(E-164) Acute downregulation of emerin alters actomyosin cytoskeleton connectivity and function

Introduction: Cyclic stretch induced by breathing is important for fetal lung growth and maturation to prepare for adaptation to breathing air. Factors that limit the force or frequency of fetal lung cycling lead to incomplete lung morphogenesis or maturation.1 In tissue under dynamic loading, mechanical forces are transmitted from extracellular matrix, via matrix adhesions and cytoskeleton, to the nuclear envelope (NE) and nuclei. NE not only provides the physical connection for nucleo-cytoskeletal coupling, but also participates in mechanosensitive transcriptional regulation.2 The NE protein emerin interacts with cytoskeletal F-actin, microtubules and myosin, and is a key regulator of the MKL1-SRF signaling pathway. Recent studies implicate emerin in efficient cell migration and nucleus polarity.3-4 We hypothesize that emerin plays a crucial role in the fetal lung cell’s mechanical processes, including responses to mechanical stretch and cell spreading.

Materials and Methods: Fetal lung fibroblast WI-38 were used in this study. Acute emerin knockdown (emerin KD) was achieved by adenoviral transduction of shRNA. For mechanical stretch, control and emerin KD cells were stretched on a flexible stretch chamber with strains ranging in 2%, 10%, 15% and 20% at physiological frequency and duration. The alignment of cytoskeleton and nuclei, cell shape, and circularity were analyzed by quantitative image analysis in Python and ImageJ. For cell spreading and matrix production, cells were plated on gelatin-coated coverslips and assayed at 8 h and 24 h.

Results and Discussion: We found that emerin expression in WI-38 cells was reduced to 5% of control, 4 days after shRNA transduction. In contrast to control cells, which briskly realigned their nuclei, actin cytoskeleton, and extracellular matrices in response to cyclic stretch, emerin KD fibroblasts showed incomplete reorientation of both nuclei (odds 37% lower) and actin cytoskeleton (odds 46% lower). Emerin KD fibroblasts were also aberrantly circular with circularity three times higher than the spindle-shaped controls, and were ~60% larger. Emerin KD cells exhibited an altered pattern of filamentous actin organization that was disconnected from the nucleus, with F-actin distribution to the cell edge twice that of controls. During cell spreading, emerin KD cells showed a 90% reduction in myosin light chain phosphorylation at 8 h and a 50% reduction at 24 h. Interestingly, emerin-downregulated fibroblasts also demonstrated reduced fibronectin fibrillogenesis and 80% reduced fibronectin production.5

Conclusion: We found that acute emerin knockdown perturbs the cells’ responsiveness to positioning cues, connectivity of the actomyosin network to the nucleus, and activation and harnessing of cell traction force via myosin light chain phosphorylation and matrix fibrillogenesis. Our findings advance an important paradigm that expands perspective on the role of emerin and its binding partners as mediators of mechanical continuity via a direct linkage between the nuclear envelope and the cytoskeleton during development and disease.

Acknowledgements: We acknowledge the support from the National Science Foundation under Award Number DMR1709349, from a grant from Kley Dom Biomimetics and from a StAAR award from the Department of ACCM at the Johns Hopkins University School of Medicine. We would also like to acknowledge partial support for the statistical analysis from the National Center for Research Resources and the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health through Grant Number 1UL1TR001079.

References:
1. Liu, M., and M. Post. 2000. Invited review: mechanochemical signal transduction in the fetal lung. J. Appl. Physiol. 89(5):2078-2084.
2. Maurer, M., and J. Lammerding. 2019. The Driving Force: Nuclear Mechanotransduction in Cellular Function, Fate, and Disease. Annu. Rev. Biomed. Eng. 21:443-468.
3. Lavenus, S. B., K. W. Vosatka, A. P. Caruso, M. F. Ullo, A. Khan, and J. S. Logue. 2022. Emerin regulation of nuclear stiffness is required for fast amoeboid migration in confined environments. J. Cell Sci. 135(8).
4. Nastaly, P., D. Purushothaman, S. Marchesi, A. Poli, T. Lendenmann, G. R. Kidiyoor, G. V. Beznoussenko, S. Lavore, O. M. Romano, D. Poulikakos, M. C. Lagomarsino, A. A. Mironov, A. Ferrari, and P. Maiuri. 2020. Role of the nuclear membrane protein Emerin in front-rear polarity of the nucleus. Nat Commun. 11(1):2122.
5. Jin Q, Pandey D, Thompson CB, Lewis S, Sung HW, Nguyen TD, Kuo S, Wilson KL, Gracias DH, Romer LH. 2023. Acute downregulation of emerin alters actomyosin cytoskeleton connectivity and function. Biophysical Journal.