Biology subjects

Wright, E. R.

Publications and source records attributed to Wright, E. R..

18 recordsLinked to original sources

bioRxiv2026

Intrastriatal injection of amplified Lewy body dementia (LBD) fibrils in mice results in accumulation of fibrils with LBD structure and neurodegeneration

The defining neuropathologic feature of Lewy body dementia (LBD) and Parkinson's disease (PD) is alpha-synuclein (Asyn) fibril accumulation in Lewy bodies (LBs) and Lewy neurites (LNs). Recently, we developed a method to amplify Asyn fibrils derived from human post-mortem LBD brain tissue and determined that the structure of amplified Asyn fibrils is highly similar to the structure of fibrils directly isolated from LBD brain tissue. We examined whether injection of these fibrils into humanized Asyn mice recapitulates pathologic Asyn fibril accumulation with human LBD Asyn fibril structure. We utilized mice containing a null allele in the mouse Snca gene and a human SNCA transgene. Intrastriatal injection of amplified Asyn fibrils derived from 3 different LBD autopsy cases resulted in cytoplasmic and neuritic phospho-Asyn accumulation in the striatum, cortex, and amygdala bilaterally at 3mpi. Fibril injections produced a reduction in the number of dopaminergic neurons in the substantia nigra, with LB-like inclusions in surviving neurons, and produced impaired sensorimotor function as measured on the accelerating rotarod test. Fibril accumulation increased 3.6-fold between 1 and 3 months post-injection (mpi) and was relatively stable between 3 and 5 mpi. We amplified fibrils from mouse brain tissue 5 mpi and collected single-particle cryo-EM data. High resolution 3D structural information could not be obtained because of the lack of helical twist, but comparison of the cryo-EM 2D class averages with those derived from amplified fibrils prepared from human LBD tissue revealed multiple matching classes, indicating that the Lewy fold is maintained in the fibrils that accumulate in mouse brain tissue. Our findings demonstrate an approach to model the accumulation of Asyn fibrils with LBD structure in mice, providing a platform for investigating disease mechanisms, imaging biomarkers, and therapeutics targeting Asyn in PD/LBD.

neuroscience↗

bioRxiv2026

In vitro-prepared A30P alpha-synuclein fibrils adopt the conserved and disease-relevant Greek key fold

The pathological hallmark of Parkinson Disease (PD) is the formation of the protein alpha-synuclein (Asyn) into {beta}-sheet rich, self-templating fibrils in the brain. Since the first atomic structure of wild-type Asyn fibrils was determined nearly a decade ago, several other in vitro structures of hereditary mutant fibrils and structures derived from post-mortem diseased patient tissue have been determined by solid-state nuclear magnetic resonance (SSNMR) spectroscopy and cryo-electron microscopy. These structures have not only expanded the library of structures available for computational modeling of drug binding and therapeutics development but have also given unprecedented insight into the disease specificity and structural polymorphism of Asyn fibrils. Here, we report the high-resolution SSNMR structure of the A30P hereditary mutant Asyn fibril, associated with early-onset PD. Our structural model is calculated using several thousand distance restraints derived from one sample, primarily sourced through 3D 13C-13C-13C correlation experiments. The structure adopts a Greek key topology yet does not include the P30 mutation site within the fibril core. We also introduce a comprehensive method for the rapid comparison of SSNMR spectra between Asyn polymorphs of known structure and validate the A30P fold. Lastly, we find that the structure is highly similar to many other experimental structures of both in vitro and ex vivo Asyn fibrils, including those with other hereditary point mutations, suggesting a conserved accessible fold.

