Cryo-electron microscopy in the fight against COVID-19-mechanism of virus entry.

Cryogenic electron microscopy (cryo-EM) and electron tomography (cryo-ET) have become a critical tool for studying viral particles. Cryo-EM has enhanced our understanding of viral assembly and replication processes at a molecular resolution. Meanwhile, in situ cryo-ET has been used to investigate how viruses attach to and invade host cells. These advances have significantly contributed to our knowledge of viral biology. Particularly, prompt elucidations of structures of the SARS-CoV-2 spike protein and its variants have directly impacted the development of vaccines and therapeutic measures. This review discusses the progress made by cryo-EM based technologies in comprehending the severe acute respiratory syndrome coronavirus-2 (SARS-Cov-2), the virus responsible for the devastating global COVID-19 pandemic in 2020 with focus on the SARS-CoV-2 spike protein and the mechanisms of the virus entry and replication.

Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 spike protein.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging shows that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Cellular cryo-electron tomography reveals dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and find that S protein recognizes integrin αvß3. Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

Direct Cryo-ET observation of platelet deformation induced by SARS-CoV-2 Spike protein.

SARS-CoV-2 is a novel coronavirus responsible for the COVID-19 pandemic. Its high pathogenicity is due to SARS-CoV-2 spike protein (S protein) contacting host-cell receptors. A critical hallmark of COVID-19 is the occurrence of coagulopathies. Here, we report the direct observation of the interactions between S protein and platelets. Live imaging showed that the S protein triggers platelets to deform dynamically, in some cases, leading to their irreversible activation. Strikingly, cellular cryo-electron tomography revealed dense decorations of S protein on the platelet surface, inducing filopodia formation. Hypothesizing that S protein binds to filopodia-inducing integrin receptors, we tested the binding to RGD motif-recognizing platelet integrins and found that S protein recognizes integrin α v ß 3 . Our results infer that the stochastic activation of platelets is due to weak interactions of S protein with integrin, which can attribute to the pathogenesis of COVID-19 and the occurrence of rare but severe coagulopathies.

H-ABC- and dystonia-causing TUBB4A mutations show distinct pathogenic effects.

Mutations in the brain-specific ß-tubulin 4A (TUBB4A) gene cause a broad spectrum of diseases, ranging from dystonia (DYT-TUBB4A) to hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC). Currently, the mechanisms of how TUBB4A variants lead to this pleiotropic manifestation remain elusive. Here, we investigated whether TUBB4A mutations causing either DYT-TUBB4A (p.R2G and p.Q424H) or H-ABC (p.R2W and p.D249N) exhibit differential effects at the molecular and cellular levels. Using live-cell imaging of disease-relevant oligodendrocytes and total internal reflection fluorescence microscopy of whole-cell lysates, we observed divergent impact on microtubule polymerization and microtubule integration, partially reflecting the observed pleiotropy. Moreover, in silico simulations demonstrated that the mutants rarely adopted a straight heterodimer conformation in contrast to wild type. In conclusion, for most of the examined variants, we deciphered potential molecular disease mechanisms that may lead to the diverse clinical manifestations and phenotype severity across and within each TUBB4A-related disease.

In situ cryo-electron tomography reveals local cellular machineries for axon branch development.

Neurons are highly polarized cells forming an intricate network of dendrites and axons. They are shaped by the dynamic reorganization of cytoskeleton components and cellular organelles. Axon branching allows the formation of new paths and increases circuit complexity. However, our understanding of branch formation is sparse due to the lack of direct in-depth observations. Using in situ cellular cryo-electron tomography on primary mouse neurons, we directly visualized the remodeling of organelles and cytoskeleton structures at axon branches. Strikingly, branched areas functioned as hotspots concentrating organelles to support dynamic activities. Unaligned actin filaments assembled at the base of premature branches accompanied by filopodia-like protrusions. Microtubules and ER comigrated into preformed branches to support outgrowth together with accumulating compact, ∼500-nm mitochondria and locally clustered ribosomes. We obtained a roadmap of events supporting the hypothesis of local protein synthesis selectively taking place at axon branches, allowing them to serve as unique control hubs for axon development and downstream neural network formation.

