Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons

Nature Cell Biology volume 13, pages 40–48 (2011)Cite this article

Subjects

Abstract

Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va. We show here that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into Purkinje neuron spines. Specifically, myosin-Va accumulates at the ER tip as the organelle moves into spines, and hydrolysis of ATP by myosin-Va is required for spine ER targeting. Moreover, myosin-Va is responsible for almost all of the spine ER insertion events. Finally, attenuation of the ability of myosin-Va to move along actin filaments reduces the maximum velocity of ER movement into spines, providing direct evidence that myosin-Va drives ER motility. Thus, we have established that an actin-based motor moves ER within animal cells, and have uncovered the mechanism for ER localization to Purkinje neuron spines, a prerequisite for synaptic plasticity.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The delayed mGluR1-dependent Ca2+ transient, but not the fast AMPA receptor-dependent Ca2+ transient, is absent in dl20J/dl20J Purkinje neuron spines.
Figure 2: Models of how myosin-Va might function to localize ER to Purkinje neuron spines.
Figure 3: Translocation of ER into spines is disrupted in dl20J/dl20J Purkinje neurons.
Figure 4: Quantification of ER dynamics indicates that ER movement into Purkinje neuron spines depends critically on myosin-Va.
Figure 5: ER insertion into control dv/dl20J Purkinje neuron spines does not depend significantly on microtubule entry into spines.
Figure 6: mGFP–myosin-Va expressed in dl20J/dl20J Purkinje neurons rescues ER targeting and accumulates at the tip of the spine ER.
Figure 7: Myosin-Va does not colocalize with the PSD marker PSD-93 and is present at the leading tip of the ER tubule as the organelle translocates into a spine.
Figure 8: Decreasing the step size or ATPase activity of myosin-Va reduces the efficiency of ER targeting to Purkinje neurons spines and the maximum velocity of ER movement into spines.

Similar content being viewed by others

References

  1. Sellers, J. R. & Weisman, L. S. Myosin V. in Myosins: A Superfamily of Molecular Motors (Springer Netherlands, 2008).

    Google Scholar 

  2. Schott, D. H., Collins, R. N. & Bretscher, A. Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length. J. Cell Biol. 156, 35–39 (2002).

    Article  CAS  Google Scholar 

  3. Jung, G., Titus, M. A. & Hammer, J. A., 3rd The Dictyostelium type V myosin MyoJ is responsible for the cortical association and motility of contractile vacuole membranes. J. Cell Biol. 186, 555–570 (2009).

    Article  CAS  Google Scholar 

  4. Woolner, S. & Bement, W. M. Unconventional myosins acting unconventionally. Trends Cell Biol. 19, 245–252 (2009).

    Article  CAS  Google Scholar 

  5. Desnos, C. et al. Myosin Va mediates docking of secretory granules at the plasma membrane. J. Neurosci. 27, 10636–10645 (2007).

    Article  CAS  Google Scholar 

  6. Provance, D. W., Jr et al. Myosin-Vb functions as a dynamic tether for peripheral endocytic compartments during transferrin trafficking. BMC Cell Biol. 9, 44 (2008).

    Article  Google Scholar 

  7. Wu, X., Bowers, B., Rao, K., Wei, Q. & Hammer, J. A., 3rd Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin V function in vivo. J. Cell Biol. 143, 1899–1918 (1998).

    Article  CAS  Google Scholar 

  8. Mercer, J. A., Seperack, P. K., Strobel, M. C., Copeland, N. G. & Jenkins, N. A. Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349, 709–713 (1991).

    Article  CAS  Google Scholar 

  9. Fukuda, M., Kuroda, T. S. & Mikoshiba, K. Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport. J. Biol. Chem. 277, 12432–12436 (2002).

    Article  CAS  Google Scholar 

  10. Wu, X. S. et al. Identification of an organelle receptor for myosin-Va. Nat. Cell Biol. 4, 271–278 (2002).

    Article  CAS  Google Scholar 

  11. Strom, M., Hume, A. N., Tarafder, A. K., Barkagianni, E. & Seabra, M. C. A family of Rab27-binding proteins. Melanophilin links Rab27a and myosin Va function in melanosome transport. J. Biol. Chem. 277, 25423–25430 (2002).

    Article  CAS  Google Scholar 

  12. Moore, K. J. et al. Dilute suppressor dsu acts semidominantly to suppress the coat color phenotype of a deletion mutation, dl20J, of the murine dilute locus. Proc. Natl Acad. Sci. USA 85, 8131–8135 (1988).

    Article  CAS  Google Scholar 

  13. Strobel, M. C., Seperack, P. K., Copeland, N. G. & Jenkins, N. A. Molecular analysis of two mouse dilute locus deletion mutations: spontaneous dilute lethal20J and radiation-induced dilute prenatal lethal Aa2 alleles. Mol. Cell. Biol. 10, 501–509 (1990).

