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Vinculin-mediated axon growth requires interaction with actin but not talin in mouse neocortical neurons

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Abstract

The actin-binding protein vinculin is a major constituent of focal adhesion, but its role in neuronal development is poorly understood. We found that vinculin deletion in mouse neocortical neurons attenuated axon growth both in vitro and in vivo. Using functional mutants, we found that expression of a constitutively active vinculin significantly enhanced axon growth while the head-neck domain had an inhibitory effect. Interestingly, we found that vinculin-talin interaction was dispensable for axon growth and neuronal migration. Strikingly, expression of the tail domain delayed migration, increased branching, and stunted axon. Inhibition of the Arp2/3 complex or abolishing the tail domain interaction with actin completely reversed the branching phenotype caused by tail domain expression without affecting axon length. Super-resolution microscopy showed increased mobility of actin in tail domain expressing neurons. Our results provide novel insights into the role of vinculin and its functional domains in regulating neuronal migration and axon growth.

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References

  1. Dent EW, Gupton SL, Gertler FB (2011) The growth cone cytoskeleton in axon outgrowth and guidance. Cold Spring Harb Perspect Biol 3(3):a001800

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Lian G, Sheen VL (2015) Cytoskeletal proteins in cortical development and disease: actin associated proteins in periventricular heterotopia. Front Cell Neurosci 9:99

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. Luo L (2002) Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu Rev Cell Dev Biol 18:601–635

    Article  CAS  PubMed  Google Scholar 

  4. Ishikawa R, Kohama K (2007) Actin-binding proteins in nerve cell growth cones. J Pharmacol Sci 105:6–11

    Article  CAS  PubMed  Google Scholar 

  5. Dent EW et al (2007) Filopodia are required for cortical neurite initiation. Nat Cell Biol 9:1347–1359

    Article  CAS  PubMed  Google Scholar 

  6. Kwiatkowski AV et al (2007) Ena/VASP is required for neuritogenesis in the developing cortex. Neuron 56:441–455

    Article  CAS  PubMed  Google Scholar 

  7. Letourneau PC, Pech IV, Rogers SL, Palm SL, McCarthy JB, Furcht LT (1988) Growth cone migration across extracellular matrix components depends on integrin, but migration across glioma cells does not. J Neurosci Res 21:286–297

    Article  CAS  PubMed  Google Scholar 

  8. Mierke CT (2009) The role of vinculin in the regulation of the mechanical properties of cells. Cell Biochem Biophys 53:115–126

    Article  CAS  PubMed  Google Scholar 

  9. Romero S, Le Clainche C, Gautreau AM (2020) Actin polymerisation downstream of integrins: signaling pathways and mechanotransduction. Biochem J 477:1–21

    Article  CAS  PubMed  Google Scholar 

  10. Calderwood DA, Yan B, de Pereda JM, Alvarez BG, Fujioka Y, Liddington RC, Ginsberg MH (2002) The phosphotyrosine binding-like domain of talin activates integrins. J Biol Chem 277:21749–21758

    Article  CAS  PubMed  Google Scholar 

  11. Garcia-Alvarez B, de Pereda JM, Calderwood DA, Ulmer TS, Critchley D, Campbell ID, Ginsberg MH, Liddington RC (2003) Structural determinants of integrin recognition by talin. Mol Cell 11:49–58

    Article  CAS  PubMed  Google Scholar 

  12. Atherton P et al (2015) Vinculin controls talin engagement with the actomyosin machinery. Nat Commun 6:10038

    Article  CAS  PubMed  Google Scholar 

  13. Ziegler WH, Liddington RC, Critchley DR (2006) The structure and regulation of vinculin. Trends Cell Biol 16:453–460

    Article  CAS  PubMed  Google Scholar 

  14. Johnson RP, Craig SW (1994) An intramolecular association between the head and tail domains of vinculin modulates talin binding. J Biol Chem 269:12611–12619

    Article  CAS  PubMed  Google Scholar 

  15. Johnson RP, Craig SW (1995) F-actin binding site masked by the intramolecular association of vinculin head and tail domains. Nature 373:261–264

    Article  CAS  PubMed  Google Scholar 

  16. Bakolitsa C et al (2004) Structural basis for vinculin activation at sites of cell adhesion. Nature 430:583–586

    Article  CAS  PubMed  Google Scholar 

  17. Borgon RA, Vonrhein C, Bricogne G, Bois PR, Izard T (2004) Crystal structure of human vinculin. Structure 12:1189–1197

    Article  CAS  PubMed  Google Scholar 

  18. Letourneau PC, Shattuck TA (1989) Distribution and possible interactions of actin-associated proteins and cell adhesion molecules of nerve growth cones. Development 105:505–519

    Article  CAS  PubMed  Google Scholar 

  19. Burridge K, Mangeat P (1984) An interaction between vinculin and talin. Nature 308:744–746

    Article  CAS  PubMed  Google Scholar 

  20. Menkel AR, Kroemker M, Bubeck P, Ronsiek M, Nikolai G, Jockusch BM (1994) Characterisation of an F-actin-binding domain in the cytoskeletal protein vinculin. J Cell Biol 126:1231–1240

