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Concomitant binding of Afadin to LGN and F-actin directs planar spindle orientation

Nature Structural & Molecular Biology volume 23, pages 155–163 (2016)Cite this article

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Abstract

Polarized epithelia form by oriented cell divisions in which the mitotic spindle aligns parallel to the epithelial plane. To orient the mitotic spindle, cortical cues trigger the recruitment of NuMA–dynein–based motors, which pull on astral microtubules via the protein LGN. We demonstrate that the junctional protein Afadin is required for spindle orientation and correct epithelial morphogenesis of Caco-2 cysts. Molecularly, Afadin binds directly and concomitantly to F-actin and to LGN. We determined the crystallographic structure of human Afadin in complex with LGN and show that it resembles the LGN–NuMA complex. In mitosis, Afadin is necessary for cortical accumulation of LGN and NuMA above the spindle poles, in an F-actin–dependent manner. Collectively, our results depict Afadin as a molecular hub governing the enrichment of LGN and NuMA at the cortex. To our knowledge, Afadin is the first-described mechanical anchor between dynein and cortical F-actin.

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Figure 1: Afadin is required to orient the mitotic spindle in HeLa cells.
Figure 2: Afadin binds directly to LGN competitively with NuMA.
Figure 3: Structure and analysis of the interface between LGNTPR and AfadinPEPT.
Figure 4: Afadin mediates the interaction of LGN with the actomyosin cortex.
Figure 5: The interaction of Afadin with LGN is required for cortical targeting of microtubule motors and for spindle orientation in HeLa cells.
Figure 6: Role of Afadin in Caco-2 planar cell divisions and cystogenesis.

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Acknowledgements

We are grateful to S. Pasqualato and V. Cecatiello of the Crystallography Unit of the European Institute of Oncology for technical support. We thank E. Martini and D. Parazzoli of the Cogentech Facility for help with image analysis. We thank scientists at the X06DA beamline at the Swiss Light Source and the ID23-2 beamline at the European Synchrotron Radiation Facility for valuable help with data collection. We are grateful to all members of the Mapelli laboratory, F. Villa, G. Scita, A. Disanza, S. Santaguida and A. De Antoni for scientific discussions, and to A. Musacchio for careful reading of the manuscript. We thank Y. Takai (Kobe University Graduate School of Medicine, Japan) for sharing the rat Afadin cDNA. This work was supported by grants from the Italian Association for Cancer Research (AIRC) to M.M., the Italian Ministry of Health to M.M. and BioStruct-X (FP7/2007-2013, grant agreement 283570).

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  1. Andrea Alfieri

    Present address: Present address: Department of Biosciences, Università degli Studi di Milano, Milan, Italy.,

  2. Manuel Carminati and Sara Gallini: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

    Manuel Carminati, Sara Gallini, Laura Pirovano, Andrea Alfieri & Marina Mapelli

  2. Fondazione Italiana Ricerca sul Cancro Institute of Molecular Oncology, Milan, Italy

    Sara Bisi

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Contributions

M.C. conducted the biochemical and structural biology experiments, and S.G. performed cell biology experiments and analysis. A.A. designed Afadin mutants, L.P. helped with the cell biology, and S.B. provided purified actin. M.M. supervised the project and wrote the manuscript.

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Correspondence to Marina Mapelli.

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Integrated supplementary information

Supplementary Figure 1 Characterization of Afadin-, LGN- and NuMA-specific antibodies, and spindle angle analysis in LGN-depleted and NuMA-depleted HeLa cells.

(a) Scheme of human Afadin (also known as AF-6 or MLLT4) gene, and its splice variants. The AF-6 gene is located on human chromosome 6, and consists of 32 exons. Alternative splicing produces six transcripts differing in their C-terminal region. Human Afadin isoforms 1 and 6 stop at exons 28 and 29 respectively, and are similar to short variant of rat Afadin (also known as s-Afadin), which was reported to be unable to bind to F-actin. Human Afadin isoforms 2, 3, 4 and 5 are similar to long rat Afadin (l-Afadin), which binds to F-actin. Isoforms 4 and 5 differ for the presence of additional 11 residues between exon 28 and 29. The LGN-binding site of Afadin characterized in this study is coded by exon 30 (highlighted in orange), and is present in all human long isoforms except isoform-3. (b) Specificity of the Afadin antibody. Confocal sections of mitotic HeLa cells expressing a control shRNA and shRNA targeting Afadin (Afadin shRNA-2), fixed and stained for endogenous Afadin. Afadin staining was lost in Afadin shRNA-2 expressing cells. The scale bar corresponds to 5 μm. (c) Immunoblot analysis of mitotic lysates of HeLa cells transiently interfered for LGN (left) or stably depleted of NuMA (left) showing the efficiency of the protein depletions. (d) Representative confocal z-sections of mitotic HeLa cells depleted for LGN (left) or NuMA (right), with the corresponding controls. LGN-depleted cells are stained with NuMA (green) and DAPI (blue), while NuMA-ablated cells are visualized with γ-tubulin (dark yellow) and DAPI (blue). The plane of the coverslip is visible as a white line. (e) Quantification of mitotic spindle alignment performed as in Fig. 1 (with means ± SEM), showing mean angular tilts of about 14.9° for NuMA-depleted cells, and 12.5° for LGN-ablated cells. **** indicates a statistical difference of P < 0.0001 by Mann-Whitney test between interfered and control cells from three independent experiments with n > 50. Scale bars correspond to 5 μM.

