RESUMEN
Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Chlamydomonas/metabolismo , Multimerización de Proteína , Synechocystis/metabolismo , Tilacoides/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Membrana Celular/metabolismo , Chlamydomonas/ultraestructura , Microscopía por Crioelectrón , Proteínas Fluorescentes Verdes/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Luz , Lípidos/química , Modelos Moleculares , Nucleótidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estrés Fisiológico/efectos de la radiación , Synechocystis/ultraestructura , Tilacoides/ultraestructuraRESUMEN
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.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Cilios/ultraestructura , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/ultraestructura , Citoesqueleto de Actina/metabolismo , Animales , Técnicas de Cultivo de Célula , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Cilios/metabolismo , Microscopía por Crioelectrón , Perros , Tomografía con Microscopio Electrónico , Expresión Génica , Humanos , Células de Riñón Canino Madin Darby , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismoRESUMEN
The cytotoxicity of cadmium (Cd), arsenate (As(V)), and arsenite (As(III)) on a strain of Chlamydomonas acidophila, isolated from the Rio Tinto, an acidic environment containing high metal(l)oid concentrations, was analyzed. We used a broad array of methods to produce complementary information: cell viability and reactive oxygen species (ROS) generation measures, ultrastructural observations, transmission electron microscopy energy dispersive x-ray microanalysis (TEM-XEDS), and gene expression. This acidophilic microorganism was affected differently by the tested metal/metalloid: It showed high resistance to arsenic while Cd was the most toxic heavy metal, showing an LC50 = 1.94 µM. Arsenite was almost four-fold more toxic (LC50= 10.91 mM) than arsenate (LC50 = 41.63 mM). Assessment of ROS generation indicated that both arsenic oxidation states generate superoxide anions. Ultrastructural analysis of exposed cells revealed that stigma, chloroplast, nucleus, and mitochondria were the main toxicity targets. Intense vacuolization and accumulation of energy reserves (starch deposits and lipid droplets) were observed after treatments. Electron-dense intracellular nanoparticle-like formation appeared in two cellular locations: inside cytoplasmic vacuoles and entrapped into the capsule, around each cell. The chemical nature (Cd or As) of these intracellular deposits was confirmed by TEM-XEDS. Additionally, they also contained an unexpected high content in phosphorous, which might support an essential role of poly-phosphates in metal resistance.
Asunto(s)
Arsénico , Cadmio , Chlamydomonas , Contaminantes del Agua/toxicidad , Arsénico/toxicidad , Cadmio/toxicidad , Chlamydomonas/efectos de los fármacos , Chlamydomonas/fisiología , Chlamydomonas/ultraestructura , ExtremófilosRESUMEN
The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo-electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.
Asunto(s)
Centriolos/química , Centriolos/metabolismo , Centriolos/ultraestructura , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Complejos Multiproteicos/metabolismo , Paramecium tetraurelia/metabolismo , Paramecium tetraurelia/ultraestructura , Unión Proteica , Combinación Trimetoprim y Sulfametoxazol/metabolismoRESUMEN
The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of Chlamydomonas wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits," namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating.
Asunto(s)
Chlamydomonas/metabolismo , Dineínas/metabolismo , Flagelos/ultraestructura , Proteínas Asociadas a Microtúbulos/metabolismo , Chlamydomonas/genética , Chlamydomonas/ultraestructura , Estructura Cuaternaria de ProteínaRESUMEN
In addition to bend propagation for swimming, Chlamydomonas cells use their flagella to glide along a surface. When polystyrene microspheres are added to cells, they attach to and move along the flagellar surface, thus serving as a proxy for gliding that can be used to assay for the flagellar components required for gliding motility. Gliding and microsphere movement are dependent on intraflagellar transport (IFT). Circumstantial evidence suggests that mechanical coupling of the IFT force-transducing machinery to a substrate is mediated by the flagellar transmembrane glycoprotein FMG-1B. Here, we show that cells carrying an insertion in the 5'-UTR of the FMG-1B gene lack FMG-1B protein, yet assemble normal-length flagella despite the loss of the major protein component of the flagellar membrane. Transmission electron microscopy shows a complete loss of the glycocalyx normally observed on the flagellar surface, suggesting it is composed of the ectodomains of FMG-1B molecules. Microsphere movements and gliding motility are also greatly reduced in the 5'-UTR mutant. Together, these data provide the first rigorous demonstration that FMG-1B is necessary for the normal expression of force at the flagellar surface in ChlamydomonasThis article has an associated First Person interview with authors from the paper.