biophysics↗

bioRxiv2025

Capsid flexibility during Ty1 virus-like particle assembly

The Ty1/Copia family (Pseudoviridae) is a widely studied group of long terminal repeat (LTR) retrotransposons present in diverse eukaryotes that share a common ancestor with retroviruses. The founding member Ty1 of Saccharomyces assembles into heterogeneous virus-like particles (VLPs) that are composed of the Gag and Gag-Pol proteins. We used cryo-electron tomography (cryo-ET) of Ty1 VLPs purified from a Ty1-less yeast strain expressing a functional Ty1 element to determine the organization and interactions of Gag in Ty1 VLPs. Using sub-tomogram averaging (STA), we observed unique capsomere structures and obtained EM density maps for pentagonal (7.5 [A]) and hexagonal (7.9 [A]) capsomeres. Through iterative rounds of alignment and classification, we demonstrate that Ty1 VLPs are composed of diverse arrangements of capsomeres, unlike true icosahedral virions. We modeled the structure of the conserved Ty1 Gag capsid C-terminal domain (CA-CTD), previously determined using X-ray crystallography into the capsomeric arrangements, and identified roles for the Dimer-1 interface present in the asymmetric unit, as well as additional interfaces involved in intra- and inter-capsomere assembly. We show that pentagonal or hexagonal capsomere assembly results from flexibility of CA-CTD - CA-CTD interactions at Interface-2. Additionally, we determined the structure of the Ty1 Gag capsid N-terminal domain (CA-NTD) using solution NMR and fitted the CA-NTD into the cryo-ET density maps. Our results indicate that the heterogeneous population of VLP size and morphology arises, at least in part, from the ability of the capsomeres to assemble in diverse organizations. (238 words)

biophysics↗

bioRxiv2025

Structural and Binding Properties of Dps: A Nucleoid Associated Protein

Prokaryotic chromosomal DNA is compacted and protected by nucleoid-associated proteins (NAPs). Among these, the DNA-binding proteins from starved cells (Dps) combine ferroxidase activity with nonspecific DNA binding to form dodecameric assemblies that condense and safeguard the genome under stress. Here, we present an integrated structural and biophysical analysis of Escherichia coli (E. coli) Dps across a pH range from 3 to 11, combining (i) single-particle cryo-EM reconstructions at [~]1.75 [A] resolution, (ii) adaptive Poisson-Boltzmann electrostatic modelling, (iii) electrophoretic mobility shift assays (EMSAs) on a 448 bp DNA fragment, and (iv) cryo-electron tomography (cryo-ET) of Dps-DNA assemblies. Through this analysis, we show that (1) the canonical ferritin-like fold is maintained at all pH values; (2) surface electrostatics shift from highly positive to net negative as the pH increases, modulating DNA affinity (EC50 values of 73.4 nM at the lowest measurement of pH 5, to 815.5 nM at the highest measurement of pH 11); and (3) short-fragment Dps-DNA complexes form amorphous [~]50 nm globular or tubular complexes rather than lattice co-crystals observed on kilobase-length DNA scaffolds. The findings reveal a possible two-stage assembly model, in which protonation-driven nucleation occurs via the Dps N-terminal lysine residues, followed by lattice ordering on long DNA. Our results further define Dps as a pH-responsive nucleoid compaction factor in bacteria.

biophysics↗

bioRxiv2025

Complementary Structural and Chemical Biology Methods Reveal the Basis for Selective Radioligand Binding to α-Synuclein in MSA Tissue

Fibrillar aggregation of -synuclein (Syn) is a hallmark of Parkinsons disease (PD) and related disorders, including multiple system atrophy (MSA) and dementia with Lewy bodies (DLB). Despite advances in Syn fibril structural characterization, the relevance of in vitro and ex vivo structures to patient aggregates remains unclear, particularly for developing therapeutic or diagnostic molecules. Cryo-electron microscopy (cryo-EM) studies of Syn fibrils with ligands often reveal binding at multiple sites, likely due to high ligand concentrations. Here, various structural and chemical biology techniques were used to characterize Syn fibrils in the presence of EX-6, a candidate ligand for positron emission tomography (PET) imaging of synucleinopathies. Transmission electron microscopy (TEM) and cryo-EM revealed no significant fibril core changes upon binding. Forster resonance energy transfer (FRET) further demonstrated that the disordered C-terminus was unaltered. Cryo-EM and crosslinking mass spectrometry (XL-MS) identified consistent binding sites, with one (Site 2*) providing a well-defined pocket for high-resolution analysis. Site 2* showed similar residue positioning in MSA patient-derived structures, suggesting MSA selectivity. [3H]-EX-6 binding assays demonstrated a 10-fold preference for MSA over PD tissue, with autoradiography further confirming MSA selectivity. Taken together, the combined use of structural and chemical biology techniques provides a comprehensive understanding of EX-6 binding that would not be possible with any single method. Optimization of ligand-protein and ligand-ligand interactions observed in the cryo-EM structure will enable the development of EX-6 as a PET imaging probe for MSA.

