Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development.

Nuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation.

Integrated structural analysis of the human nuclear pore complex scaffold.

The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.

Chloroplast biogenesis involves spatial coordination of nuclear and organellar gene expression in Chlamydomonas.

The localization of translation can direct the polypeptide product to the proper intracellular compartment. Our results reveal translation by cytosolic ribosomes on a domain of the chloroplast envelope in the unicellular green alga Chlamydomonas (Chlamydomonas reinhardtii). We show that this envelope domain of isolated chloroplasts retains translationally active ribosomes and mRNAs encoding chloroplast proteins. This domain is aligned with localized translation by chloroplast ribosomes in the translation zone, a chloroplast compartment where photosystem subunits encoded by the plastid genome are synthesized and assembled. Roles of localized translation in directing newly synthesized subunits of photosynthesis complexes to discrete regions within the chloroplast for their assembly are suggested by differences in localization on the chloroplast of mRNAs encoding either subunit of the light-harvesting complex II or the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Transcription of the chloroplast genome is spatially coordinated with translation, as revealed by our demonstration of a subpopulation of transcriptionally active chloroplast nucleoids at the translation zone. We propose that the expression of chloroplast proteins by the nuclear-cytosolic and organellar genetic systems is organized in spatially aligned subcompartments of the cytoplasm and chloroplast to facilitate the biogenesis of the photosynthetic complexes.

Structures of the mannose-6-phosphate pathway enzyme, GlcNAc-1-phosphotransferase.

The mannose-6-phosphate (M6P) pathway is responsible for the transport of hydrolytic enzymes to lysosomes. N-acetylglucosamine-1-phosphotransferase (GNPT) catalyzes the first step of tagging these hydrolases with M6P, which when recognized by receptors in the Golgi diverts them to lysosomes. Genetic defects in the GNPT subunits, GNPTAB and GNPTG, cause the lysosomal storage diseases mucolipidosis types II and III. To better understand its function, we determined partial three-dimensional structures of the GNPT complex. The catalytic domain contains a deep cavity for binding of uridine diphosphate-N-acetylglucosamine, and the surrounding residues point to a one-step transfer mechanism. An isolated structure of the gamma subunit of GNPT reveals that it can bind to mannose-containing glycans in different configurations, suggesting that it may play a role in directing glycans into the active site. These findings may facilitate the development of therapies for lysosomal storage diseases.

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo-EM.

Cilia are thin microtubule-based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo-electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse-grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.

Tubulin lattice in cilia is in a stressed form regulated by microtubule inner proteins.

Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic resolution map of the Tetrahymena doublet microtubule by cryoelectron microscopy. The map demonstrates that the network of microtubule inner proteins weaves into the tubulin lattice and forms an inner sheath. From mass spectrometry data and de novo modeling, we identified Rib43a proteins as the filamentous microtubule inner proteins in the protofilament ribbon region. The Rib43a-tubulin interaction leads to an elongated tubulin dimer distance every 2 dimers. In addition, the tubulin lattice structure with missing microtubule inner proteins (MIPs) by sarkosyl treatment shows significant longitudinal compaction and lateral angle change between protofilaments. These results are evidence that the MIPs directly affect and stabilize the tubulin lattice. It suggests that the doublet microtubule is an intrinsically stressed filament and that this stress could be manipulated in the regulation of ciliary waveforms.

In situ structural analysis of the human nuclear pore complex.

Nuclear pore complexes are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Determining their 110-megadalton structure imposes a formidable challenge and requires in situ structural biology approaches. Of approximately 30 nucleoporins (Nups), 15 are structured and form the Y and inner-ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ~60 nm in diameter. The scaffold is decorated with transport-channel Nups that often contain phenylalanine-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here we combine cryo-electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modelling to generate, to our knowledge, the most comprehensive architectural model of the human nuclear pore complex to date. Our data suggest previously unknown protein interfaces across Y complexes and to inner-ring complex members. We show that the transport-channel Nup358 (also known as Ranbp2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport-channel Nups. We conclude that, similar to coated vesicles, several copies of the same structural building block--although compositionally identical--engage in different local sets of interactions and conformations.

Microtubule Inner Proteins: A Meshwork of Luminal Proteins Stabilizing the Doublet Microtubule.

Motile eukaryotic cilia and flagella are hair-like organelles responsible for cell motility and mucociliary clearance. Using cryo-electron tomography, it has been shown that the doublet microtubule, the cytoskeleton core of the cilia and flagella, has microtubule inner protein structures binding periodically inside its lumen. More recently, single-particle cryo-electron microscopy analyses of isolated doublet microtubules have shown that microtubule inner proteins form a meshwork inside the doublet microtubule. High-resolution structures revealed new types of interactions between the microtubule inner proteins and the tubulin lattice. In addition, they offered insights into the potential roles of microtubule inner proteins in the stabilization and assembly of the doublet microtubule. Herein, we review our new insights into microtubule inner proteins from the doublet microtubule together with the current body of literature on microtubule inner proteins.

Cryo-electron microscopy structures of ArnA, a key enzyme for polymyxin resistance, revealed unexpected oligomerizations and domain movements.

Gram-negative bacteria evade the attack of cationic antimicrobial peptides through modifying their lipid A structure in their outer membranes with 4-amino-4-deoxy-L-arabinose (Ara4N). ArnA is a crucial enzyme in the lipid A modification pathway and its deletion abolishes the polymyxin resistance of gram-negative bacteria. Previous studies by X-ray crystallography have shown that full-length ArnA forms a three-bladed propeller-shaped hexamer. Here, the structures of ArnA determined by cryo-electron microscopy (cryo-EM) reveal that ArnA exists in two 3D architectures, hexamer and tetramer. This is the first observation of a tetrameric ArnA. The hexameric cryo-EM structure is similar to previous crystal structures but shows differences in domain movements and conformational changes. We propose that ArnA oligomeric states are in a dynamic equilibrium, where the hexamer state is energetically more favorable, and its domain movements are important for cooperating with downstream enzymes in the lipid A-Ara4N modification pathway. The results provide us with new possibilities to explore inhibitors targeting ArnA.

Capturing protein communities by structural proteomics in a thermophilic eukaryote.

The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization.

DomainFit: Identification of protein domains in cryo-EM maps at intermediate resolution using AlphaFold2-predicted models.

Cryoelectron microscopy (cryo-EM) has revolutionized the structural determination of macromolecular complexes. With the paradigm shift to structure determination of highly complex endogenous macromolecular complexes ex vivo and in situ structural biology, there are an increasing number of structures of native complexes. These complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 Å remains challenging because side chains cannot be visualized reliably. Here, we present DomainFit, a program for semi-automated domain-level protein identification from cryo-EM maps, particularly at resolutions lower than 4 Å. By fitting domains from AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the domains and protein candidates forming the density. Using DomainFit, we identified two microtubule inner proteins, one of which contains a CCDC81 domain and is exclusively localized in the proximal region of the doublet microtubule in Tetrahymena thermophila.

Effect of α-tubulin acetylation on the doublet microtubule structure.

Acetylation of α-tubulin at the lysine 40 residue (αK40) by αTAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage resistant. αK40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule (DMT) in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that αK40 acetylation exerts a small-scale effect on the DMT structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between αK40 acetylation and phosphorylation in cilia.

Fourier ring correlation as a resolution criterion for super-resolution microscopy.

Optical nanoscopy techniques using localization based image reconstruction, also termed super-resolution microscopy (SRM), have become a standard tool to bypass the diffraction limit in fluorescence light microscopy. The localization precision measured for the detected fluorophores is commonly used to describe the maximal attainable resolution. However, this measure takes not all experimental factors, which impact onto the finally achieved resolution, into account. Several other methods to measure the resolution of super-resolved images were previously suggested, typically relying on intrinsic standards, such as molecular rulers, or on a priori knowledge about the specimen, e.g. its spatial frequency content. Here we show that Fourier ring correlation provides an easy-to-use, laboratory consistent standard for measuring the resolution of SRM images. We provide a freely available software tool that combines resolution measurement with image reconstruction.

Revisiting the supramolecular organization of photosystem II in Chlamydomonas reinhardtii.

