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MAP4K4 regulates integrin-FERM binding to control endothelial cell motility



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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Figure 1: MAP4K4 regulates endothelial cell membrane dynamics.
Figure 2: Map4k4 is essential for vascular development.
Figure 3: MAP4K4 and moesin regulate membrane retraction.
Figure 4: MAP4K4 and moesin regulate FA length.
Figure 5: MAP4K4 and moesin promote FA disassembly.
Figure 6: Anti-INTα5β1 rescues Map4k4 and MSN loss of function defects.


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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

Authors and Affiliations



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.

Corresponding author

Correspondence to Weilan Ye.

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Competing interests

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

Extended data figures and tables

Extended Data Figure 1 The roles of a subset of MAP4Ks and Notch in the HUVEC sprouting assay.

a, A diagram illustrating the cellular and sub-cellular structures in the three-dimensional (3D) culture of HUVECs coated on beads35. A sprout refers to the multi-cell structure that resembles a capillary. These sprouts grow out of the HUVECs coated on the surface of the plastic beads, and their length increased over time. The structures associated with each individual endothelial cell in this culture system are indicated on the diagram. Subcellular protrusions are membrane structures between 5 and 10 μm in width and are irregularly shaped. Filopodia are significantly thinner than subcellular protrusions (<1 μm) and are linear. b, Representative bright field images of HUVEC sprouts treated with the indicated siRNA, GNE-220, or a Notch pathway inhibitor DBZ (Notchi) after 1 and 4 days in culture. These images were taken from similar cultures that generated data presented in Fig. 1a. These figures showed the two types of images we used to monitor and quantify HUVEC sprouting behaviours. Unlike siMAP4K4 or GNE-220, DBZ did not increase subcellular protrusions and accumulation of aberrant structures near the beads; instead, it increased branching of capillary-like sprouts. c, Distribution of subcellular protrusion lengths in the HUVEC sprouting assay after 1 day in culture. Treatment with siMAP4K4 or GNE-220 significantly increased the number of subcellular protrusions longer than 40 μm. Data represent means from 4 experiments. d, The number of total subcellular protrusions per bead from the same experiments as b. e, Dose–response curve relating the number of long protrusions (>40 μm) to GNE-220 concentration after 24 h with inhibitor treatment. n = 4 independent cultures. f, Western blot analysis showing MAP4K4 knockdown efficiency in HUVEC for three independent siRNAs 72 h after transfection. g, Representative bright field images of HUVEC sprouts after 1 day in culture with MAP4K4 knockdown using three independent siRNA. h, Quantitative PCR measuring mRNA levels of four closely related human MAP4K genes after knockdown with the indicated siRNA pools. Knockdown of MAP4K4 does not affect other kinases. i, Representative bright field images of HUVEC sprouts after knockdown with the indicated siRNA after 1 day in culture. In b, g and i, red arrows indicate sprouts consisting of cell bodies, white arrows indicate subcellular membrane protrusions, and asterisks indicate beads coated with HUVEC. j, Quantification of experiment shown in i showing only MAP4K4 knockdown results in increase of long protrusions (>40 μm). Scale bars, 50 μm. For all extended figures: error bars represent standard error of the mean (s.e.m.); statistical significance between the indicated sample versus control, or the marked pairs are: P < 0.05, P < 0.01, P < 0.001, or NS (P ≥ 0.05) by statistical analysis described in the Methods.

