(H-299) Deciphering Force-induced Protein Interactions Surrounding Keratin Fibers Using Microneedle Stretch

Epithelial cells are constantly exposed to physical stimuli during embryogenesis, tissue regeneration, and cancer progression. Physical stimuli are converted to biochemical signals that influence cell migration, proliferation, or differentiation; a process known as mechano-transduction. The cytoskeleton of epithelial tissues serves as a critical force-sensor during this process. Among the constituents of the cytoskeleton, actin filaments have been extensively studied as the cell’s principal responders to external forces. Many LIM domain containing proteins interact with these actin fibers in a force-sensitive manner, therefore, serving as an initiator of mechano-induced signal transduction. However, the actin cytoskeleton is not the only mechano-transducer in cells. Recently, we found that the keratin network also responds to mechanical forces by recruiting focal adhesion protein, tensin 4. Therefore, the keratin network may also serve as the force-sensing element in cells. Using a microneedle-based cell stretch assay, we investigated how the members of the LIM protein family interact with the actin and keratin cytoskeletal networks. Furthermore, we developed a technique to study protein interactions under mechanical strain in vitro using reconstituted keratin network.

To visualize stretch-dependent protein interactions, a microneedle was gently placed onto cells neighboring Madin-Darby canine kidney (MDCK) cells expressing GFP-tagged LIM proteins. The microneedle moved the adjacent cells away from the LIM protein expressing cells, thus pulling and straining the expressing cells via cell-cell contacts. The recombinant His-tagged keratin 8 and keratin 18 were assembled into filaments and adsorbed onto a clean coverslip. The filaments were visualized using fluorescently labeled anti-His antibody. Similar to GFP-tagged LIM protein expressing MDCK cells, the recombinant keratin filaments were stretched using a microneedle under the control of a micro-manipulator. Purified GFP-LMO1-LIM proteins were pre-diluted at a concentration of 1 μM in PBS (+BSA/+Zn). The relative accumulation of GFP-tagged proteins and the intensity ratio of LMO1 binding to the keratin fibers (GFP-LMO/His-k8) were analyzed using ImageJ and Microsoft Excel.

Using an in vivo microneedle stretch assay, GFP-tagged LIM proteins expressing MDCK cells were exposed to mechanical strain and the co-localization of LIM proteins along the force-bearing cytoskeletal filaments were observed. Among the members of the LIM protein family, LIM-domain proteins containing at least three LIM domains, e.g., zyxin, accumulated along actin fibers as previously shown. However, two members of the LIM protein family, LMO1 and LIMK1, with only two LIM domains, accumulated along fibers, but they did not co-localize with actin filaments or zyxin. Instead, LMO1 and LIMK1 accumulated around keratin filaments (Figure 1A and B), suggesting that at least some LIM proteins are recruited to the keratin network in a force-dependent manner.

To investigate whether LMO1 and LIMK1 bind directly or indirectly to the keratin filaments, we developed an in vitro protein interaction assay using reconstituted keratin filaments. The surface bound keratin filament bundles were stretched using a microneedle (Figure 2A) so that one section of the fibers was strained while other parts of the fibers remained relaxed. Purified GFP-tagged LMO1 accumulated along both unstretched (control) and stretched keratin filaments, but significantly more along stretched keratin filaments (Figure 2B and C). Hence, GFP-tagged LMO1 recruited along the force-bearing keratin filament bundles, but not along the force-free keratin fibers. We hypothesize that the densely packed keratin filaments elongate under force application and expose cryptic binding sites for mechano-sensing proteins such as LMO1. These data collectively suggest that the keratin network recruits a unique set of proteins under mechanical force, thereby playing an integral role in mechano-transduction, complementing that of the actin network.