Lysate-based pipeline to characterize microtubule-associated proteins uncovers unique microtubule behaviours.

The microtubule cytoskeleton forms complex macromolecular assemblies with a range of microtubule-associated proteins (MAPs) that have fundamental roles in cell architecture, division and motility. Determining how an individual MAP modulates microtubule behaviour is an important step in understanding the physiological roles of various microtubule assemblies. To characterize how MAPs control microtubule properties and functions, we developed an approach allowing for medium-throughput analyses of MAPs in cell-free conditions using lysates of mammalian cells. Our pipeline allows for quantitative as well as ultrastructural analyses of microtubule-MAP assemblies. Analysing 45 bona fide and potential mammalian MAPs, we uncovered previously unknown activities that lead to distinct and unique microtubule behaviours such as microtubule coiling or hook formation, or liquid-liquid phase separation along the microtubule lattice that initiates microtubule branching. We have thus established a powerful tool for a thorough characterization of a wide range of MAPs and MAP variants, thus opening avenues for the determination of mechanisms underlying their physiological roles and pathological implications.

Mutations in the most divergent α-tubulin isotype, α8-tubulin, cause defective platelet biogenesis.

BACKGROUND: In the panel of genes commonly associated with inherited macrothrombocytopenia, an important fraction encodes key cytoskeletal proteins such as tubulin isotypes, the building blocks of microtubules. Macrothrombocytopenia-causing mutations have been identified in the TUBB1 and TUBA4A genes, emphasizing their importance in the formation of platelets and their marginal band, a unique microtubule ring-like structure that supports the platelet typical disc-shaped morphology. This raised the hypothesis that other tubulin isotypes normally expressed in platelets could play a similar role in their formation. OBJECTIVES: To assess whether tubulin isotype genes other than TUBA4A and TUBB1 could be implicated in inherited macrothrombocytopenia. METHODS: We used high throughput sequencing to screen a cohort of 448 French blood donors with mild thrombocytopenia for mutations in a panel of selected genes known or suspected to be involved in platelet biogenesis. RESULTS: We identified six distinct novel mutations in TUBA8, which encodes the most-divergent α-tubulin, as the causative determinant of macrothrombocytopenia and platelet marginal band defects. Functionally, all TUBA8 mutations were found to fully or partially inhibit the incorporation of the mutated α8-tubulin in the microtubule network. CONCLUSION: This study provides strong support for a key role of multiple tubulin genes in platelet biogenesis by discovering variants in a tubulin gene that was previously not known to be important for platelets.

Cytoskeleton and Membrane Organization at Axon Branches.

Axon branching is a critical process ensuring a high degree of interconnectivity for neural network formation. As branching occurs at sites distant from the soma, it is necessary that axons have a local system to dynamically control and regulate axonal growth. This machinery depends on the orchestration of cellular functions such as cytoskeleton, subcellular transport, energy production, protein- and membrane synthesis that are adapted for branch formation. Compared to the axon shaft, branching sites show a distinct and dynamic arrangement of cytoskeleton components, endoplasmic reticulum and mitochondria. This review discusses the regulation of axon branching in the context of cytoskeleton and membrane remodeling.

Distinct roles of α- and ß-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration.

Tubulin polyglutamylation is a post-translational modification of the microtubule cytoskeleton, which is generated by a variety of enzymes with different specificities. The "tubulin code" hypothesis predicts that modifications generated by specific enzymes selectively control microtubule functions. Our recent finding that excessive accumulation of polyglutamylation in neurons causes their degeneration and perturbs axonal transport provides an opportunity for testing this hypothesis. By developing novel mouse models and a new glutamylation-specific antibody, we demonstrate here that the glutamylases TTLL1 and TTLL7 generate unique and distinct glutamylation patterns on neuronal microtubules. We find that under physiological conditions, TTLL1 polyglutamylates α-tubulin, while TTLL7 modifies ß-tubulin. TTLL1, but not TTLL7, catalyses the excessive hyperglutamylation found in mice lacking the deglutamylase CCP1. Consequently, deletion of TTLL1, but not of TTLL7, prevents degeneration of Purkinje cells and of myelinated axons in peripheral nerves in these mice. Moreover, loss of TTLL1 leads to increased mitochondria motility in neurons, while loss of TTLL7 has no such effect. By revealing how specific patterns of tubulin glutamylation, generated by distinct enzymes, translate into specific physiological and pathological readouts, we demonstrate the relevance of the tubulin code for homeostasis.