Asunto(s)
Chlamydomonas , Flagelos , Glicoproteínas , Proteínas de Plantas , Chlamydomonas/genética , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Flagelos/genética , Flagelos/metabolismo , Flagelos/ultraestructura , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
WDR92 associates with a prefoldin-like cochaperone complex and known dynein assembly factors. WDR92 has been very highly conserved and has a phylogenetic signature consistent with it playing a role in motile ciliary assembly or activity. Knockdown of WDR92 expression in planaria resulted in ciliary loss, reduced beat frequency and dyskinetic motion of the remaining ventral cilia. We have now identified a Chlamydomonas wdr92 mutant that encodes a protein missing the last four WD repeats. The wdr92-1 mutant builds only â¼0.7-µm cilia lacking both inner and outer dynein arms, but with intact doublet microtubules and central pair. When cytoplasmic extracts prepared by freeze/thaw from a control strain were fractionated by gel filtration, outer arm dynein components were present in several distinct high molecular weight complexes. In contrast, wdr92-1 extracts almost completely lacked all three outer arm heavy chains, while the IFT dynein heavy chain was present in normal amounts. A wdr92-1 tpg1-2 double mutant builds â¼7-µm immotile flaccid cilia that completely lack dynein arms. These data indicate that WDR92 is a key assembly factor specifically required for the stability of axonemal dynein heavy chains in cytoplasm and suggest that cytoplasmic/IFT dynein heavy chains use a distinct folding pathway.
Asunto(s)
Proteínas Algáceas/metabolismo , Axonema/metabolismo , Chlamydomonas/metabolismo , Dineínas/metabolismo , Repeticiones WD40 , Proteínas Algáceas/química , Secuencia de Aminoácidos , Axonema/ultraestructura , Secuencia de Bases , Chlamydomonas/ultraestructura , Cilios/metabolismo , Cilios/ultraestructura , Ritmo Circadiano , Ácido Glutámico/metabolismo , Modelos Biológicos , Mutación/genética , Estabilidad ProteicaRESUMEN
Many secreted peptides used for cell-cell communication require conversion of a C-terminal glycine to an amide for bioactivity. This reaction is catalyzed only by the integral membrane protein peptidylglycine α-amidating monooxygenase (PAM). PAM has been highly conserved and is found throughout the metazoa; PAM-like sequences are also present in choanoflagellates, filastereans, unicellular and colonial chlorophyte green algae, dinoflagellates and haptophytes. Recent studies have revealed that in addition to playing a key role in peptidergic signaling, PAM also regulates ciliogenesis in vertebrates, planaria and chlorophyte algae, and is required for the stability of actin-based microvilli. Here we briefly introduce the basic principles involved in ciliogenesis, the sequential reactions catalyzed by PAM and the trafficking of PAM through the secretory and endocytic pathways. We then discuss the multi-faceted roles this enzyme plays in the formation and maintenance of cytoskeleton-based cellular protrusions and propose models for how PAM protein and amidating activity might contribute to ciliogenesis. Finally, we consider why some ciliated organisms lack PAM, and discuss the potential ramifications of ciliary localized PAM for the endocrine features commonly observed in patients with ciliopathies.
Asunto(s)
Chlamydomonas/enzimología , Cilios/metabolismo , Oxigenasas de Función Mixta/metabolismo , Complejos Multienzimáticos/metabolismo , Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Actinas/metabolismo , Chlamydomonas/citología , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Cilios/ultraestructura , Oxigenasas de Función Mixta/análisis , Modelos Moleculares , Complejos Multienzimáticos/análisis , Proteínas de Plantas/análisis , Biosíntesis de Proteínas , Transporte de Proteínas , Transducción de SeñalRESUMEN
The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins, which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors (DNAAFs) before being transported to and assembled on the ciliary axoneme. Here, we characterize the function of WDR92 in Chlamydomonas. Loss of WDR92, a cytoplasmic protein, in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella, disappearance of axonemal dynein arms, and diminishment of dynein arm heavy chains in the cytoplasm, suggesting that WDR92 is a DNAAF. Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBL1, RPAP3, MOT48, ODA7, and DYX1C. The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBL1/2 and RPAP3, while PF13, another PIH1 domain-containing protein with function in dynein preassembly, did not. Interestingly, the third PIH1 domain-containing protein TWI1 was not related to flagellar motility. WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs. Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.
Asunto(s)
Chlamydomonas/fisiología , Cilios/genética , Cilios/metabolismo , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Chlamydomonas/citología , Chlamydomonas/ultraestructura , Citoplasma , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Mutación , Fenotipo , Unión ProteicaRESUMEN
We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate-tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.
Asunto(s)
Microtúbulos/metabolismo , Tubulina (Proteína)/fisiología , Animales , Arabidopsis/metabolismo , Arabidopsis/ultraestructura , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/ultraestructura , Línea Celular , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Tomografía con Microscopio Electrónico , Guanosina Trifosfato/metabolismo , Microtúbulos/química , Microtúbulos/ultraestructura , Potoroidae/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestructura , Tubulina (Proteína)/metabolismoRESUMEN
Facing adverse conditions such as nitrogen (N) deprivation, microalgae enter cellular quiescence, a reversible cell cycle arrest with drastic changes in metabolism allowing cells to remain viable. Recovering from N deprivation and quiescence is an active and orderly process as we are showing here for Chlamydomonas reinhardtii We conducted comparative transcriptomics on this alga to discern processes relevant to quiescence in the context of N deprivation and recovery following refeeding. A mutant with slow recovery from N deprivation, compromised hydrolysis of triacylglycerols7 (cht7), was included to better define the regulatory processes governing the respective transitions. We identified an ordered set of biological processes with expression patterns that showed sequential reversal following N resupply and uncovered acclimation responses specific to the recovery phase. Biochemical assays and microscopy validated selected inferences made based on the transcriptional analyses. These comprise (1) the restoration of N source preference and cellular bioenergetics during the early stage of recovery; (2) flagellum-based motility in the mid to late stage of recovery; and (3) recovery phase-specific gene groups cooperating in the rapid replenishment of chloroplast proteins. In the cht7 mutant, a large number of programmed responses failed to readjust in a timely manner. Finally, evidence is provided for the involvement of the cAMP-protein kinase A pathway in gating the recovery. We conclude that the recovery from N deprivation represents not simply a reversal of processes directly following N deprivation, but a distinct cellular state.
Asunto(s)
Chlamydomonas/genética , Chlamydomonas/metabolismo , Nitrógeno/deficiencia , Transcripción Genética , Aclimatación , Ciclo Celular , Chlamydomonas/ultraestructura , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Galactolípidos/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Metabolismo de los Lípidos/genética , Metaboloma/genética , Mutación/genética , Oxidación-Reducción , Análisis de Secuencia de ARN , Transcriptoma/genéticaRESUMEN
The confinement of Rubisco in a chloroplast microcompartment, or pyrenoid, is a distinctive feature of most microalgae, and contributes to perhaps ~30 Pg of carbon fixed each year, yet our understanding of pyrenoid composition, regulation, and function remains fragmentary. Recently, significant progress in understanding the pyrenoid has arisen from studies using mutant lines, mass spectrometric analysis of isolated pyrenoids, and advanced ultrastructural imaging of the microcompartment in the model alga Chlamydomonas. The emergence of molecular details in other lineages provides a comparative framework for this review, and evidence that most pyrenoids function similarly, even in the absence of a common ancestry. The objective of this review is to explore pyrenoid diversity throughout key algal lineages and discuss whether common ultrastructural and cellular features are indicative of common functional processes. By characterizing pyrenoid origins in terms of mechanistic and structural parallels, we hope to provide key unanswered questions which will inform future research directions.