neuroscience↗

bioRxiv2025

FLUID-CELL: Flow-enabled Light and Ultrastructural Imaging Device for Correlative Electron and Light Localization

We present a novel microfluidic flow cell, or Flow-enabled Light and Ultrastructural Imaging Device for Correlative Electron and Light Localization (FLUID-CELL), that connects fluorescence light microscopy (FLM) and cryo-electron microscopy (cryo-EM) for advanced biological imaging and ultrastructural and structural analyses. The design of the FLUID-CELL features a precisely engineered microchannel that maintains native cell culturing conditions, supporting correlation and enabling real-time observation by FLM, as well as subsequent cryo-EM analysis. In this study, this device enabled practical FLM imaging over extended experimental periods, consistent sample handling, and the ability to perform correlative imaging. This capability connects dynamic cellular events imaged by fluorescence light microscopy with high-resolution ultrastructural data collected with cryo-EM. Our dual-modality approach streamlines the workflow and opens new possibilities for investigating the relationship between cellular function and molecular architecture at the nanoscale.

biophysics↗

bioRxiv2025

Alpha-Synuclein Fibril Structures Cluster into Distinct Classes

The accumulation of Alpha-synuclein (Asyn) fibrils is the defining pathologic feature in Parkinson Disease (PD), Lewy Body Dementia (LBD), and Multiple System Atrophy (MSA). As such, the process of Asyn fibril formation has been an important research area and fibrils themselves have become attractive targets for disease diagnosis and therapeutic intervention. Due to the presence of mixed populations of fibrillar proteins associated with neurodegenerative diseases in brain tissue, high-resolution structures of Asyn fibrils are essential for the design of high-specificity imaging and therapeutic agents. Approximately one hundred high-resolution solid-state NMR (SSNMR) spectroscopy and cryo-electron microscopy (cryo-EM) structures of Asyn fibrils have been deposited to the Protein Databank (PDB); intriguingly there is significant polymorphism among them. Understanding the molecular makeup and characteristic features of each structural polymorph can determine conserved structural motifs which can be used as templates to design ligands with high specificity for clinical use. Utilizing standard alignment tools and density-based clustering approaches, we objectively classify fibril structures by tertiary structure type. We find that 81% of the structures cluster into two polymorph classes. Within each class, additional subtle variations are observed which position sidechains in specific, conserved orientations, well poised as druggable targets. Furthermore, we find that the conserved structural motifs associated with each class are found in all but one published Asyn fibril structure. We consider these classifications and conserved motifs in the context of disease-relevant fibril structures and offer a perspective on the utility of in vitro fibrils as substrates for drug development and models for disease pathogenesis.

biophysics↗

bioRxiv2024

In situ crystalline structure of the Eosinophil major basic protein-1

Eosinophils are white blood cells that participate in innate immune responses and have an essential role in the pathogenesis of inflammatory and neoplastic disorders. Upon activation, eosinophils release cytotoxic proteins such as major basic protein-1 (MBP-1) from cytoplasmic secretory granules (SGr) wherein MBP-1 is stored as nanocrystals. How the MBP-1 nanocrystalline core is formed, stabilized, and subsequently mobilized remains unknown. Here, we report the in-situ structure of crystalline MBP-1 within SGrs of human eosinophils. The structure reveals a mechanism for intragranular crystal packing and stabilization of MBP-1 via a structurally conserved loop region that is associated with calcium-dependent carbohydrate binding in other C-type lectin (CTL) proteins. Single-cell and single-SGr profiling correlating real-space three-dimensional information from cellular montage cryo-electron tomography (cryo-ET) and microcrystal electron diffraction (MicroED) data obtained from non-activated and IL33-activated eosinophils revealed activation-dependent crystal expansion and extrusion of expanded crystals from SGr. These results suggest that MBP-1 crystals play a dynamic role in the release of SGr contents. Collectively, this research demonstrates the importance of in-situ macromolecular structure determination.

