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Cytoskeletal Alterations and Biomechanical Properties of parkin-Mutant Human Primary Fibroblasts

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Abstract

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient’s fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young’s modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

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Abbreviations

α-Tub:

α-Tubulin

AFM:

Atomic force microscopy

CLSM:

Confocal laser scanning microscopy

ABPs:

Actin-binding proteins

COF:

Cofilin

F-actin:

Filamentous actin

Gsk-3:

Glycogen synthase kinase-3

PD:

Parkinson’s disease

CTR:

Healthy control

P1:

PD patient

siRNAs:

Interfering RNAs

LIMK:

LIM kinase

MLC:

Myosin light chain

p-COF:

Phosphorylated cofilin

Akt:

Protein kinase B

PAK:

Rac-Cdc42 p21-activated kinase

ROI:

Region of interest

ROCK:

Rho associated kinase

References

  1. Klein, C., & Westenberger, A. (2012). Genetics of Parkinson’s disease. Cold Spring Harbor Perspectives in Medicine, 2(1), a008888. doi:10.1101/cshperspect.a008888.

    Article  PubMed Central  PubMed  Google Scholar 

  2. Kahle, P. J., & Haass, C. (2004). How does parkin ligate ubiquitin to Parkinson’s disease? EMBO Reports, 5(7), 681–685. doi:10.1038/sj.embor.7400188.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Pacelli, C., De Rasmo, D., Signorile, A., Grattagliano, I., di Tullio, G., D’Orazio, A., et al. (2011). Mitochondrial defect and PGC-1α dysfunction in parkin-associated familial Parkinson’s disease. Biochimica et Biophysica Acta, 1812(8), 1041–1053. doi:10.1016/j.bbadis.2010.12.022.

    Article  CAS  PubMed  Google Scholar 

  4. Winklhofer, K. F. (2014). Parkin and mitochondrial quality control: Toward assembling the puzzle. Trends in Cell Biology, 24(6), 332–334. doi:10.1016/j.tcb.2014.01.001.

    Article  CAS  PubMed  Google Scholar 

  5. Zanon, A., Rakovic, A., Blankenburg, H., Doncheva, N. T., Schwienbacher, C., Serafin, A., et al. (2013). Profiling of Parkin-binding partners using tandem affinity purification. PLoS ONE, 8(11), 78648. doi:10.1371/journal.pone.0078648.

    Article  Google Scholar 

  6. Sarraf, S. A., Raman, M., Guarani-Pereira, V., Sowa, M. E., Huttlin, E. L., Gygi, S. P., et al. (2013). Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature, 496(7445), 372–376. doi:10.1038/nature12043.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  7. Gillardon, F. (2009). Leucine-rich repeat kinase 2 phosphorylates brain tubulin-beta isoforms and modulates microtubule stability a point of convergence in parkinsonian neurodegeneration? Journal of Neurochemistry, 110(5), 1514–1522. doi:10.1111/j.1471-4159.2009.06235.

    Article  CAS  PubMed  Google Scholar 

  8. Häbig, K., Gellhaar, S., Heim, B., Djuric, V., Giesert, F., Wurst, W., et al. (2013). LRRK2 guides the actin cytoskeleton at growth cones together with ARHGEF7 and tropomyosin 4. Biochimica et Biophysica Acta, 1832(12), 2352–2367. doi:10.1016/j.bbadis.2013.09.009.

    Article  PubMed  Google Scholar 

  9. Cartelli, D., Goldwurm, S., Casagrande, F., Pezzoli, G., & Cappelletti, G. (2012). Microtubule destabilization is shared by genetic and idiopathic Parkinson’s disease patient fibroblasts. PLoS ONE, 7(5), e37467. doi:10.1371/journal.pone.0037467.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  10. Kubo, S. I., Kitami, T., Noda, S., Shimura, H., Uchiyama, Y., Asakawa, S., et al. (2001). Parkin is associated with cellular vesicles. Journal of Neurochemistry, 78(1), 42–54. doi:10.1046/j.1471-4159.2001.00364.x.

    Article  CAS  PubMed  Google Scholar 

  11. Revenu, C., Athman, R., Robine, S., & Louvard, D. (2004). The co-workers of actin filaments: from cell structures to signals. Nature Reviews Molecular Cell Biology, 8, 635–646. doi:10.1038/nrm1437.

    Article  Google Scholar 

  12. Vergara, D., Simeone, P., del Boccio, P., Toto, C., Pieragostino, D., Tinelli, A., et al. (2013). Comparative proteome profiling of breast tumor cell lines by gel electrophoresis and mass spectrometry reveals an epithelial mesenchymal transition associated protein signature. Molecular BioSystems, 6, 1127–1138. doi:10.1039/c2mb25401h.

