Nuclear envelope wrinkling predicts mesenchymal progenitor cell mechano-response in 2D and 3D microenvironments
Introduction
The micromechanical niche of the cell modulates phenotype and function, from proliferation and extracellular matrix deposition to downstream lineage specification [[1], [2], [3], [4]]. During these processes, exogenous forces are transmitted through the cytoskeleton, where they converge at the nucleus and are converted to changes in gene expression through a variety of mechanisms [2,[5], [6], [7]]. One well-described mechanotransductive mechanism involves the force-induced translocation of proteins into the nucleus, where they initiate downstream transcriptional programs. This paradigm has been best described for the YAP/TAZ protein complex, which functions as a transcriptional co-activator that initiates gene expression patterns that define both development and disease [2,8]. Outside of YAP/TAZ, similar force-sensitive nuclear translocation mechanisms have been reported for other factors, including RARγ [6], MRTF-A (also known as Mkl1) [5,9], HDAC3 [10], β-catenin [11], TWIST [12], and NKX2.5 [13]. For some of these factors, the mechanisms driving force-sensitive nuclear translocation are well described, such as the role of G-actin binding to MRTF-A causing its sequestration in the cytosol [5], while for others, the mechanism governing force-induced transcriptional co-activator translocation has not yet been fully elucidated.
The nuclear envelope is itself a mechano-responsive element of the cell, and may regulate force-induced nuclear translocation of transcription factors [6,14,15]. Lamin-A/C, an intermediate filament that forms an extensive network at the inner nuclear envelope, functions to maintain nuclear morphology, organization, and stiffness [[16], [17], [18]]. Lamin-A/C is regulated at the protein level, with the ratio of lamin-A/C to lamin-B1/B2 scaling with the stiffness of the cellular niche [6]. This expression-based regulation is enabled by a feedback circuit, wherein mechanical force causes RARγ nuclear translocation and binding to the promoter of lamin-A/C, increasing its expression. Upregulation of lamin A/C promotes its incorporation into the nuclear envelope in a force-dependent fashion, where persistent mechanical stimulation of progenitor cells (in the form of increased substrate stiffness or dynamic mechanical loading) directs accumulation of lamin-A/C at the nuclear periphery, thereby stiffening the nucleus and sensitizing the cell to additional exogenous mechanical inputs [19]. This force-induced assembly of lamin-A/C results from structural polarization of the lamina, where cryptic lamin-A/C domains become exposed or buried as a consequence of force generation within the cell [20,21]. These cryptic residues within lamin-A/C contain binding sites for other inner nuclear proteins, including emerin, which is phosphorylated in a mechano-responsive fashion and is key for mechano-adaptation and nuclear stiffening of isolated nuclei with applied force [14]. Besides emerin, lamin-A/C can also act as an anchoring scaffold to other proteins within the nuclear envelope, allowing for the transmission of mechanical force to complexed proteins. In addition to regulating nuclear mechanics, mechanical stress in the NE can also control mechano-signaling directly. Recent work shows that mechanical engagement of the nuclear envelope (i.e., direct compression via atomic force microscopy, “AFM”) can elicit a mechano-signaling response. Mechanical stretch of the NE increased the permissivity of nuclear pore complexes, resulting in increased nuclear import of YAP/TAZ, independent of the polymerization state of the actin cytoskeleton [22]. These findings suggest that the nuclear envelope (NE) is a key mechano-responsive signaling hub, providing structural support for the nucleus while also playing key roles in mechano-adaptation that ultimately dictate how extracellular forces are sensed and transmitted.
Despite this important role in cellular mechanotransduction, how the morphology and function of the nuclear envelope varies in response to inputs from the microenvironment is not well defined. Commonly, the nucleus is represented as an ellipsoidal structure, with a radius of curvature that varies slowly along its perimeter. However, this ellipsoidal morphology is not present in every cell type and microenvironmental context. Rather, recent work across a variety of biomaterial platforms has illustrated several scenarios in which the nuclear envelope can show a markedly “wrinkled” morphology, including on soft substrates [6,21,23,24], after treatment with pharmacologic agents that alter nuclear structure or cytoskeletal contractility [25], during cellular attachment and detachment [20,21,[25], [26], [27]], in breast tissue and engineered acini [28], during confined migration [29,30], and following lamin-A/C knockdown [27,31]. Super resolution and electron microscopy of cells cultured on 2D glass substrates has further shown that various elements of the cytoskeleton can physically occupy these invaginations, though it is unclear if these nuclear invaginations are caused by cytoskeletal interactions or if these cytoskeletal elements are simply anchored to the nuclear envelope and deform along with the nucleus [28,32]. To date, most observations of a wrinkled nuclear morphology have been qualitative in nature, and there is a clear gap of knowledge with respect to understanding how wrinkled nuclear envelope conformations arise in response to exogenous cues, and critically, how the wrinkled nuclear morphology may regulate progenitor cell behavior and mechano-adaptation. Previous studies have proposed that the wrinkled nuclear lamina is unfurled during cellular spreading, and bulk nuclear flattening reaches steady state only when the wrinkled lamina becomes maximally-unfurled [26,27,33]. Here, we systematically examined the functionality of this nuclear envelope morphology and correlated this feature with progenitor cell mechano-responses across a variety of microenvironmental contexts using engineered hyaluronic acid hydrogel systems.
