Assembly of recombinant tau into filaments identical to those of Alzheimer's disease and chronic traumatic encephalopathy

Abundant filamentous inclusions of tau are characteristic of more than 20 neurodegenerative diseases that are collectively termed tauopathies. Electron cryo-microscopy (cryo-EM) structures of tau amyloid filaments from human brain revealed that different tau folds characterise many different diseases. A lack of laboratory-based model systems to generate these structures has hampered efforts to uncover the molecular mechanisms that underlie tauopathies. Here, we report in vitro assembly conditions with recombinant tau that replicate the structures of filaments from both Alzheimer's disease (AD) and chronic traumatic encephalopathy (CTE), as determined by cryo-EM. Our results suggest that post-translational modifications of tau modulate filament assembly, and that previously observed additional densities in AD and CTE filaments may arise from the presence of inorganic salts, like phosphates and sodium chloride. In vitro assembly of tau into disease-relevant filaments will facilitate studies to determine their roles in different diseases, as well as the development of compounds that specifically bind to these structures or prevent their formation.

Structure of the bacterial flagellar hook cap provides insights into a hook assembly mechanism

Assembly of bacterial flagellar hook requires FlgD, a protein known to form the hook cap. Symmetry mismatch between the hook and the hook cap is believed to drive efficient assembly of the hook in a way similar to the filament cap helping filament assembly. However, the hook cap dependent mechanism of hook assembly has remained poorly understood. Here, we report the crystal structure of the hook cap composed of five subunits of FlgD from Salmonella enterica at 3.3 Å resolution. The pentameric structure of the hook cap is divided into two parts: a stalk region composed of five N-terminal domains; and a petal region containing five C-terminal domains. Biochemical and genetic analyses show that the N-terminal domains of the hook cap is essential for the hook-capping function, and the structure now clearly reveals why. A plausible hook assembly mechanism promoted by the hook cap is proposed based on the structure.

Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

Cryo-EM Structures of Amyloid-β 42 Filaments from Human Brain

Filament assembly of amyloid-β peptides ending at residue 42 (Aβ42) is a central event in Alzheimer's disease. We report the cryo-EM structures of Aβ42 filaments from brain. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains of individuals with sporadic Alzheimer's disease and Type II filaments in individuals with familial Alzheimer's disease and other conditions. The structures of Aβ42 filaments from brain differ from those of filaments assembled in vitro. By contrast, in App NL-F knock-in mice, Aβ42 deposits were made of Type II filaments. Knowledge of Aβ42 filament structures from human brain may lead to the development of inhibitors of assembly and improved imaging agents.

PIAS1 Regulates Hepatitis C Virus-Induced Lipid Droplet Accumulation by Controlling Septin 9 and Microtubule Filament Assembly

Chronic hepatitis C virus (HCV) infection often leads to fibrosis and chronic hepatitis, then cirrhosis and ultimately hepatocellular carcinoma (HCC). The processes of the HVC life cycle involve intimate interactions between viral and host cell proteins and lipid metabolism. However, the molecules and mechanisms involved in this tripartite interaction remain poorly understood. Herein, we show that the infection of HCC-derived Huh7.5 cells with HCV promotes upregulation of the protein inhibitor of activated STAT1 (PIAS1). Reciprocally, PIAS1 regulated the expression of HCV core protein and HCV-induced LD accumulation and impaired HCV replication. Furthermore, PIAS1 controlled HCV-promoted septin 9 filament formation and microtubule polymerization. Subsequently, we found that PIAS1 interacted with septin 9 and controlled its assembly on filaments, which thus affected septin 9-induced lipid droplet accumulation. Taken together, these data reveal that PIAS1 regulates the accumulation of lipid droplets and offer a meaningful insight into how HCV interacts with host proteins.