Tubulin polyglutamylation, a regulator of microtubule functions, can cause neurodegeneration.

Neurodegenerative diseases lead to a progressive demise of neuronal functions that ultimately results in neuronal death. Besides a large variety of molecular pathways that have been linked to the degeneration of neurons, dysfunctions of the microtubule cytoskeleton are common features of many human neurodegenerative disorders. Yet, it is unclear whether microtubule dysfunctions are causative, or mere bystanders in the disease progression. A so-far little explored regulatory mechanism of the microtubule cytoskeleton, the posttranslational modifications of tubulin, emerge as candidate mechanisms involved in neuronal dysfunction, and thus, degeneration. Here we review the role of tubulin polyglutamylation, a prominent modification of neuronal microtubules. We discuss the current understanding of how polyglutamylation controls microtubule functions in healthy neurons, and how deregulation of this modification leads to neurodegeneration in mice and humans.

Purification of Tubulin with Controlled Posttranslational Modifications and Isotypes from Limited Sources by Polymerization-Depolymerization Cycles.

One important aspect of studies of the microtubule cytoskeleton is the investigation of microtubule behavior in in vitro reconstitution experiments. They allow the analysis of the intrinsic properties of microtubules, such as dynamics, and their interactions with microtubule-associated proteins (MAPs). The "tubulin code" is an emerging concept that points to different tubulin isotypes and various posttranslational modifications (PTMs) as regulators of microtubule properties and functions. To explore the molecular mechanisms of the tubulin code, it is crucial to perform in vitro reconstitution experiments using purified tubulin with specific isotypes and PTMs. To date, this was technically challenging as brain tubulin, which is widely used in in vitro experiments, harbors many PTMs and has a defined isotype composition. Hence, we developed this protocol to purify tubulin from different sources and with different isotype compositions and controlled PTMs, using the classical approach of polymerization and depolymerization cycles. Compared to existing methods based on affinity purification, this approach yields pure, polymerization-competent tubulin, as tubulin resistant to polymerization or depolymerization is discarded during the successive purification steps. We describe the purification of tubulin from cell lines, grown either in suspension or as adherent cultures, and from single mouse brains. The method first describes the generation of cell mass in both suspension and adherent settings, the lysis step, followed by the successive stages of tubulin purification by polymerization-depolymerization cycles. Our method yields tubulin that can be used in experiments addressing the impact of the tubulin code on the intrinsic properties of microtubules and microtubule interactions with associated proteins.

Genetically encoded live-cell sensor for tyrosinated microtubules.

Microtubule cytoskeleton exists in various biochemical forms in different cells due to tubulin posttranslational modifications (PTMs). Tubulin PTMs are known to affect microtubule stability, dynamics, and interaction with MAPs and motors in a specific manner, widely known as tubulin code hypothesis. At present, there exists no tool that can specifically mark tubulin PTMs in living cells, thus severely limiting our understanding of their dynamics and cellular functions. Using a yeast display library, we identified a binder against terminal tyrosine of α-tubulin, a unique PTM site. Extensive characterization validates the robustness and nonperturbing nature of our binder as tyrosination sensor, a live-cell tubulin nanobody specific towards tyrosinated microtubules. Using this sensor, we followed nocodazole-, colchicine-, and vincristine-induced depolymerization events of tyrosinated microtubules in real time and found each distinctly perturbs the microtubule polymer. Together, our work describes a novel tyrosination sensor and its potential applications to study the dynamics of microtubule and their PTM processes in living cells.

Tubulin polyglutamylation is a general traffic-control mechanism in hippocampal neurons.

