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Updated: Aug 9, 2025
Peptide Scanning-assisted Identification of a Monoclonal Antibody-recognized Linear B-cell Epitope
Published on: March 24, 2017
The lamin A/C Ig-fold undergoes cell density-dependent changes that alter epitope binding
Melanie Wallace1,2, Gregory R Fedorchak1,2, Richa Agrawal1,2
1Meinig School of Biomedical Engineering, Cornell University, Ithaca, NY, USA.
Insights
Antibody recognition of Lamins A/C changes with cell density due to conformational shifts, not protein levels. This impacts immunofluorescence data interpretation for nuclear intermediate filament proteins.
Area of Science:
- Cell Biology
- Biochemistry
- Biophysics
Background:
- Lamins A/C are nuclear intermediate filament proteins crucial for cellular mechanics and biochemistry.
- Antibody-based detection is vital for studying Lamin A/C localization and function.
Purpose of the Study:
- To investigate the cell density-dependent recognition of Lamins A/C by antibodies.
- To understand the underlying mechanisms and implications for immunofluorescence studies.
Main Methods:
- Immunofluorescence using JOL-2 antibody targeting Lamin A/C Ig-fold.
- Cell density manipulation and cell spreading assays.
- Cytoskeletal disruption and Linker of Nucleoskeleton and Cytoskeleton (LINC) complex perturbation.
- Nuclear stiffness and force transmission measurements.
Main Results:
- Antibody JOL-2 recognition of Lamin A/C is highly dependent on cell density, despite unchanged Lamin A/C levels.
- Proposed mechanism involves conformational changes (unfolding/masking) of Ig-fold loops (C'E, EF) upon cell spreading.
- Labeling was unaffected by cytoskeletal or LINC complex disruption.
- Nuclear stiffness and force transmission remained constant across cell densities.
Conclusions:
- Cell density influences Lamin A/C antibody accessibility, likely via conformational changes.
- Findings necessitate careful interpretation of immunofluorescence data for Lamins A/C.
- Conformational plasticity of Lamins A/C may be functionally relevant in cellular processes.
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.
