RESUMO
Bidirectional transport in cilia is carried out by polymers of the IFTA and IFTB protein complexes, called anterograde and retrograde intraflagellar transport (IFT) trains. Anterograde trains deliver cargoes from the cell to the cilium tip, then convert into retrograde trains for cargo export. We set out to understand how the IFT complexes can perform these two directly opposing roles before and after conversion. We use cryoelectron tomography and in situ cross-linking mass spectrometry to determine the structure of retrograde IFT trains and compare it with the known structure of anterograde trains. The retrograde train is a 2-fold symmetric polymer organized around a central thread of IFTA complexes. We conclude that anterograde-to-retrograde remodeling involves global rearrangements of the IFTA/B complexes and requires complete disassembly of the anterograde train. Finally, we describe how conformational changes to cargo-binding sites facilitate unidirectional cargo transport in a bidirectional system.
Assuntos
Cílios , Microscopia Crioeletrônica , Flagelos , Flagelos/metabolismo , Flagelos/ultraestrutura , Cílios/metabolismo , Transporte Biológico , Chlamydomonas reinhardtii/metabolismo , Modelos Moleculares , Transporte ProteicoRESUMO
Cilia are ubiquitous microtubule-based eukaryotic organelles that project from the cell to generate motility or function in cellular signaling. Motile cilia or flagella contain axonemal dynein motors and other complexes to achieve beating. Primary cilia are immotile and act as signaling hubs, with receptors shuttling between the cytoplasm and ciliary compartment. In both cilia types, an intraflagellar transport (IFT) system powered by unique kinesin and dynein motors functions to deliver the molecules required to build cilia and maintain their functions. Cryo-electron tomography has helped to reveal the organization of protein complex arrangement along the axoneme and the structure of anterograde IFT trains as well as the structure of primary cilia. Only recently, single-particle analysis (SPA) cryo-electron microscopy has provided molecular details of the protein organization of ciliary components, helping us to understand how they bind to microtubule doublets and how mechanical force propagated by dynein conformational changes is converted into ciliary beating. Here we highlight recent structural advances that are leading to greater knowledge of ciliary function.
Assuntos
Dineínas do Axonema , Cílios , Dineínas do Axonema/genética , Dineínas do Axonema/metabolismo , Transporte Biológico/fisiologia , Biologia , Cílios/metabolismo , Microscopia Crioeletrônica , Flagelos/metabolismo , CinesinasRESUMO
Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.
Assuntos
Proteínas de Transporte/metabolismo , Cílios/fisiologia , Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Plantas/metabolismo , Axonema/metabolismo , Proteínas de Transporte/química , Chlamydomonas/fisiologia , Cílios/ultraestrutura , Flagelos/fisiologia , Flagelos/ultraestrutura , Imunofluorescência , Proteínas Associadas aos Microtúbulos/química , Complexos Multiproteicos/ultraestrutura , Conformação Proteica , Transporte Proteico , Relação Estrutura-Atividade , Tetrahymena thermophila/fisiologiaRESUMO
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.
Assuntos
Cílios , Dineínas , Transporte Biológico , Proteínas de Transporte/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Flagelos/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Microtúbulos/metabolismo , Transporte ProteicoRESUMO
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.
Assuntos
Chlamydomonas , Dineínas , Transporte Biológico , Chlamydomonas/metabolismo , Cílios/metabolismo , Dineínas/genética , Dineínas/metabolismo , Flagelos/metabolismo , Transporte ProteicoRESUMO
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.
Assuntos
Nanopartículas , Nanofios , Ouro/química , Microtúbulos/química , Nanofios/química , Tubulina (Proteína)RESUMO
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.
Assuntos
Dineínas do Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Pulmão/metabolismo , Sequência de Bases , Cílios/ultraestrutura , Dineínas/ultraestrutura , Homozigoto , Humanos , Síndrome de Kartagener/genética , Mutação/genética , Transporte ProteicoRESUMO
We demonstrate absolute quantitative mass density mapping in three dimensions of frozen-hydrated biological matter with an isotropic resolution of 180 nm. As model for a biological system we use Chlamydomonas cells in buffer solution confined in a microcapillary. We use ptychographic X-ray computed tomography to image the entire specimen, including the 18 µm-diameter capillary, thereby providing directly an absolute mass density measurement of biological matter with an uncertainty of about 6%. The resulting maps have sufficient contrast to distinguish cells from the surrounding ice and several organelles of different densities inside the cells. Organelles are identified by comparison with a stained, resin-embedded specimen, which can be compared with established transmission electron microscopy results. For some identified organelles, the knowledge of their elemental composition reduces the uncertainty of their mass density measurement down to 1% with values consistent with previous measurements of dry weight concentrations in thin cellular sections by scanning transmission electron microscopy. With prospects of improving the spatial resolution in the near future, we expect that the capability of non-destructive three-dimensional mapping of mass density in biological samples close to their native state becomes a valuable method for measuring the packing of organic matter on the nanoscale.
Assuntos
Chlamydomonas reinhardtii/fisiologia , Chlamydomonas reinhardtii/ultraestrutura , Imageamento Tridimensional/métodos , Organelas/ultraestrutura , Tomografia Computadorizada por Raios X/métodos , CongelamentoRESUMO
Zernike phase contrast microscopy is a well-established method for imaging specimens with low absorption contrast. It has been successfully implemented in full-field microscopy using visible light and X-rays. In microscopy Cowley's reciprocity principle connects scanning and full-field imaging. Even though the reciprocity in Zernike phase contrast has been discussed by several authors over the past thirty years, only recently it was experimentally verified using scanning X-ray microscopy. In this paper, we investigate the image and contrast formation in scanning Zernike phase contrast microscopy with a particular and detailed focus on the origin of imaging artifacts that are typically associated with Zernike phase contrast. We demonstrate experimentally with X-rays the effect of the phase mask design on the contrast and halo artifacts and present an optimized design of the phase mask with respect to photon efficiency and artifact reduction. Similarly, due to the principle of reciprocity the observations and conclusions of this work have direct applicability to Zernike phase contrast in full-field microscopy as well.
RESUMO
The Tubulin Code revolutionizes our understanding of microtubule dynamics and functions, proposing a nuanced system governed by tubulin isotypes, posttranslational modifications (PTMs) and microtubule-associated proteins (MAPs). Tubulin isotypes, diverse across species, contribute structural complexity, and are thought to influence microtubule functions. PTMs encode dynamic information on microtubules, which are read by several microtubule interacting proteins and impact on cellular processes. Here we discuss recent technological and methodological advances, such as in genome engineering, live cell imaging, expansion microscopy, and cryo-electron microscopy that reveal new elements and levels of complexity of the tubulin code, including new modifying enzymes and nanopatterns of PTMs on individual microtubules. The Tubulin Code's exploration holds transformative potential, guiding therapeutic strategies and illuminating connections to diseases like cancer and neurodegenerative disorders, underscoring its relevance in decoding fundamental cellular language.
Assuntos
Processamento de Proteína Pós-Traducional , Tubulina (Proteína) , Humanos , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Animais , Microtúbulos/metabolismo , Microtúbulos/químicaRESUMO
CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line. We further empower this genetic-editing approach by efficient crossing and highly robust screening protocols. For complete details on the use and execution of this protocol, please refer to Nievergelt et al. (2023).1.
Assuntos
Sistemas CRISPR-Cas , Chlamydomonas reinhardtii , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Mutagênese , GenomaRESUMO
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.
Assuntos
Chlamydomonas reinhardtii , Flagelos , Flagelos/metabolismo , Estrutura Molecular , Transporte Biológico , Cílios/metabolismo , Proteínas de Transporte/metabolismoRESUMO
Expansion microscopy is an innovative method that enables super-resolution imaging of biological materials using a simple confocal microscope. The principle of this method relies on the physical isotropic expansion of a biological specimen cross-linked to a swellable polymer, stained with antibodies, and imaged. Since its first development, several improved versions of expansion microscopy and adaptations for different types of samples have been produced. Here, we show the application of ultrastructure expansion microscopy (U-ExM) to investigate the 3D organization of the green algae Chlamydomonas reinhardtii cellular ultrastructure, with a particular emphasis on the different types of sample fixation that can be used, as well as compatible staining procedures including membranes. Graphical overview.
RESUMO
CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line. We also present methods for high-throughput crossing of transformants and a custom quantitative PCR (qPCR)-based high-throughput screening of mutants as well as meiotic progeny. We demonstrate how to use this pipeline to facilitate the generation of mutant lines without residual selectable markers by co-targeted insertion. Finally, we describe how insertional cassettes can be erroneously mutated during insertion and suggest strategies to select for lines that are modified as designed.
Assuntos
Chlamydomonas reinhardtii , Chlamydomonas , Sistemas CRISPR-Cas , Cultura , EletroporaçãoRESUMO
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.
