Dobson, C. | Search bioRxiv

bioRxiv2024

The open to closed D-loop conformational switch determines length in filopodia-like actin bundles

Filopodia, microspikes and cytonemes are implicated in sensing the environment and in dissemination of morphogens, organelles and pathogens across tissues. Their major structural component is parallel bundles of actin filaments that assemble from the cell membrane. Whilst the length of filopodia is central to their function, it is not known how their lengths are determined by actin bundle dynamics. Here, we identified a set of monoclonal antibodies that lengthen filopodia-like structures formed in a cell-free reconstitution system, and used them to uncover a key molecular switch governing length regulation. Using immunolabelling, enzyme-linked immunosorbent assays, immunoprecipitation and immunoblock experiments, we identified four antibodies that lengthen actin bundles by selectively binding the open DNase 1-binding loop (D-loop) of actin filaments. The antibodies inhibit actin disassembly and their effects can be alleviated by providing additional actin or cofilin. This work indicates that maintaining an open state of the actin filament D-loop is a mechanism of generating the long projections of filopodia-like actin bundles.

biochemistry↗

bioRxiv2022

Spectral Interferences Impede the High-Resolution Mass Analysis of Recombinant Adeno-Associated Viruses

Recombinant adeno-associated viruses (rAAVs) are the leading platform for in vivo delivery of gene therapies, with several already approved for clinical use. However, the heterogeneity and structural complexity of these viral particles render them challenging targets to characterize. Orbitrap-based native mass spectrometry (MS) is a method capable of directly characterizing intact megadalton protein assemblies. Here we used such an approach to characterize four different preparations of rAAV8 (two empty and two filled) differing in both their transgene and relative capsid protein isoform (i.e. VP1, VP2 and VP3) content. Interestingly, in native MS measurements of these samples, we observe complicated, unusual, and dramatically different spectral appearances between the four rAAV preparations that cannot be rationalized or interpreted using conventional approaches (i.e. charge state deconvolution). By combining high-resolution native MS, single particle charge detection MS, and spectral simulations, we reveal that these unexpected features result from a combination of stochastic assembly-induced heterogeneity and divergent gas phase charging behaviour between the four rAAV preparations. Our results stress the often-neglected heterogeneity of rAAVs, but also highlight the pitfalls of standard high-resolution mass analysis for such particles. Finally, we show that charge detection MS and spectral simulations can be used to tackle these challenges.

biochemistry↗