biophysics↗

bioRxiv2024

High-Resolution Cryo-EM Structure Determination of α-Synuclein - A Prototypical Amyloid Fibril

The physiological role of -synuclein (-syn), an intrinsically disordered presynaptic neuronal protein, is believed to impact the release of neurotransmitters through interactions with the SNARE complex. However, under certain cellular conditions that are not well understood, -syn will self-assemble into {beta}-sheet rich fibrils that accumulate and form insoluble neuronal inclusions. Studies of patient derived brain tissues have concluded that these inclusions are associated with Parkinsons disease, the second most common neurodegenerative disorder, and other synuclein related diseases called synucleinopathies. In addition, repetitions of and specific mutations to the SNCA gene, the gene that encodes -syn, results in an increased disposition for synucleinopathies. The latest advances in cryo-EM structure determination and real-space helical reconstruction methods have resulted in over 60 in vitro structures of -syn fibrils solved to date, with a handful of these reaching a resolution below 2.5 [A]. Here, we provide a protocol for -syn protein expression, purification, and fibrilization. We detail how sample quality is assessed by negative stain transmission electron microscopy (NS-TEM) analysis and followed by sample vitrification using the Vitrobot Mark IV vitrification robot. We provide a detailed step by step protocol for high resolution cryo-EM structure determination of -syn fibrils using RELION and a series of specialized helical reconstruction tools that can be run within RELION. Finally, we detail how ChimeraX, Coot, and Phenix are used to build and refine a molecular model into the high resolution cryo-EM map. This workflow resulted in a 2.04 [A] structure of -syn fibrils with excellent resolution of residues 36 to 97 and an additional island of density for residues 15 to 22 that had not been previously reported. This workflow should serve as a starting point for individuals new to the neurodegeneration and structural biology fields. Together, this procedure lays the foundation for advanced structural studies of -synuclein and other amyloid fibrils. Key FeaturesO_LIIn vitro fibril amplification method yielding twisting fibrils that span several micrometers in length and are suitable for cryo-EM structure determination. C_LIO_LIHigh-throughput cryo-EM data collection of neurodegenerative fibrils, such as alpha-synuclein. C_LIO_LIUse of RELION implementations of helical reconstruction algorithms to generate high-resolution 3D structures of a-synuclein fibrils. C_LIO_LIBrief demonstration of the use of ChimeraX, Coot, and Phenix for molecular model building and refinement.s C_LI Graphical overview of -synuclein fibrilization and cryo-EM structure determination-synuclein protein expression and purification is followed by a fibrilization protocol yielding twisting filaments that span several micrometers in length and are validated by negative stain transmission electron microscopy (NS-TEM). The sample is then vitrified, followed by cryo-EM data collection. Real-space helical reconstruction is performed in RELION to generate an electron potential map that is used for model building. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/613698v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1b008forg.highwire.dtl.DTLVardef@c1c1e1org.highwire.dtl.DTLVardef@9fc539org.highwire.dtl.DTLVardef@14d3918_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Integrated Fluorescence Microscopy (iFLM) for Cryo-FIB-milling and In-situ Cryo-ET

Correlative cryo-FLM-FIB milling is a powerful sample preparation technique for in situ cryo-ET. However, correlative workflows that incorporate precise targeting remain challenging. Here, we demonstrate the development and use of an integrated Fluorescence Light Microscope (iFLM) module within a cryo-FIB-SEM to enable a coordinate-based two-point 3D correlative workflow. The iFLM guided targeting of regions of interest coupled with an automated milling process of the cryo-FIB-SEM instrument allows for the efficient preparation of 9-12 [~]200 nm thick lamellae within 24 hours. Using regular and montage-cryo-ET data collection schemes, we acquired data from FIB-milled lamellae of HeLa cells to examine cellular ultrastructure. Overall, this workflow facilitates on-the-fly targeting and automated FIB-milling of cryo-preserved cells, bacteria, and possibly high pressure frozen tissue, to produce lamellae for downstream cryo-ET data collection.

biophysics↗

bioRxiv2023

Handling difficult cryo-ET samples: A study with primary neurons from Drosophila melanogaster

Cellular neurobiology has benefited from recent advances in the field of cryo-electron tomography (cryo-ET). Numerous structural and ultrastructural insights have been obtained from plunge-frozen primary neurons cultured on electron microscopy grids. With most primary neurons been derived from rodent sources, we sought to expand the breadth of sample availability by using primary neurons derived from 3rd instar Drosophila melanogaster larval brains. Ultrastructural abnormalities were encountered while establishing this model system for cryo-ET, which were exemplified by excessive membrane blebbing and cellular fragmentation. To optimize neuronal samples, we integrated substrate selection, micropatterning, montage data collection, and chemical fixation. Efforts to address difficulties in establishing Drosophila neurons for future cryo-ET studies in cellular neurobiology also provided insights that future practitioners can use when attempting to establish other cell-based model systems.

biophysics↗

bioRxiv2023

Atomic-level architecture of Caulobacter crescentus flagellar filaments provide evidence for multi-flagellin filament stabilization

Flagella are dynamic, ion-powered machines with assembly pathways that are optimized for efficient flagella production. In bacteria, dozens of genes are coordinated at specific times in the cell lifecycle to generate each component of the flagellum. This is the case for Caulobacter crescentus, but little is known about why this species encodes six different flagellin genes. Furthermore, little is known about the benefits multi-flagellin species possess over single flagellin species, if any, or what molecular properties allow for multi-flagellin filaments to assemble. Here we present an in-depth analysis of several single flagellin filaments from C. crescentus, including an extremely well-resolved structure of a bacterial flagellar filament. We highlight key molecular interactions that differ between each bacterial strain and speculate how these interactions may alleviate or impose helical strain on the overall architecture of the filament. We detail conserved residues within the flagellin subunit that allow for the synthesis of multi-flagellin filaments. We further comment on how these molecular differences impact bacterial motility and highlight how no single flagellin filament achieves wild-type levels of motility, suggesting C. crescentus has evolved to produce a filament optimized for motility comprised of six flagellins. Finally, we highlight an ordered arrangement of glycosylation sites on the surface of the filaments and speculate how these sites may protect the {beta}-hairpin located on the surface exposed domain of the flagellin subunit.

biophysics↗

bioRxiv2022

Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

biophysics↗

bioRxiv2021

Respiratory syncytial virus matrix protein assembles as a lattice with local and extended order that coordinates the position of the fusion glycoprotein

Respiratory syncytial virus (RSV) is a significant cause of respiratory illness in young children and adults worldwide. There is currently no vaccine or targeted antiviral for RSV. RSV is an enveloped, filamentous, negative-strand RNA virus. Individual virions vary in both diameter and length, with an average diameter of [~]130 nm and ranging from [~]500 nm to over 10 m in length. The RSV matrix (M) protein is peripherally associated with the interior of the viral membrane. Though the general arrangement of structural proteins within the virion is known, the molecular organization of M and other structural proteins was previously unknown. Here, using whole-cell cryo-electron tomography and sub-tomogram averaging, we show that M is arranged in a packed helical-like lattice of M-dimers ordered at an angle of [~]47{degrees} to the viral long axis. Sub-tomogram averages including F and M indicate that the position of F on the viral surface is correlated with the underlying M lattice. Finally, we report that RSV F is frequently observed as pairs, with the F trimers oriented in an anti-parallel conformation to support potential interaction between trimers. These results provide insight into RSV assembly and virion organization and may aid in the identification and development of RSV vaccines and anti-viral targets.

biophysics↗

bioRxiv2021

Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technique that can provide nanometer-level resolution of biological structures within the cellular context and in a near-native frozen-hydrated state. It remains a challenge to culture or adhere cells on TEM grids in a manner that is suitable for tomography while preserving the physiological state of the cells. Here, we demonstrate the versatility of micropatterning to direct and promote growth of both cultured and primary eukaryotic cells on TEM grids. We show that micropatterning is compatible with and can be used to enhance studies of host-pathogen interactions using respiratory syncytial virus infected BEAS-2B cells as an example. We demonstrate the ability to use whole-cell tomography of primary Drosophila neuronal cells to identify organelles and cytoskeletal stuctures in cellular axons and the potential for micropatterning to dramatically increase throughput for these studies. During micropatterning, cell growth is targeted by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design make micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (FIB-SEM).

