Westphal, K. | Search bioRxiv

bioRxiv2024

Dysfunctional mechanotransduction regulates the progression of PIK3CA-driven vascular malformations

Somatic activating mutations in PIK3CA are common drivers of vascular and lymphatic malformations. Despite common biophysical signatures of tissues susceptible to lesion formation, including compliant extracellular matrix and low rates of perfusion, lesions vary in clinical presentation from localized cystic dilatation to diffuse and infiltrative vascular dysplasia. The mechanisms driving the differences in disease severity and variability in clinical presentation and the role of the biophysical microenvironment in potentiating progression are poorly understood. Here, we investigate the role of hemodynamic forces and the biophysical microenvironment in the pathophysiology of vascular malformations, and we identify hemodynamic shear stress and defective endothelial cell mechanotransduction as key regulators of lesion progression. We found that constitutive PI3K activation impaired flow-mediated endothelial cell alignment and barrier function. We show that defective shear stress sensing in PIK3CAE542Kendothelial cells is associated with reduced myosin light chain phosphorylation, junctional instability, and defective recruitment of vinculin to cell-cell junctions. Using 3D microfluidic models of the vasculature, we demonstrate that PIK3CAE542Kmicrovessels apply reduced traction forces and are unaffected by flow interruption. We further found that draining transmural flow resulted in increased sprouting and invasion responses in PIK3CAE542K microvessels. Mechanistically, constitutive PI3K activation decreased cellular and nuclear elasticity resulting in defective cellular tensional homeostasis in endothelial cells which may underlie vascular dilation, tissue hyperplasia, and hypersprouting in PIK3CA-driven venous and lymphatic malformations. Together, these results suggest that defective nuclear mechanics, impaired cellular mechanotransduction, and maladaptive hemodynamic responses contribute to the development and progression of PIK3CA-driven vascular malformations.

bioengineering↗

bioRxiv2022

Mapping Hsp104 structure and substrate interactions using crosslinking mass spectrometry

Molecular machines from the AAA+ family play important roles in protein folding, disaggregation and DNA processing. Recent cryo-EM structures of AAA+ molecular machines have uncovered nuanced changes in conformation that underlie their specialized functions. Furthermore, complexes between these machines and substrates begin to explain their mechanism of activity. Here we explore how crosslinking mass spectrometry (XL-MS) can be used to interpret changes in conformation induced by ATP and how substrates are associated. We applied a panel of crosslinking reagents to produce high-resolution crosslinking maps and interpret our data on previously determined X-ray and cryo-EM structures of Hsp104 from a thermophilic yeast, Calcarisporiella thermophila. We developed an analysis pipeline to differentiate between intra-subunit and inter-subunit contacts within the hexameric homo-oligomer. We identify crosslinks that break the asymmetry that are only present when ATP is bound and are absent in an ATP-binding deficient mutant. Finally, we identify contacts between Hsp104 and a model substrate to identify contacts on the central channel of Hsp104 across the length of the substrate indicating that we have trapped interactions consistent with translocation of the substrate. Our simple and robust XL-MS-based experiments and methods help interpret how these molecular machines change conformation and bind to substrates even in the context of homo-oligomeric assemblies.

biophysics↗