Biology subjects

Howes, S.

Publications and source records attributed to Howes, S..

5 recordsLinked to original sources

bioRxiv2026

In-cell structural insights into fungal ER stress responses

Approximately a third of a cells proteome is folded in the endoplasmic reticulum (ER). The accumulation of misfolded proteins in the ER results in ER stress. The unfolded protein response (UPR) has evolved to attenuate ER stress by increasing the cells folding capacity and reducing the folding load in the ER. In metazoans, the latter is notably achieved through mRNA translation inhibition. However, S. cerevisiae lacks the corresponding pathways, and its reliance on translational downregulation in response to ER stress remains largely unexplored. In this study we combined cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to investigate the effect of ER stress on the translational machinery of S. cerevisiae. At the ultrastructural level, we describe changes in organelle morphology, confirming ER volume expansion and the activation of autophagy. Further structural analysis reveals that ER stress triggers an increase in the relative abundance of inactive 80S ribosomes (up to 25% of the total), both in the cytosol and at the ER. Finally, we visualise the binding of the elongation factors eEF2, eIF5A and eEF3 to inactive ribosomes. Our work provides evidence that ER stress in S. cerevisiae leads to modest translation inhibition, which might contribute to alleviating ER stress.

molecular biology↗

bioRxiv2025

Taxane-Induced Conformational Changes in the Microtubule Lattice Activate GEF-H1-Dependent RhoA Signaling

Taxanes are widely used chemotherapeutic agents that perturb cell division. They also exert effects during interphase, but the underlying mechanisms are poorly understood. Here, we show that taxanes activate RhoA signaling and induce actin remodeling by displacing the RhoA activator GEF-H1 from microtubules. This taxane-induced release of GEF-H1 occurs rapidly, is independent of tubulin post-translational modifications, and can be recapitulated using purified proteins. In vitro reconstitution assays combined with analyses of microtubule structure revealed that microtubule binding by GEF-H1 is inhibited by microtubule-stabilizing agents that expand the microtubule lattice, such as taxanes and GMPCPP, but not by others, including GTP{gamma}S and discodermolide, which stabilize a compacted microtubule lattice. Our findings demonstrate that alterations in microtubule lattice conformation can activate key signaling pathways, offering new insights into the mode of action of taxanes and the possible origins of their side effects.

cell biology↗

bioRxiv2025

Structure of an oxygen-induced tubular nanocompartment in Pyrococcus furiosus

Reactive oxygen species (ROS) pose a significant threat to biological molecules, prompting organisms to develop systems that buffer oxidative stress and contain iron, which otherwise amplifies ROS production. Understanding oxidative stress responses requires identifying the key proteins involved and their cellular organization. Here, we combined proteomics and cryo-EM to investigate the response of the anaerobic hyperthermophilic archaeon Pyrococcus furiosus to oxygen exposure. Proteome analysis revealed a significant upregulation of the oxidoreductase Rubrerythrin (Rbr) under oxidative stress. Cryo-electron tomograms showed the formation of prominent oxidative stress-induced tubules (OSITs). Single-particle cryo-EM and mass spectrometry of enriched OSITs identified them as stacked rings of Rbr homotetramers. The 3.3 [A] structure demonstrates that rubredoxin-like domains mediate homotetramer assembly, suggesting that their oxidation drives OSIT formation. Within OSITs, we discovered virus-like particles formed by a ferritin-like/encapsulin fusion protein with iron hydroxide cores, uncovering a sophisticated organelle that protects P. furiosus from ROS through advanced compartmentalization.

molecular biology↗

bioRxiv2021

Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) can infer molecular, functional and dynamic information to ultrastructure by linking information of different imaging modalities. One of the main challenges, especially in 3D-CLEM, is the accurate registration of fluorescent signals to electron microscopy (EM). Here, we present fluorescent BSA-gold (fBSA-Au), a bimodal endocytic tracer as fiducial marker for 2D and 3D CLEM applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent bovine serum albumin (BSA). The conjugate is efficiently endocytosed and distributed throughout the 3D endo-lysosomal network of the cells, and has an excellent visibility both in fluorescence microscopy (FM) and EM. We demonstrate the use of fBSA-Au in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryo-EM. As a fiducial marker, fBSA-Au facilitates rapid registration of regions of interest between FM and EM modalities and enables accurate (50-150 nm) correlation of fluorescence to EM data. Endocytosed fBSA-Au benefits from a homogenous 3D distribution throughout the endosomal system within the cell, and does not obscure any cellular ultrastructure. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo-)CLEM applications.

cell biology↗

bioRxiv2021

In-cell structures of a conserved supramolecular array at the mitochondria-cytoskeleton interface in mammalian sperm

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbors through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types where mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

cell biology↗