed

Publication types

LinkOut - more resources