Photosystem II (PSII) is a multiprotein complex that splits water and initiates electron transfer in photosynthesis. The central part of PSII, the PSII core, is surrounded by light-harvesting complex II proteins (LHCIIs). In higher plants, two or three LHCII trimers are seen on each side of the PSII core whereas only one is seen in the corresponding positions in Chlamydomonas reinhardtii, probably due to the absence of CP24, a minor monomeric LHCII. Here, we re-examined the supramolecular organization of the C. reinhardtii PSII-LHCII supercomplex by determining the effect of different solubilizing detergents. When we solubilized the thylakoid membranes with n-dodecyl-ß-D-maltoside (ß-DM) or n-dodecyl-α-D-maltoside (α-DM) and subjected them to gel filtration, we observed a clear difference in molecular mass. The α-DM-solubilized PSII-LHCII supercomplex bound twice more LHCII than the ß-DM-solubilized supercomplex and retained higher oxygen-evolving activity. Single-particle image analysis from electron micrographs of the α-DM-solubilized and negatively stained supercomplex revealed that the PSII-LHCII supercomplex had a novel supramolecular organization, with three LHCII trimers attached to each side of the core.

Regulatory mechanisms of the dynein-2 motility by post-translational modification revealed by MD simulation.

Intraflagellar transport for ciliary assembly and maintenance is driven by dynein and kinesins specific to the cilia. It has been shown that anterograde and retrograde transports run on different regions of the doublet microtubule, i.e., separate train tracks. However, little is known about the regulatory mechanism of this selective process. Since the doublet microtubule is known to display specific post-translational modifications of tubulins, i.e., "tubulin code", for molecular motor regulations, we investigated the motility of ciliary specific dynein-2 under different post-translational modification by coarse-grained molecular dynamics. Our setup allows us to simulate the landing behaviors of dynein-2 on un-modified, detyrosinated, poly-glutamylated and poly-glycylated microtubules in silico. Our study revealed that poly-glutamylation can play an inhibitory effect on dynein-2 motility. Our result indicates that poly-glutamylation of the B-tubule of the doublet microtubule can be used as an efficient means to target retrograde intraflagellar transport onto the A-tubule.

Unveiling the intriguing role of FAP20 in tubulin recruitment at the doublet microtubule inner junctions.

In this issue of Structure, Bangera et al. investigate the role of the inner junction protein FAP20 in doublet microtubule assembly. Using cryo-EM and microtubule dynamic assays, they demonstrate that FAP20 recruits free tubulins to existing microtubule lattices, shedding light on B-tubule closure during doublet microtubule formation.

DomainFit: Identification of Protein Domains in cryo-EM maps at Intermediate Resolution using AlphaFold2-predicted Models.

Cryo-electron microscopy (cryo-EM) has revolutionized our understanding of macromolecular complexes, enabling high-resolution structure determination. With the paradigm shift to in situ structural biology recently driven by the ground-breaking development of cryo-focused ion beam milling and cryo-electron tomography, there are an increasing number of structures at sub-nanometer resolution of complexes solved directly within their cellular environment. These cellular complexes often contain unidentified proteins, related to different cellular states or processes. Identifying proteins at resolutions lower than 4 Å remains challenging because the side chains cannot be visualized reliably. Here, we present DomainFit, a program for automated domain-level protein identification from cryo-EM maps at resolutions lower than 4 Å. By fitting domains from artificial intelligence-predicted models such as AlphaFold2-predicted models into cryo-EM maps, the program performs statistical analyses and attempts to identify the proteins forming the density. Using DomainFit, we identified two microtubule inner proteins, one of them, a CCDC81 domain-containing protein, is exclusively localized in the proximal region of the doublet microtubule from the ciliate Tetrahymena thermophila. The flexibility and capability of DomainFit makes it a valuable tool for analyzing in situ structures.

Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen.

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance.

CEP104/FAP256 and associated cap complex maintain stability of the ciliary tip.

Cilia are essential organelles that protrude from the cell body. Cilia are made of a microtubule-based structure called the axoneme. In most types of cilia, the ciliary tip is distinct from the rest of the cilium. Here, we used cryo-electron tomography and subtomogram averaging to obtain the structure of the ciliary tip of the ciliate Tetrahymena thermophila. We show that the microtubules at the tip are highly crosslinked with each other and stabilized by luminal proteins, plugs, and cap proteins at the plus ends. In the tip region, the central pair lacks typical projections and twists significantly. By analyzing cells lacking a ciliary tip-enriched protein CEP104/FAP256 by cryo-electron tomography and proteomics, we discovered candidates for the central pair cap complex and explained the potential functions of CEP104/FAP256. These data provide new insights into the function of the ciliary tip and the mechanisms of ciliary assembly and length regulation.

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes.

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 19,000 XLs, we identified 4,757 unique amino acid interactions among 1,143 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease.