title: Detection of microtubules in vivo using antibody-immobilized nanoneedles.
abstract: We present here an alternative, force-based measurement method for the detection of intracellular cytoskeletal proteins in the live cell. High aspect ratio nanoneedles of 200 nm in diameter were functionalized with anti-tubulin antibodies and inserted, using an atomic force microscope (AFM), into live NIH3T3 cells, without affecting cell viability. Force curves were recorded during insertion and evacuation of nanoneedles from the cells, and used to analyse intracellular interactions of the nanoneedles with the microtubule cytoskeleton during evacuation from the cell. Disruption of microtubules led to a correlated time-dependent decrease in the measured intracellular binding forces, pointing to the high-sensitivity and high-specificity of this detection method. This analytical technique allows for real-time evaluation of the microtubule network in the live cell, without the need to use potentially harmful molecular markers as do conventional detection methods, and may prove beneficial in the diagnosis and investigation of cytoskeleton-associated diseases.
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan; Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Yaron R
authors.initials: YR
authors.lastname: Silberberg
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
authors.forename: Ryuzo
authors.initials: R
authors.lastname: Kawamura
authors.affiliation: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan.
authors.forename: Seunghwan
authors.initials: S
authors.lastname: Ryu
authors.affiliation: Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Fukazawa
authors.affiliation: Department of Materials Engineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8654, Japan.
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishihara
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan; Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan. Electronic address: chikashi-nakamura@aist.go.jp.
authors.forename: Chikashi
authors.initials: C
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D051379:Mice; D018625:Microscopy, Atomic Force; D008870:Microtubules / Q000378:metabolism*; D015336:Molecular Probe Techniques / Q000295:instrumentation*; D041681:NIH 3T3 Cells; D036103:Nanotechnology; D053770:Nanowires; D013314:Stress, Mechanical; D014404:Tubulin / Q000378:metabolism*
chemical_list: D000906:Antibodies; D014404:Tubulin
keywords: Atomic force microscopy; Force measurement; Intracellular protein detection; Microtubules; Nanoneedle