    Article  Google Scholar 

  13. De Masi, R., Vergara, D., Pasca, S., Acierno, R., Greco, M., Spagnolo, L., et al. (2009). PBMCs protein expression profile in relapsing IFN-treated multiple sclerosis: A pilot study on relation to clinical findings and brain atrophy. Journal of Neuroimmunology, 210(1–2), 80–86. doi:10.1016/j.jneuroim.2009.03.002.

    Article  PubMed  Google Scholar 

  14. Uribe, R., & Jay, D. (2009). A review of actin binding proteins: New perspectives. Molecular Biology Reports, 36(1), 121–125. doi:10.1007/s11033-007-9159-2.

    Article  CAS  PubMed  Google Scholar 

  15. Dent, E. W., & Gertler, F. B. (2003). Cytoskeletal dynamics and transport in growth cone motility and axon guidance. Neuron, 40(2), 209–227. doi:10.1016/S0896-6273(03)00633-0.

    Article  CAS  PubMed  Google Scholar 

  16. Sarmiere, P. D., & Bamburg, J. R. (2004). Regulation of the neuronal actin cytoskeleton by ADF/cofilin. Journal of Neurobiology, 58(1), 103–117. doi:10.1002/neu.10267.

    Article  CAS  PubMed  Google Scholar 

  17. Schonhofen, P., de Medeiros, L. M., Chatain, C. P., Bristot, I. J., & Klamt, F. (2014). Cofilin/actin rod formation by dysregulation of cofilin-1 activity as a central initial step in neurodegeneration. Mini Review in Medicinal Chemistry, 14(5), 393–400. doi:10.2174/1389557514666140506161458.

    Article  CAS  Google Scholar 

  18. Mizuno, K. (2013). Signaling mechanisms and functional roles of cofilin phosphorylation and dephosphorylation. Cellular Signalling, 25(2), 457–469. doi:10.1016/j.cellsig.2012.11.001.

    Article  CAS  PubMed  Google Scholar 

  19. Riento, K., & Ridley, A. J. (2003). Rocks: Multifunctional kinases in cell behaviour. Nature Reviews Molecular Cell Biology, 4(6), 446–456. doi:10.1038/nrm1128.

    Article  CAS  PubMed  Google Scholar 

  20. Ferretta, A., Gaballo, A., Tanzarella, P., Piccoli, C., Capitanio, N., Nico, B., et al. (2014). Effect of resveratrol on mitochondrial function: Implications in parkin-associated familiar parkinson’s disease. Biochimica et Biophysica Acta, 1842(7), 902–915. doi:10.1016/j.bbadis.2014.02.010.

    Article  CAS  PubMed  Google Scholar 

  21. Boudaoud, A., Burian, A., Borowska-Wykręt, D., Uyttewaal, M., Wrzalik, R., Kwiatkowska, D., et al. (2014). FibrilTool, an ImageJ plug-into quantify fibrillar structures in raw microscopy images. Nature Protocols, 9, 457–463. doi:10.1038/nprot.2014.024.

    Article  CAS  PubMed  Google Scholar 

  22. Leporatti, S., Vergara, D., Zacheo, A., Vergaro, V., Maruccio, G., Cingolani, R., et al. (2009). Cytomechanical and topological investigation of MCF-7 cells by scanning force microscopy. Nanotechnology, 20(5), 55103–55108. doi:10.1088/0957-4484/20/5/055103.

    Article  Google Scholar 

  23. Vergara, D., Martignago, R., Leporatti, S., Bonsegna, S., Maruccio, G., De Nuccio, F., et al. (2009). Biomechanical and proteomic analysis of INF- beta-treated astrocytes. Nanotechnology, 20(45), 455106–455115. doi:10.1088/0957-4484/20/45/455106.

    Article  PubMed  Google Scholar 

  24. Butt, H. J., & Jaschke, M. (1995). Calculation of thermal noise in atomic force microscopy. Nanotechnology, 6, 1–7. doi:10.1088/0957-4484/6/1/001.

    Article  Google Scholar 

  25. Takahashi-Yanaga, F., & Sasaguri, T. (2008). GSK-3beta regulates cyclin D1 expression: A new target for chemotherapy. Cellular Signalling, 4, 581–589. doi:10.1016/j.cellsig.2007.10.018.

    Article  Google Scholar 

  26. Rawal, N., Corti, O., Sacchetti, P., Ardilla-Osorio, H., Sehat, B., Brice, A., et al. (2009). Parkin protects dopaminergic neurons from excessive Wnt/beta-catenin signaling. Biochemical and Biophysical Research Communications, 388(3), 473–478. doi:10.1016/j.bbrc.2009.07.014.

    Article  CAS  PubMed  Google Scholar 

  27. Sousa, V. L., Bellani, S., Giannandrea, M., Yousuf, M., Valtorta, F., Meldolesi, J., et al. (2009). {alpha}-synuclein and its A30P mutant affect actin cytoskeletal structure and dynamics. Molecular Biology of the Cell, 16, 3725–3739. doi:10.1091/mbc.E08-03-0302.

    Article  Google Scholar 

  28. Esposito, A., Dohm, C. P., Kermer, P., Bähr, M., & Wouters, F. S. (2007). Alpha-synuclein and its disease-related mutants interact differentially with the microtubule protein tau and associate with the actin cytoskeleton. Neurobiology of Diseases, 3, 521–531. doi:10.1016/j.nbd.2007.01.014.

    Article  Google Scholar 

  29. Kim, K. H., & Son, J. H. (2010). PINK1 gene knockdown leads to increased binding of parkin with actin filament. Neuroscience Letters, 468(3), 272–276. doi:10.1016/j.neulet.2009.11.011.

    Article  CAS  PubMed  Google Scholar 

  30. Lim, M. K., Kawamura, T., Ohsawa, Y., Ohtsubo, M., Asakawa, S., Takayanagi, A., et al. (2007). Parkin interacts with LIM Kinase 1 and reduces its cofilin-phosphorylation activity via ubiquitination. Experimental Cell Research, 313(13), 2858–2874. doi:10.1016/j.yexcr.2007.04.016.

    Article  CAS  PubMed  Google Scholar 

  31. Bradke, F., & Dotti, C. G. (1999). The role of local actin instability in axon formation. Science, 283(5409), 1931–1934. doi:10.1126/science.283.5409.1931.

    Article  CAS  PubMed  Google Scholar 

  32. Geraldo, S., & Gordon-Weeks, P. R. (2009). Cytoskeletal dynamics in growth-cone steering. Journal of Cell Science, 122(20), 3595–3604. doi:10.1242/jcs.042309.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Parisiadou, L., Xie, C., Cho, H. J., Lin, X., Gu, X. L., Long, C. X., et al. (2009). Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis. Journal of Neuroscience, 29(44), 13971–13980. doi:10.1523/JNEUROSCI.3799-09.2009.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Fierro-González, J. C., White, M. D., Silva, J. C., & Plachta, N. (2013). Cadherin-dependent filopodia control preimplantation embryo compaction. Nature Cell Biology, 15(12), 1424–1433. doi:10.1038/ncb2875.

    Article  PubMed  Google Scholar 

  35. Narumiya, S., Ishizaki, T., & Ufhata, M. (2000). Use and properties of ROCK-specific inhibitor Y-27632. In W. E. Balch, J. D. Channing, & H. Alan (Eds.), Methods in Enzymology (pp. 273–284). San Diego: Academic Press. doi:10.1016/S0076-6879(00)25449-9.

    Google Scholar 

Download references

Acknowledgments

This work was supported by the PON project 254/Ric. “Implementation of human and environment health research center”, Cod. PONa3_00334, REA research Grant no PITN-GA-2012-316549 (IT LIVER) from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013), PRIN 2010FPTBSH “NANO Molecular technologies for Drug delivery - NANOMED”, Cod. PON02_00563_3448479-F “RINOVATIS”, and Cod. PONa3_00134 “Omics and nanotechnology for early diagnosis of human diseases - ONEV”. LLdM thanks Prof. Arezki Boudaoud for useful discussions about FibrilTool.

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Authors and Affiliations

  1. Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy

    Daniele Vergara, Marzia M. Ferraro & Michele Maffia

  2. Institute of Nanoscience-NNL, CNR, Via Arnesano, 16, Lecce, 73100, Italy

    Marzia M. Ferraro, Mariafrancesca Cascione, Loretta L. del Mercato, Stefano Leporatti, Ross Rinaldi & Antonio Gaballo

  3. Department of Mathematics and Physics “Ennio De Giorgi”, University of Salento, Lecce, Italy

    Mariafrancesca Cascione & Ross Rinaldi

  4. Department of Basic Medical Sciences, Neurosciences and Organs of Senses, University of Bari ‘A. Moro’, Bari, Italy

    Anna Ferretta, Paola Tanzarella & Tiziana Cocco

  5. Department of Pharmacology, Faculty of Medicine, Universitè de Montreal, 2900 Boulevard Edouard-Montpetit, Montreal, QC, H3T1J4, Canada

    Consiglia Pacelli

  6. Institute of Science of Food Production, CNR, Lecce, Italy

    Angelo Santino

  7. Laboratory of Clinical Proteomic, ‘‘Giovanni Paolo II’’ Hospital, ASL-Lecce, Italy

    Daniele Vergara & Michele Maffia

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  1. Daniele Vergara
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Corresponding author

Correspondence to Antonio Gaballo.

Additional information

Daniele Vergara, Marzia M. Ferraro and Mariafrancesca Cascione have contributed equally to this work.

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Vergara, D., Ferraro, M.M., Cascione, M. et al. Cytoskeletal Alterations and Biomechanical Properties of parkin-Mutant Human Primary Fibroblasts. Cell Biochem Biophys 71, 1395–1404 (2015). https://doi.org/10.1007/s12013-014-0362-1

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