    Article  CAS  Google Scholar 

  14. Tilelli, C. Q., Martins, A. R., Larson, R. E. & Garcia-Cairasco, N. Immunohistochemical localization of myosin Va in the adult rat brain. Neuroscience 121, 573–586 (2003).

    Article  CAS  Google Scholar 

  15. Miyata, M. et al. Local calcium release in dendritic spines required for long-term synaptic depression. Neuron 28, 233–244 (2000).

    Article  CAS  Google Scholar 

  16. Takagishi, Y. et al. The dilute-lethal (dl) gene attacks a Ca2+ store in the dendritic spine of Purkinje cells in mice. Neurosci. Lett. 215, 169–172 (1996).

    Article  CAS  Google Scholar 

  17. Bourne, J. N. & Harris, K. M. Balancing structure and function at hippocampal dendritic spines. Annu. Rev. Neurosci. 31, 47–67 (2008).

    Article  CAS  Google Scholar 

  18. English, A. R., Zurek, N. & Voeltz, G. K. Peripheral ER structure and function. Curr. Opin. Cell Biol. 21, 596–602 (2009).

    Article  CAS  Google Scholar 

  19. Harris, K. M. & Stevens, J. K. Dendritic spines of rat cerebellar Purkinje cells: serial electron microscopy with reference to their biophysical characteristics. J. Neurosci. 8, 4455–4469 (1988).

    Article  CAS  Google Scholar 

  20. Matsumoto, M. et al. Ataxia and epileptic seizures in mice lacking type 1 inositol 1, 4, 5-trisphosphate receptor. Nature 379, 168–171 (1996).

    Article  CAS  Google Scholar 

  21. Street, V. A. et al. The type 1 inositol 1, 4, 5-trisphosphate receptor gene is altered in the opisthotonos mouse. J. Neurosci. 17, 635–645 (1997).

    Article  CAS  Google Scholar 

  22. Hartmann, J. & Konnerth, A. Mechanisms of metabotropic glutamate receptor-mediated synaptic signaling in cerebellar Purkinje cells. Acta Physiol. 195, 79–90 (2008).

    Article  Google Scholar 

  23. Kano, M., Hashimoto, K. & Tabata, T. Type-1 metabotropic glutamate receptor in cerebellar Purkinje cells: a key molecule responsible for long-term depression, endocannabinoid signalling and synapse elimination. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 363, 2173–2186 (2008).

    Article  CAS  Google Scholar 

  24. Takechi, H., Eilers, J. & Konnerth, A. A new class of synaptic response involving calcium release in dendritic spines. Nature 396, 757–760 (1998).

    Article  CAS  Google Scholar 

  25. Finch, E. A. & Augustine, G. J. Local calcium signalling by inositol-1, 4, 5-trisphosphate in Purkinje cell dendrites. Nature 396, 753–756 (1998).

    Article  CAS  Google Scholar 

  26. Bittins, C. M., Eichler, T. W. & Gerdes, H. H. Expression of the dominant-negative tail of myosin Va enhances exocytosis of large dense core vesicles in neurons. Cell. Mol. Neurobiol. 29, 597–608 (2009).

    Article  CAS  Google Scholar 

  27. Watanabe, M. et al. Myosin-Va regulates exocytosis through the submicromolar Ca2+-dependent binding of syntaxin-1A. Mol. Biol. Cell 16, 4519–4530 (2005).

    Article  CAS  Google Scholar 

  28. Xiao, B. et al. Homer regulates the association of group 1 metabotropic glutamate receptors with multivalent complexes of homer-related, synaptic proteins. Neuron 21, 707–716 (1998).

    Article  CAS  Google Scholar 

  29. Shiraishi-Yamaguchi, Y. & Furuichi, T. The Homer family proteins. Genome Biol. 8, 206 (2007).

    Article  Google Scholar 

  30. Shiraishi, Y., Mizutani, A., Yuasa, S., Mikoshiba, K. & Furuichi, T. Differential expression of Homer family proteins in the developing mouse brain. J. Comp. Neurol. 473, 582–599 (2004).

    Article  CAS  Google Scholar 

  31. Jaworski, J. et al. Dynamic microtubules regulate dendritic spine morphology and synaptic plasticity. Neuron 61, 85–100 (2009).

    Article  CAS  Google Scholar 

  32. Hu, X., Viesselmann, C., Nam, S., Merriam, E. & Dent, E. W. Activity-dependent dynamic microtubule invasion of dendritic spines. J. Neurosci. 28, 13094–13105 (2008).

