Biology subjects

Howes, S. C.

Publications and source records attributed to Howes, S. C..

4 recordsLinked to original sources

bioRxiv2024

A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

Cilia are essential motile or sensory organelles found on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, however the underlying biochemical mechanisms are largely unexplored. Here, we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins, CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules, and, when mutated, cause ciliopathies such as Joubert syndrome. CEP104, a protein containing a tubulin-binding TOG domain, is an inhibitor of microtubule growth and shortening that interacts with EBs on the microtubule surface and with a luminal microtubule-pausing factor CSPP1. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics directly but act as scaffolds for their partners. Cryo-electron tomography showed that together, ciliary tip module members form plus-end-specific cork-like structures which reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.

cell biology↗

bioRxiv2022

Increased microtubule lattice spacing correlates with selective binding of kinesin-1 in cells

Within the cell cargo is transported via motor proteins walking along microtubules. The affinity of motor proteins for microtubules is controlled by various layers of regulation like tubulin isoforms, post- translational modifications and microtubule associated proteins. Recently, the conformation of the microtubule lattice has also emerged as a potential regulatory factor, but to what extent it acts as an additional layer of regulation has remained unclear. In this study, we used cryo-correlative light and electron microscopy to study microtubule lattices inside cells. We find that, while most microtubules have a compacted lattice ([~]41 [A]), a significant proportion of the microtubule cores have expanded lattice spacings and that these lattice spacings could be modulated by the microtubule stabilizing drug Taxol. Furthermore, kinesin-1 predominantly binds microtubules with a more expanded lattice spacing ([~]41.6 [A]). The different lattice spacings present in the cell can thus act as an additional factor that modulates the binding of motor proteins to specific microtubule subsets.

molecular biology↗

bioRxiv2020

The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility

Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures rein-forcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.

cell biology↗

bioRxiv2020

A human monoclonal antibody targeting a conserved pocket in the SARS-CoV-2 receptor-binding domain core

SARS-CoV-2 has caused a global outbreak of severe respiratory disease (COVID-19), leading to an unprecedented public health crisis. To date, there has been over thirty-three million diagnosed infections, and over one million deaths. No vaccine or targeted therapeutics are currently available. We previously identified a human monoclonal antibody, 47D11, capable of cross-neutralising SARS-CoV-2 and the related 2002/2003 SARS-CoV in vitro, and preventing SARS-CoV-2 induced pneumonia in a hamster model. Here we present the structural basis of its neutralization mechanism. We describe cryo-EM structures of trimeric SARS-CoV and SARS-CoV-2 spike ectodomains in complex with the 47D11 Fab. These data reveal that 47D11 binds specifically to the closed conformation of the receptor binding domain, distal to the ACE2 binding site. The CDRL3 stabilises the N343 glycan in an upright conformation, exposing a conserved and mutationally constrained hydrophobic pocket, into which the CDRH3 loop inserts two aromatic residues. Interestingly, 47D11 preferentially selects for the partially open conformation of the SARS-CoV-2 spike, suggesting that it could be used effectively in combination with other antibodies that target the exposed receptor-binding motif. Taken together, these results expose a cryptic site of vulnerability on the SARS-CoV-2 RBD and provide a structural roadmap for the development of 47D11 as a prophylactic or post-exposure therapy for COVID-19.

immunology↗