Extended Data Figure 2 The roles of MAP4K4 and Dll4-Notch in regulating HUVEC proliferation, migration, and subcellular structures in the HUVEC sprouting assay.

a, Quantification of HUVEC nuclei number per bead in the 3D culture at the indicated time points in the presence or absence of GNE-220 or anti-Dll4 antibody. While inhibition of Notch-Dll4 signalling increased cell number, GNE-220 had no significant effect. n = 16 beads per condition. Representative of 4 experiments. b, Representative images of HUVEC sprouts stained with DAPI (blue) and EdU (green). After 4 days in culture, cells were incubated with EdU for 16 h before staining and imaging. c, Quantification of percentage of nuclei that score as EdU positive. n = 4 experiments. d, Wound area as a function of time in the HUVEC scratch wound healing assay treated with control siRNA, siMAP4K4 or GNE-220. n = 6 independent cultures. e, Representative images of HUVEC sprouts after 1 day in culture with the indicated treatments. siMAP4K4 and GNE-220 increased long subcellular protrusions but anti-Dll4 had no effect on these structures. Red arrows indicate sprouts, white arrows indicate protrusions. f, Quantification of images shown in e. n = 3 experiments. g, Quantification of filopodia number and dynamics in HUVEC sprouts from experiments described in Supplementary Video 3. Scale bars, 100 μm.

Extended Data Figure 3 Additional characterizations of Map4k4 conditional knockout mice.

a, Schematic representation of the Map4k4 floxed allele used in this study. When this floxed allele is present in cells expressing Cre, recombination results in the deletion of sequence encoding the MAP4K4 catalytic domain and introduces a stop codon at amino acid 144. If the truncated protein (amino acids 1–144) could be translated, it is expected to be non-functional and probably unstable. b, Summary of genotypes detected from progenies of the indicated cross. Genotype distribution is consistent with embryonic lethality based on Chi-squared analysis. P value indicates the probability of obtaining a distribution similar to the expected distributions for lethality or non-lethality. c, Quantitative RT–PCR analysis of Map4k4 normalized to mRPS13 in isolated endothelial cells and non-endothelial cells from Map4k4fl/fl::Tie2-Cre positive (+) embryos versus Map4k4fl/fl::Tie2-Cre negative (−) littermates. n = 3–4 embryos per genotype. d, Map4k4 CKO embryos and wild-type littermates harvested at the indicated stages. Arrows indicate focal haemorrhage and oedema. Scale bar, 1 mm. e, Quantification of ERG-positive endothelial cell nuclei normalized to vascularized areas in E14.5 head skins derived from control or CKO embryos. n = 7 control embryos, 4 CKO embryos. f, Representative confocal images of E14.5 embryonic head skin after 4 h labelling with EdU. Vessels stained with CD31 (blue), endothelial cell nuclei stained with ERG (green), proliferating nuclei marked as EdU positive (red). White arrows indicate proliferating endothelial cells. g, endothelial cell proliferation in the embryonic head skins were quantified as % of EdU+ cells in all ERG+ cells. n = 9 control embryos, 7 CKO embryos. h, Fraction of membrane protrusions along the vascular front with indicated lengths in control and CKO embryonic head skins. n = 3 animals per genotype. Asterisks indicate statistics between control and CKO. i, Total number of protrusions along the vascular front in control and CKO head skins per mm vascular front. n = 3 animals per genotype.

Extended Data Figure 4 Additional characterizations of Map4k4 conditional and inducible knockout mice.

a, Confocal images of E14.5 head skin vasculature stained with VE-Cadherin in control and CKO embryos. Bottom panels are enlarged view of the boxed areas. b, Quantification of endothelial cell junction morphology in control and CKO animals shown in panel a. As defined in Bentley et al.43, the “active” junction refers to diffused or serrated junction that reflects junctional remodelling, the “inhibited” morphology refers to linear junctions that reflect relative junctional stability, and the “mixed” junction contains both morphologies within the defined length. n > 13 regions per group, 2 embryos per genotype. Scale bars represent 12 μm. c, Permeability of confluent HUVEC monolayers with the indicated treatments was measured by FITC-dextran trans-well diffusion over time. n = 3 experiments. d, Quantitative RT–PCR results depicting Map4k4 expression levels relative to mRPS13 as a function of time after birth for control and iKO animals. Pups were injected with 80 mg per kg (body weight) tamoxifen once daily starting on P1, and mRNA were isolated and measured from tail clips on P3, P5, and P7. n = 4 control mice, 6 iKO mice for all time points. e, Representative confocal images of P7 retinal vasculature 16 h after EdU injection. Vessels stained with IsoB4 (blue), endothelial cell nuclei stained with ERG (green), proliferating nuclei marked as EdU positive (red). Arrows indicate EdU+ endothelial cells. f, Quantification of EdU+ endothelial cells normalized to total ERG+ cells in the retina. n = 5 control animals, 6 iKO animals. g, Representative confocal images of control and iKO retina on P7 stained with desmin to highlight pericytes (green) and IsoB4 to indicate endothelial cells (red). h, Quantification of pericyte coverage from experiments shown in d. n = 6 animals per genotype. Scale bars in e and g, 50 μm.

