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FHOD1 interaction with nesprin-2G mediates TAN line formation and nuclear movement

Nature Cell Biology volume 16, pages 708–715 (2014)Cite this article

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Abstract

Active positioning of the nucleus is integral to division, migration and differentiation of mammalian cells1. Fibroblasts polarizing for migration orient their centrosomes by actin-dependent nuclear movement2. This nuclear movement depends on nesprin-2 giant (N2G), a large, actin-binding outer nuclear membrane component of transmembrane actin-associated (TAN) lines that couple nuclei to moving actin cables3. Here, we identify the diaphanous formin FHOD1 as an interaction partner of N2G. Silencing FHOD1 expression or expression of fragments containing binding sites for N2G or FHOD1 disrupted nuclear movement and centrosome orientation in polarizing fibroblasts. Unexpectedly, silencing of FHOD1 expression did not affect the formation or rearward flow of dorsal actin cables required for nuclear positioning. Rather, N2G–FHOD1 interaction provided a second connection to actin cables essential for TAN line formation and thus nuclear movement. These results reveal a unique function for a formin in coupling an organelle to actin filaments for translocation, and suggest that TAN lines require multi-point attachments to actin cables to resist the large forces necessary to move the nucleus.

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Figure 1: FHOD1 interacts with N2G.
Figure 2: FHOD1 is required for nuclear movement.
Figure 3: FHOD1 is dispensable for formation of dorsal actin cables and retrograde actin flow.
Figure 4: FHOD1 is essential for TAN line formation.
Figure 5: The N-terminal actin-binding site of FHOD1 provides N2G with an additional contact to actin filaments required for TAN line formation.

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  • 06 June 2014

    In the version of this Letter originally published online, the fourth name in the author list was written incorrectly; it should have read 'G. W. Gant Luxton'. This has been corrected in all versions of the Letter.

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Acknowledgements

We thank D. Discher and M. Geyer for helpful discussion. This work was financially supported in part by the Deutsche Forschungsgemeinschaft (GRK1188 to S.K., grant FA 378/6-2 to O.T.F.) and NIH (grant GM099481 to G.G.G.). O.T.F. is a member of the CellNetworks Cluster of Excellence EXC81.

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Author notes

  1. G. W. Gant Luxton

    Present address: Present address: Department of Biology, University of Minnesota, Minneapolis, Minnesota 55455, USA.,

  2. Stefan Kutscheidt, Ruijun Zhu and Susumu Antoku: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, Heidelberg, INF 324, 69120 Heidelberg, Germany

    Stefan Kutscheidt & Oliver T. Fackler

  2. Department of Pathology and Cell Biology, Columbia University, New York, New York 10032, USA

    Ruijun Zhu, Susumu Antoku, G. W. Gant Luxton & Gregg G. Gundersen

  3. Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5S 3E1, Canada

    Igor Stagljar

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Contributions

G.G.G. and O.T.F. conceived the study, designed experiments and wrote the manuscript. S.K., R.Z., S.A., G.W.L. and I.S. designed and conducted experiments and discussed and interpreted the data together with O.T.F. and G.G.G.

Corresponding authors

Correspondence to Oliver T. Fackler or Gregg G. Gundersen.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Additional evidence that the interaction between FHOD1s N-terminus and nesprin-2G (N2G) is specific and phylogenetic comparison of the FHOD1 interacting region of N2G.

(a) Schematic of N2G and FHOD1 with boundaries for constructs used in membrane yeast-two hybrid. (b) Legend for yeast two-hybrid indicating the FHOD1 fragments used as a bait for the experiment shown in panel c. (c) Yeast two-hybrid results for the interaction between N2G J, H and I fragments and FHOD1 fragments indicated in panel b. Bar, 5 mm. (d) N2G HI fragment interacts with HA-FHOD WT in cell lysates. GFP-N2G HI was expressed alone or co-expressed with HA-FHOD1 WT in 293T cells and lysates were immunoprecipitated with HA antibody. Western blots were probed with antibodies for HA and GFP. Input shows level of expression of transfected proteins. (e) Phylogenetic comparison between spectrin repeats (SRs) 9–13 of N2G. Red indicates residues conserved between at least four of the five species; pink indicates residues that are conserved in at least three of the species. Consensus residues are shown below for highly conserved positions. Sequences were obtained from the following sources. Human (H. sapiens, NP_878918.2, NCBI), Mouse (M. musculus, NIH3T3 fibroblast cDNA), Chicken (G. gallus, XP_003641488, NCBI), Frog (X. tropicanis, XP_002933763, NCBI), Fish (D. rerio, F1QVC9, Uniport).

Supplementary Figure 2 FHOD1 knock down by siRNAs.

Western blot of FHOD1 levels in NIH3T3 fibroblasts after knockdown with four different siRNAs targeting FHOD1. Control siRNA knockdown of GAPDH is shown for comparison. Vinculin is a loading control.

Supplementary Figure 3 FHOD1 knockdown does not affect actin structures induced by serum.

Starved NIH3T3 fibroblasts were stimulated with 20% FCS, fixed at indicated time points and stained with rhodamine phalloidin for F-actin (red) and DAPI for DNA (blue). (a) Fluorescence images of dorsal actin cables over the nucleus. Bar, 10 m. (b) Quantification of the number of actin cables over the nucleus per cell in control siRNA cells at various time points after serum stimulation. (c) Quantification of centrosome orientation in serum-stimulated wound edge NIH3T3 fibroblasts. (d) Comparison of dorsal actin cables over nuclei in siFHOD1 treated cells with oriented and non-oriented centrosomes. Data in b–d are from 3 experiments; n, number of cells analysed per experiment is shown in b–d. Error bars: s.d. ∗∗, P < 0.01; ∗, P < 0.05; ns, not significantly different by two-tailed t-test.

Supplementary Figure 4 Localization of FHOD1 ΔC with endogenous TAN lines and effect of FHOD1 knockdown on endogenous TAN lines.

(a) Immunofluorescence images of GFP-FHOD1 ΔC and endogenous N2G on the dorsal surface of nuclei in LPA-stimulated NIH3T3 fibroblasts. Arrowheads, TAN lines containing N2G and dorsal actin cables and GFP-FHOD1 ΔC (bottom panels). Leading edge of the cell is toward the top. Bar, 10 m. (b) Immunofluorescence images of endogenous N2G (N2G antibody-stained) and F-actin (rhodamine phalloidin) in NIH3T3 fibroblasts treated with the indicated siRNAs. Arrowheads, TAN lines containing N2G co-localized with dorsal actin cables in control siRNA-treated cells. TAN lines are not observed in FHOD1 siRNA-treated cells. Bar, 5 m. (c) Quantification of endogenous TAN lines in control siRNA- and FHOD1 siRNA-treated cells. Data are from 3 experiments; n, number of cells analysed per experiment shown in c. ∗∗, P < 0.01 by Fishers exact test.

Supplementary Figure 5 The I705A mutation in active FHOD1 ΔC disrupts its induction of and localization with thick actin filament bundles.

NIH3T3 cells expressing the indicated GFP-FHOD1 variants were stained for F-actin with rhodamine phalloidin. Note that active FHOD1 ΔC induces the formation of thick F-actin bundles associates with them. Actin bundle formation and actin filament association of FHOD1 is potently disrupted by the I705A mutation. Bar, 20 μm.

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Kutscheidt, S., Zhu, R., Antoku, S. et al. FHOD1 interaction with nesprin-2G mediates TAN line formation and nuclear movement. Nat Cell Biol 16, 708–715 (2014). https://doi.org/10.1038/ncb2981

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