Gentry, J. | Search bioRxiv

bioRxiv2026

Structural basis of caveolin-driven membrane bending

Caveolins are monotopic membrane proteins essential for caveolae formation and have key roles in signaling and lipid regulation. Caveolins assemble into amphipathic discs with a central {beta}-barrel, an architecture distinct from other membrane-remodeling proteins. These discs embed in the membrane inducing membrane curvature. However, the mechanism of disc-driven bending remains unresolved. Using cryo-electron tomography, structure-guided mutagenesis, and mammalian cell studies, we show that evolutionarily distinct caveolins differ dramatically in their ability to curve membranes despite their conserved architecture. Through computational and theoretical analyses, we demonstrate that patterning of hydrophobic residues along the rim of the disc of human Caveolin-1 induces the deformation of the surrounding leaflet, dictating membrane bending. Finally, we determine a 4.1[A] resolution structure of Caveolin-1 within heterologous caveolae in situ, showing the disc adopts a funnel-like conformation, further shaping membrane architecture. Together, these findings reveal fundamental structural principles that empower caveolins to sculpt and remodel cellular membranes.

cell biology↗

bioRxiv2026

Evaluation of an Oxford Nanopore sequencing workflow for mycobacteria from primary MGIT culture

Illumina sequencing of primary MGIT cultures is an established workflow in several reference mycobacteriology laboratories. Oxford Nanopore Technologies (ONT) provides real-time genetic sequencing yielding long reads which help resolve repetitive genomes and is being explored for in-house implementation within diagnostic laboratories. However, low DNA yields from primary MGIT cultures frequently limit the application of ONT workflows, due to high minimum DNA input requirements for library preparation. We validated a modified ONT workflow combining rapid, semi-automated DNA extraction from MGIT cultures with Rapid PCR-Barcoding for whole-genome amplification, and compared its performance with Illumina sequencing for species identification and Mycobacterium tuberculosis complex (MTBC) single-nucleotide polymorphism (SNP) detection. A platform-agnostic analysis pipeline enabled consistent human read removal, taxonomic assignment, and MTBC genomic characterisation. ONT sequencing data was subsampled at 1, 6, and 72 hours to determine the earliest time point for reliable species identification. The concordance between sequencing platforms on species classification was 98.3% (95.8-99.5%) with all differences arising from potential mixed infections. SNP agreement was high, with a mean of 0.3 and a median of 0 SNP differences between sequencing platforms after masking. These findings demonstrate the feasibility of PCR-amplified ONT sequencing as a reliable alternative for routine genomic characterisation of MGIT cultures. IMPORTANCERapid identification of mycobacterial infections is essential for timely patient care and infection control. Many clinical laboratories currently rely on outsourcing sequencing to external reference centres, which adds time and delays the return of clinically actionable results. These services commonly use Illumina sequencing, which, while highly accurate, involves complex workflows and longer turnaround times. Newer technologies offer the potential to generate results in real time, but their use has been limited by the low amount of DNA available from routine culture samples. In this study, we developed an improved workflow that increases the amount of usable DNA and enables reliable, rapid sequencing directly from these samples. This approach allows multiple samples to be processed together and reduces the time needed to obtain results. Importantly, it could enable clinical laboratories to perform sequencing in-house, reducing reliance on external services and improving turnaround times.

molecular biology↗