High efficiency penetration of antibody-immobilized nanoneedle thorough plasma membrane for in situ detection of cytoskeletal proteins in living cells.
BACKGROUND
The field of structural dynamics of cytoskeletons in living cells is gathering wide interest, since better understanding of cytoskeleton intracellular organization will provide us with not only insights into basic cell biology but may also enable development of new strategies in regenerative medicine and cancer therapy, fields in which cytoskeleton-dependent dynamics play a pivotal role. The nanoneedle technology is a powerful tool allowing for intracellular investigations, as it can be directly inserted into live cells by penetrating through the plasma membrane causing minimal damage to cells, under the precise manipulation using atomic force microscope. Modifications of the nanoneedles using antibodies have allowed for accurate mechanical detection of various cytoskeletal components, including actin, microtubules and intermediate filaments. However, successful penetration of the nanoneedle through the plasma membrane has been shown to vary greatly between different cell types and conditions. In an effort to overcome this problem and improve the success rate of nanoneedle insertion into the live cells, we have focused here on the fluidity of the membrane lipid bilayer, which may hinder nanoneedle penetration into the cytosolic environment.


RESULTS
We aimed to reduce apparent fluidity of the membrane by either increasing the approach velocity or reducing experimental temperatures. Although changes in approach velocity did not have much effect, lowering the temperature was found to greatly improve the detection of unbinding forces, suggesting that alteration in the plasma membrane fluidity led to increase in nanoneedle penetration.


CONCLUSIONS
Operation at a lower temperature of 4 °C greatly improved the success rate of nanoneedle insertion to live cells at an optimized approach velocity, while it did not affect the binding of antibodies immobilized on the nanoneedle to vimentins for mechanical detection. As these experimental parameters can be applied to various cell types, these results may improve the versatility of the nanoneedle technology to other cell lines and platforms.
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Central5 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
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Kawamura
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-26 Naka-cho, Koganei, Tokyo, 184-8588, Japan.
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Shimizu
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-26 Naka-cho, Koganei, Tokyo, 184-8588, Japan.
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Matsumoto
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Central5 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
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A
Yamagishi
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Central5 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan.
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Silberberg
Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan.
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Iijima
Department of Biomolecular Science and Reaction, The Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, 567-0047, Japan.
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Kuroda
Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8654, Japan.
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Fukazawa
Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8654, Japan.
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Ishihara
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Central5 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8565, Japan. chikashi-nakamura@aist.go.jp.|Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-26 Naka-cho, Koganei, Tokyo, 184-8588, Japan. chikashi-nakamura@aist.go.jp.
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http://orcid.org/0000-0002-8012-2192
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Nakamura
D055910:Antibodies, Immobilized / Q000737:chemistry* / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D036103:Nanotechnology / Q000295:instrumentation*; D009339:Needles; D059010:Single-Cell Analysis / Q000295:instrumentation* / Q000379:methods
D055910:Antibodies, Immobilized; D003598:Cytoskeletal Proteins
Atomic force microscopy; Cytoskeleton; Intermediate filament; Mechanobiology; Nanoneedle; Single cell analysis