Asunto(s)
Chlamydomonas , Cloroplastos/metabolismo , Cloroplastos/ultraestructura , Microalgas , Algas Marinas , Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Microalgas/metabolismo , Microalgas/ultraestructura , Ribulosa-Bifosfato Carboxilasa/metabolismo , Algas Marinas/metabolismo , Algas Marinas/ultraestructuraRESUMEN
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.
Asunto(s)
Células/metabolismo , Células/ultraestructura , Microscopía Electrónica/métodos , Chlamydomonas/citología , Chlamydomonas/ultraestructura , Flagelos/ultraestructura , Factores de TiempoRESUMEN
Electron microscopy (EM) for biological samples, developed in the 1940-1950s, changed our conception about the architecture of eukaryotic cells. It was followed by a period where EM applied to cell biology had seemingly fallen asleep, even though new methods with important implications for modern EM were developed. Among these was the discovery that samples can be preserved by chemical fixation and most importantly by rapid freezing without the formation of crystalline ice, giving birth to the world of cryo-EM. The past 15-20 years are hallmarked by a tremendous interest in EM, driven by important technological advances. Cryo-EM, in particular, is now capable of revealing structures of proteins at a near-atomic resolution owing to improved sample preparation methods, microscopes and cameras. In this review, we focus on the challenges associated with the imaging of membranes by EM and give examples from the field of host-pathogen interactions, in particular of virus-infected cells. Despite the advantages of imaging membranes under native conditions in cryo-EM, conventional EM will remain an important complementary method, in particular if large volumes need to be imaged.
Asunto(s)
Membrana Celular/ultraestructura , Chlamydomonas/ultraestructura , Microscopía por Crioelectrón/métodos , Nervio Óptico/ultraestructura , Virus Vaccinia/ultraestructura , Virión/ultraestructura , Resinas Acrílicas , Animales , Membrana Celular/virología , Microscopía por Crioelectrón/historia , Microscopía por Crioelectrón/instrumentación , Células HeLa , Historia del Siglo XX , Historia del Siglo XXI , Interacciones Huésped-Patógeno , Humanos , Imagenología Tridimensional , Ratones , VitrificaciónRESUMEN
Intraflagellar transport (IFT) is a form of motor-dependent cargo transport that is essential for the assembly, maintenance, and length control of cilia, which play critical roles in motility, sensory reception, and signal transduction in virtually all eukaryotic cells. During IFT, anterograde kinesin-2 and retrograde IFT dynein motors drive the bidirectional transport of IFT trains that deliver cargo, for example, axoneme precursors such as tubulins as well as molecules of the signal transduction machinery, to their site of assembly within the cilium. Following its discovery in Chlamydomonas, IFT has emerged as a powerful model system for studying general principles of motor-dependent cargo transport and we now appreciate the diversity that exists in the mechanism of IFT within cilia of different cell types. The absence of heterotrimeric kinesin-2 function, for example, causes a complete loss of both IFT and cilia in Chlamydomonas, but following its loss in Caenorhabditis elegans, where its primary function is loading the IFT machinery into cilia, homodimeric kinesin-2-driven IFT persists and assembles a full-length cilium. Generally, heterotrimeric kinesin-2 and IFT dynein motors are thought to play widespread roles as core IFT motors, whereas homodimeric kinesin-2 motors are accessory motors that mediate different functions in a broad range of cilia, in some cases contributing to axoneme assembly or the delivery of signaling molecules but in many other cases their ciliary functions, if any, remain unknown. In this review, we focus on mechanisms of motor action, motor cooperation, and motor-dependent cargo delivery during IFT.
