Structure and function of FAP47 in the central pair apparatus of Chlamydomonas flagella.

Motile cilia have a so-called "9 + 2" structure, which consists of nine doublet microtubules and a central pair apparatus. The central pair apparatus (CA) is thought to interact mechanically with radial spokes and to control the flagellar beating. Recently, the components of the CA have been identified by proteomic and genomic analyses. Still, the mechanism of how the CA contributes to ciliary motility has much to be revealed. Here, we focused on one CA component with a large molecular weight: FAP47, and its relationship with two other CA components with large molecular weight: HYDIN, and CPC1. The analyses of motility of the Chlamydomonas mutants revealed that in contrast to cpc1 or hydin, which swam more slowly than the wild type, fap47 cells displayed wild-type swimming velocity and flagellar beat frequency, yet interestingly, fap47 cells have phototaxis defects and swim straighter than the wild-type cells. Furthermore, the double mutant fap47cpc1 and fap47hydin showed significantly slower swimming than cpc1 and hydin cells, and the motility defect of fap47cpc1 was rescued to the cpc1 level with GFP-tagged FAP47, indicating that the lack of FAP47 makes the motility defect of cpc1 worse. Cryo-electron tomography demonstrated that the fap47 lacks a part of the C1-C2 bridge of CA. Taken together, these observations indicate that FAP47 maintains the structural stiffness of the CA, which is important for flagellar regulation.

Assembly of FAP93 at the proximal axoneme in Chlamydomonas cilia.

To identify proteins specific to the proximal ciliary axoneme, we used iTRAQ to compare short (~2 µm) and full-length (~11 µm) axonemes of Chlamydomonas. Known compoents of the proximal axoneme such as minor dynein heavy chains and LF5 kinase as well as the ciliary tip proteins FAP256 (CEP104) and EB1 were enriched in short axonemes whereas proteins present along the length of the axoneme were of similar abundance in both samples. The iTRAQ analysis revealed that FAP93, a protein of unknown function, and protein phosphatase 2A (PP2A) are enriched in the short axonemes. Consistently, immunoblots show enrichment of FAP93 and PP2A in short axonemes and immunofluorescence confirms the localization of FAP93 and enrichment of PP2A at the proximal axoneme. Ciliary regeneration reveals that FAP93 assembles continuously but more slowly than other axonemal structures and terminates at 1.03 µm in steady-state axonemes. The length of FAP93 assembly correlates with ciliary length, demonstrating ciliary length-dependent assembly of FAP93. Dikaryon rescue experiments show that FAP93 can assemble independently of IFT transport. In addition, FRAP analysis of GFP-tagged FAP93 demonstrates that FAP93 is stably anchored in axoneme. FAP93 may function as a scaffold for assembly of other specific proteins at the proximal axoneme.

Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane.

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

Unveiling Liquid-Crystalline Lipids in the Urothelial Membrane through Cryo-EM.

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

Unveiling Liquid-Crystalline Lipids in the Urothelial Membrane through Cryo-EM.

The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this investigation, we employed cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex embedded within the porcine AUM at a resolution of 3.5 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane.

α- and ß-tubulin C-terminal tails with distinct modifications are crucial for ciliary motility and assembly.

α- and ß-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTTs). The function of this region in cilia and flagella is still unclear, except that glutamates in CTTs act as the sites for post-translational modifications that affect ciliary motility. The unicellular alga Chlamydomonas possesses only two α-tubulin and two ß-tubulin genes, each pair encoding an identical protein. This simple gene organization might enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutant strains expressing tubulins with modified CTTs. We found that the mutant strain in which four glutamate residues in the α-tubulin CTT had been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant strain lacking the glutamate-rich region of the ß-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to mutant strains harboring a mutation in a subunit of katanin, the function of which has been shown to depend on the ß-tubulin CTT. Therefore, our study reveals distinct and important roles of α- and ß-tubulin CTTs in the formation and function of cilia.

ATP-Responsive Nanoparticles Covered with Biomolecular Machine "Chaperonin GroEL".

Herein, we report an ATP-responsive nanoparticle (GroEL NP) whose surface is fully covered with the biomolecular machine "chaperonin protein GroEL". GroEL NP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroEL NP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine-like function and enable GroEL NP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroEL NP per GroEL was 4.8 and 4.0 times greater than those of precursor cys GroEL and its DNA-functionalized analogue, respectively. Finally, we confirmed that GroEL NP could be iteratively extended to double-layered ( GroEL ) 2 ${{^{({\rm GroEL}){_{2}}}}}$ NP.

