Biology subjects

Hurst, L. R.

Publications and source records attributed to Hurst, L. R..

4 recordsLinked to original sources

bioRxiv2024

3D nanoscale architecture of the respiratory epithelium reveals motile cilia-rootlets-mitochondria axis of communication

A major frontier in single cell biology is decoding how transcriptional states result in cellular-level architectural changes, ultimately driving function. A remarkable example of this cellular remodelling program is the differentiation of airway stem cells into the human respiratory multiciliated epithelium, a tissue barrier protecting against bacteria, viruses and particulate matter. Here, we present the first isotropic three-dimensional map of the airway epithelium at the nanometre scale unveiling the coordinated changes in cellular organisation, organelle topology and contacts, occurring during multiciliogenesis. This analysis led us to discover a cellular mechanism of communication whereby motile cilia relay mechanical information to mitochondria through striated cytoskeletal fibres, the rootlets, to promote effective ciliary motility and ATP generation. Altogether, this study integrates nanometre-scale structural, functional and dynamic insights to elucidate fundamental mechanisms responsible for airway defence.

cell biology↗

bioRxiv2020

Sphingolipids with Very Long-chain Fatty Acids Regulate Vacuole Fusion During Tethering and Docking

Sphingolipids are essential in membrane trafficking and cellular homeostasis. Here, we show that sphingolipids containing very long-chain fatty acids (VLCFAs) promote robust homotypic vacuolar fusion in Saccharomyces cerevisiae. The elongase Elo3 adds two carbons to 24-carbon (C24) acyl chains to make C26 VLCFAs that are incorporated into sphingolipids. Vacuoles isolated from elo3{Delta} cells had increased fluidity relative to the wild-type and were attenuated for fusion. Upon further testing we found that vesicle the tethering stage was affected as elo3{Delta} vacuole clusters contained fewer vesicles versus the WT. Vacuole tethering requires the interactions of late endosomal Rab GTPase Ypt7 and the HOPS tethering complex. Pulldown assays using GST-Ypt7 showed that HOPS from elo3{Delta} vacuole extracts failed to bind Ypt7 while HOPS from WT extracts interacted with GST-Ypt7. Furthermore GFP-Ypt7 failed to localize at vertex microdomains of elo3{Delta} vacuoles relative to the WT, whereas HOPS and regulatory lipids did accumulate at vertices. Finally, we found that elo3{Delta} vacuoles had reduced V-ATPase. Together these data show that C26-VLCFA containing sphingolipids are important for Ytp7 function and vacuole homeostasis.

cell biology↗

bioRxiv2019

Copper Blocks V-ATPase Activity and SNARE Complex Formation to Inhibit Yeast Vacuole Fusion

The accumulation of Copper in organisms can lead to altered functions of various pathways, and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg+, Cd2+, and Cu2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu2+ to Cu+. Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V-ATPase H+ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that that Cu2+ is responsible for the inhibition of vacuole fusion and V-ATPase function. This notion is supported by the differential effects chelators. The Cu2+-specific chelator TETA rescued fusion, whereas the Cu+-specific chelator BCS had no effect on the inhibited fusion.

biochemistry↗

bioRxiv2018

Phosphatidic acid inhibits SNARE priming by inducing conformational changes in Sec18 protomers

Eukaryotic homeostasis relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are re-activated by Sec18/NSF driven disassembly to enable a new round of fusion. We previously found that phosphatidic acid (PA) binds Sec18 to sequester it from SNAREs. Dephosphorylation of PA dissociates Sec18 from the membrane allowing it to engage SNARE complexes. We now report that PA induces conformational changes in Sec18 protomers, while hexameric Sec18 cannot bind PA membranes. The association of Sec18 with PA was shown to be sensitive to membrane curvature, suggesting that regulation could vary on different organelles in a curvature dependent manner. Molecular dynamics showed that PA binding sites exist on the D1 and D2 domains of Sec18 and that residues needed for binding were masked in the hexameric form of the protein. Together these data indicate that PA regulates Sec18 function through altering protein architecture and stabilizing membrane-bound protomers.

biochemistry↗