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Gold nanowires have great potential use as interconnects in electronic, photonic, and optoelectronic devices. To date, there are various fabrication strategies for gold nanowires, each one associated with particular drawbacks as they utilize high temperatures, toxic chemicals, or expensive compounds to produce nanowires of suboptimal quality. Inspired by nanowire fabrication strategies that used higher-order biopolymer structures as molds for electroless deposition of gold, we here report a strategy for the growth of gold nanowires from seed nanoparticles within the lumen of microtubules. Luminal targeting of seed particles occurs through covalently linked Fab fragments of an antibody recognizing the acetylated lysine 40 on the luminal side of α-tubulin. Gold nanowires grown by electroless deposition within the microtubule lumen exhibit a homogeneous morphology and high aspect ratios with a mean diameter of 20 nm. Our approach is fast, simple, and inexpensive and does not require toxic chemicals or other harsh conditions.

Anterograde intraflagellar transport (IFT) trains are essential for cilia assembly and maintenance. These trains are formed of 22 IFT-A and IFT-B proteins that link structural and signaling cargos to microtubule motors for import into cilia. It remains unknown how the IFT-A/-B proteins are arranged into complexes and how these complexes polymerize into functional trains. Here we use in situ cryo-electron tomography of Chlamydomonas reinhardtii cilia and AlphaFold2 protein structure predictions to generate a molecular model of the entire anterograde train. We show how the conformations of both IFT-A and IFT-B are dependent on lateral interactions with neighboring repeats, suggesting that polymerization is required to cooperatively stabilize the complexes. Following three-dimensional classification, we reveal how IFT-B extends two flexible tethers to maintain a connection with IFT-A that can withstand the mechanical stresses present in actively beating cilia. Overall, our findings provide a framework for understanding the fundamental processes that govern cilia assembly.

Anterograde intraflagellar transport trains are essential for cilia assembly and maintenance. These trains are formed of 22 IFTA and IFTB proteins that link structural and signalling cargoes to microtubule motors for import into cilia. It remains unknown how the IFTA/B proteins are arranged into complexes and how these complexes polymerise into functional trains. Here, we use in situ cryo-electron tomography and Alphafold2 protein structure predictions to generate the first molecular model of the entire anterograde train. We show how the conformation of both IFTA and IFTB is dependent on lateral interactions with neighbouring repeats, suggesting that polymerization is required to cooperatively stabilize the complexes. The retrograde dynein motor binding site is a composite surface involving multiple IFTB repeats, ensuring that dynein can only form a strong interaction with IFTB upon train assembly. Finally, we reveal how IFTB extends two flexible tethers to maintain a connection with IFTA that can withstand the mechanical stresses present in actively beating cilia. Overall, our findings provide a framework for understanding the fundamental processes that are involved in cilia assembly.

Cells need to be able to sense different types of signals, such as chemical and mechanical stimuli, from the extracellular environment in order to properly function. Most eukaryotic cells sense these signals in part through a specialized hair-like organelle, the cilium, that extends from the cell body as a sort of antenna. The signaling and sensory functions of cilia are fundamental during the early stages of embryonic development, when cilia coordinate the establishment of the internal left-right asymmetry that is typical of the vertebrate body. Later, cilia continue to be required for the correct development and function of specific tissues and organs, such as the brain, heart, kidney, liver, and pancreas. Sensory cilia allow us to sense the environment that surrounds us; for instance, we see as a result of the connecting cilia of photoreceptors in our retina, we smell through the sensory cilia at the tips of our olfactory neurons, and we hear thanks to the kinocilia of our sensory hair cells. Motile cilia, which themselves have sensory functions, also work as propeller-like extensions that allow us to breathe because they keep our lungs clean, to reproduce because they propel sperm cells, and even to properly reason because they contribute to the flow of cerebrospinal fluid in our brain ventricles. Not surprisingly, defects in the assembly and function of these tiny organelles result in devastating pathologies, collectively known as ciliopathies (Box 1). Thus, the proper function of cilia is fundamental for human health.