    Article  CAS  PubMed  Google Scholar 

  21. Xu W, Baribault H, Adamson ED (1998) Vinculin knockout results in heart and brain defects during embryonic development. Development 125:327–337

    Article  CAS  PubMed  Google Scholar 

  22. Xu W, Coll JL, Adamson ED (1998) Rescue of the mutant phenotype by reexpression of full-length vinculin in null F9 cells; effects on cell locomotion by domain deleted vinculin. J Cell Sci 111(Pt 11):1535–1544

    Article  CAS  PubMed  Google Scholar 

  23. Demali KA (2004) Vinculin–a dynamic regulator of cell adhesion. Trends Biochem Sci 29:565–567

    Article  CAS  PubMed  Google Scholar 

  24. Thievessen I et al (2013) Vinculin-actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth. J Cell Biol 202:163–177

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Coll JL, Ben-Ze’ev A, Ezzell RM, Rodriguez Fernandez JL, Baribault H, Oshima RG, Adamson ED (1995) Targeted disruption of vinculin genes in F9 and embryonic stem cells changes cell morphology, adhesion, and locomotion. Proc Natl Acad Sci U S A 92:9161–9165

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Varnum-Finney B, Reichardt LF (1994) Vinculin-deficient PC12 cell lines extend unstable lamellipodia and filopodia and have a reduced rate of neurite outgrowth. J Cell Biol 127:1071–1084

    Article  CAS  PubMed  Google Scholar 

  27. Sydor AM, Su AL, Wang FS, Xu A, Jay DG (1996) Talin and vinculin play distinct roles in filopodial motility in the neuronal growth cone. J Cell Biol 134:1197–1207

    Article  CAS  PubMed  Google Scholar 

  28. Li CL, Sathyamurthy A, Oldenborg A, Tank D, Ramanan N (2014) SRF phosphorylation by glycogen synthase kinase-3 promotes axon growth in hippocampal neurons. J Neurosci 34:4027–4042

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. Nadarajah B, Parnavelas JG (2002) Modes of neuronal migration in the developing cerebral cortex. Nat Rev Neurosci 3:423–432

    Article  CAS  PubMed  Google Scholar 

  30. Humphries JD, Wang P, Streuli C, Geiger B, Humphries MJ, Ballestrem C (2007) Vinculin controls focal adhesion formation by direct interactions with talin and actin. J Cell Biol 179:1043–1057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Cohen DM, Chen H, Johnson RP, Choudhury B, Craig SW (2005) Two distinct head-tail interfaces cooperate to suppress activation of vinculin by talin. J Biol Chem 280:17109–17117

    Article  CAS  PubMed  Google Scholar 

  32. Cohen DM, Kutscher B, Chen H, Murphy DB, Craig SW (2006) A conformational switch in vinculin drives formation and dynamics of a talin-vinculin complex at focal adhesions. J Biol Chem 281:16006–16015

    Article  CAS  PubMed  Google Scholar 

  33. Chandrasekar I, Stradal TE, Holt MR, Entschladen F, Jockusch BM, Ziegler WH (2005) Vinculin acts as a sensor in lipid regulation of adhesion-site turnover. J Cell Sci 118:1461–1472

    Article  CAS  PubMed  Google Scholar 

  34. Ferrere A, Vitalis T, Gingras H, Gaspar P, Cases O (2006) Expression of Cux-1 and Cux-2 in the developing somatosensory cortex of normal and barrel-defective mice. Anat Rec A Discov Mol Cell Evol Biol 288:158–165

    Article  PubMed  CAS  Google Scholar 

  35. Atherton P, Stutchbury B, Jethwa D, Ballestrem C (2016) Mechanosensitive components of integrin adhesions: role of vinculin. Exp Cell Res 343:21–27

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Roca-Cusachs P, del Rio A, Puklin-Faucher E, Gauthier NC, Biais N, Sheetz MP (2013) Integrin-dependent force transmission to the extracellular matrix by alpha-actinin triggers adhesion maturation. Proc Natl Acad Sci USA 110:E1361–E1370

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Le S et al (2017) Mechanotransmission and Mechanosensing of Human alpha-Actinin 1. Cell Rep 21:2714–2723

    Article  CAS  PubMed  Google Scholar 

  38. Mierke CT, Kollmannsberger P, Zitterbart DP, Smith J, Fabry B, Goldmann WH (2008) Mechano-coupling and regulation of contractility by the vinculin tail domain. Biophys J 94:661–670

    Article  CAS  PubMed  Google Scholar 

  39. Craig AM, Banker G (1994) Neuronal polarity. Annu Rev Neurosci 17:267–310

    Article  CAS  PubMed  Google Scholar 

  40. Bays JL, DeMali KA (2017) Vinculin in cell-cell and cell-matrix adhesions. Cell Mol Life Sci 74:2999–3009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Stevens GR, Zhang C, Berg MM, Lambert MP, Barber K, Cantallops I, Routtenberg A, Klein WL (1996) CNS neuronal focal adhesion kinase forms clusters that co-localise with vinculin. J Neurosci Res 46:445–455