    Article  CAS  Google Scholar 

  33. Bannai, H., Inoue, T., Nakayama, T., Hattori, M. & Mikoshiba, K. Kinesin dependent, rapid, bi-directional transport of ER sub-compartment in dendrites of hippocampal neurons. J. Cell Sci. 117, 163–175 (2004).

    Article  CAS  Google Scholar 

  34. Grigoriev, I. et al. STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER. Curr. Biol. 18, 177–182 (2008).

    Article  CAS  Google Scholar 

  35. Serinagaoglu, Y. et al. A promoter element with enhancer properties, and the orphan nuclear receptor RORalpha, are required for Purkinje cell-specific expression of a Gi/o modulator. Mol. Cell. Neurosci. 34, 324–342 (2007).

    Article  CAS  Google Scholar 

  36. Altan-Bonnet, N. et al. Golgi inheritance in mammalian cells is mediated through endoplasmic reticulum export activities. Mol. Biol. Cell 17, 990–1005 (2006).

    Article  CAS  Google Scholar 

  37. Petralia, R. S. et al. Glutamate receptor targeting in the postsynaptic spine involves mechanisms that are independent of myosin Va. Eur. J. Neurosci. 13, 1722–1732 (2001).

    Article  CAS  Google Scholar 

  38. Linden, D. J. & Ahn, S. Activation of presynaptic cAMP-dependent protein kinase is required for induction of cerebellar long-term potentiation. J. Neurosci. 19, 10221–10227 (1999).

    Article  CAS  Google Scholar 

  39. Hisatsune, C. et al. Inositol 1, 4, 5-trisphosphate receptor type 1 in granule cells, not in Purkinje cells, regulates the dendritic morphology of Purkinje cells through brain-derived neurotrophic factor production. J. Neurosci. 26, 10916–10924 (2006).

    Article  CAS  Google Scholar 

  40. Yengo, C. M., De la Cruz, E. M., Safer, D., Ostap, E. M. & Sweeney, H. L. Kinetic characterization of the weak binding states of myosin V. Biochemistry 41, 8508–8517 (2002).

    Article  CAS  Google Scholar 

  41. Shimada, T., Sasaki, N., Ohkura, R. & Sutoh, K. Alanine scanning mutagenesis of the switch I region in the ATPase site of Dictyostelium discoideum myosin II. Biochemistry 36, 14037–14043 (1997).

    Article  CAS  Google Scholar 

  42. Walikonis, R. S. et al. Identification of proteins in the postsynaptic density fraction by mass spectrometry. J. Neurosci. 20, 4069–4080 (2000).

    Article  CAS  Google Scholar 

  43. Forgacs, E. et al. Switch 1 mutation S217A converts myosin V into a low duty ratio motor. J. Biol. Chem. 284, 2138–2149 (2009).

    Article  CAS  Google Scholar 

  44. Sakamoto, T., Yildez, A., Selvin, P. R. & Sellers, J. R. Step-size is determined by neck length in myosin V. Biochemistry 44, 16203–16210 (2005).

    Article  CAS  Google Scholar 

  45. Sakamoto, T. et al. Neck length and processivity of myosin V. J. Biol. Chem. 278, 29201–29207 (2003).

    Article  CAS  Google Scholar 

  46. Purcell, T. J., Morris, C., Spudich, J. A. & Sweeney, H. L. Role of the lever arm in the processive stepping of myosin V. Proc. Natl Acad. Sci. USA 99, 14159–14164 (2002).

    Article  CAS  Google Scholar 

  47. Tabb, J. S., Molyneaux, B. J., Cohen, D. L., Kuznetsov, S. A. & Langford, G. M. Transport of ER vesicles on actin filaments in neurons by myosin V. J. Cell Sci. 111, 3221–3234 (1998).

    CAS  PubMed  Google Scholar 

  48. Wollert, T., Weiss, D. G., Gerdes, H. H. & Kuznetsov, S. A. Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis. J. Cell Biol. 159, 571–577 (2002).

    Article  CAS  Google Scholar 

  49. Estrada, P. et al. Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae. J. Cell Biol. 163, 1255–1266 (2003).

    Article  CAS  Google Scholar 

  50. Sparkes, I., Runions, J., Hawes, C. & Griffing, L. Movement and remodeling of the endoplasmic reticulum in nondividing cells of tobacco leaves. Plant Cell 21, 3937–3949 (2009).

    Article  CAS  Google Scholar 

  51. Yokota, E. et al. An isoform of myosin XI is responsible for the translocation of endoplasmic reticulum in tobacco cultured BY-2 cells. J. Exp. Bot. 60, 197–212 (2009).

    Article  CAS  Google Scholar 

  52. Ueda, H. et al. Myosin-dependent endoplasmic reticulum motility and F-actin organization in plant cells. Proc. Natl Acad. Sci. USA 107, 6894–6899.