CRISPR/Cas genome engineering in the unicellular green algal modelChlamydomonas reinhardtiihas until now only been applied to targeted gene disruption, whereas scar-less knock-in transgenesis has generally been considered infeasible. We have developed highly efficient homology-directed knock-in mutagenesis in cell-walled strains ofChlamydomonas. Our method allows scarless integration of fusion tags and sequence modifications of near arbitrary proteins without need for a preceding mutant line.

Cilia or eukaryotic flagella are microtubule-based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavorable to diffusive transport of most components required for their assembly and maintenance. Instead, a system of intraflagellar transport (IFT) trains moves cargo rapidly up and down the cilium (Figure 1A).

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß′ COPI coatomer subunits and demonstrate an accumulation of ‘coat-less’ vesicles that fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation., eLife digest Most human cells have at least one small hair-like structure on their surface called a cilium. These structures can act as antennae and allow the cell to sense signals from the rest of the body. To do this, they contain proteins that differ from the rest of the cell. The content of cilia depends on regulated delivery of these proteins in and out of cilia by a process called the intraflagellar transport or IFT, which involves a large complex made of several proteins. This complex shuttles the cargo proteins back and forth between the base and the tip of the cilia. However, ciliary proteins are not produced in the cilia; instead, they are made in a different part of the cell and then they are transported to the ciliary base. At the point where they enter the cilia, they were thought to bind to the assembling IFT ‘trains’ and be transported across the ciliary gate to the positions where they are needed in cilia. One of the components of the IFT machinery is a protein called WDR35, also known as IFT121. If the gene that codes for this protein is faulty or missing, it results in severe disorders in both humans and mice including a range of potentially lethal skeletal dysplasias. Interestingly, without WDR35, cells cannot build functional cilia. The absence of this protein not only disrupts IFT, stopping certain ciliary proteins and their associated membranes from entering cilia; it also causes a ‘traffic jam’ with a pile-up of transport intermediates from the place in cell where they are made to the cilia. It is unclear why a mutation in one of the components of the IFT would have this effect, raising the question of whether WDR35, or IFTs a whole, has another role in bringing the cargo proteins into the cilia. To understand this phenomenon, Quidwai et al. analysed the structure of WDR35 and other IFT proteins and found that they are very similar to a protein complex called COPI, which is involved in transporting membrane proteins around the cell. When certain proteins are newly made, they are stored in small lipid bubbles – called vesicles – that then selectively move to where the proteins are needed. COPI coats these vesicles, helping them get to where they need to go in a process called vesicular transport. Quidwai et al. found that WDR35 and other IFT proteins are able to bind to specific types of lipid molecules, suggesting that they might be assisting in a form of vesicle transport too. Indeed, when mouse cells grown in the lab were genetically engineered so they could not produce WDR35, coatless vesicles accumulated around the base of the cilia. Adding back WDR35 to these mutant cells rescued these defects in vesicle transport to cilia as well as allowed functional cilia to be formed. These results provide evidence that WDR35, likely with other IFT proteins, acts as a COPI-like complex to deliver proteins to growing cilia. Further research will investigate the composition of these vesicles that transport proteins to cilia, and help pinpoint where they originate. Quidwai et al.’s findings not only shed light on how different genetic mutations found in patients with cilia dysfunction affect different steps of transporting proteins to and within cilia. They also increase our understanding of the cellular roadmap by which cells shuttle building blocks around in order to assemble these important ‘antennae’.

The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. Here, we use in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base. We combine this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which IFT-A, then dynein-1b, and finally kinesin-2 sequentially bind before entry into the cilium.One Sentence SummaryNative molecular structure of the ciliary transition zone and hierarchical order of IFT assembly visualized within Chlamydomonas cells.