Asunto(s)
Caenorhabditis elegans/metabolismo , Chlamydomonas/metabolismo , Cilios/metabolismo , Flagelos/metabolismo , Animales , Axonema/metabolismo , Axonema/ultraestructura , Cuerpos Basales/metabolismo , Cuerpos Basales/ultraestructura , Transporte Biológico , Caenorhabditis elegans/genética , Caenorhabditis elegans/ultraestructura , Chlamydomonas/genética , Chlamydomonas/ultraestructura , Cilios/ultraestructura , Dineínas/química , Dineínas/genética , Dineínas/metabolismo , Flagelos/ultraestructura , Regulación de la Expresión Génica , Cinesinas/química , Cinesinas/genética , Cinesinas/metabolismo , Multimerización de Proteína , Transducción de Señal , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMEN
While cryo-electron tomography (cryo-ET) can reveal biological structures in their native state within the cellular environment, it requires the production of high-quality frozen-hydrated sections that are thinner than 300nm. Sample requirements are even more stringent for the visualization of membrane-bound protein complexes within dense cellular regions. Focused ion beam (FIB) sample preparation for transmission electron microscopy (TEM) is a well-established technique in material science, but there are only few examples of biological samples exhibiting sufficient quality for high-resolution in situ investigation by cryo-ET. In this work, we present a comprehensive description of a cryo-sample preparation workflow incorporating additional conductive-coating procedures. These coating steps eliminate the adverse effects of sample charging on imaging with the Volta phase plate, allowing data acquisition with improved contrast. We discuss optimized FIB milling strategies adapted from material science and each critical step required to produce homogeneously thin, non-charging FIB lamellas that make large areas of unperturbed HeLa and Chlamydomonas cells accessible for cryo-ET at molecular resolution.
Asunto(s)
Secciones por Congelación/métodos , Proteínas de la Membrana/ultraestructura , Manejo de Especímenes/métodos , Chlamydomonas/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Células HeLa , Humanos , Microscopía Electrónica de Transmisión/métodosRESUMEN
Chlamydomonas acidophila LAFIC-004 is an acidophilic strain of green microalgae isolated from coal mining drainage. In the present work, this strain was cultivated in acidic medium (pH 3.6) under phototrophic, mixotrophic, and heterotrophic regimes to determine the best condition for growth and lipid production, simultaneously assessing possible morphological and ultrastructural alterations in the cells. For heterotrophic and mixotrophic treatments, two organic carbon sources were tested: 1 % glucose and 1 % sodium acetate. Lipid content and fatty acid profiles were only determined in phototrophic condition. The higher growth rates were achieved in phototrophic conditions, varying from 0.18 to 0.82 day-1. Glucose did not result in significant growth increase in either mixotrophic or heterotrophic conditions, and acetate proved to be toxic to the strain in both conditions. Oil content under phototrophic condition was 15.9 % at exponential growth phase and increased to 54.63 % at stationary phase. Based on cell morphology (flow cytometry and light microscopy) and ultrastructure (transmission electron microscopy), similar characteristics were observed between phototrophic and mixotrophic conditions with glucose evidencing many lipid bodies, starch granules, and intense fluorescence. Under the tested conditions, mixotrophic and heterotrophic modes did not result in increased neutral lipid fluorescence. It can be concluded that the strain is a promising lipid producer when grown until stationary phase in acidic medium and under a phototrophic regime, presenting a fatty acid profile suitable for biodiesel production. The ability to grow this strain in acidic mining residues suggests a potential for bioremediation with production of useful biomass.