    Article  CAS  Google Scholar 

  53. Wang, Z. et al. Myosin Vb mobilizes recycling endosomes and AMPA receptors for postsynaptic plasticity. Cell 135, 535–548 (2008).

    Article  CAS  Google Scholar 

  54. Langford, G. M. Actin- and microtubule-dependent organelle motors: interrelationships between the two motility systems. Curr. Opin. Cell Biol. 7, 82–88 (1995).

    Article  CAS  Google Scholar 

  55. Toresson, H. & Grant, S. G. Dynamic distribution of endoplasmic reticulum in hippocampal neuron dendritic spines. Eur. J. Neurosci. 22, 1793–1798 (2005).

    Article  Google Scholar 

  56. Holbro, N., Grunditz, A. & Oertner, T. G. Differential distribution of endoplasmic reticulum controls metabotropic signaling and plasticity at hippocampal synapses. Proc. Natl Acad. Sci. USA 106, 15055–15060 (2009).

    Article  CAS  Google Scholar 

  57. Hansel, C. et al. alphaCaMKII Is essential for cerebellar LTD and motor learning. Neuron 51, 835–843 (2006).

    Article  CAS  Google Scholar 

  58. De Zeeuw, C. I. et al. Expression of a protein kinase C inhibitor in Purkinje cells blocks cerebellar LTD and adaptation of the vestibulo-ocular reflex. Neuron 20, 495–508 (1998).

    Article  CAS  Google Scholar 

  59. Seperack, P. K., Mercer, J. A., Strobel, M. C., Copeland, N. G. & Jenkins, N. A. Retroviral sequences located within an intron of the dilute gene alter dilute expression in a tissue-specific manner. EMBO J. 14, 2326–2332 (1995).

    Article  CAS  Google Scholar 

  60. Tabata, T. et al. A reliable method for culture of dissociated mouse cerebellar cells enriched for Purkinje neurons. J. Neurosci. Methods 104, 45–53 (2000).

    Article  CAS  Google Scholar 

  61. Alami, N. H., Jung, P. & Brown, A. Myosin Va increases the efficiency of neurofilament transport by decreasing the duration of long-term pauses. J. Neurosci. 29, 6625–6634 (2009).

    Article  CAS  Google Scholar 

  62. Johnson, H. W. & Schell, M. J. Neuronal IP3 3-kinase is an F-actin bundling protein: role in dendritic targeting and regulation of spine morphology. Mol. Biol. Cell 20, 5166–5180 (2009).

    Article  CAS  Google Scholar 

  63. McCroskery, S., Chaudhry, A., Lin, L. & Daniels, M. P. Transmembrane agrin regulates filopodia in rat hippocampal neurons in culture. Mol. Cell. Neurosci. 33, 15–28 (2006).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Bock and D. J. Linden for teaching us cerebellar culture preparation; J. Oberdick, E. L. Snapp, J. Lippincott-Schwartz, H. D. White, J. R. Sellers, J. A. Martina, S. McCroskery, M. J. Schell and D. S. Bredt for DNA constructs; R. E. Cheney for advice and X. Wu for microscopy support.

Author information

Authors and Affiliations

  1. Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, Maryland, USA

    Wolfgang Wagner & John A. Hammer III

  2. Section on Synaptic Transmission, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892, Maryland, USA

    Stephan D. Brenowitz

Authors
  1. Wolfgang Wagner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stephan D. Brenowitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John A. Hammer III
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.W. and J.A.H. designed the project; W.W. carried out the experiments, except the Ca2+ imaging, which was carried out by S.D.B.; J.A.H. contributed new reagents and W.W., S.D.B. and J.A.H. analysed the data and wrote the manuscript.

Corresponding author

Correspondence to John A. Hammer III.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1635 kb)

Supplementary Movie 1

Supplementary Information (MOV 10581 kb)

Supplementary Movie 2

Supplementary Information (MOV 474 kb)

Supplementary Movie 3

Supplementary Information (MOV 11555 kb)

Supplementary Movie 4

Supplementary Information (MOV 352 kb)

Supplementary Movie 5

Supplementary Information (MOV 4145 kb)

Supplementary Movie 6

Supplementary Information (MOV 1134 kb)

Supplementary Movie 7

Supplementary Information (MOV 12918 kb)

Supplementary Movie 8

Supplementary Information (MOV 12563 kb)

Supplementary Movie 9

Supplementary Information (MOV 12766 kb)

Supplementary Movie 10

Supplementary Information (MOV 5617 kb)

Supplementary Movie 11

Supplementary Information (MOV 1678 kb)

Supplementary Movie 12

Supplementary Information (MOV 521 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wagner, W., Brenowitz, S. & Hammer, J. Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons. Nat Cell Biol 13, 40–48 (2011). https://doi.org/10.1038/ncb2132

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2132

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing