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Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells

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Abstract

Femtosecond-pulsed laser irradiation was found to initiate giant plasma membrane vesicle (GPMV) formation on individual cells. Laser-induced GPMV formation resulted from intracellular cavitation and did not require the addition of chemical stressors to the cellular environment. The viscosity, structure, and contents of laser-induced GPMVs were measured with fluorescence microscopy and single-particle tracking. These GPMVs exhibit the following properties: (1) GPMVs grow fastest immediately after laser irradiation; (2) GPMVs contain barriers to free diffusion of incorporated fluorescent beads; (3) materials from both the cytoplasm and surrounding media flow into the growing GPMVs; (4) the GPMVs are surrounded by phospholipids, including phosphatidylserine; (5) F-actin is incorporated into the vesicles; and (6) caspase activity is not essential for GPMV formation. The effective viscosity of 65 nm polystyrene nanoparticles within GPMVs ranged from 32 to 434 cP. The nanoparticle diffusion was commonly affected by relatively large, macromolecular structures within the bleb.

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Acknowledgments

Partial support for this project was provided by the Michigan Nanotechnology Institute for Medicine and Biological Sciences, the National Institute of Biomedical Imaging and Bioengineering (R01-EB005028), and by the NSF Frontiers in Physics FOCUS Center under grant PHY-0114336. C.V.K. received fellowship support from the NIH Michigan Molecular Biophysics Training Program (T32 GM008270-20), the Applied Physics program, and the Graham Environmental Sustainability Institute. The authors thank Meghan Liroff, Tom Dunham, Pascale Leroueil, Kevin McDonough, Alina Kotlya, and Kathryn Kelly.

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Authors and Affiliations

  1. Applied Physics Program, University of Michigan, Ann Arbor, MI, USA

    Christopher V. Kelly, David A. Reis, Bradford G. Orr & Mark M. Banaszak Holl

  2. Biophysics, University of Michigan, Ann Arbor, MI, USA

    Christopher V. Kelly & Mark M. Banaszak Holl

  3. Graham Environmental Sustainability Institute, University of Michigan, Ann Arbor, MI, USA

    Christopher V. Kelly & Mark M. Banaszak Holl

  4. Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Ann Arbor, MI, USA

    Christopher V. Kelly, Mary-Margaret T. Kober, Päivö Kinnunen, Bradford G. Orr & Mark M. Banaszak Holl

  5. Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

    Mary-Margaret T. Kober & Mark M. Banaszak Holl

  6. Department of Physics, University of Michigan, Ann Arbor, MI, USA

    Päivö Kinnunen, David A. Reis & Bradford G. Orr

  7. 450 Church Street, Ann Arbor, MI, 48109, USA

    Bradford G. Orr

  8. 930 North University Avenue, Ann Arbor, MI, 48109, USA

    Mark M. Banaszak Holl

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  1. Christopher V. Kelly
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Mary-Margaret T. Kober and Päivö Kinnunen gave equal contribution to this manuscript.

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Kelly, C.V., Kober, MM.T., Kinnunen, P. et al. Pulsed-laser creation and characterization of giant plasma membrane vesicles from cells. J Biol Phys 35, 279–295 (2009). https://doi.org/10.1007/s10867-009-9167-7

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