Multiple Roles of Flagellar Export Chaperones for Efficient and Robust Flagellar Filament Formation in Salmonella

FlgN, FliS, and FliT are flagellar export chaperones specific for FlgK/FlgL, FliC, and FliD, respectively, which are essential component proteins for filament formation. These chaperones facilitate the docking of their cognate substrates to a transmembrane export gate protein, FlhA, to facilitate their subsequent unfolding and export by the flagellar type III secretion system (fT3SS). Dynamic interactions of the chaperones with FlhA are thought to determine the substrate export order. To clarify the role of flagellar chaperones in filament assembly, we constructed cells lacking FlgN, FliS, and/or FliT. Removal of either FlgN, FliS, or FliT resulted in leakage of a large amount of unassembled FliC monomers into the culture media, indicating that these chaperones contribute to robust and efficient filament formation. The ∆flgN ∆fliS ∆fliT (∆NST) cells produced short filaments similarly to the ∆fliS mutant. Suppressor mutations of the ∆NST cells, which lengthened the filament, were all found in FliC and destabilized the folded structure of FliC monomer. Deletion of FliS inhibited FliC export and filament elongation only after FliC synthesis was complete. We propose that FliS is not involved in the transport of FliC upon onset of filament formation, but FliS-assisted unfolding of FliC by the fT3SS becomes essential for its rapid and efficient export to form a long filament when FliC becomes fully expressed in the cytoplasm.

BB0259 Encompasses a Peptidoglycan Lytic Enzyme Function for Proper Assembly of Periplasmic Flagella in Borrelia burgdorferi

Assembly of the bacterial flagellar rod, hook, and filament requires penetration through the peptidoglycan (PG) sacculus and outer membrane. In most β- and γ-proteobacteria, the protein FlgJ has two functional domains that enable PG hydrolyzing activity to create pores, facilitating proper assembly of the flagellar rod. However, two distinct proteins performing the same functions as the dual-domain FlgJ are proposed in δ- and ε-proteobacteria as well as spirochetes. The Lyme disease spirochete Borrelia burgdorferi genome possesses a FlgJ and a PG lytic SLT enzyme protein homolog (BB0259). FlgJ in B. burgdorferi is crucial for flagellar hook and filament assembly but not for the proper rod assembly reported in other bacteria. However, BB0259 has never been characterized. Here, we use cryo-electron tomography to visualize periplasmic flagella in different bb0259 mutant strains and provide evidence that the E580 residue of BB0259 is essential for PG-hydrolyzing activity. Without the enzyme activity, the flagellar hook fails to penetrate through the pores in the cell wall to complete assembly of an intact periplasmic flagellum. Given that FlgJ and BB0259 interact with each other, they likely coordinate the penetration through the PG sacculus and assembly of a functional flagellum in B. burgdorferi and other spirochetes. Because of its role, we renamed BB0259 as flagellar-specific lytic transglycosylase or LTaseBb.

Molecular Interactions Driving Intermediate Filament Assembly

Given the role of intermediate filaments (IFs) in normal cell physiology and scores of IF-linked diseases, the importance of understanding their molecular structure is beyond doubt. Research into the IF structure was initiated more than 30 years ago, and some important advances have been made. Using crystallography and other methods, the central coiled-coil domain of the elementary dimer and also the structural basis of the soluble tetramer formation have been studied to atomic precision. However, the molecular interactions driving later stages of the filament assembly are still not fully understood. For cytoplasmic IFs, much of the currently available insight is due to chemical cross-linking experiments that date back to the 1990s. This technique has since been radically improved, and several groups have utilized it recently to obtain data on lamin filament assembly. Here, we will summarize these findings and reflect on the remaining open questions and challenges of IF structure. We argue that, in addition to X-ray crystallography, chemical cross-linking and cryoelectron microscopy are the techniques that should enable major new advances in the field in the near future.

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging

Intermediate filaments (IF) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a ~49 nm axial repeat both in cells and in vitro. As unit-length-filaments (ULFs) precursors were measured at ~59 nm, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical and structural properties in cells.