RESUMEN
RATIONALE: The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates. OBJECTIVE: We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid. METHODS AND RESULTS: Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics. CONCLUSIONS: Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.
Asunto(s)
Acoplamiento Excitación-Contracción , Frecuencia Cardíaca , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Sarcolema/metabolismo , Potenciales de Acción , Animales , Difusión , Tomografía con Microscopio Electrónico , Femenino , Recuperación de Fluorescencia tras Fotoblanqueo , Miocitos Cardíacos/ultraestructura , Conejos , Sarcolema/ultraestructuraRESUMEN
During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.
Asunto(s)
Cinesinas/metabolismo , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Motoras Moleculares , Mutación , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Basal bodies (BBs) are microtubule-based organelles that act as a template for and stabilize cilia at the cell surface. Centrins ubiquitously associate with BBs and function in BB assembly, maturation and stability. Human POC5 (hPOC5) is a highly conserved centrin-binding protein that binds centrins through Sfi1p-like repeats and is required for building full-length, mature centrioles. Here, we use the BB-rich cytoskeleton of Tetrahymena thermophila to characterize Poc5 BB functions. Tetrahymena Poc5 (TtPoc5) uniquely incorporates into assembling BBs and is then removed from mature BBs prior to ciliogenesis. Complete genomic knockout of TtPOC5 leads to a significantly increased production of BBs, yet a markedly reduced ciliary density, both of which are rescued by reintroduction of TtPoc5. A second Tetrahymena POC5-like gene, SFR1, is similarly implicated in modulating BB production. When TtPOC5 and SFR1 are co-deleted, cell viability is compromised and BB overproduction is exacerbated. Overproduced BBs display defective transition zone formation and a diminished capacity for ciliogenesis. This study uncovers a requirement for Poc5 in building mature BBs, providing a possible functional link between hPOC5 mutations and impaired cilia.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Cuerpos Basales , Tetrahymena thermophila , Proteínas Portadoras , Centriolos/genética , Cilios/genética , Humanos , Microtúbulos , Tetrahymena thermophila/genéticaRESUMEN
Motile cilia generate directed hydrodynamic flow that is important for the motility of cells and extracellular fluids. To optimize directed hydrodynamic flow, motile cilia are organized and oriented into a polarized array. Basal bodies (BBs) nucleate and position motile cilia at the cell cortex. Cytoplasmic BB-associated microtubules are conserved structures that extend from BBs. By using the ciliate, Tetrahymena thermophila, combined with EM-tomography and light microscopy, we show that BB-appendage microtubules assemble coincidently with new BB assembly and that they are attached to the cell cortex. These BB-appendage microtubules are specifically marked by post translational modifications of tubulin, including glycylation. Mutations that prevent glycylation shorten BB-appendage microtubules and disrupt BB positioning and cortical attachment. Consistent with the attachment of BB-appendage microtubules to the cell cortex to position BBs, mutations that disrupt the cellular cortical cytoskeleton disrupt the cortical attachment and positioning of BBs. In summary, BB-appendage microtubules promote the organization of ciliary arrays through attachment to the cell cortex.
Asunto(s)
Cuerpos Basales/metabolismo , Cilios/metabolismo , Microtúbulos/metabolismo , Tetrahymena thermophila/metabolismo , Cuerpos Basales/ultraestructura , Cilios/genética , Glicosilación , Microtúbulos/genética , Microtúbulos/ultraestructura , Mutación , Tetrahymena thermophila/genética , Tetrahymena thermophila/ultraestructuraRESUMEN
During sexual reproduction or conjugation, ciliates form a specialized cell adhesion zone for the purpose of exchanging gametic pronuclei. Hundreds of individual membrane fusion events transform the adhesion zone into a perforated membrane curtain, the mating junction. Pronuclei from each mating partner are propelled through this fenestrated membrane junction by a web of short, cris-crossing microtubules. Pronuclear passage results in the formation of two breaches in the membrane junction. Following pronuclear exchange and karyogamy (fertilization), cells seal these twin membrane breaches thereby re-establishing cellular independence. This would seem like a straightforward problem: simply grow membrane in from the edges of each breach in a fashion similar to how animal cells "grow" their cytokinetic furrows or how plant cells construct a cell wall during mitosis. Serial section electron microscopy and 3-D electron tomography reveal that the actual mechanism is less straightforward. Each of the two membrane breaches transforms into a bowed membrane assembly platform. The resulting membrane protrusions continue to grow into the cytoplasm of the mating partner, traverse the cytoplasm in anti-parallel directions and make contact with the plasma membrane that flanks the mating junction. This investigation reveals the details of a novel, developmentally-induced mechanism of membrane disruption and restoration associated with pronuclear exchange and fertilization in the ciliate, Tetrahymena thermophila.
Asunto(s)
Conjugación Genética/fisiología , Fusión de Membrana/fisiología , Tetrahymena thermophila/fisiología , Animales , Adhesión Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Cilióforos , Conjugación Genética/genética , Citoplasma , Microscopía Electrónica , Microtúbulos , Mitosis , Reproducción/fisiología , Tetrahymena/genética , Tetrahymena thermophila/genéticaRESUMEN
Electrophysiological studies of excitable organs usually focus on action potential (AP)-generating cells, whereas nonexcitable cells are generally considered as barriers to electrical conduction. Whether nonexcitable cells may modulate excitable cell function or even contribute to AP conduction via direct electrotonic coupling to AP-generating cells is unresolved in the heart: such coupling is present in vitro, but conclusive evidence in situ is lacking. We used genetically encoded voltage-sensitive fluorescent protein 2.3 (VSFP2.3) to monitor transmembrane potential in either myocytes or nonmyocytes of murine hearts. We confirm that VSFP2.3 allows measurement of cell type-specific electrical activity. We show that VSFP2.3, expressed solely in nonmyocytes, can report cardiomyocyte AP-like signals at the border of healed cryoinjuries. Using EM-based tomographic reconstruction, we further discovered tunneling nanotube connections between myocytes and nonmyocytes in cardiac scar border tissue. Our results provide direct electrophysiological evidence of heterocellular electrotonic coupling in native myocardium and identify tunneling nanotubes as a possible substrate for electrical cell coupling that may be in addition to previously discovered connexins at sites of myocyte-nonmyocyte contact in the heart. These findings call for reevaluation of cardiac nonmyocyte roles in electrical connectivity of the heterocellular heart.
Asunto(s)
Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Sistema de Conducción Cardíaco/metabolismo , Miocardio/citología , Miocitos Cardíacos/metabolismo , Optogenética , Potenciales de Acción , Animales , Proteínas Bacterianas/metabolismo , Comunicación Celular , Recuento de Células , Membrana Celular/metabolismo , Conductividad Eléctrica , Femenino , Fibroblastos/metabolismo , Corazón/fisiología , Proteínas Luminiscentes/metabolismo , Masculino , Potenciales de la Membrana , Ratones , Ratones Transgénicos , Células Musculares/metabolismoRESUMEN
Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Glicoproteínas/genética , Síndrome de Kartagener/genética , Pez Cebra/genética , Animales , Chlamydomonas/genética , Cilios/genética , Análisis Mutacional de ADN/métodos , Dineínas/genética , Femenino , Humanos , Masculino , Mutación , Sistemas de Lectura Abierta , Planarias/genética , Proteoma/genéticaRESUMEN
Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.
Asunto(s)
Membrana Celular/metabolismo , Extensiones de la Superficie Celular/metabolismo , Uniones Intercelulares/metabolismo , Tetrahymena thermophila/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/fisiología , Espacio Extracelular/metabolismo , Uniones Intercelulares/ultraestructura , Metabolismo de los Lípidos , Vesículas Secretoras/metabolismo , Tetrahymena thermophila/fisiología , Tetrahymena thermophila/ultraestructuraRESUMEN
Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.
Asunto(s)
Axonema/metabolismo , Cilios/metabolismo , Dineínas/metabolismo , Proteínas de Peces/metabolismo , Oryzias , Proteínas/metabolismo , Animales , Axonema/química , Axonema/genética , Axonema/patología , Chlamydomonas/genética , Chlamydomonas/metabolismo , Cilios/química , Cilios/genética , Cilios/patología , Clonación Molecular , Células Epiteliales/citología , Proteínas de Peces/genética , Genes Recesivos/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Síndrome de Kartagener/genética , Síndrome de Kartagener/patología , Masculino , Ratones , Datos de Secuencia Molecular , Mutación/genética , Oryzias/embriología , Oryzias/genética , Oryzias/metabolismo , Unión Proteica , Proteínas/genética , Homología de Secuencia de Aminoácido , Motilidad Espermática , Testículo/citologíaRESUMEN
Karyotypes, composed of chromosomes, must be accurately partitioned by the mitotic spindle for optimal cell health. However, it is unknown how underlying characteristics of karyotypes, such as chromosome number and size, govern the scaling of the mitotic spindle to ensure accurate chromosome segregation and cell proliferation. We utilize budding yeast strains engineered with fewer chromosomes, including just two "mega chromosomes," to study how spindle size and function are responsive to, and scaled by, karyotype. We determined that deletion and overexpression of spindle-related genes are detrimental to the growth of strains with two chromosomes, suggesting that mega chromosomes exert altered demands on the spindle. Using confocal microscopy, we demonstrate that cells with fewer but longer chromosomes have smaller spindle pole bodies, fewer microtubules, and longer spindles. Moreover, using electron tomography and confocal imaging, we observe elongated, bent anaphase spindles with fewer core microtubules in strains with mega chromosomes. Cells harboring mega chromosomes grow more slowly, are delayed in mitosis, and a subset struggle to complete chromosome segregation. We propose that the karyotype of the cell dictates the microtubule number, type, spindle pole body size, and spindle length, subsequently influencing the dynamics of mitosis, such as the rate of spindle elongation and the velocity of pole separation. Taken together, our results suggest that mitotic spindles are highly plastic ultrastructures that can accommodate and adjust to a variety of karyotypes, even within a species.
Asunto(s)
Saccharomyces cerevisiae , Huso Acromático , Huso Acromático/metabolismo , Saccharomyces cerevisiae/genética , Microtúbulos/metabolismo , Segregación Cromosómica , Mitosis , Cromosomas Fúngicos/genética , CariotipoRESUMEN
Trisomy 21, the genetic cause of Down syndrome, disrupts primary cilia formation and function, in part through elevated Pericentrin, a centrosome protein encoded on chromosome 21. Yet how trisomy 21 and elevated Pericentrin disrupt cilia-related molecules and pathways, and the in vivo phenotypic relevance remain unclear. Utilizing ciliogenesis time course experiments combined with light microscopy and electron tomography, we reveal that chromosome 21 polyploidy elevates Pericentrin and microtubules away from the centrosome that corral MyosinVA and EHD1, delaying ciliary membrane delivery and mother centriole uncapping essential for ciliogenesis. If given enough time, trisomy 21 cells eventually ciliate, but these ciliated cells demonstrate persistent trafficking defects that reduce transition zone protein localization and decrease sonic hedgehog signaling in direct anticorrelation with Pericentrin levels. Consistent with cultured trisomy 21 cells, a mouse model of Down syndrome with elevated Pericentrin has fewer primary cilia in cerebellar granule neuron progenitors and thinner external granular layers at P4. Our work reveals that elevated Pericentrin from trisomy 21 disrupts multiple early steps of ciliogenesis and creates persistent trafficking defects in ciliated cells. This pericentrosomal crowding mechanism results in signaling deficiencies consistent with the neurological phenotypes found in individuals with Down syndrome.
Human cells typically have 23 pairs of structures known as chromosomes. Each chromosome contains a unique set of genes which provide the instructions needed to make proteins and other essential molecules found in the body. Individuals with Down syndrome have an extra copy of chromosome 21. This genetic alteration is known as trisomy 21 and affects many different organs in the body, leading to various medical conditions including intellectual disability, heart defects, and immune deficiencies. A recent study showed that cells from individuals with Down syndrome had defects in forming primary cilia structures on the surface of cells which work as signaling hubs to control how cells grow and develop. These cilia defects were in large part due to excess levels of a protein known as Pericentrin, which is encoded by a gene found on chromosome 21. But it is unclear how Pericentrin disrupts cilia assembly, and how this may contribute to the medical conditions observed in individuals with Down syndrome. To address these questions, Jewett et al. studied human cells that had been engineered to have trisomy 21. The experiments found that trisomy 21 led to higher levels of Pericentrin and altered the way molecules were organized at the sites where primary cilia form. This caused the components required to build and maintain the primary cilium to become trapped in the wrong locations. The trisomy 21 cells were eventually able to rearrange the molecules and build a primary cilium, but it took them twice as long as cells with 23 pairs of chromosomes and their primary cilium did not properly work. Further experiments were then conducted on mice that had been engineered to have an extra copy of a portion of genes on human chromosome 21, including the gene for Pericentrin. Jewett et al. found that these mice assembled cilia later and had defects in cilia signaling, similar to the human trisomy 21 cells. This resulted in mild abnormalities in brain development that were consistent with what occurs in individuals with Down syndrome. These findings suggest that the elevated levels of Pericentrin in trisomy 21 causes changes in cilia formation and function which, in turn, may alter how the mouse brain develops. Further studies will be required to find out whether defects in primary cilia may contribute to other medical conditions observed in individuals with Down syndrome.
Asunto(s)
Síndrome de Down , Ratones , Animales , Proteínas Hedgehog/metabolismo , Centriolos/metabolismo , Centrosoma/metabolismo , Cilios/metabolismoRESUMEN
Polarized cells, such as neuronal, epithelial, and fungal cells, all display a specialized organization of their microtubules (MTs). The interphase MT cytoskeleton of the rod-shaped fission yeast, Schizosaccharomyces pombe, has been extensively described by fluorescence microscopy. Here, we describe a large-scale, electron tomography investigation of S. pombe, including a 3D reconstruction of a complete eukaryotic cell volume at sufficient resolution to show both how many MTs there are in a bundle and their detailed architecture. Most cytoplasmic MTs are open at one end and capped at the other, providing evidence about their polarity. Electron-dense bridges between the MTs themselves and between MTs and the nuclear envelope were frequently observed. Finally, we have investigated structure/function relationships between MTs and both mitochondria and vesicles. Our analysis shows that electron tomography of well-preserved cells is ideally suited for describing fine ultrastructural details that were not visible with previous techniques.
Asunto(s)
Polaridad Celular/fisiología , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestructura , Tomografía Computarizada por Rayos X/métodos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestructura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Interfase/fisiología , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Modelos Moleculares , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Polímeros/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Centrioles are necessary for flagella and cilia formation, cytokinesis, cell-cycle control and centrosome organization/spindle assembly. They duplicate once per cell cycle, but the mechanisms underlying their duplication remain unclear. Here we show using electron tomography of staged C. elegans one-cell embryos that daughter centriole assembly begins with the formation and elongation of a central tube followed by the peripheral assembly of nine singlet microtubules. Tube formation and elongation is dependent on the SAS-5 and SAS-6 proteins, whereas the assembly of singlet microtubules onto the central tube depends on SAS-4. We further show that centriole assembly is triggered by an upstream signal mediated by SPD-2 and ZYG-1. These results define a structural pathway for the assembly of a daughter centriole and should have general relevance for future studies on centriole assembly in other organisms.
Asunto(s)
Caenorhabditis elegans/fisiología , Centriolos/fisiología , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/fisiología , Centriolos/ultraestructura , Femenino , Masculino , Proteínas Quinasas/fisiología , Interferencia de ARNRESUMEN
Polyploidy, increased sets of chromosomes, occurs during development, cellular stress, disease and evolution. Despite its prevalence, little is known about the physiological alterations that accompany polyploidy. We previously described 'ploidy-specific lethality', where a gene deletion that is not lethal in haploid or diploid budding yeast causes lethality in triploids or tetraploids. Here we report a genome-wide screen to identify ploidy-specific lethal functions. Only 39 out of 3,740 mutations screened exhibited ploidy-specific lethality. Almost all of these mutations affect genomic stability by impairing homologous recombination, sister chromatid cohesion, or mitotic spindle function. We uncovered defects in wild-type tetraploids predicted by the screen, and identified mechanisms by which tetraploidization affects genomic stability. We show that tetraploids have a high incidence of syntelic/monopolar kinetochore attachments to the spindle pole. We suggest that this defect can be explained by mismatches in the ability to scale the size of the spindle pole body, spindle and kinetochores. Thus, geometric constraints may have profound effects on genome stability; the phenomenon described here may be relevant in a variety of biological contexts, including disease states such as cancer.
Asunto(s)
Genes Letales/genética , Genoma Fúngico/genética , Genómica , Poliploidía , Saccharomyces cerevisiae/genética , Aurora Quinasas , Cromátides/genética , Cromátides/metabolismo , Emparejamiento Cromosómico , Diploidia , Genes Fúngicos/genética , Inestabilidad Genómica/genética , Cinetocoros/metabolismo , Mitosis , Mutación/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Recombinación Genética/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genética , Huso Acromático/metabolismoRESUMEN
Motile cilia beat with an asymmetric waveform consisting of a power stroke that generates a propulsive force and a recovery stroke that returns the cilium back to the start. Cilia are anchored to the cell cortex by basal bodies (BBs) that are directly coupled to the ciliary doublet microtubules (MTs). We find that, consistent with ciliary forces imposing on BBs, bending patterns in BB triplet MTs are responsive to ciliary beating. BB bending varies as environmental conditions change the ciliary waveform. Bending occurs where striated fibers (SFs) attach to BBs and mutants with short SFs that fail to connect to adjacent BBs exhibit abnormal BB bending, supporting a model in which SFs couple ciliary forces between BBs. Finally, loss of the BB stability protein Poc1, which helps interconnect BB triplet MTs, prevents the normal distributed BB and ciliary bending patterns. Collectively, BBs experience ciliary forces and manage mechanical coupling of these forces to their surrounding cellular architecture for normal ciliary beating.
