Biology subjects

Dillard, L.

Publications and source records attributed to Dillard, L..

6 recordsLinked to original sources

bioRxiv2026

Cryo-EM reveals a right-handed double-helix dimer architecture of PCDH15 critical for mechanotransduction

Tip links connect the stereocilia of mechanosensory hair cells in the inner ear and transmit force onto mechanotransduction (MET) channels. Tip links consist of protocadherin 15 (PCDH15) and cadherin 23 (CDH23), which assemble into an extracellular filament approximately 150 nm in length. Rare freeze-etched electron microscopy (EM) images have suggested that tip links could be right-handed double helices in vivo, but direct structural evidence has been lacking. Using cryo-EM we determined the structure of a large part of the extracellular PCDH15 domain. Two PCDH15 molecules form a parallel cis dimer stabilized by several dimerization interfaces, including two strand crossovers and two parallel contacts, yielding a right-handed double helix. Functional studies show that mutations in PCDH15 dimerization-domains impair MET. Our results establish the molecular foundation for how PCDH15 forms a right-handed double helix to enable mechanical sensing.

neuroscience↗

bioRxiv2024

A higher order PUF complex is central to regulation of C. elegans germline stem cells

PUF RNA-binding proteins are broadly conserved stem cell regulators. Nematode PUF proteins maintain germline stem cells (GSCs) and, with key partner proteins, repress differentiation mRNAs, including gld-1. Here we report that PUF protein FBF-2 and its partner LST-1 form a ternary complex that represses gld-1 via a pair of adjacent FBF-2 binding elements (FBEs) in its 3UTR. One LST-1 molecule links two FBF-2 molecules via motifs in the LST-1 intrinsically-disordered region; the gld-1 FBE pair includes a well-established canonical FBE and a newly-identified noncanonical FBE. Remarkably, this FBE pair drives both full RNA repression in GSCs and full RNA activation upon differentiation. Discovery of the LST-1-FBF-2 ternary complex, the gld-1 adjacent FBEs, and their in vivo significance predicts an expanded regulatory repertoire of different assemblies of PUF-partner complexes in nematode germline stem cells. It also suggests analogous PUF controls may await discovery in other biological contexts and organisms.

biochemistry↗

bioRxiv2022

Structural and functional landscape of α-synuclein fibril conformations amplified from cerebrospinal fluid

Lewy body dementias are pathologically defined by the deposition of -synuclein fibrils into inclusions throughout the brain. Cerebrospinal fluid(CSF) in disease harbors circulating -synuclein-fibril seeds, and parental -synuclein fibrils can template core structure into amplified fibrils. Using cryo-electron microscopy, we identify six novel -synuclein fibril assemblies amplified from ten CSF samples (3.8[A] to 2.9[A] nominal resolutions). Fibrils are classified based on two types of filament interaction, two types of {beta}-sheet stacking, and two types of hydrophobic pocket. CSF-amplified fibril products have one, two, or three distinct assemblies each. Six of ten samples share a common fibril assembly. Within this classification, the fibrils have distinct profiles in amyloid dye binding, and dramatically different potencies in both seeding new inclusions in neurons and evoked microglial pro-inflammatory responses. However, no single structural feature predicts functional phenotypes. Our results highlight CSF as a valuable resource to identify novel -synuclein assemblies potentially important in disease.

neuroscience↗

bioRxiv2021

Untwisted α-synuclein Filaments formed in the Presence of Lipid Vesicles

Accumulation of filamentous aggregates of -synuclein is a pathological hallmark of several neurodegenerative diseases including Parkinsons disease (PD). Interaction between -synuclein and lipids has been shown to play a critical role in aggregation of -synuclein. Most structural studies have, however, been focused on -synuclein filaments formed in the absence of lipids. Here, we report structural investigation of -synuclein filaments assembled under the quiescent conditions in the presence of anionic lipid vesicles using electron microscopy (EM) including cryo-EM. Our transmission electron microscopy (TEM) analyses reveal that -synuclein forms curly protofilaments at an early stage of aggregation. The flexible protofilaments were then converted to long filaments after a longer incubation of 30 days. More detailed structural analyses using cryo-EM reveal that the long filaments adopt untwisted structures with different diameters, which have not been observed in previous -synuclein filaments formed in vitro. The untwisted filaments are rather similar to straight filaments with no observable twist that are extracted from patients with dementia with Lewy bodies. Our structural studies highlight the conformational diversity of -synuclein filaments, requiring additional structural investigation of not only more ex vivo -synuclein filaments, but also in vitro -synuclein filaments formed in the presence of diverse co-factors to better understand the molecular basis of diverse molecular conformations of -synuclein filaments.

biochemistry↗

bioRxiv2021

Distinct cryo-EM Structure of α-synuclein Filaments derived by Tau

Recent structural studies of ex vivo amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations distinct from those in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo -synuclein filaments extracted from multiple system atrophy (MSA) patients adopt quite distinct molecular structures from those of in vitro -synuclein filaments, suggesting the presence of co-factors for -synuclein aggregation in vivo. Here, we report structural characterizations of -synuclein filaments derived by a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted -synuclein filament at 4.0 [A] resolution is somewhat similar to one of the polymorphs of in vitro -synuclein filaments. However, the N- and C-terminal regions of the tau-promoted -synuclein filament have different molecular conformations. Our structural studies highlight the conformational plasticity of -synuclein filaments, requiring additional structural investigation of not only more ex vivo -synuclein filaments, but also in vitro -synuclein filaments formed in the presence of diverse co-factors to better understand molecular basis of diverse molecular conformations of -synuclein filaments.

biochemistry↗

bioRxiv2020

Cryo-EM Structures of the SARS-CoV-2 Endoribonuclease Nsp15

New therapeutics are urgently needed to inhibit SARS-CoV-2, the virus responsible for the on-going Covid-19 pandemic. Nsp15, a uridine-specific endoribonuclease found in all coronaviruses, processes viral RNA to evade detection by RNA-activated host defense systems, making it a promising drug target. Previous work with SARS-CoV-1 established that Nsp15 is active as a hexamer, yet how Nsp15 recognizes and processes viral RNA remains unknown. Here we report a series of cryo-EM reconstructions of SARS-CoV-2 Nsp15. The UTP-bound cryo-EM reconstruction at 3.36 [A] resolution provides molecular details into how critical residues within the Nsp15 active site recognize uridine and facilitate catalysis of the phosphodiester bond, whereas the apo-states reveal active site conformational heterogeneity. We further demonstrate the specificity and mechanism of nuclease activity by analyzing Nsp15 products using mass spectrometry. Collectively, these findings advance understanding of how Nsp15 processes viral RNA and provide a structural framework for the development of new therapeutics.

biochemistry↗