Extended Data Figure 5 HUVEC sprouting assay with individual MSN siRNAs and MAP4K4 kinase activity on moesin.

a, Representative bright field images of HUVEC bead sprouts transfected with three independent siRNA targeting MSN after one day in culture. Red arrows indicate sprouts consisting of cell bodies, white arrows indicate subcellular protrusions, and asterisks indicate beads coated with HUVEC. Scale bar, 50 μm. b, Western blot confirmed knockdown of MSN 72 h after transfection with three independent siRNAs. c, ATP consumption rates of recombinant activated MAP4K4 kinase domain against full-length moesin or a peptide corresponding to amino acids surrounding T558 in moesin. d, Western blot of reaction products from c showing moesin phosphorylation at T558. t, total. e, Western blot of the indicated total and phosphorylated (p) proteins from HUVEC transfected with the indicated siRNA. n = 3 experiments.

Extended Data Figure 6 The roles of MAP4K4 and moesin on myosin and focal adhesions.

a, Western blot analysis of HUVEC lysates 72 h after transfection with MAP4K4 siRNA with (+) or without (−) 24 h treatment with GNE-220. b, Representative images of HUVEC treated with siMAP4K4 or GNE-220 and stained with p-myosin. c, A representative image of HUVEC stained with phalloidin to highlight actin (red), active INTβ1 (green), and DAPI (blue) (left). Right, automated segmentation of long focal adhesions (red) overlaying on top of active INTβ1 staining (green) and the outline of the cell (blue). d, Epifluorescent images of HUVEC transfected with the indicated siRNA pools. Active β1 (green) and β3 (red) integrins mark mature and nascent focal adhesions, respectively. DAPI staining is shown in blue. e, Confocal images of HUVEC treated with control siRNA, siMAP4K4 or GNE-220 and stained with paxillin (left) and integrin αVβ5 (right). f, Epifluorescent images of HUVEC transfected with control siRNA or siRNA targeting the 3′ UTR of MSN. siRNA-treated cells were electroporated with constructs expressing GFP, or GFP-tagged wild-type moesin or moesin(T558A) (green) and stained with active INTβ1 antibody (red). Arrows indicate cells expressing GFP or GFP-tagged proteins. g, Quantification of long FAs in GFP positive cells shown in f. Interestingly, expression of the moesin(T558A) construct moderately increased long FAs, indicating that this construct may have weak dominant negative activity. n = 3 experiments for all panels. Scale bars, 10 μm.

Extended Data Figure 7 Further characterizations of MAP4K4 and moesin in retraction fibres, FA dynamics, and integrin activity.

a, Confocal images of retraction fibres in HUVEC infected with empty viral vector (bottom) or viral construct expressing HA-MAP4K4 and stained with anti-HA antibody (red) and pERM (green). We evaluated 13 commercially available anti-MAP4K4 antibodies in HUVECs with and without siMAP4K4, but failed to identify any antibody that specifically stained MAP4K4 on cells. Overexpressed MAP4K4 was then used to evaluate its distribution. b, c, FA assembly rates (b) and average FA decay time (c) in HUVEC expressing paxillin–GFP. Quantification was done using the Focal Adhesion Analysis Server ( n = 8 videos per condition. d, TIRF images of retraction fibres in HUVEC stained with total INTβ1 (red) and pERM (green). e, TIRF images of retraction fibres in HUVEC stained with pERM (green) and talin (red). f, FACs analysis of active and total INTβ1 in CHO cells expressing mCherry alone, mCherry-tagged moesin FERM domain, or mCherry-tagged Band4.1 FERM domain. Each bar represents the mean of more than 3 independent pools of CHO cells transfected with the indicated constructs. Integrin activation in this experiment relied on endogenously expressed Talin. g, Recombinant wild-type or mutant integrin β1ICD coated beads were incubated with talin for one hour (1 h) except the sample labelled O/N (overnight, lane 3 from the left), followed by the addition of the indicated competitor proteins. The pulled-down or input proteins were analysed by western blotting. Comparison between lanes 3 and 5 indicates that one-hour incubation allowed maximal talin binding with integrin β1ICD similar to overnight incubation. IP, immunoprecipitation. h, Quantification of talin and moesin associated with β1ICD-coated beads in the presence of increasing concentrations of moesin. n = 3 experiments. i, Talin and moesin at the indicated quantities were co-incubated with beads coated with INTβ1ICD. The immunoprecipitated proteins were analysed by western blotting. Increased talin input reduced moesin binding to β1ICD, suggesting that talin competes with moesin for binding to β1ICD. j, FACs analysis of active and total INTβ1 in CHO cells expressing mCherry alone, mCherry-tagged wild-type talin or moesin FERM domain, or the indicated mCherry-tagged talin-moesin chimaeric FERM domain. To avoid the confounding effect of moesin-FERM’s inhibitory activity, we gated for cells with low FERM expression where moesin-FERM was insufficient to inhibit INTβ1. Cells expressing the same levels of mCherry were gated and analysed for total and active INTβ1. n = 3 experiments. Scale bars, 5 μm.

Extended Data Figure 8 Additional information about the integrin-α5β1 antibodies, MAP4K4 inhibitor GNE-495, and a model depicting how MAP4K4 and moesin regulate FA disassembly.

a, HUVEC migration assay results plotted as number of cells migrated through the membrane (y-axis) versus the concentrations of antibodies (x-axis). The graph shows that the anti-human INTα5β1 MAb 18C12 dose dependently inhibited migration of HUVECs on fibronectin. b, CHOB2-mα5β1 cells migration assay results plotted as number of cells migrated through the membrane (y-axis) versus the concentrations of antibodies (x-axis). The graph shows that the anti-murine INTα5β1 monoclonal antibody 10E7 dose-dependently inhibited migration of CHOB2-mα5β1 on fibronectin. Data presented in a and b were derived from 6 independent samples per condition. Detail information about MAbs 18C12 and 10E7 can be found in the Methods. Note that since HUVEC expressed several fibronectin receptors, inhibition of migration by an anti-α5β1 MAb was partial even at high MAb concentrations, whereas CHOB2 cells are deficient for many integrins44, inhibition of CHOB2-mα5β1 cells migration by an anti-α5β1 MAb was more profound. c, Quantitative PCR measurement of Map4k4 normalized to mRPS13 using cDNA from neonatal tail clips of P7 mice with the indicated Map4k4 genotypes (control or iKO) after injection with tamoxifen and the indicated antibodies. d, Model for MAP4K4 regulation of membrane retraction. Upon phosphorylation by MAP4K4, the FERM domain of activated moesin competes with talin-FERM for binding to active integrin, leading to integrin inactivation and FA disassembly. These events promote efficient membrane retraction to enable cell migration. Additional FA components omitted for simplicity. eg, Characterization of the MAP4K4 selective inhibitor GNE-495: dose response curves relating GNE-495 concentration to long membrane protrusions in HUVEC bead sprouting assay (e), pERM-positive spikes (f), and long FAs in 2D HUVEC culture 24 h after GNE-495 treatment (g). Data represent average of 4 independent cultures.

Extended Data Figure 9 The role of MAP4K4 in pathologic angiogenesis.

a, b, Murine pancreatic (KPP-1) and lung (TC-1) cancer cells were implanted subcutaneously in control and Map4k4iKO/iKO sibling mice. Mean volumes of KPP-1 tumours from 10 mice per genotype (a) and TC-1 tumours from 15 mice per genotype (b) were measured over time. Per IACUC guidance, at the later time points, mice with tumour volumes exceeding 1,000 mm3 (KPP-1) or 2,500 mm3 (TC-1) were euthanized and no longer included in the mean tumour volume calculation. On day 20 for the KPP-1 study: n = 9 for the control group, n = 10 for the iKO group. On day 24 for the TC-1 study: n = 11 for the control group, n = 14 for the iKO group. c, Representative confocal image of a KPP-1 tumour section stained with FITC-lectin (green), CD31 (red) to indicate how functional vessels were analysed. White arrowhead indicates a perfused vessel (double positive for FITC-lectin and CD31, shown as yellow), blue arrow indicates a non-perfused vessel (single positive for CD31). d, Quantification of perfused tumour vessels on whole tumour sections from the KPP-1 model. Each dot represents the mean value of an entire tumour from a mouse. e, Quantification of tumour vessel areas normalized to the viable tumour areas on whole tumour sections from the KPP-1 model revealed no significant change in tumour vascular density in the iKO host. Each dot represents the mean value of an entire tumour from a mouse. f, Representative confocal projection image of a KPP-1 tumour thick section stained with CD31 to indicate how long subcellular protrusions were analysed. Arrow indicates a sprout, yellow lines indicate the subcellular protrusions. g, Quantification of long protrusions in each tumour. Each dot represents the mean value of multiple micrographs from the tumour of one mouse. A total of 170 protrusions were analysed. Scale bars represent 100 μm for c and f. Regarding e, the lack of significant change in tumour vascular density is not surprising as we observed a delay in vascularization balanced by the accumulation of endothelial cells in the already vascularized areas (Figs 2b, f, 6b), resulting in a lack of overall density change. Decreased perfusion may reflect the blood vessel structural alteration due to the aforementioned endothelial cell accumulation (Fig. 1a red arrowheads). hl, Effects of the MAP4K4 inhibitor GNE-495 was evaluated in an oxygen-induced retinopathy (OIR) model that mimics vascular pathologies in human proliferative diabetic retinopathy and retinopathy of prematurity. h, Confocal images of P17 retinas stained for isolectin-B4 from mice subjected to the OIR procedure and treated with either vehicle or GNE-495. Red asterisks mark areas of vaso-obliteration (avascular area) resulting from high oxygen damage. Right, close-up views of the boxed areas. Red arrows mark pathologic vascular tufts. i, Areas of pathologic vascular tufts normalized to retinal areas. Control retinas contain numerous pathological neovascular tufts, which is largely absent from the GNE-495 treated retinas, revealing the inhibitory effect of GNE-495 on pathologic angiogenesis. j, Avascular areas normalized to retinal areas. GNE-495 increased avascular area, indicating an inhibition of vascular regrowth into the oxygen damaged avascular area. k, Autofluorescence of red blood cells indicative of haemorrhage (white asterisks) in retinas from the same experiment shown in hj. l, Haemorrhagic areas normalized to retinal area illustrates that GNE-495 reduced haemorrhage. Each dot represents one animal. Scale bar, 50 μm.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2. (PDF 274 kb)

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. (MOV 2130 kb)

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. (MOV 4758 kb)

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. (MOV 547 kb)

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. (MOV 203 kb)

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. (MOV 983 kb)

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Vitorino, P., Yeung, S., Crow, A. et al. MAP4K4 regulates integrin-FERM binding to control endothelial cell motility. Nature 519, 425–430 (2015).

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