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Here, we performed two-color imaging of fluorescent protein-tagged IFT components, which indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC–GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B and IFT dynein typically remain associated at the tip and segments of the anterograde trains convert directly into retrograde trains. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit, preventing the build-up of IFT material in flagella.

The intraflagellar transport (IFT) system is a remarkable molecular machine used by cells to assemble and maintain the cilium, a long organelle extending from eukaryotic cells that gives rise to motility, sensing and signaling. IFT plays a critical role in building the cilium by shuttling structural components and signaling receptors between the ciliary base and tip. To provide effective transport, IFT-A and IFT-B adaptor protein complexes assemble into highly repetitive polymers, called IFT trains, that are powered by the motors kinesin-2 and IFT-dynein to move bidirectionally along the microtubules. This dynamic system must be precisely regulated to shuttle different cargo proteins between the ciliary tip and base. In this Cell Science at a Glance article and the accompanying poster, we discuss the current structural and mechanistic understanding of IFT trains and how they function as macromolecular machines to assemble the structure of the cilium.

Cilia and flagella are microtubule doublet based organelles found across the eukaryotic tree of life. Their very high aspect ratio and crowded interior are unfavourable to diffusive transport for their assembly and maintenance. Instead, a highly dynamic system of intraflagellar transport (IFT) trains moves rapidly up and down the cilium. However, the mechanism of how these trains turn around upon reaching the ciliary tip has remained elusive. It has been hypothesized that there exists a dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate this conversion. In this work, we use physical and chemical methods to manipulate IFT in the cilia of the unicellular green alga Chlamydomonas reinhardtii to show that no such stationary tip-machinery is required for IFT turnaround. Instead, we demonstrate that the conversion from anterograde to retrograde IFT trains is a calcium independent intrinsic ability of the IFT system.

Glycylation regulates axonemal dyneins Physiological functions of the microtubule cytoskeleton are expected to be regulated by a variety of posttranslational tubulin modifications. For instance, tubulin glycylation is almost exclusively found in cilia and flagella, but its role in the function of these organelles remains unclear. Gadadhar et al. now demonstrate in mice that glycylation, although nonessential for the formation of cilia and flagella, coordinates the beat waveform of sperm flagella. This activity is a prerequisite for progressive sperm swimming and thus for male fertility. At the ultrastructural level, lack of glycylation perturbed the distribution of axonemal dynein conformations, which may explain the observed defects in flagellar beat. Science , this issue p. eabd4914

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia and across the diffusion barrier is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. InWdr35mouse mutants, the IFT-A peripheral components are degraded and core components accumulate at the transition zone. We reveal deep sequence homology and structural similarity of WDR35 and other IFT-As to the coatomer COPI proteinsaandβ′, and demonstrate an accumulation of ‘coat-less’ vesicles which fail to fuse withWdr35mutant cilia. Our data provides the firstin situevidence of a novel coatomer function for WDR35 likely with other IFT-A proteins in delivering ciliary membrane cargo from the Golgi necessary for cilia elongation.

Primary cilia are microtubule-based organelles involved in key signaling and sensing processes in eukaryotic cells. Unlike motile cilia, which have been thoroughly studied, the structure and the composition of primary cilia remain largely unexplored despite their fundamental role in development and homeostasis. They have for long been falsely regarded as simplified versions of motile cilia because they lack distinctive elements such as dynein arms, radial spokes, and central pair complex. However, revealing the detailed molecular composition and 3D structure of primary cilia is necessary in order to understand the mechanisms that govern their functions. Such structural investigations are so far being precluded by the challenging preparation of primary cilia for cryo-electron microscopy. Here, we developed an enabling method for investigating the structure of primary cilia at molecular resolution by cryo-electron tomography. We show that the well-known “9+0” arrangement of microtubule doublets is present only at the base of the primary cilium. A few microns away from the base the ciliary architecture changes into an unstructured bundle of EB1-decorated microtubule singlets and some actin filaments. Our results suggest the existence of a previously unobserved crosstalk between actin filaments and microtubules in the primary cilium. Our work provides unprecedented insights into the molecular structure of primary cilia and a general framework for uncovering their molecular composition and function in health and disease. This opens up new possibilities to study aspects of this important organelle that have so far been out of reach.

