ABSTRACT
Lamins A/C are nuclear intermediate filament proteins that are involved in diverse cellular mechanical and biochemical functions. Here, we report that recognition of Lamins A/C by a commonly used antibody (JOL-2) that binds the Lamin A/C Ig-fold and other antibodies targeting similar epitopes is highly dependent on cell density, even though Lamin A/Clevels do not change. We propose that the effect is caused by partial unfolding or masking of the C’E and/or EF loops of the Ig-fold in response to cell spreading. Surprisingly, JOL-2 antibody labeling was insensitive to disruption of cytoskeletal filaments or the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Furthermore, neither nuclear stiffness nor nucleo-cytoskeletal force transmission changed with cell density. These findings are important for the interpretation of immunofluorescence data for Lamin A/C and also raise the intriguing prospect that the conformational changes may play a role in Lamin A/C mediated cellular function.
Acknowledgments
We thank the Biotechnology Resource Center (BRC) Flow Cytometry Facility (RRID:SCR_021740) and sequencing facility (RRID:SCR_021727) at the Cornell Institute of Biotechnology for their resources and technical assistance. This work was performed in part at the Cornell NanoScale Science & Technology Facility, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (award NNCI-2025233). This work was supported by awards from the National Institutes of Health (R01 HL082792, R01 GM137605, U54 CA210184 to J.L.), the National Science Foundation (CBET 1715606, MCB-1715606, and URoL-2022048 to J.L.; Graduate Research Fellowships 2016229710 to M.W. and 2014163403 to G.R.F.), the Department of Defense Breast Cancer Research Program (Breakthrough Award BC150580 to J.L.), the American Heart Association (20PRE35080179 to M.M.), and the Volkswagen Foundation (A130142 to J.L.). The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Supplementary material
Supplemental data for this article can be accessed online at https://doi.org/10.1080/19491034.2023.2180206
Data Availability
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.