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11. Antibody-based analysis reveals 'filamentous vs. non-filamentous' and 'cytoplasmic vs. nuclear' crosstalk of cytoskeletal proteins

Author

Kunio Takeyasu, Masahiro Kumeta, Shige H. Yoshimura, Yuya Hirai, and Tsuneyoshi Horigome

Subjects
Monoclonal antibody, Cytoplasm, Nuclear scaffold, Actinin, macromolecular substances, Chemical Fractionation, Biology, medicine, Humans, Nuclear protein, Cytoskeleton, Nuclear export signal, Nuclear matrix, Cell Nucleus, Antibodies, Monoclonal, Cell Biology, Subcellular localization, Cell biology, Cytoskeletal Proteins, medicine.anatomical_structure, Keratin, Nuclear transport, Nucleus, Lamin, HeLa Cells
Abstract

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus.

Published
2013

14. Antibody-based analysis reveals 'filamentous vs. non-filamentous' and 'cytoplasmic vs. nuclear' crosstalk of cytoskeletal proteins.

Author

00582181, 90710369, 90346106, Kumeta, Masahiro, Hirai, Yuya, Yoshimura, Shige H, Horigome, Tsuneyoshi, Takeyasu, Kunio, 00582181, 90710369, 90346106, Kumeta, Masahiro, Hirai, Yuya, Yoshimura, Shige H, Horigome, Tsuneyoshi, and Takeyasu, Kunio

Abstract

To uncover the molecular composition and dynamics of the functional scaffold for the nucleus, three fractions of biochemically-stable nuclear protein complexes were extracted and used as immunogens to produce a variety of monoclonal antibodies. Many helix-based cytoskeletal proteins were identified as antigens, suggesting their dynamic contribution to nuclear architecture and function. Interestingly, sets of antibodies distinguished distinct subcellular localization of a single isoform of certain cytoskeletal proteins; distinct molecular forms of keratin and actinin were found in the nucleus. Their nuclear shuttling properties were verified by the apparent nuclear accumulations under inhibition of CRM1-dependent nuclear export. Nuclear keratins do not take an obvious filamentous structure, as was revealed by non-filamentous cytoplasmic keratin-specific monoclonal antibody. These results suggest the distinct roles of the helix-based cytoskeletal proteins in the nucleus.

Published
2013

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