Assuntos
Chlamydomonas/ultraestrutura , Cílios/ultraestrutura , Flagelos/ultraestrutura , Ouriços-do-Mar/ultraestrutura , Tetrahymena/ultraestrutura , Animais , Axonema/ultraestrutura , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Processamento de Imagem Assistida por ComputadorRESUMO
Intraflagellar transport (IFT) is a conserved process of cargo transport in cilia that is essential for development and homeostasis in organisms ranging from algae to vertebrates. In humans, variants in genes encoding subunits of the cargo-adapting IFT-A and IFT-B protein complexes are a common cause of genetic diseases known as ciliopathies. While recent progress has been made in determining the atomic structure of IFT-B, little is known of the structural biology of IFT-A. Here, we combined chemical cross-linking mass spectrometry and cryo-electron tomography with AlphaFold2-based prediction of both protein structures and interaction interfaces to model the overall architecture of the monomeric six-subunit IFT-A complex, as well as its polymeric assembly within cilia. We define monomer-monomer contacts and membrane-associated regions available for association with transported cargo, and we also use this model to provide insights into the pleiotropic nature of human ciliopathy-associated genetic variants in genes encoding IFT-A subunits. Our work demonstrates the power of integration of experimental and computational strategies both for multi-protein structure determination and for understanding the etiology of human genetic disease.
Assuntos
Cílios , Ciliopatias , Humanos , Animais , Transporte Biológico , Tomografia com Microscopia Eletrônica , HomeostaseRESUMO
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).1-3 Anterograde IFT, from the cell body to the ciliary tip, is driven by kinesin-II motors, whereas retrograde IFT is powered by cytoplasmic dynein-1b motors.4 Both motors are associated with long chains of IFT protein complexes, known as IFT trains, and their cargoes.5-8 The conversion from anterograde to retrograde motility at the ciliary tip involves (1) the dissociation of kinesin motors from trains,9 (2) a fundamental restructuring of the train from the anterograde to the retrograde architecture,8,10,11 (3) the unloading and reloading of cargo,2 and (4) the activation of the dynein motors.8,12 A prominent hypothesis is that there is dedicated calcium-dependent protein-based machinery at the ciliary tip to mediate these processes.4,13 However, the mechanisms of IFT turnaround have remained elusive. In this study, we use mechanical and chemical methods to block IFT at intermediate positions along the cilia of the green algae Chlamydomonas reinhardtii, in normal and calcium-depleted conditions. We show that IFT turnaround, kinesin dissociation, and dynein-1b activation can consistently be induced at arbitrary distances from the ciliary tip, with no stationary tip machinery being required. Instead, we demonstrate that the anterograde-to-retrograde conversion is a calcium-independent intrinsic ability of IFT.
Assuntos
Dineínas , Cinesinas , Transporte Biológico , Cálcio/metabolismo , Cílios/metabolismo , Dineínas do Citoplasma/metabolismo , Dineínas/metabolismo , Flagelos/fisiologiaRESUMO
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. In this work, we used in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas. We combined 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 bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.
Assuntos
Chlamydomonas , Cílios , Flagelos , Chlamydomonas/metabolismo , Cílios/metabolismo , Microscopia Crioeletrônica/métodos , Dineínas/metabolismo , Tomografia com Microscopia Eletrônica/métodos , Flagelos/metabolismo , Flagelos/ultraestrutura , Cinesinas/metabolismo , Transporte Proteico , Transdução de SinaisRESUMO
Electron cryo-tomography is a potential approach to analyzing the three-dimensional conformation of frozen hydrated biological macromolecules using electron microscopy. Since projections of each individual object illuminated from different orientations are merged, electron tomography is capable of structural analysis of such heterogeneous environments as in vivo or with polymorphism, although radiation damage and the missing wedge are severe problems. Here, recent results on the structure of eukaryotic flagella, which is an ATP-driven bending organelle, from green algae Chlamydomonas are presented. Tomographic analysis reveals asymmetric molecular arrangements, especially that of the dynein motor proteins, in flagella, giving insight into the mechanism of planar asymmetric bending motion. Methodological challenges to obtaining higher-resolution structures from this technique are also discussed.
Assuntos
Cílios/ultraestrutura , Flagelos/ultraestrutura , Chlamydomonas/efeitos da radiação , Chlamydomonas/ultraestrutura , Microscopia Crioeletrônica/métodos , Dineínas/química , Dineínas/ultraestrutura , Tomografia com Microscopia Eletrônica , Imageamento Tridimensional/métodos , Microtúbulos/química , Microtúbulos/ultraestrutura , Conformação ProteicaRESUMO
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.