CONCLUSION

Much of the original work exploring focal adhesions (cell-ECM interaction) and adherens junctions (cell-cell interaction) has been accomplished on rigid and planar 2D culture systems. Over time the culture platforms used have moved towards physiologically soft planar 2D systems. While these physiologic planar substrates unveiled new understanding of how cells probe and sense their microenvironment, they still lack the dimensionality that is characteristic of the native tissue niche in which most mesenchymal tissues reside. As new imaging modalities have been developed that enable imaging of these interactions in native tissue sections, it has been noted that some of the core principles and structures have translated favorably into the 3rd _{dimension, while others have not (Baker and Chen}

2012). The work detailed in this chapter began with the examination of nuclear envelope wrinkling and its role in mechanoadaptation of the cell in soft 2D planar microenvironments. Through this work, we established a clear mechanism in which nuclear envelope wrinkles had to be unfurled before contractile stress could build up in the cytoskeleton resulting in cell spreading and nuclear translocation of mechano- responsive transcription factors. However, when these same mechanisms were queried within native tissue sections, these 2D predictions did not hold up, and in fact the exact opposite response was observed. That is, in the native tissue niche, cells with more contractility, as determined by increased YAP/TAZ localization, exhibited increased nuclear wrinkling. We hypothesized that this arose from the influence of dimensionality and the manner by which nuclear stress was built up in an environment where there are ECM interactions present in all dimensions. To better recapitulate these interactions, we cultured cells in MMP-degradable HA hydrogels and found that these remodelable systems that enabled 3D cell spreading could accurately model the native niche scenario,

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wherein increased YAP/TAZ localization was observed in conjunction with a wrinkled NE phenotype.

Moving forward, this work highlights the need to consider our in vitro choice of synthetic microenvironments with data from actual tissue samples. More specifically, studying nuclear mechanobiology on 2D planar morphologies might not be the most appropriate context relative to the native in vivo microenvironment. Instead 3D systems that can be remodeled might potentially provide more faithful predictions when compared to this native context. This choice in platforms does not solely center around hydrogels crosslinked with MMP-degradable domains, but can additionally include collagen-1 gels, which have already been widely used in studying the role of the nucleus in 3D migratory contexts (Petrie, Koo et al. 2014, Skau, Fischer et al. 2016). Comparatively though, this MMP- degradable NorHA system does allow for the precise and independent tuning of multiple mechanobiologic parameters, such as substrate stiffness and ligand presentation, which will hopefully allow for additional conclusions to be drawn regarding the role of various components in driving nuclear mechanobiology in native-like contexts.

In addition to the above, this work provided new insight into the importance of a wrinkled nuclear envelope morphology in understanding the mechanobiologic state of the cell. While many groups have previously noted this morphology (Swift, Ivanovska et al. 2013, Kim and Wirtz 2015, Kim, Li et al. 2016, Jorgens, Inman et al. 2017), its role and function has not been well-described to date. In particular, the finding that the wrinkled morphology correlates with an increased contractile state in tissues is in disagreement with other

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hypotheses about how a wrinkled nuclear envelope could result in a reduced contractile state, such as through the reduction of stretch-sensitive calcium signaling into the nucleus (Itano, Okamoto et al. 2003) or through cessation certain protein-protein interactions (Poh, Shevtsov et al. 2012). As such, this necessitates new investigations into the manner by which nuclear wrinkles can be generated and understood as a contractile morphological element. One thought is that these wrinkles play no active role and merely are the result of how nuclear deformation occurs in 3D environments. Importantly, in 2D microenvironments, nuclear stiffening is tied to the build-up of contractile stress and increased polarization/formation of F-actin bundles, so by the time there is a clear perinuclear actin cap, the nucleus is too stiff and does not allow for actin protrusion into the nucleus and non-protrusive TAN lines are formed. In 3D, nuclear stress can be built up independently from other cytoskeletal events in a variety of ways, and the prevalence of wrinkles along with increased nuclear YAP/TAZ translocation suggests that F-actin protrusion into a non pre-stressed nucleus can result in the buildup of nuclear pre-stress. Yet, with previous reports of nesprin clustering happening within actin-protrusions into the nucleus (‘TAN Lines’), it is more likely that these wrinkles in 3D play multiple roles, including providing increased nesprin coupling to the actin cytoskeleton and allowing for mechanical reinforcement, thereby resulting in increases in the contractile state of the cell (Driscoll+ Biophysical Journal 2015). This implies that previous descriptions of TAN line slippage in 2D might not be as prevalent in 3D, and anchors like SUN2 that were essential to prevent against this slippage play a different role in 3D, as the wrinkled nuclear morphology may act to keep these interactions in place and increase the stability of LINC complex formation (Luxton, Gomes et al. 2010, Folker, Östlund et al. 2011). In a way, these nuclear wrinkles in 3D could be directly analogous to some of the original

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descriptions of TAN lines being the predominant structure of nesprin-nuclear interactions on 2D planar substrates.

Our future work in this system will focus around defined pharmacologic perturbations of cellular contractility, both in tissue (using an explanted mouse meniscus culture system) and in these MMP-degradable NorHA hydrogels across various substrate mechanics and hydrogel degradability potentials. These studies will shed further light on how cells in native and native-like contexts can respond to exogenous mechanical cues, and further define the role that nuclear structure and architecture play in mediating these changes.

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