Supplementary Figure 2 Canoe (Afadin) binds directly to Pins (LGN) and competes with Mud (NuMA).

SEC analysis and corresponding Coomassie-blue stained SDS-PAGE of the competitive interactions between the C-terminal portion of Drosophila Canoe (encompassing residues 1755-2051), Pins(LGN)TPR (spanning residues 25-406) and Mud1895-2094. At equimolar concentration, Canoe(Afadin)Cter enters a stoichiometric complex with Pins(LGN)TPR eluting in fractions 3-6 (black trace). Addition to the mix of equimolar amounts of Mud1895-2094 results in a complex of Mud1895-2094–Pins(LGN)TPR eluting slightly earlier than Canoe(Afadin)Cter in isolation (fractions 4-7). This analysis demonstrates that Canoe(Afadin) and Mud(NuMA) are mutually exclusive interactors of Pins(LGN)TPR, with Mud(NuMA) displaying higher affinity.

Supplementary Figure 3 Sequence alignment of the C terminus of Afadin long isoforms.

Afadin residues are colored based on the conservation calculated on the alignment of seven orthologues of Homo sapiens, Rattus norvegicus, Falco cherrug, Chelonia mydas, Xenopus laevis, Danio rerio and Drosophila melanogaster. The orange line highlights the Afadin fragment directly in contact with the LGNTPR domain according to the crystallographic structure, with the Phe1730AF and Glu1735AF labeled as red circles. The F-actin binding region of Afadin determined experimentally (Supplementary Figure 4b) is upstream of the LGN-binding stretch, and is predicted to adopt a helical conformation, as depicted in light gray (secondary structure prediction was performed using the server https://www.predictprotein.org/).

Supplementary Figure 4 Biochemical characterization of the interaction between AfadinCter and actin.

(a) Afadin does not bind to globular actin (G-actin). SEC analysis and corresponding SDS-PAGE showing that at 150 μM concentration of both species AfadinCter and monomeric G-actin do not form a complex. The elution profiles of AfadinCter and G-actin are also shown as references for the individual runs. Despite having similar molecular weight, AfadinCter elutes near the globular 158 kDa marker (lanes 3-6 of the SDS-PAGE), whereas G-actin elutes in the later fractions 7-10. (b) Afadin binds to F-actin through a region lying upstream to the LGN binding site. High-speed cosedimentation assay of two complementary fragments of AfadinCter (residues 1514-1824) with F-actin, showing that the actin-binding domain of human Afadin spans residues 1514-1682, upstream of the LGN-binding domain (colored in purple in the top scheme). Supernatant and pellet fractions of an analogous high-speed sedimentation experiment performed in the absence of F-actin are shown as a control of cosedimentation specificity.

Supplementary Figure 5 Analysis of spindle motors in Afadin-depleted HeLa anaphases, localization of Afadin rescue constructs and NuMA rescue experiments.

(a) Ablation of Afadin in HeLa cells impairs cortical localization of LGN, NuMA and Dynactin in anaphase. Confocal sections of control shRNA (top) and Afadin shRNA-2 expressing HeLa cells (bottom) in anaphase stained for LGN, NuMA, and p150Glued. Chromosomes are visualized with DAPI (blue). (b) Immunostaining of endogenous Afadin in mitotic HeLa cells interfered for LGN (left) or NuMA (right) with the corresponding controls. DNA is stained with DAPI. Quantification of cortical signals revealed that the cortical accumulation of Afadin is not altered by loss of LGN nor NuMA. (c-d) mCherry-Afadin rescue constructs. Immunoblot and immunostaining of HeLa cells transiently transfected with mCherry-Afadin, mCherry-Afadin-ΔLGN, or mCherry-Afadin-ΔACTIN showing the expression levels and the cortical localization of the shRNA-2 resistant rescue constructs. (e) Quantification of cortical mCherry-Afadin constructs analyzed in (d) showing that the F-actin binding domain of Afadin is required for the correct localization of the protein at the cortex. **** indicates P < 0.0001 by Kruskal-Wallis test from three independent experiments with n > 40. (f) Rescue experiment of cortical NuMA in HeLa cells stably depleted of Afadin, and transfected with shRNA-2 resistant mCherry-tagged rat Afadin wild-type, Afadin-ΔLGN, or Afadin-ΔACTIN. Quantification of cortical NuMA signals indicates that only Afadin wild-type rescues the cortical localization of NuMA in metaphase. The bottom panels show the mCherry-signal of the transfected cells. Under the conditions of methanol fixation used to visualize cortical NuMA, the cortical mCherry signal of all the rescue constructs is lost. **** indicates P < 0.0001 by Kruskal-Wallis test from three independent experiments with n > 32. Scale bars correspond to 5 μm.

Supplementary Figure 6 Distribution of cortical F-actin in metaphases of Caco-2 cells, either wild type or lacking Afadin in three-dimensional cysts.

Cysts of Caco-2 cells wild-type or stably interfered for Afadin stained with DAPI (blue) and TRITC-conjugated Phalloidin (red). Confocal sections of the equatorial region of the cysts show that in mitotic cells (indicated by grey arrows) cortical F-actin distributes uniformly all around the plasma membrane regardless of the presence of Afadin.

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Carminati, M., Gallini, S., Pirovano, L. et al. Concomitant binding of Afadin to LGN and F-actin directs planar spindle orientation. Nat Struct Mol Biol 23, 155–163 (2016). https://doi.org/10.1038/nsmb.3152

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