Bioconjugation of Au25 Nanocluster to Monoclonal Antibody at Tryptophan.

We report the first bioconjugation of Au25 nanocluster to a monoclonal antibody at scarcely exposed tryptophan (Trp) residues toward the development of high-resolution probes for cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET). To achieve this, we improved the Trp-selective bioconjugation using hydroxylamine (ABNOH) reagents instead of previously developed N-oxyl radicals (ABNO). This new protocol allowed for the application of Trp-selective bioconjugation to acid-sensitive proteins such as antibodies. We found that a two-step procedure utilizing first Trp-selective bioconjugation for the introduction of azide groups to the protein and then strain-promoted azide-alkyne cycloaddition (SPAAC) to attach a bicyclononyne (BCN)-presenting redox-sensitive Au25 nanocluster was essential for a scalable procedure. Covalent labeling of the antibody with gold nanoclusters was confirmed by various analytical methods, including cryo-EM analysis of the Au25 nanocluster conjugates.

Parallel cryo electron tomography on in situ lamellae.

In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. However, the possibilities for recording tomographic tilt series in a high-throughput manner are limited, in part by the lamella-shaped samples. Here we utilize a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining access to sample areas conventionally used for tracking specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows for the collection of sample areas that were previously unreachable, thus maximizing the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high throughput and quality of PACE-tomo.

Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation.

Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C-type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 Å resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258-263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection.

Identifying proteins in the cell by tagging techniques for cryo-electron microscopy.

Cryo-electron microscopy (cryo-EM) is currently expanding its application from molecular structures to cellular structures. The cellular environment is heterogeneous, containing many different proteins, and very crowded. This environment is in sharp contrast to the specimens for single particle analysis, by which purified homogeneous samples are analyzed. To answer biological questions from the structural studies of cells, it is crucial to identify biological molecules (typically, proteins) of interest and tagging is becoming the critical technique for cryo-EM. In this review, we explain the requirements for tags and review recent advances of tagging and identification methods for cryo-EM.

Cryo-EM performance testing of hardware and data acquisition strategies.

The increasing popularity and adoption rate of cryo-electron microscopy (cryo-EM) is evidenced by a growing number of new microscope installations around the world. The quality and reliability of the instruments improved dramatically in recent years, but site-specific issues or unnoticed problems during installation could undermine productivity. Newcomers to the field may also have limited experience and/or low confidence in the capabilities of the equipment or their own skills. Therefore, it is recommended to perform an initial test of the complete cryo-EM workflow with an 'easy' test sample, such as apoferritin, before starting work with real and challenging samples. Analogous test experiments are also recommended for the quantification of new data acquisition approaches or imaging hardware. Here, we present the results from our initial tests of a recently installed Krios G4 electron microscope equipped with two latest generation direct electron detector cameras-Gatan K3 and Falcon 4. Three beam-image shift-based data acquisition strategies were also tested. We detail the methodology and discuss the critical parameters and steps for performance testing. The two cameras performed equally, and the single- and multi-shot per-hole acquisition schemes produced comparable results. We also evaluated the effects of environmental factors and optical flaws on data quality. Our results reaffirmed the exceptional performance of the software aberration correction in Relion in dealing with severe coma aberration. We hope that this work will help cryo-EM teams in their testing and troubleshooting of hardware and data collection approaches.

Alternating Heterochiral Supramolecular Copolymerization.

In stacking-based supramolecular polymerization, chiral hydrogen bonding (H-bonding) monomers often prefer to adapt a homochiral monomer sequence. Herein, we investigated the polymerization of a chiral thiophene-fused cyclooctatetraene (COT) as a novel nonplanar-core monomer and found the first example of the formation of an alternating heterochiral supramolecular copolymer. Although single enantiomer (-) or (+)-COT alone did not polymerize, when (-) and (+)-COT were mixed together, supramolecular polymerization took place to give a stereochemically alternating copolymer. By means of the microcrystal electron crystallography of a shorter side-chained COT analogue, we found that the resulting heterochiral supramolecular copolymer adapted an alternating arrangement of H-bonded and polar π-stacked parts. A computational study using density-functional theory (DFT) suggested that such an alternating heterochiral preference occurs because it allows two thiophene amide moieties facing each other to effectively cancel their in-plane dipole moments.