    Article  CAS  PubMed  Google Scholar 

  42. Le Clainche C, Dwivedi SP, Didry D, Carlier MF (2010) Vinculin is a dually regulated actin filament barbed end-capping and side-binding protein. J Biol Chem 285:23420–23432

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. Blanchoin L, Pollard TD, Mullins RD (2000) Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks. Curr Biol 10:1273–1282

    Article  CAS  PubMed  Google Scholar 

  44. Mullins RD, Heuser JA, Pollard TD (1998) The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc Natl Acad Sci USA 95:6181–6186

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Sturner, T. et al. (2019). Transient localisation of the Arp2/3 complex initiates neuronal dendrite branching in vivo. Development 146(7)

  46. Hetrick B, Han MS, Helgeson LA, Nolen BJ (2013) Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem Biol 20:701–712

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Huttelmaier S, Bubeck P, Rudiger M, Jockusch BM (1997) Characterisation of two F-actin-binding and oligomerisation sites in the cell-contact protein vinculin. Eur J Biochem 247:1136–1142

    Article  CAS  PubMed  Google Scholar 

  48. Thompson PM et al (2014) Identification of an actin binding surface on vinculin that mediates mechanical cell and focal adhesion properties. Structure 22:697–706

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Riedl J et al (2008) Lifeact: a versatile marker to visualise F-actin. Nat Methods 5:605–607

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Zhang M et al (2012) Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods 9:727–729

    Article  CAS  PubMed  Google Scholar 

  51. Wozniak MA, Modzelewska K, Kwong L, Keely PJ (2004) Focal adhesion regulation of cell behavior. Biochim Biophys Acta 1692:103–119

    Article  CAS  PubMed  Google Scholar 

  52. De Pascalis C, Etienne-Manneville S (2017) Single and collective cell migration: the mechanics of adhesions. Mol Biol Cell 28:1833–1846

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Carisey A, Ballestrem C (2011) Vinculin, an adapter protein in control of cell adhesion signalling. Eur J Cell Biol 90:157–163

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Ezzell RM, Goldmann WH, Wang N, Parashurama N, Ingber DE (1997) Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton. Exp Cell Res 231:14–26

    Article  CAS  PubMed  Google Scholar 

  55. Carisey A et al (2013) Vinculin regulates the recruitment and release of core focal adhesion proteins in a force-dependent manner. Curr Biol 23:271–281

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ziegler WH, Gingras AR, Critchley DR, Emsley J (2008) Integrin connections to the cytoskeleton through talin and vinculin. Biochem Soc Trans 36:235–239

    Article  CAS  PubMed  Google Scholar 

  57. Nair D, Hosy E, Petersen JD, Constals A, Giannone G, Choquet D, Sibarita JB (2013) Super-resolution imaging reveals that AMPA receptors inside synapses are dynamically organised in nanodomains regulated by PSD95. J Neurosci 33:13204–13224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank the microscopy facility and the Central Animal Facility in the division of biological sciences for confocal imaging and animal care, respectively.

Funding

This work was supported by the Department of Biotechnology (DBT)-IISc Partnership Program (N.R. and D.N.), Department of Biotechnology Genomics Engineering Taskforce (N.R. and D.N.), STAR program grant (D.N.), Indian Institute of Science Eminence Program, and University Grants Commission, India (D.N.). P.M. was supported by a fellowship from Council for Scientific and Industrial Research. V.B. was supported by a fellowship from the University Grants Commission, India.

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Authors and Affiliations

  1. Centre for Neuroscience, Indian Institute of Science, Bangalore, 560012, Karnataka, India

    Pranay Mandal, Vivek Belapurkar, Deepak Nair & Narendrakumar Ramanan

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  1. Pranay Mandal
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  2. Vivek Belapurkar
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  3. Deepak Nair
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  4. Narendrakumar Ramanan
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Contributions

NR conceived of the project and designed the experiments. PM performed all the experiments and analysed the data; VB and DN performed the super-resolution microscopy experiments and analysed the data. NR wrote the manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Narendrakumar Ramanan.

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The authors declare that they have no conflict of interest.

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All the procedures in this study were performed according to the rules and guidelines declared in the Compendium of CPCSEA 2018 by the Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA), Ministry of Fisheries, Animal Husbandry and Dairying, India. The research protocol was approved by the Institutional Animal Ethics Committee (IAEC) of the Indian Institute of Science (Project No. CAF/Ethics/516/2016), allowing the use of animals for in vitro and in vivo studies.

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Mandal, P., Belapurkar, V., Nair, D. et al. Vinculin-mediated axon growth requires interaction with actin but not talin in mouse neocortical neurons. Cell. Mol. Life Sci. 78, 5807–5826 (2021). https://doi.org/10.1007/s00018-021-03879-7

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