cell biology↗

bioRxiv2020

CorRelator: An interactive and flexible toolkit for high-precision cryo-correlative light and electron microscopy

Cryo-correlative light and electron microscopy (CLEM) is a technique that uses the spatiotemporal cues from fluorescence light microscopy (FLM) to investigate the high-resolution ultrastructure of biological samples by cryo-electron microscopy (cryo-EM). Cryo-CLEM provides advantages for identifying and distinguishing fluorescently labeled proteins, macromolecular complexes, and organelles from the cellular environment. Challenges remain on how correlation workflows and software tools are implemented on different microscope platforms to support microscopy-driven structural studies. Here, we present an open-source desktop application tool, CorRelator, to bridge between cryo-FLM and cryo-EM/ET data collection instruments. CorRelator was designed to be flexible for both on-the-fly and post-acquisition correlation schemes. The CorRelator workflow is easily adapted to any fluorescence and transmission electron microscope (TEM) system configuration. CorRelator was benchmarked under cryogenic and ambient temperature conditions using several FLM and TEM instruments, demonstrating that CorRelator is a rapid and efficient application for image and position registration in CLEM studies. CorRelator is a cross-platform software featuring an intuitive Graphical User Interface (GUI) that guides the user through the correlation process. CorRelator source code is available at: https://github.com/wright-cemrc-projects/corr.

biophysics↗

bioRxiv2020

Flagellar structures from the bacterium Caulobacter crescentus and implications for phage {Phi}CbK predation of multi-flagellin bacteria

Caulobacter crescentus is a gram-negative alpha-proteobacterium that commonly lives in oligotrophic fresh and salt-water environments. C. crescentus is a host to many bacteriophages, including {phi}CbK and {phi}CbK-like bacteriophages, which first adsorb to cells by interaction with the bacterial flagellum. It is commonly thought that the six paralogs of the flagellin gene present in C. crescentus are important for bacteriophage evasion. Here, we show that deletion of specific flagellins in C. crescentus can indeed attenuate {phi}CbK adsorption efficiency, although no single deletion completely ablates {phi}CbK adsorption. Thus, bacteriophage {phi}CbK likely recognizes a common motif amongst the six known flagellins in C. crescentus with varying degrees of efficiency. Interestingly, we observe that most deletion strains still generate flagellar filaments, with the exception of a strain that contains only the most divergent flagellin, FljJ, or a strain that contains only FljN and FljO. To visualize the surface residues that are likely recognized by {phi}CbK, we determined two high-resolution structures of the FljK filament, with and without an amino acid substitution that induces straightening of the filament. We observe post-translational modifications on conserved surface threonine residues of FljK that are likely O-linked glycans. The possibility of interplay between these modifications and {phi}CbK adsorption is discussed. We also determined the structure of a filament composed of a heterogeneous mixture of FljK and FljL, the final resolution of which was limited to approximately 4.6 [A]. Altogether, this work builds a platform for future investigation of how phage {phi}CbK infects C. crescentus at the molecular level.

microbiology↗

bioRxiv2017

Obstruction of pilus retraction stimulates bacterial surface sensing

Surface association provides numerous fitness advantages to bacteria. Thus, it is critical for bacteria to recognize surface contact and to consequently initiate physiological changes required for a surface-associated lifestyle (1). Ubiquitous microbial appendages called pili are involved in sensing surfaces and mediating downstream surface-associated behaviors (2-6). The mechanism by which pili mediate surface sensing remains unknown, largely due to the difficulty to visualize their dynamic nature and to directly modulate their activity without genetic modification. Here, we show that Caulobacter crescentus pili undergo dynamic cycles of extension and retraction that cease within seconds of surface contact, and this arrest of pilus activity coincides with surface-stimulated holdfast synthesis. By physically blocking pili, we show that imposing resistance to pilus retraction is sufficient to stimulate holdfast synthesis in the absence of surface contact. Thus, resistance to type IV pilus retraction upon surface attachment is used for surface sensing.\n\nOne Sentence SummaryBacteria use the tension imparted on retracting pilus fibers upon their binding to a surface for surface sensing.

microbiology↗