Inducible LAP-tagged Stable Cell Lines for Investigating Protein Function, Spatiotemporal Localization and Protein Interaction Networks

Abstract

Multi-protein complexes, rather than single proteins acting in isolation, often govern molecular pathways regulating cellular homeostasis. Based on this principle, the purification of critical proteins required for the functioning of these pathways along with their native interacting partners has not only allowed the mapping of the protein constituents of these pathways, but has also provided a deeper understanding of how these proteins coordinate to regulate these pathways. Within this context, understanding a protein's spatiotemporal localization and its protein-protein interaction network can aid in defining its role within a pathway, as well as how its misregulation may lead to disease pathogenesis. To address this need, several approaches for protein purification such as tandem affinity purification (TAP) and localization and affinity purification (LAP) have been designed and used successfully. Nevertheless, in order to apply these approaches to pathway-scale proteomic analyses, these strategies must be supplemented with modern technological developments in cloning and mammalian stable cell line generation. Here, we describe a method for generating LAP-tagged human inducible stable cell lines for investigating protein subcellular localization and proteinprotein interaction networks. This approach has been successfully applied to the dissection of multiple cellular pathways including cell division and is compatible with high-throughput proteomic analyses.

The myosin regulatory light chain Myl5 localizes to mitotic spindle poles and is required for proper cell division

“…To begin to understand the cellular role of Myl5 and its link to tumorigenesis, we analyzed its subcellular localization throughout the cell cycle. First, we generated a LAP(GFP-TEV-S-Peptide)-Myl5 inducible stable cell line that expressed GFP-Myl5 upon induction with Dox ( Figure S1a; Bradley, Ramirez, Cheung, Gholkar, & Torres, 2016;Torres, Miller, & Jackson, 2009). The LAP-Myl5 cell line was treated with Dox for 16 hr to express GFP-Myl5 and cells were fixed, stained with Hoechst 33342 DNA dye, and anti-α-Tubulin and anti-GFP antibodies, and imaged by immunofluorescence microscopy.…”

“…The HeLa LAP(GFP-TEV-S-Peptide)-Myl5, -Myl5-AA, -Myl5-20E, -Myl5-21E, -Myl5-CAD, -Myl5-siRes, and -MYO10 inducible stable cell lines were generated as described previously (Bradley et al, 2016;Torres et al, 2009). Briefly, full-length MYL5 (coding for amino acid residues 1-173) and mutant derivatives (alanine mutations at both…”

The myosin regulatory light chain Myl5 localizes to mitotic spindle poles and is required for proper cell division

“…First, we generated a LAP(GFP-TEV-S-Peptide)-Myl5 inducible stable cell line that expressed GFP-Myl5 upon induction with Dox ( Fig. S1A) 31,32 . The LAP-Myl5 cell line was treated with Dox for 16 hours to express GFP-Myl5 and cells were fixed, stained with Hoechst 33342 DNA dye, and anti-a-Tubulin and anti-GFP antibodies and imaged by immunofluorescence microscopy.…”

“…The HeLa LAP(GFP-TEV-S-Peptide)-Myl5 stable cell line was generated according to Torres et al 31,32 Briefly, full-length MYL5 (coding for amino acid residues 1-173) was cloned into pDONR221 and transferred to pGLAP1 through a Gateway reaction to generate the pGLAP1-MYL5 vector that was transfected into HeLa Flp-In T-Rex cells to generate the LAP-Myl5 inducible stable cell line as described previously. 31,32 Immunoblotting For Myl5 cell cycle protein expression analysis, HeLa cells were synchronized in G1/S with 2 mM thymidine for 18-hours. Cells were then washed with PBS three times and twice with F12:DMEM media with 10% FBS and released into the cell cycle.…”

Dissecting the mechanisms of cell division

“…In affinity purifications, a tagged protein is expressed within cells, and the protein complexes are immunoprecipitated via antibodies that target the protein tag and are analyzed by MS ( Fig. 1, upper right) (51,57). We have used this approach to study various protein complexes of the cell division machinery, including enzymes that regulate the length of the mitotic spindle (58), ubiquitylation complexes that regulate cytokinesis (59), novel light chains of the dynein machinery (60), and a novel kinesin involved in centrosome cohesion (49, 61, 62).…”