Movement of cargos along microtubules plays key roles in diverse cellular processes, from signalling to mitosis. In cilia, rapid movement of ciliary components along the microtubules to and from the assembly site is essential for the assembly and disassembly of the structure itself1. This bidirectional transport, known as intraflagellar transport (IFT)2, is driven by the anterograde motor kinesin-23 and the retrograde motor dynein-1b (dynein-2 in mammals)4,5. However, to drive retrograde transport, dynein-1b must first be delivered to the ciliary tip by anterograde IFT6. Although, the presence of opposing motors in bidirectional transport processes often leads to periodic stalling and slowing of cargos7, IFT is highly processive1,2,8. Using cryo-electron tomography, we show that a tug-of-war between kinesin-2 and dynein-1b is prevented by loading dynein-1b onto anterograde IFT trains in an autoinhibited form and by positioning it away from the microtubule track to prevent binding. Once at the ciliary tip, dynein-1b must transition into an active form and engage microtubules to power retrograde trains. These findings provide a striking example of how coordinated structural changes mediate the behaviour of complex cellular machinery.

Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. Many mechanistic details about how starved cells regulate the process of protein synthesis are still unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through filament assembly is an efficient mechanism that allows yeast cells to adapt to fluctuating environments., Summary: Filament formation by eIF2B is an adaptive mechanism that allows for efficient shutdown of translation in starved yeast cells.

Primary cilia are microtubule-based organelles that are important for signaling and sensing in eukaryotic cells. Unlike the thoroughly studied motile cilia, the three-dimensional architecture and molecular composition of primary cilia are largely unexplored. Yet, studying these aspects is necessary to understand how primary cilia function in health and disease. We developed an enabling method for investigating the structure of primary cilia isolated from MDCK-II cells at molecular resolution by cryo-electron tomography. We show that the textbook ‘9 + 0’ arrangement of microtubule doublets is only present at the primary cilium base. A few microns out, the architecture changes into an unstructured bundle of EB1-decorated microtubules and actin filaments, putting an end to a long debate on the presence or absence of actin filaments in primary cilia. Our work provides a plethora of insights into the molecular structure of primary cilia and offers a methodological framework to study these important organelles. Primary cilia are isolated from monolayers of mammalian cells in culture for cryo-ET analyses, revealing new features as well as how they differ from motile cilia.

Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, extend laterally, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that microridges are formed of F-actin networks and require the function of the Arp2/3 complex for their maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.

Multiple approaches to use deep neural networks for image restoration have recently been proposed. Training such networks requires well registered pairs of high and low-quality images. While this is easily achievable for many imaging modalities, e.g., fluorescence light microscopy, for others it is not. Here we summarize on a number of recent developments in the fast-paced field of Content-Aware Image Restoration (CARE), in particular, and the associated area of neural network training, more in general. We then give specific examples how electron microscopy data can benefit from these new technologies.

In situ cryo-electron tomography (cryo-ET) and subtomogram averaging are powerful tools, able to provide 3D structures of biological samples at sub-nanometer resolution, while preserving information about cellular context and higher-order assembly. Best results are typically achieved, when applied to highly repetitive structures, such as viruses. Other typical examples are protein complexes that decorate long stretches along ciliary microtubules at stereotypical and precise repeats, such as axonemal dyneins. For such cases, a plethora of subtomogram averaging protocols exist. In this chapter, we show how we use cryo-ET and subtomogram averaging to study the architecture of the intraflagellar transport (IFT) machinery, a more challenging target that appears only in low copy numbers per tomogram. In the IFT trains, repeating units of IFT adaptor proteins engage two oppositely directed molecular motors to quickly shuttle ciliary building blocks and other proteins to the tip of the cilium and/or back to the base. This dynamic and sporadic nature of IFT trains poses challenges for determining the localization or precise orientation of the particles to be averaged. Solutions to these problems are described in this chapter.

Yeast essential enzymes are able to assemble and form membrane-less compartments in the cytoplasm during stress conditions (Narayanaswamy et al., 2009). These microcompartments form rapidly under ATP-depletion upon cellular regulation of pH and molecular crowding (Munder et al., 2016). So far, the behavior of most of these enzymes has been characterized by live imaging using fluorescence microscopy. The ultrastructure of only a few enzymes in the assembled form has been solved, thus revealing their capacity of forming highly ordered self-assemblies (Barry et al., 2014; Petrovska et al., 2014; Prouteau et al., 2017). Here we show how we used correlative light and electron microscopy (CLEM) and transmission electron tomography (ET) to reveal the in situ arrangement adopted by the yeast enzyme eIF2B (eukaryotic translation initiation 2B) in its compartmentalized form upon energy depletion.

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.

Cytoplasmic dyneins transport cellular components from the periphery toward the center of the cell. By moving cargoes along microtubules, dyneins ensure proper cell division, regulate exchange of materials between organelles, and contribute to the internal organization of eukaryotic cells. Two recent studies show that, upon dimerization, cytoplasmic dyneins intrinsically adopt an autoinhibited configuration that can be relieved by other factors to precisely control motor activity and regulate dynein-based transport.

Yeast essential enzymes are able to assemble and form membrane-less compartments in the cytoplasm during stress conditions ( Narayanaswamy et al., 2009 ). These microcompartments form rapidly under ATP-depletion upon cellular regulation of pH and molecular crowding ( Munder et al., 2016 ). So far, the behavior of most of these enzymes has been characterized by live imaging using fluorescence microscopy. The ultrastructure of only a few enzymes in the assembled form has been solved, thus revealing their capacity of forming highly ordered self-assemblies ( Barry et al., 2014 ; Petrovska et al., 2014 ; Prouteau et al., 2017 ). Here we show how we used correlative light and electron microscopy (CLEM) and transmission electron tomography (ET) to reveal the in situ arrangement adopted by the yeast enzyme eIF2B (eukaryotic translation initiation 2B) in its compartmentalized form upon energy depletion.

A series of recent hardware and software developments have transformed cryo-electron microscopy (cryoEM) from a niche tool into a method that has become indispensable in structural and functional biology. Samples that are rapidly frozen are encased in a near-native state inside a layer of amorphous ice, and then imaged in an electron microscope cooled to cryogenic temperatures. Despite being conceptually simple, cryoEM owns its success to a plethora of technological developments from numerous research groups. Here, we review the key technologies that have made this astonishing transformation possible and highlight recent trends with a focus on cryo-electron tomography. Additionally, we discuss how correlated microscopy is an exciting and perpendicular development route forward in this already rapidly growing field. We specifically discuss microscopy techniques that allow to complement time-dependent information of dynamic processes to the unique high resolution obtained in cryoEM.

In situ cryo-electron tomography (cryo-ET) and subtomogram averaging are powerful tools, able to provide 3D structures of biological samples at sub-nanometer resolution, while preserving information about cellular context and higher-order assembly. Best results are typically achieved, when applied to highly repetitive structures, such as viruses. Other typical examples are protein complexes that decorate long stretches along ciliary microtubules at stereotypical and precise repeats, such as axonemal dyneins. For such cases, a plethora of subtomogram averaging protocols exist. In this chapter, we show how we use cryo-ET and subtomogram averaging to study the architecture of the intraflagellar transport (IFT) machinery, a more challenging target that appears only in low copy numbers per tomogram. In the IFT trains, repeating units of IFT adaptor proteins engage two oppositely directed molecular motors to quickly shuttle ciliary building blocks and other proteins to the tip of the cilium and/or back to the base. This dynamic and sporadic nature of IFT trains poses challenges for determining the localization or precise orientation of the particles to be averaged. Solutions to these problems are described in this chapter.

