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MAP4K4 regulates integrin-FERM binding to control endothelial cell motility

Nature volume 519, pages 425430 (26 March 2015) | Download Citation

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Abstract

Cell migration is a stepwise process that coordinates multiple molecular machineries. Using in vitro angiogenesis screens with short interfering RNA and chemical inhibitors, we define here a MAP4K4–moesin–talin–β1-integrin molecular pathway that promotes efficient plasma membrane retraction during endothelial cell migration. Loss of MAP4K4 decreased membrane dynamics, slowed endothelial cell migration, and impaired angiogenesis in vitro and in vivo. In migrating endothelial cells, MAP4K4 phosphorylates moesin in retracting membranes at sites of focal adhesion disassembly. Epistasis analyses indicated that moesin functions downstream of MAP4K4 to inactivate integrin by competing with talin for binding to β1-integrin intracellular domain. Consequently, loss of moesin (encoded by the MSN gene) or MAP4K4 reduced adhesion disassembly rate in endothelial cells. Additionally, α5β1-integrin blockade reversed the membrane retraction defects associated with loss of Map4k4 in vitro and in vivo. Our study uncovers a novel aspect of endothelial cell migration. Finally, loss of MAP4K4 function suppressed pathological angiogenesis in disease models, identifying MAP4K4 as a potential therapeutic target.

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Acknowledgements

We thank L. Parker, R. Tam, K. Lyle, and B. Haley for contributing DNA constructs and siRNA design; H. Lu for initiating the chemical screen; J. Sudhamsu, P. Hass, J. Payandeh, and P. Lupardus for assisting with construct design and protein purification; J. Nonomiya, P. Wu, J. Wu, M. Lorenzo, H. Li, S. Schmidt for assay optimization, R. Ybarra and L. Magee for animal husbandry; S. Warming and M. Roose-Girma for generating floxed Map4k4 mice; J. Boggs, T. Crawford, L. Wang, J. Drobnick and L. Gazzard for compound synthesis and pharmacokinetics studies; M. Sagolla for input on image acquisition and analyses; L. dePalatis, C. Reed for antibody generation, V. Pham, D. Kirkpatrick for kinase substrate screen, L. Murray, J. Burton and C. Wilson for discussions. H. Gerhardt for advice.

Author information

Affiliations

  1. Molecular Biology Department, Genentech, Inc., South San Francisco, California 94080, USA

    • Philip Vitorino
    • , Stacey Yeung
    • , Ailey Crow
    • , Tanya Smyczek
    •  & Weilan Ye
  2. Chemical Biology and Therapeutics Department, St Jude Children’s Research Hospital, Memphis, Tennessee 38105, USA

    • Jesse Bakke
  3. Translational Oncology Department, Genentech, Inc., South San Francisco, California 94080, USA

    • Kristina West
    • , Erin McNamara
    •  & Stephen Gould
  4. Pathology Department, Genentech, Inc., South San Francisco, California 94080, USA

    • Jeffrey Eastham-Anderson
  5. Structural Biology Department, Genentech, Inc., South San Francisco, California 94080, USA

    • Seth F. Harris
  6. Discovery Chemistry Department, Genentech, Inc., South San Francisco, California 94080, USA

    • Chudi Ndubaku

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Contributions

P.V. and W.Y. conceived the concept of the paper, designed most experiments, and co-wrote the paper. P.V. carried out the majority of the experiments. S.Y., E.M., S.G. carried out and supervised many in vivo studies. J.B., T.S., K.W. carried out several in vitro studies. S.F.H. carried out structural analysis of MAP4K4. C.N. is responsible for all experiments related to the generation and characterization of chemical inhibitors. A.C. and J.E.-A. wrote automated image analysis code and did some of the image data analyses. All authors contributed to the writing and proof-reading of the manuscript.

Competing interests

All authors except J.B. are Genentech/Roche employees.

Corresponding author

Correspondence to Weilan Ye.

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Supplementary information

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  1. 1.

    Supplementary Information

    This file contains Supplementary Tables 1-2.

Videos

  1. 1.

    HUVEC sprouting in 3D culture: Day 1-2 after imbedding in fibrin

    Left Cells treated with vehicle (DMSO) control show frequent extension and retraction of subcellular protrusions. Right Cells treated with GNE-220 showing reduced retraction of subcellular protrusions.

  2. 2.

    HUVEC sprouting in 3D culture: Day 3-5 after imbedding in fibrin

    Left Cells treated with DMSO show frequent extension and retraction of subcellular protrusions, and growth of capillary‐like sprouts. Right Cells treated with GNE-220 (GNE-220) show reduced retraction of subcellular protrusions, defective growth of capillary-like sprouts, and increased membrane encounter and fusion near the bead.

  3. 3.

    Time-lapse recording of HUVEC membrane dynamics

    HUVEC stably expressing Lck-RFP (yellow) and stained with Hoechst (blue) in the 3D sprouting assay. Left Sprouts transfected with siControl in the presence of DMSO show frequent extension and retraction of subcellular protrusions at the tip of the sprout. Middle Sprouts transfected with siMAP4K4 in the presence of DMSO show infrequent retraction of long subcellular protrusions. Right Sprouts transfected with siControl in the presence of GNE-220 show infrequent retraction of subcellular protrusions. Very small spikes that are frequently extending and retracting in all panels are filopodia.

  4. 4.

    Migration of a single endothelial cells in 2D culture

    Migration of a HUVEC stably expressing Lck-RFP (yellow) and stained with Hoechst (blue) on 2D culture showing cluster of retraction fibers at the back of the cell. Images were taken every 5 minutes for 5 hours.

  5. 5.

    Time-lapse recording of endothelial cell Fas in 2D culture

    HUVEC expressing paxillin-GFP undergoing random movement. Left HUVEC treated with DMSO show rapid membrane retraction and extension, as well as FA assembly and disassembly. Right HUVEC treated with GNE-220 (GNE-220) showing rapid membrane extension and FA assembly, but very slow membrane retraction and FA disassembly. GNE-220 was added before imaging to evaluate the acute effect of MAP4K4 inhibition. Arrows indicate representative membrane retraction events.

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DOI

https://doi.org/10.1038/nature14323

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