Biology subjects

Wozny, M. R.

Publications and source records attributed to Wozny, M. R..

5 recordsLinked to original sources

bioRxiv2025

In situ Architecture of the Tad Pilus Machine in Caulobacter crescentus

The Tight adherence (Tad) pilus is a broadly distributed and evolutionarily distinct subclass of type IV pili that mediates cell adhesion, biofilm formation, predation, and surface sensing in many bacteria, including Caulobacter crescentus, Myxococcus xanthus, Vibrio vulnificus, and Bifidobacterium breve. Tad pili undergo cycles of extension and retraction powered by a cell-envelope-embedded nanomachine. Despite their biological importance, the architecture and assembly mechanism of the Tad pilus system have remained poorly understood. Although cryo-electron tomography (cryo-ET) has elucidated the in situ structures of other type IV pilus systems, no intact Tad machine structure has previously been reported. Here, we use cryo-ET and subtomogram averaging to resolve the near-native architecture of the C. crescentus Tad pilus within the bacterial cell envelope. 3D classification further reveals multiple assembly intermediates, and integrative modelling incorporating AlphaFold3 predictions help define the spatial arrangement of all core components. The resulting structural framework gives insight into the stepwise assembly process of the C. crescentus Tad pilus machine. Altogether, our results provide an in situ architectural model of the Tad pilus machine, establishing a foundation for understanding homologous systems across a broad range of bacteria. ImportanceInvestigating the Tad pilus nanomachine in a genetically tractable, non-pathogenic organism like Caulobacter crescentus provides a powerful model for elucidating the architecture and functional dynamics of this widespread system. Insights gained from studying the Tad machinery can improve our understanding of related Tad pilus systems in pathogenic bacteria such as Aggregatibacter actinomycetemcomitans, where Tad pili are a key determinant of biofilm formation and chronic infection. Additionally, the remarkable functional diversity of Tad systems, ranging from surface sensing in C. crescentus to bacterial predation in M. xanthus, highlights their broad biological relevance. By revealing the in situ structure and assembly mechanism of the Tad pilus biosynthetic machinery, this study advances our understanding of a major class of bacterial nanomachines and may thus provide structural insights that could inform the development of new therapeutic strategies targeting pilus-mediated virulence.

microbiology↗

bioRxiv2023

Microfibril-associated glycoprotein 4 forms octamers that mediate interactions with elastogenic proteins and cells

Microfibrillar-associated protein 4 (MFAP4) is a 36-kDa extracellular glycoprotein with critical roles in human pathologies, including fibrosis in several organ systems, chronic obstructive pulmonary disease, and cardiovascular disorders. In elastic tissues such as arteries, lungs, and skin, MFAP4 associates with microfibrils and elastic fibres, which are the central extracellular fibres affected in thoracic aneurysms. MFAP4 directly interacts with elastogenic proteins, including fibrillin-1 and tropoelastin, and with cells via integrins. MFAP4 multimerisation represents a critical hallmark required for its physiological and pathological properties. However, molecular details and functional consequences of MFAP4 multimerisation are lacking. Here we present a cryo-electron microscopy structure of human MFAP4. In the presence of calcium, MFAP4 assembles as an octamer with D2 point group symmetry, where two sets of homodimers constitute the top and bottom halves of each octamer. Each of the homodimers is linked together by an inter-molecular disulfide bond. An engineered C34S missense mutation in MFAP4 prevented disulfide-bond formation between monomers, but the mutant formed octamers similar to wild type MFAP4. The atomic model, built into the 3.55 [A] cryo-EM map, suggests that several salt-bridges are important for interactions within and between homodimers, while non-polar interactions are important for octamer halves to assemble. In the absence of calcium, MFAP4 dissociates into tetramers, representing the top/bottom halves of the octamers. Binding studies with elastogenic proteins, including fibrillin-1, tropoelastin, LTBP4, and small fibulins showed that MFAP4 has multiple surfaces for protein-protein interactions, which depend upon the higher-order assembly of MFAP4. While the disulfide-bond mediated by C34S contributes little to those protein interactions, it modulated cell interaction. When MFAP4 forms assemblies with fibrillin-1, it abrogates MFAP4 interactions with cells. Overall, the study provides detailed molecular structure-function relationships of MFAP4 interactions with elastogenic proteins and cells.

cell biology↗

bioRxiv2022

Supramolecular architecture of the ER-mitochondria encounter structure in its native environment

The endoplasmic reticulum and mitochondria are main hubs of eukaryotic membrane biogenesis which rely on lipid exchange via membrane contact sites, but the underpinning mechanisms remain poorly understood. In yeast, tethering and lipid transfer between the two organelles is mediated by the ER-mitochondria encounter structure ERMES, a four-subunit complex of unclear stoichiometry and architecture. We determined the molecular organization of ERMES within cells using integrative structural biology, combining quantitative live-imaging, cryo-correlative microscopy, subtomogram averaging and molecular modeling. ERMES assembles into approximately 25 discrete bridge-like complexes distributed irregularly across a contact site. Each bridge consists of three lipid-binding SMP domains arranged in zig-zag fashion. Our molecular model of ERMES reveals an unconventional restrained pathway for lipids. These findings resolve a supramolecular architecture controlling interorganelle lipid fluxes.

cell biology↗

bioRxiv2020

The supramolecular landscape of growing human axons

During brain development, human axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly inaccessible to imaging at molecular resolution in a near-native context. Here we apply cryo-correlative light microscopy and electron tomography to growing axonal tracts from human cerebral organoids. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These data provide an unprecedented resource and reveal a molecular architecture that helps explain the unique biology of growing human axons.

cell biology↗

bioRxiv2019

Rod-shaped tricalbins contribute to PM asymmetry at curved ER-PM contact sites

Lipid flow between cellular organelles occurs via membrane contact sites that form dynamic conduits. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate the membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography and high-throughput genetics we address this interplay of architecture and function in budding yeast. We find that ER-PM contacts are diverse in protein composition and membrane morphology, not in intermembrane distance. In situ cryo-EM of tricalbins reveals their molecular organisation that suggests an unexpected structural framework for lipid transfer. Genetic analysis identifies functional redundancies, both for tricalbin domains and cellular lipid routes, and points to tricalbin function in maintenance of PM asymmetry. These results uncover a modularity of molecular and structural functions of tricalbins, and of their roles within the network of cellular lipid fluxes.

cell biology↗