Yeast cells, when exposed to stress, can enter a protective state in which cell division, growth and metabolism are downregulated. They remain viable in this state until nutrients become available again. How cells enter this protective survival state and what happens at a cellular and subcellular level is largely unknown. In this study, we used electron tomography to investigate the stress-induced ultrastructural changes in the cytoplasm of yeast cells. After ATP depletion, we observed a significant cytosolic compaction and an extensive cytoplasmic reorganization, as well as the emergence of distinct membrane-bound and membrane-less organelles. By using correlative light and electron microscopy (CLEM), we further demonstrate that one of these membrane-less organelles is generated by the reversible polymerization into large bundles of filaments of the eukaryotic translation initiation factor 2B (eIF2B), an essential enzyme in the initiation of protein synthesis. The changes we observe are part of a stress-induced survival strategy, allowing yeast cells to save energy, protect proteins from degradation, and inhibit protein functionality by forming assemblies of said proteins.

Apical projections are integral functional units of epithelial cells. Microvilli and stereocilia are cylindrical apical projections that are formed of bundled actin. Microridges on the other hand, grow laterally long, forming labyrinthine patterns on surfaces of various kinds of squamous epithelial cells. So far, the structural organization and functions of microridges have remained elusive. We have analyzed microridges on zebrafish epidermal cells using confocal and electron microscopy methods including electron tomography, to show that a microridge is formed of a network of F-actin and requires the function of the Arp2/3 complex for its maintenance. During development, microridges begin as F-actin punctae showing signatures of branching and requiring an active Arp2/3 complex. Using inhibitors of actin polymerization and the Arp2/3 complex, we show that microridges organize the surface glycan layer. Our analyses have unraveled the F-actin organization supporting the most abundant and evolutionarily conserved apical projection, which functions in glycan organization.

Multiple approaches to use deep learning for image restoration have recently been proposed. Training such approaches requires well registered pairs of high and low quality images. While this is easily achievable for many imaging modalities, e.g. fluorescence light microscopy, for others it is not. Cryo-transmission electron microscopy (cryo-TEM) could profoundly benefit from improved denoising methods, unfortunately it is one of the latter. Here we show how recent advances in network training for image restoration tasks, i.e. denoising, can be applied to cryo-TEM data. We describe our proposed method and show how it can be applied to single cryo-TEM projections and whole cryo-tomographic image volumes. Our proposed restoration method dramatically increases contrast in cryo-TEM images, which improves the interpretability of the acquired data. Furthermore we show that automated downstream processing on restored image data, demonstrated on a dense segmentation task, leads to improved results., Comment: This work has been submitted to the IEEE for possible publication. Copyright may be transferred without notice, after which this version may no longer be accessible. This version fixed flipped graph labels in Figure 5

Molecular motors propel cellular components at velocities up to microns per second with nanometer precision. Imaging techniques combining high temporal and spatial resolution are therefore indispensable to understand the cellular mechanics at the molecular level. For example, intraflagellar transport (IFT) trains constantly shuttle ciliary components between the base and tip of the eukaryotic cilium. 3-D electron microscopy has revealed IFT train morphology and position, but was unable to correlate these features with the direction of train movement. Here, we present the methodology required to combine live-cell imaging at millisecond frame rates with electron tomography. Using this approach, we were able to correlate the direction of movement of every IFT train in a flagellum with its morphology and microtubule track. The method is ready to be further adapted for other experimental systems, including studies of single molecule dynamics.

