Fedorchak, G. R. | Search bioRxiv

bioRxiv2022

The Lamin A/C Ig-fold undergoes cell density-dependent changes that alter epitope accessibility

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/C protein levels do not change with cell density. The density-dependent Lamin A/C labeling was distinct from previously reported differential apico-basal labeling, which was independent of cell density. Comparison of the density-dependent labeling effects of antibodies recognizing different Lamin A/C epitopes suggests that the effect is caused by partial unfolding or masking of the CE and/or EF loops of the Ig-fold in response to cell spreading. Seeding cells on micropatterned surfaces with different areas confirmed that increased cell spreading resulted in reduced Lamin A/C labeling with the JOL-2 antibody. Surprisingly, JOL-2 antibody labeling was insensitive to depolymerization of cytoskeletal filaments or disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Although the density-dependent changes of the Ig-fold did not alter nuclear stiffness or nucleo-cytoskeletal force transmission, they may nonetheless modulate interaction with lamin binding partners and thereby affect cellular functions. Taken together, our results point to a previously unrecognized change in the Lamin A/C Ig-fold that affects recognition by the JOL-2 antibody. These findings are not only important for the interpretation of immunofluorescence data for Lamin A/C, but also raise the intriguing prospect that the conformational changes may play a role in Lamin A/C mediated cellular function.

cell biology↗

bioRxiv2018

Mutant lamins cause mechanically-induced nuclear envelope rupture, DNA damage, and DNA-PK activation in muscle

Mutations in the human LMNA gene, which encodes the nuclear envelope (NE) proteins lamins A and C, cause autosomal dominant Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, limb-girdle muscular dystrophy, and other diseases collectively known as laminopathies. The molecular mechanisms responsible for these diseases remain incompletely understood, but the muscle-specific defects suggest that mutations may render nuclei more susceptible to mechanical stress. Using three mouse models of muscle laminopathies, we found that Lmna mutations caused extensive NE abnormalities, consisting of chromatin protrusions into the cytoplasm and transient rupture of the NE in skeletal muscle cells. NE damage was associated with DNA damage, activation of DNA damage response pathways, and reduced viability. Intriguingly, NE damage resulted from nuclear migration in maturing skeletal muscle cells, rather than actomyosin contractility. NE damage and DNA damage was reduced by either depletion of kinesin-1 or disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. LINC complex disruption rescued myofiber function and viability in Lmna mutant myofibers, indicating that the myofiber dysfunction is the result of mechanically induced NE damage. The extent of NE damage and DNA damage in Lmna mouse models correlated with the disease onset and severity in vivo. Moreover, inducing DNA damage in wild-type muscle cells was sufficient to phenocopy the reduced cell viability of lamin A/C-deficient muscle cells, suggesting a causative role of DNA damage in disease pathogenesis. Corroborating the mouse model data, muscle biopsies from patients with LMNA muscular dystrophy revealed significant DNA damage compared to age-matched controls, particularly in severe cases of the disease. Taken together, these findings point to a new and important role of DNA damage as a pathogenic contributor for LMNA skeletal muscle diseases.

cell biology↗