Nano- and Microspheres Containing Inorganic and Biological Nanoparticles: Self-Assembly and Electron Tomographic Analysis.

Organofullerene amphiphiles show diverse behaviors in water, forming vesicles, micelles, Langmuir-Blodgett films, and anisotropic nanostructures. We found that gradual in situ protonation of an organic solution of (4-heptylphenyl)5C60-K+ by water or buffer generates the corresponding protonated molecule, (4-heptylphenyl)5C60H, which self-assembles to form nano- and microspheres of organofullerene (fullerspheres) with uniform diameters ranging from 30 nm to 2.5 µm that are controlled by the preparation or pH of the buffer. By using an aqueous solution of an organic dye, inorganic nanoparticle, protein, and virus, we encapsulated these entities in the fullersphere. This approach via self-assembly is distinct from other preparations of organic core-shell particles that generally require polymerization for the construction of a robust shell. The sphere is entirely amorphous, thermally stable up to 300 °C under vacuum, and resistant to electron irradiation, and we found the unconventional utility of the sphere for electron tomographic imaging of nanoparticles and biomaterials.

Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs.

The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has remained elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actin-actinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin dimers showed contraction-dependent swinging and sliding motions in response to a global twist in the F-actin lattice. Our observation suggests that the actin-actinin complex constitutes a molecular lattice spring, which maintains the integrity of the Z-disc during the muscle contraction cycle.

B/N-Doped p-Arylenevinylene Chromophores: Synthesis, Properties, and Microcrystal Electron Crystallographic Study.

Linearly conjugated systems have long served as an archetype of conjugated materials, but suffer from two intrinsic structural problems: potential instability due to intermolecular interactions and the flexibility of the C-C bonds connecting C═C bonds. Efforts to solve these problems have included the insertion of aromatic units as a part of the conjugation and the introduction of carbon bridges to stop the bond rotation. We report here B/N-doped p-arylenevinylene chromophores synthesized through the incorporation of a cyclopenta[c][1,2]azaborole framework as a part of the conjugated system. The ring strain intrinsic to this new skeleton both flattens and rigidifies the conjugation, and the B--N+ dative bond is much easier to form than a C-C bond, which simplifies the synthetic design. The B-N dative bond also reduces the HOMO-LUMO gap, thereby causing a significant redshift of the absorption and emission compared with their all-carbon congeners while retaining high photostability and high fluorescence quantum yield in both solution and film states. A doubly B/N-doped compound showed emission peaks at 540 nm with a small Stokes shift of 20 nm and a fluorescence quantum yield of 98%. The molecules serve as excellent lipophilic fluorescent dyes for live-cell imaging, showing a higher photostability than that of commercially available BODIPY-based dyes.

TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii.

Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.

Cryo-EM structures of cardiac thin filaments reveal the 3D architecture of troponin.

Troponin is an essential component of striated muscle and it regulates the sliding of actomyosin system in a calcium-dependent manner. Despite its importance, the structure of troponin has been elusive due to its high structural heterogeneity. In this study, we analyzed the 3D structures of murine cardiac thin filaments using a cryo-electron microscope equipped with a Volta phase plate (VPP). Contrast enhancement by a VPP enabled us to reconstruct the entire repeat of the thin filament. We determined the orientation of troponin relative to F-actin and tropomyosin, and characterized the interactions between troponin and tropomyosin. This study provides a structural basis for understanding the molecular mechanism of actomyosin system.

Cryo-Electron Microscopy Methodology: Current Aspects and Future Directions.

Cryo-electron microscopy (cryo-EM) has emerged as a powerful structure determination technique. Its most prolific branch is single particle analysis (SPA), a method being used in a growing number of laboratories worldwide to determine high-resolution protein structures. Cryo-electron tomography (cryo-ET) is another powerful approach that enables visualization of protein complexes in their native cellular environment. Despite the wide-ranging success of cryo-EM, there are many methodological aspects that could be improved. Those include sample preparation, sample screening, data acquisition, image processing, and structure validation. Future developments will increase the reliability and throughput of the technique and reduce the cost and skill level barrier for its adoption.

Inner lumen proteins stabilize doublet microtubules in cilia and flagella.

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.