Neurons are highly complex cells that heavily rely on intracellular transport to distribute a range of functionally essential cargoes within the cell. Post-translational modifications of tubulin are emerging as mechanisms for regulating microtubule functions, but their impact on neuronal transport is only marginally understood. Here, we have systematically studied the impact of post-translational polyglutamylation on axonal transport. In cultured hippocampal neurons, deletion of a single deglutamylase, CCP1 (also known as AGTPBP1), is sufficient to induce abnormal accumulation of polyglutamylation, i.e. hyperglutamylation. We next investigated how hyperglutamylation affects axonal transport of a range of functionally different neuronal cargoes: mitochondria, lysosomes, LAMP1 endosomes and BDNF vesicles. Strikingly, we found a reduced motility for all these cargoes, suggesting that polyglutamylation could act as a regulator of cargo transport in neurons. This, together with the recent discovery that hyperglutamylation induces neurodegeneration, makes it likely that perturbed neuronal trafficking could be one of the central molecular causes underlying this novel type of degeneration.This article has an associated First Person interview with the first author of the paper.

Knocking Out Multiple Genes in Cultured Primary Neurons to Study Tubulin Posttranslational Modifications.

Microtubules, as integral part of the eukaryotic cytoskeleton, exert numerous essential functions in cells. A mechanism to control these diverse functions are the posttranslational modifications of tubulin. Despite being known for decades, relatively little insight into the cellular functions of these modifications has been gained so far. The discovery of tubulin-modifying enzymes and a growing number of available knockout mice now allow working with primary cells from those mouse models to address biological functions and molecular mechanisms behind those modifications. However, a number of those mouse models show either lethality or sterility, making it difficult to impossible to obtain a sufficient number of animals for a systematic study with primary cells. Moreover, many of those modifications are controlled by several redundant enzymes, and it is often necessary to knock out several enzymes in parallel to obtain a significant change in a given tubulin modification. Here we describe a method to generate primary cells with combinatorial knockout genotypes using conditional knockout mice. The conditional alleles are converted into knockout in the cultured primary cells by transduction with a lentivirus encoding cre-recombinase. This approach has allowed us to knock out the two main brain deglutamylases in mouse primary neurons, which leads to strongly increased polyglutamylation in these cells. Our method can be applied to measure different cellular processes, such as axonal transport, for which it can be combined with the expression of different fluorescent reporters to label intracellular proteins. Using a panel of conditional knockout mice, our method can further be applied to study the functions of a variety of tubulin modifications that require simultaneous knockout of multiple genes.

Measuring the Impact of Tubulin Posttranslational Modifications on Axonal Transport.

Axonal transport is a process essential for neuronal function and survival that takes place on the cellular highways-the microtubules. It requires three major components: the microtubules that serve as tracks for the transport, the motor proteins that drive the movement, and the transported cargoes with their adaptor proteins. Axonal transport could be controlled by tubulin posttranslational modifications, which by decorating specific microtubule tracks could determine the specificity of cargo delivery inside neurons. However, it appears that the effects of tubulin modifications on transport can be rather subtle, and might thus be easily overlooked depending on which parameter of the transport process is analyzed. Here we propose an analysis paradigm that allows detecting rather subtle alterations in neuronal transport, as induced for instance by accumulation of posttranslational polyglutamylation. Analyzing mitochondria movements in axons, we found that neither the average speed nor the distance traveled were affected by hyperglutamylation, but we detected an about 50% reduction of the overall motility, suggesting that polyglutamylation controls the efficiency of mitochondria transport in axons. Our protocol can readily be expanded to the analysis of the impact of other tubulin modifications on the transport of a range of different neuronal cargoes.

Microtubule-Associated Proteins: Structuring the Cytoskeleton.

Microtubule-associated proteins (MAPs) were initially discovered as proteins that bind to and stabilize microtubules. Today, an ever-growing number of MAPs reveals a more complex picture of these proteins as organizers of the microtubule cytoskeleton that have a large variety of functions. MAPs enable microtubules to participate in a plethora of cellular processes such as the assembly of mitotic and meiotic spindles, neuronal development, and the formation of the ciliary axoneme. Although some subgroups of MAPs have been exhaustively characterized, a strikingly large number of MAPs remain barely characterized other than their interactions with microtubules. We provide a comprehensive view on the currently known MAPs in mammals. We discuss their molecular mechanisms and functions, as well as their physiological role and links to pathologies.

Purification of tubulin with controlled post-translational modifications by polymerization-depolymerization cycles.

In vitro reconstitutions of microtubule assemblies have provided essential mechanistic insights into the molecular bases of microtubule dynamics and their interactions with associated proteins. The tubulin code has emerged as a regulatory mechanism for microtubule functions, which suggests that tubulin isotypes and post-translational modifications (PTMs) play important roles in controlling microtubule functions. To investigate the tubulin code mechanism, it is essential to analyze different tubulin variants in vitro. Until now, this has been difficult, as most reconstitution experiments have used heavily post-translationally modified tubulin purified from brain tissue. Therefore, we developed a protocol that allows purification of tubulin with controlled PTMs from limited sources through cycles of polymerization and depolymerization. Although alternative protocols using affinity purification of tubulin also yield very pure tubulin, our protocol has the unique advantage of selecting for fully functional tubulin, as non-polymerizable tubulin is excluded in the successive polymerization cycles. It thus provides a novel procedure for obtaining tubulin with controlled PTMs for in vitro reconstitution experiments. We describe specific procedures for tubulin purification from adherent cells, cells grown in suspension cultures and single mouse brains. The protocol can be combined with drug treatment, transfection of cells before tubulin purification or enzymatic treatment during the purification process. The amplification of cells and their growth in spinner bottles takes ~13 d; the tubulin purification takes 6-7 h. The tubulin can be used in total internal reflection fluorescence (TIRF)-microscopy-based experiments or pelleting assays for the investigation of intrinsic properties of microtubules and their interactions with associated proteins.

Loss of the deglutamylase CCP5 perturbs multiple steps of spermatogenesis and leads to male infertility.

Sperm cells are highly specialized mammalian cells, and their biogenesis requires unique intracellular structures. Perturbation of spermatogenesis often leads to male infertility. Here, we assess the role of a post-translational modification of tubulin, glutamylation, in spermatogenesis. We show that mice lacking the tubulin deglutamylase CCP5 (also known as AGBL5) do not form functional sperm. In these mice, spermatids accumulate polyglutamylated tubulin, accompanied by the occurrence of disorganized microtubule arrays, in particular in the sperm manchette. Spermatids further fail to re-arrange their intracellular space and accumulate organelles and cytosol, while nuclei condense normally. Strikingly, spermatids lacking CCP5 show supernumerary centrioles, suggesting that glutamylation could control centriole duplication. We show that most of these observed defects are also present in mice in which CCP5 is deleted only in the male germ line, strongly suggesting that they are germ-cell autonomous. Our findings reveal that polyglutamylation is, beyond its known importance for sperm flagella, an essential regulator of several microtubule-based functions during spermatogenesis. This makes enzymes involved in glutamylation prime candidates for being genes involved in male sterility.

Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport.

Posttranslational modifications of tubulin are emerging regulators of microtubule functions. We have shown earlier that upregulated polyglutamylation is linked to rapid degeneration of Purkinje cells in mice with a mutation in the deglutamylating enzyme CCP1. How polyglutamylation leads to degeneration, whether it affects multiple neuron types, or which physiological processes it regulates in healthy neurons has remained unknown. Here, we demonstrate that excessive polyglutamylation induces neurodegeneration in a cell-autonomous manner and can occur in many parts of the central nervous system. Degeneration of selected neurons in CCP1-deficient mice can be fully rescued by simultaneous knockout of the counteracting polyglutamylase TTLL1. Excessive polyglutamylation reduces the efficiency of neuronal transport in cultured hippocampal neurons, suggesting that impaired cargo transport plays an important role in the observed degenerative phenotypes. We thus establish polyglutamylation as a cell-autonomous mechanism for neurodegeneration that might be therapeutically accessible through manipulation of the enzymes that control this posttranslational modification.