Asunto(s)
Cuerpos Basales , Cilios , Cuerpos Basales/metabolismo , Cilios/metabolismo , Microtúbulos/metabolismo , Fenómenos MecánicosRESUMEN
Electron tomography of the chemical synapse provides important architectural information regarding the organization of synaptic organelles including synaptic vesicles, Nissl bodies, and early endosomes. Here, we describe methods for the preparation of select murine brain regions for high-pressure freezing, freeze substitution, and EM tomographic analysis of synaptic structures. The method uses fresh brain slices prepared using a vibratome and biopsy punches to collect specific brain regions of interest suitable for subsequent preservation and EM tomographic imaging.
Asunto(s)
Tomografía con Microscopio Electrónico , Electrones , Animales , Tomografía con Microscopio Electrónico/métodos , Substitución por Congelación , Ratones , Orgánulos , SinapsisRESUMEN
The Caenorhabditis elegans one-cell embryo is a powerful system in which to study microtubule organization because this large cell assembles both meiotic and mitotic spindles within the same cytoplasm over the course of 1 h in a stereotypical manner. The fertilized oocyte assembles two consecutive acentrosomal meiotic spindles that function to reduce the replicated maternal diploid set of chromosomes to a single-copy haploid set. The resulting maternal DNA then unites with the paternal DNA to form a zygotic diploid complement, around which a centrosome-based mitotic spindle forms. The early C. elegans embryo is amenable to live-cell imaging and electron tomography, permitting a detailed structural comparison of the meiotic and mitotic modes of spindle assembly.
Asunto(s)
Caenorhabditis elegans/embriología , Huso Acromático/fisiología , Adenosina Trifosfatasas/metabolismo , Animales , Centriolos/fisiología , Centrosoma/fisiología , Katanina , Meiosis , Microtúbulos/metabolismo , MitosisRESUMEN
Mice lacking expression of dynamin 1, a GTPase implicated in the fission reaction of synaptic vesicle endocytosis, fail to thrive and exhibit severe activity-dependent endocytic defects at their synapses. Here, we have used electron tomography to investigate the massive increase in clathrin-coated pit abundance that is selectively observed at a subset of synapses in dynamin 1 KO primary neuron cultures under conditions of spontaneous network activity. This increase, leading to branched tubular plasma membrane invaginations capped by clathrin-coated buds, occurs selectively at inhibitory synapses. A similar massive increase of clathrin-coated profiles (in this case, of clathrin-coated vesicles) is observed at inhibitory synapses of neurons that lack expression of synaptojanin 1, a phosphoinositide phosphatase involved in clathrin-coated vesicle uncoating. Thus, although excitatory synapses are largely spared under these conditions, inhibitory synapses are uniquely sensitive to perturbation of endocytic proteins, probably as a result of their higher levels of tonic activity leading to a buildup of clathrin-coated intermediates in these synapses. In contrast, the predominant endocytic structures observed at the majority of dynamin 1 KO synapses after acute stimulation are endosome-like intermediates that originate by a dynamin 1-independent form of endocytosis. These findings reveal a striking heterogeneity in the mode of synaptic vesicle recycling in different synapses and functional states.
Asunto(s)
Dinamina I/fisiología , Endocitosis , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Dinamina I/genética , Ratones , Ratones Noqueados , Microscopía Electrónica , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/fisiologíaRESUMEN
BRR6 and BRL1 are two paralogs that encode transmembrane proteins of the nuclear envelope (NE) involved in membrane fluidity and nuclear pore complex biogenesis in organisms that undergo a closed mitosis. We show that mutation of a conserved cysteine in the intralumenal domain of Saccharomyces cerevisiae Brr6p results in a novel temperature sensitive allele, brr6-Y100H, that arrests growth due to defects in spindle formation. Analysis of brr6-Y100H cells by electron tomography and Brr6p localization by super-resolution imaging supports the idea that Brr6p is involved in insertion of the newly duplicated spindle pole body into the NE.
RESUMEN
Control of centrosome assembly is critical for cell division, intracellular trafficking, and cilia. Regulation of centrosome number occurs through the precise duplication of centrioles that reside in centrosomes. Here we explored transcriptional control of centriole assembly and find that the RNA splicing factor SON is specifically required for completing procentriole assembly. Whole genome mRNA sequencing identified genes whose splicing and expression are affected by the reduction of SON, with an enrichment in genes involved in the microtubule (MT) cytoskeleton, centrosome, and centriolar satellites. SON is required for the proper splicing and expression of CEP131, which encodes a major centriolar satellite protein and is required to organize the trafficking and MT network around the centrosomes. This study highlights the importance of the distinct MT trafficking network that is intimately associated with nascent centrioles and is responsible for procentriole development and efficient ciliogenesis.