Asunto(s)
Chlamydomonas/metabolismo , Chlamydomonas/ultraestructura , Ácidos Grasos/biosíntesis , Glucosa/metabolismo , Procesos Heterotróficos/fisiología , Procesos Fototróficos/fisiología , Biodegradación Ambiental , Biocombustibles , Chlamydomonas/crecimiento & desarrollo , Minas de Carbón , Gotas Lipídicas/metabolismo , Microalgas/clasificación , Microalgas/metabolismo , Microscopía Electrónica de TransmisiónRESUMEN
The cilium is a large macromolecular machine that is vital for motility, signaling, and sensing in most eukaryotic cells. Its conserved core structure, the axoneme, contains nine microtubule doublets, each comprising a full A-microtubule and an incomplete B-microtubule. However, thus far, the function of this doublet geometry has not been understood. We developed a time-resolved correlative fluorescence and three-dimensional electron microscopy approach to investigate the dynamics of intraflagellar transport (IFT) trains, which carry ciliary building blocks along microtubules during the assembly and disassembly of the cilium. Using this method, we showed that each microtubule doublet is used as a bidirectional double-track railway: Anterograde IFT trains move along B-microtubules, and retrograde trains move along A-microtubules. Thus, the microtubule doublet geometry provides direction-specific rails to coordinate bidirectional transport of ciliary components.
Asunto(s)
Axonema/metabolismo , Chlamydomonas/metabolismo , Flagelos/metabolismo , Axonema/ultraestructura , Transporte Biológico , Chlamydomonas/ultraestructura , Cilios/metabolismo , Cilios/ultraestructura , Flagelos/ultraestructura , Imagenología Tridimensional/métodos , Microscopía Electrónica/métodos , Microscopía Fluorescente/métodosRESUMEN
Cilia and flagella often exhibit synchronized behavior; this includes phase locking, as seen in Chlamydomonas, and metachronal wave formation in the respiratory cilia of higher organisms. Since the observations by Gray and Rothschild of phase synchrony of nearby swimming spermatozoa, it has been a working hypothesis that synchrony arises from hydrodynamic interactions between beating filaments. Recent work on the dynamics of physically separated pairs of flagella isolated from the multicellular alga Volvox has shown that hydrodynamic coupling alone is sufficient to produce synchrony. However, the situation is more complex in unicellular organisms bearing few flagella. We show that flagella of Chlamydomonas mutants deficient in filamentary connections between basal bodies display markedly different synchronization from the wild type. We perform micromanipulation on configurations of flagella and conclude that a mechanism, internal to the cell, must provide an additional flagellar coupling. In naturally occurring species with 4, 8, or even 16 flagella, we find diverse symmetries of basal body positioning and of the flagellar apparatus that are coincident with specific gaits of flagellar actuation, suggesting that it is a competition between intracellular coupling and hydrodynamic interactions that ultimately determines the precise form of flagellar coordination in unicellular algae.
Asunto(s)
Chlamydomonas/fisiología , Flagelos/fisiología , Chlamydomonas/ultraestructura , Flagelos/ultraestructura , Modelos Biológicos , MovimientoRESUMEN
Intraflagellar transport (IFT) is responsible for the bidirectional trafficking of molecular components required for the elongation and maintenance of eukaryotic cilia and flagella. Cargo is transported by IFT 'trains', linear rows of multiprotein particles moved by molecular motors along the axonemal doublets. We have previously described two structurally distinct categories of 'long' and 'short' trains. Here, we analyse the relative number of these trains throughout flagellar regeneration and show that long trains are most abundant at the beginning of flagellar growth whereas short trains gradually increase in number as flagella elongate. These observations are incompatible with the previous hypothesis that short trains are derived solely from the reorganization of long trains at the flagellar tip. We demonstrate with electron tomography the existence of two distinct ultrastructural organizations for the short trains, we name these 'narrow' and 'wide', and provide the first 3D model of the narrow short trains. These trains are characterized by tri-lobed units, which repeat longitudinally every 16â nm and contact protofilament 7 of the B-tubule. Functional implications of the new structural evidence are discussed.