Stay on the right track Cilia contain a well-ordered array of microtubule doublets along their length. A longstanding question in cilium structure and function is why the microtubule arrangement in cilia is so complex. Stepanek and Pigino developed a time-resolved correlative fluorescence and three-dimensional electron microscopy method to show that the doublets provide directionality to intraflagellar transport. One microtubule in the pair moves cargoes up to the ciliary tip. Meanwhile, the other microtubule moves cargoes back to the cell body. These results explain why the axoneme is built out of microtubule doublets and suggest a mechanistic picture of how the logistics of bidirectional intraflagellar transport are regulated. Science , this issue p. 721

Although eukaryotic flagella and cilia all share the basic 9+2 microtubule-organization of their internal axonemes, and are capable of generating bending-motion, the waveforms, amplitudes, and velocities of the bending-motions are quite diverse. To explore the structural basis of this functional diversity of flagella and cilia, we here compare the axonemal structure of three different organisms with widely divergent bending-motions by electron cryo-tomography. We reconstruct the 3D structure of the axoneme of Tetrahymena cilia, and compare it with the axoneme of the flagellum of sea urchin sperm, as well as with the axoneme of Chlamydomonas flagella, which we analyzed previously. This comparative structural analysis defines the diversity of molecular architectures in these organisms, and forms the basis for future correlation with their different bending-motions.

The radial spoke (RS) is a complex of at least 23 proteins that works as a mechanochemical transducer between the central‐pair apparatus and the peripheral microtubule doublets in eukaryotic flagella and motile cilia. The RS contributes to the regulation of the activity of dynein motors, and thus to flagellar motility. Despite numerous biochemical, physiological and structural studies, the mechanism of the function of the radial spoke remains unclear. Detailed knowledge of the 3D structure of the RS protein complex is needed in order to understand how RS regulates dynein activity. Here we review the most important findings on the structure of the RS, including results of our recent cryo‐electron tomographic analysis of the RS protein complex.

We present a strategy for the alignment of dual-axis tomographic series, based on reference points and simultaneous alignment of both series. Each series is first aligned individually, an affine transformation is determined to bring the two series in a unique reference system, and all experimental coordinates are combined in a single system of equations. In case of severe shrinkage, a global and a local refinement of the orientation parameters are performed to correct all minors misalignments. The strategy is illustrated on tomographic experiments performed on sections from plastic-embedded biological samples. The efficiency in correcting the misalignment of gold particles and in improving the quality of the reconstruction is documented both visually and quantitatively. In our approach every region of the tomogram is associated with its own orientation parameters and can be eventually reconstructed with the preferred algorithm. This is convenient in the computation of 3D averages of equivalent structures. A simulation experiment is presented to show that the performances of this approach are superior to those of the method of rotation in direct space.

Ultrastructural study of Chlamydomonas cilia shows that anterograde IFT particles form trains that are long and narrow, while retrograde IFT form short, compact particle trains., Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.

We are presenting a program for interactive segmentation of tomographic maps, based on objective criteria so as to yield reproducible results. The strategy starts with the automatic segmentation of the entire volume with the watershed algorithm in 3D. The watershed regions are clustered successively by supervised classification, allowing the segmentation of known organelles, such as membranes, vesicles and microtubules. These organelles are processed with topological models and input parameters manually derived from the tomograms. After known organelles are extracted from the volume, all other watershed regions can be organized into homogeneous assemblies on the basis of their densities. To complete the process, all voxels in the volume are assigned either to the background or individual structures, which can then be extracted for visualization with any rendering technique. The user interface of the program is written in Java, and computational routines are written in C. For some operations, involving the visualization of the tomogram, we refer to existing software, either open or commercial. While the program runs, a history file is created, that allows all parameters and other data to be saved for the purposes of comparison or exchange. Initially, the program was developed for the segmentation of synapses, and organelles belonging to these structures have thus far been the principal targets modeled with JUST. Since each organelle is clustered independently from the rest of the volume, however, the program can accommodate new models of different organelles as well as tomograms of other types of preparations of tissue, such as cytoskeletal components in vitreous ice.