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Control of mitochondrial function and cell growth by the atypical cadherin Fat1

Abstract

Mitochondrial products such as ATP, reactive oxygen species, and aspartate are key regulators of cellular metabolism and growth. Abnormal mitochondrial function compromises integrated growth-related processes such as development and tissue repair1,2, as well as homeostatic mechanisms that counteract ageing and neurodegeneration3, cardiovascular disease4,5, and cancer6,7. Physiologic mechanisms that control mitochondrial activity in such settings remain incompletely understood. Here we show that the atypical Fat1 cadherin acts as a molecular ‘brake’ on mitochondrial respiration that regulates vascular smooth muscle cell (SMC) proliferation after arterial injury. Fragments of Fat1 accumulate in SMC mitochondria, and the Fat1 intracellular domain interacts with multiple mitochondrial proteins, including critical factors associated with the inner mitochondrial membrane. SMCs lacking Fat1 (Fat1KO) grow faster, consume more oxygen for ATP production, and contain more aspartate. Notably, expression in Fat1KO cells of a modified Fat1 intracellular domain that localizes exclusively to mitochondria largely normalizes oxygen consumption, and the growth advantage of these cells can be suppressed by inhibition of mitochondrial respiration, which suggest that a Fat1-mediated growth control mechanism is intrinsic to mitochondria. Consistent with this idea, Fat1 species associate with multiple respiratory complexes, and Fat1 deletion both increases the activity of complexes I and II and promotes the formation of complex-I-containing supercomplexes. In vivo, Fat1 is expressed in injured human and mouse arteries, and inactivation of SMC Fat1 in mice potentiates the response to vascular damage, with markedly increased medial hyperplasia and neointimal growth, and evidence of higher SMC mitochondrial respiration. These studies suggest that Fat1 controls mitochondrial activity to restrain cell growth during the reparative, proliferative state induced by vascular injury. Given recent reports linking Fat1 to cancer, abnormal kidney and muscle development, and neuropsychiatric disease8,9,10,11,12,13, this Fat1 function may have importance in other settings of altered cell growth and metabolism.

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Figure 1: Fat1 fragments localize to SMC mitochondria and interact with inner mitochondrial membrane proteins.
Figure 2: Fat1 suppresses SMC growth by restraining mitochondrial respiration.
Figure 3: The Fat1 ICD limits complex I and II activities, and complex-I-containing supercomplex formation in SMCs.
Figure 4: Fat1 restrains SMC growth and mitochondrial respiration after vascular injury.

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Acknowledgements

We thank R.N. Kitsis for helpful discussions; A. Jenny for critical reading of the manuscript; X.L. Du for technical help with the Seahorse experiments and for scientific advice; G. Perumal at the Einstein Analytical Imaging Facility for help with electron microscopy imaging; and M.A. Gawinowicz at the Columbia Proteomics laboratory for performing the mass spectrometry analysis. This work was supported by funds from the Diabetes Training and Research Center of Albert Einstein College of Medicine (NIH P60DK20541); funds from the Medical Scientist Training Program (NIH T32-GM007288), Cellular, Molecular Biology, and, Genetics Training Grant (NIH T32-GM007491), and an American Medical Association Seed Grant (all to L.L.C.); from the American Heart Association to D.F.R-B. (pre-doctoral award 11PRE5450002) and to N.E.S.S. (Grant-in-Aid 13GRNT16950064); and from the NIH to L.H. (CA205262) and to N.E.S.S. (HL088104 and HL104518).

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L.L.C., D.F.R.-B., P.C., C.M.D., and R.H. generated reagents, performed experiments, and analysed data. B.P.O. performed confocal imaging and analysis of the co-localization studies. V.M. and L.H. imaged and analysed the redox-sensitive ratiometric sensor roGFP. L.G. performed immunohistochemistry on human coronary arteries. M.A.P. and A.F. performed the bioinformatic analysis. L.L.C., D.F.R.-B., and N.E.S.S. designed the study and wrote the paper. L.L.C. and D.F.R.-B. contributed equally to the study. All authors read and approved the final manuscript.

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Correspondence to Nicholas E. S. Sibinga.

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Nature thanks M. Bennett, R. Thorne and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Fat1 ICD associates with inner mitochondrial membrane proteins.

a, Description of subgroups within the mitochondrial cluster identified by STRING, PageRank, and DAVID enrichment analysis of TAP–MS results. GO, gene ontology. b, Western blotting for Fat1 expression in mouse aortic SMCs. c, Co-immunoprecipitation of Fat1 ICD and prohibitin (PHB) in 293T cells. IP, immunoprecipitation; performed with IgG (control) or Myc antibody, as indicated. WB, western blot. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 Loss of Fat1 increases expression of pro-proliferative gene products and ATP turnover, but does not affect basal ROS levels in SMCs.

a, AlamarBlue assay in mouse aortic SMCs plated at specified, matched cell densities, showing correlation of AlamarBlue reduction across a range of input cell numbers (n = 3). b, Expression of cyclin D1 and β-catenin in mouse aortic SMCs with indicated treatments. EV, empty vector. c, Quantification of OCR in mouse aortic SMCs from Fig. 2b (n = 10). d, Coupling efficiency calculated as percentage of reduction in OCR after oligomycin treatment (n = 10). e, Net ATP levels in mouse aortic SMCs. RU, relative units (n = 5). f, ROS levels in mouse aortic SMCs, measured with a redox-sensitive ratiometric sensor, roGFP. Left, Ratiometric (390/470 nm) value, representing the oxidative state of the sensor and intracellular ROS levels, and expressed as fold increase above the average baseline (before H2O2 stimulation) ratio (n = 15). Right: Images of cells showing representative ROS levels detected by the ratiometric sensor at baseline (t = 0) and at different time points after H2O2 stimulation. Data analysed by two-way ANOVA (a, c, f); and two-tailed t-test (d, e). NS, not significant. All data shown as mean ± s.e.m. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 Fat1 suppresses vascular SMC growth by inhibiting the electron transport chain.

a, Population growth of mouse aortic SMCs in the presence of various concentrations of rotenone, a complex I inhibitor. Addition of rotenone at concentrations from 0.1–2 μM did not compromise wild-type cell growth; by contrast, Fat1KO cell growth was suppressed to wild-type levels (n = 3); significance assessed by two-way ANOVA. b, Western blotting for NDUFS3 expression in mouse aortic SMCs treated with control siRNA (sictl) or Ndufs3 siRNA (siNdufs3). c, Proliferation of mouse aortic SMCs after siNdufs3 treatment, expressed as the ratio of EdU to Hoechst signal. RU, relative units. n = 3 for Fat1KO siNdufs3, n = 5 for other groups; significance assessed by one-way ANOVA. NS, not significant. All data shown as mean ± s.e.m. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 Loss of Fat1 does not affect overall mitochondrial structure, mass, or dynamics in vascular SMCs.

a, Electron microscopy imaging (original magnification, 5,000×) of mitochondria in mouse aortic SMCs. bf, Quantification of electron micrographs for the number of mitochondria per cytoplasmic area (b, n = 11); mitochondrial area per cytoplasmic area (c, n = 111 for wild type, n = 119 for Fat1KO); mitochondrial circularity (d, n = 111 for wild type, n = 119 for Fat1KO); mitochondrial crista length (e, n = 34 for wild type, n = 45 for Fat1KO); and crista width (f, n = 34 for wild type, n = 45 for Fat1KO). AU, area units; RU, relative units. g, Expression of representative oxidative phosphorylation proteins from each mitochondrial respiratory complex in total cell lysates of mouse aortic SMCs. h, qRT–PCR analysis of mitochondrial biogenesis and fusion/fission markers, normalized to Rpl13a expression. n = 3. i, Protein expression of biogenesis markers. j, LC3 levels in 3T3-L1 cells and Atg5 knockout cells (positive and negative controls for autophagy, respectively), as well as in wild-type and Fat1KO SMCs. Conversion of LC3 I to LC3 II is indicative of autophagic activity. k, Expression of Mitofusins 1 and 2, regulators of mitochondrial fusion. NS, not significant. All data shown as mean ± s.e.m., significance assessed by two-tailed t-tests. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 5 Mitochondria-targeted Fat1 ICD is sufficient to repress oxygen consumption in vascular SMCs.

a, Electroporation of Fat1–IL-2R in mouse aortic SMCs, followed by subcellular fraction and SDS–PAGE analysis of Fat1 ICD fragments. Green bracket, endogenous Fat1 ICD species; red bracket, Fat1 ICD products from Fat1–IL-2R; blue asterisk, non-specific signal. b, Schematic of mitochondria-targeted Fat1 ICD, Fat1mito. Asterisk, stop codon. c, Top, Detection of Fat1mito in the mitochondrial fraction of Fat1mito -transfected 293T cells. Bottom, Exclusion of Fat1mito from the nuclear fraction of Fat1mito -transfected 293T cells. d, Electroporation of Fat1mito in mouse aortic SMCs, followed by subcellular fraction and SDS–PAGE analysis of Fat1 ICD fragments. Green bracket, endogenous Fat1 ICD species; blue asterisk, non-specific signal. e, f, Quantification of baseline (e) and maximal OCR (f) after introducing Fat1–IL-2R or Fat1mito into Fat1KO cells from Fig. 3a. Data shown as mean ± s.e.m., n = 15, significance assessed by one-way ANOVA. C, cytoplasmic; EV, empty vector; Mit, mitochondrial; Ms, microsomal; −Nuc, non-nuclear fraction; +Nuc, nuclear fraction; WL, whole-cell lysate. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 6 Fat1 ICD associates with mitochondrial respiratory complexes in SMCs, and limits the incorporation of complex I into supercomplexes.

a, Rate of cytochrome C oxidation by immunocaptured complex IV from mouse aortic SMCs lysates, expressed as fold-change from wild type (n = 18). b, Rate of hydrolysis of ATP to ADP and phosphate by immunocaptured complex V from human aortic SMCs treated with control siRNA (sictl) or FAT1 siRNA (siFAT1), expressed as fold change from sictl. n = 4. c, SDS–PAGE analysis of proteins eluted from immunocaptured respiratory complexes after completion of enzymatic assays, followed by immunoblotting for Fat1 and complex II, IV, and V subunits. Red arrowhead, specific Fat1 signal; blue asterisk, non-specific signal. d, Quantification of native complex I (CI) levels from BN–PAGE analyses, including the example presented in Fig. 3e. n = 5. e, Two-dimensional BN/SDS–PAGE analysis of mouse aortic SMC mitochondrial lysates, immunoblotted for Fat1 and complexes I–V. Dashed lines indicate co-migration of Fat1 ICD species with respiratory complexes. Bottom panels are merged images of the individual western blots for complexes I–V (presented in Extended Data Fig. 7). SC, supercomplex. NS, not significant. Data shown as mean ± s.e.m. and analysed by two-tailed t-test (a, b, d). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 7 Two-dimensional BN/SDS–PAGE analysis of SMC mitochondrial lysates, immunoblotted for complexes I–V.

Individual western blots for complexes I–V used to generate the merged images in Extended Data Fig. 6e, bottom panels.

Extended Data Figure 8 FAT1 suppresses proliferation and mitochondrial respiration in human SMCs.

a, Western blotting for FAT1 expression in human aortic SMCs (HASMCs) treated with control siRNA (sictl) or FAT1 siRNAs (siFAT1) 1–3. For subsequent experiments, siFAT1 3 was used unless otherwise indicated. b, Proliferation of HASMCs after siFAT1 treatment, expressed as the ratio of EdU to Hoechst signal, normalized to sictl. n = 3, significance assessed by two-tailed t-test. c, Expression of cyclin D1 in sictl- or siFAT1-treated HASMCs. d, Oxygen consumption rate (OCR) of sictl- or siFAT1-treated HASMCs at baseline and in response to 2 μg ml−1 oligomycin (1), 3 μM FCCP (2), and 2 μM rotenone (3). e, Quantification of OCR from (d). n = 3, significance assessed by two-way ANOVA. All data shown as mean ± s.e.m. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 9 Fat1 expression is induced in SMCs after vascular injury.

a, Immunohistochemistry for Fat1 in control and Fat1SMKO carotid arteries before and after arterial injury, three (d3) or fourteen days (d14) after carotid ligation. L, lumen. The internal elastic lamina has been highlighted with a black line. Scale bar, 25 μm. b, Expression of Fat1 and Acta2 (also known as SMA) by immunofluorescence in control and Fat1SMKO carotid arteries, fourteen days after injury. White squares indicate the regions shown in higher magnification. Yellow line marks the internal and external elastic laminae. Scale bar, 20 μm. c, Nitrotyrosine (NT) staining of control and Fat1SMKO carotid arteries, seven days after injury. White line marks the internal elastic lamina. Scale bar, 50 μm. d, Cells isolated from carotid arteries three days after ligation injury expressing SMC (Acta2, calponin 1, transgelin) or endothelial cell (Pecam1) markers by immunofluorescence.

Extended Data Table 1 Fat1 interactors identified by TAP–MS that localize to the inner mitochondrial membrane

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Cao, L., Riascos-Bernal, D., Chinnasamy, P. et al. Control of mitochondrial function and cell growth by the atypical cadherin Fat1. Nature 539, 575–578 (2016). https://doi.org/10.1038/nature20170

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