A method to map the interaction network of the nuclear lamina with genetically encoded photo-crosslinkers in vivo

Abstract

Lamins are intermediate filaments that assemble in a meshwork at the inner nuclear periphery of metazoan cells. The nuclear periphery fulfils important functions by providing stability to the nuclear membrane, connecting the cytoskeleton with chromatin, and participating in signal transduction. Mutations in lamins interfere with these functions and cause severe, phenotypically diverse diseases collectively referred to as laminopathies. The molecular consequences of these mutations are largely unclear but likely include alterations in lamin-protein and lamin-chromatin interactions. These interactions are challenging to study biochemically mainly because the lamina is resistant to high salt and detergent concentrations and co-immunoprecipitation are susceptible to artefacts. Here, we used genetic code expansion to install photo-activated crosslinkers to capture direct lamin-protein interactions in vivo. Mapping the Ig-fold of laminC for interactions, we identified laminC-crosslink products with laminB1, LAP2, and TRIM28. We observed significant changes in the crosslink intensities between laminC mutants mimicking different phosphorylation states. Similarly, we found variations in laminC crosslink product intensities comparing asynchronous cells and cells synchronized in prophase. This method can be extended to other laminC domains or other lamins to reveal changes in their interactome as a result of mutations or cell cycle stages.

Genetically encoded crosslinkers to address protein–protein interactions

“…Characterization of the binding process led to the finding that VCP/p97 plays a crucial role in regulating IFITM3 trafficking, turnover, and activity. In a recent study, Bpa was incorporated into laminC variants to study the network of interacting proteins at the nuclear lamina in live mammalian cells (Neumann‐Staubitz et al, 2022 ).…”

The Multifaceted Roles of Lamins in Lung Cancer and DNA Damage Response

“…The CDK1 complex targets several sites within different lamins, including lamin A/C (Thr19, Ser22, Ser392 and Ser404), lamin B1 (Ser23 and Ser393), and lamin B2 (Thr34, Ser37 and Ser405) (Figure 1C). All of these phosphorylation events contribute to lamin depolymerization and NEBD before the mitotic spindle assem-bles [20,[33][34][35]. Upon NEBD, NPC components are distributed between the endoplasmic reticulum (ER) and cytoplasm [36].…”

“…Rubporn et al [69] found that lamin A/C and several co-regulated proteins including KAP1 (TIF1β, TRIM28) were overexpressed in the A549 lung cancer cell line compared to MRC-5 normal lung fibroblast cells. Interestingly, Neumann-Staubitz et al [33] identified KAP1 as an interactor of the Ig-domain of lamin C using genetically encoded crosslinkers. KAP1 is involved in cell survival after DNA damage and its phosphorylation by ATM, ATR, and DNA-PK recruits KAP1 to DNA strand breaks [78].…”