To accomplish this, we first developed quantitative metrics to define the degree of nuclear envelope wrinkling and used these tools to assess the morphological state of the nuclear envelope in an unbiased fashion. Short-term perturbations of cellular contractility in MSCs cultured on soft 2D hydrogels rapidly altered nuclear envelope morphology. When coupling single cell wrinkling metrics with the visualization of mechano-active transcription factor localization, strong correlations were observed between wrinkling state and accumulation of these factors in the nucleus. These data suggest that wrinkling engenders laxity in the nuclear envelope and results in a ‘toe-region’ in cellular mechano-sensing, wherein the nuclear envelope must be pulled taut prior to mechanically active nuclear shuttling of transcriptional activators. We further probed this relationship in 3D microenvironments and showed that the wrinkled morphology of the nuclear envelope can regulate these same responses, albeit in a different manner. In these 3D environments, increased cytoskeletal tension and mechano-sensitive signaling in MMP-degradable hydrogels correlated with increased nuclear envelope wrinkling. These wrinkles arose from impingements of the contractile actin cytoskeleton on the nucleus, in both engineered and native 3D environments. Accordingly, nuclear envelope wrinkling was the only significant predictor of mechano-sensitive transcription factor shuttling in 3D as compared to traditional 3D nuclear shape or volume morphometrics (which did not predict mechano-sensitive YAP shuttling). Together, these results indicate that the nuclear envelope morphology of a cell is a strong predictor of cellular mechanotransduction, and that may play a central role in how exogenous signals are experienced by the nucleus to regulate mechanotransductive signaling.
Section snippets
Materials and methods
Methacrylated Hyaluronic Acid (MeHA) Hydrogel Synthesis and Casting. MeHA was synthesized as previously reported [34], where methacrylic anhydride was reacted with 1% w/v sodium hyaluronate (70 kDa, Lifecore Biosciences) in dH2O with pH maintained at 8 ± 0.5. After reacting for 6 h, the macromer solution was purified via dialysis (MW cutoff of 6–8 kDa) and then lyophilized for storage. Methacrylation level was confirmed to be ~108% by 1H NMR (Supplemental Figure #12) unless noted otherwise.
Results
The nuclear envelope (NE) is wrinkled on soft 2D hydrogels. To characterize nuclear envelope morphology in response to various microenvironmental perturbations, mesenchymal stem cells (MSCs) were seeded onto thin (h = 100 μm) 2D methacrylated hyaluronic acid hydrogels (MeHA), functionalized with RGD to enable cell-ECM adhesion, and UV polymerized to a Young's Modulus of 10 kPa (verified by AFM) (Fig. 1A). We chose this modulus as it represents a mechanical threshold at which mechanosensitive
Discussion
Our work in 2D hydrogel platforms revealed that the degree of nuclear envelope (NE) wrinkling in mesenchymal progenitor cells can predict their focal adhesion maturation state and YAP/TAZ nuclear localization. Nuclear envelope wrinkling was evident when mesenchymal stem cells were cultured on soft planar 2D substrates (on which cells exhibit apical-basal polarity, Fig. 1). The degree of NE wrinkling changed rapidly in response to modulation of cell contractility (with changes seen as quickly as
Credit author statement
Brian D. Cosgrove: Conceptualization, Methodology, Software, Investigation, Visualization, Validation, Resources, Formal analysis, Writing – original draft, Writing – review & editing, Claudia Loebel: Conceptualization, Methodology, Investigation, Formal analysis, Validation, Visualization, Writing – original draft, Writing – review & editing, Tristan P. Driscoll: Conceptualization, Methodology, Investigation, Validation, Resources, Formal analysis, Writing – review & editing, Tonia K. Tsinman:
Data availability statement
All data are available upon request.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Institutes of Health (R01 AR077362, R01 AR056624, R01 AR071399) and the National Science Foundation-sponsored Center for Engineering Mechanobiology (CMMI-1548571). The authors would like to thank the CDB microscopy core at Penn for the use of core equipment and assistance with STED microscopy, Dr. Xi Jiang for help with tissue sectioning, Ryan Daniels for help with preliminary studies, and Dr. Steven Caliari, Dr. Benjamin Freedman, and Dr. Brianne
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