RESUMEN
Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While granule cell cilia are essential during early developmental stages, they become infrequent upon maturation. Here, we provide nanoscopic resolution of cilia in situ using large-scale electron microscopy volumes and immunostaining of mouse cerebella. In many granule cells, we found intracellular cilia, concealed from the external environment. Cilia were disassembled in differentiating granule cell neurons-in a process we call cilia deconstruction-distinct from premitotic cilia resorption in proliferating progenitors. In differentiating granule cells, cilia deconstruction involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Unlike ciliated neurons in other brain regions, our results show the deconstruction of concealed cilia in differentiating granule cells, which might prevent mitogenic hedgehog responsiveness. Ciliary deconstruction could be paradigmatic of cilia removal during differentiation in other tissues.
Asunto(s)
Diferenciación Celular , Cerebelo , Cilios , Proteínas Hedgehog , Neuronas , Cilios/metabolismo , Cilios/ultraestructura , Animales , Neuronas/metabolismo , Neuronas/citología , Neuronas/ultraestructura , Ratones , Cerebelo/metabolismo , Cerebelo/citología , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Neurogénesis , Centriolos/metabolismo , Centriolos/ultraestructura , Ratones Endogámicos C57BLRESUMEN
A primary cilium is a membrane-bound extension from the cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. Primary cilia in the brain are less accessible than cilia on cultured cells or epithelial tissues because in the brain they protrude into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs) but were absent from oligodendrocytes and microglia. Ultrastructural comparisons revealed that the base of the cilium and the microtubule organization differed between neurons and glia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting that cilia are poised to encounter locally released signaling molecules. Our analysis indicated that synapse proximity is likely due to random encounters in the neuropil, with no evidence that cilia modulate synapse activity as would be expected in tetrapartite synapses. The observed cell class differences in proximity to synapses were largely due to differences in external cilia length. Many key structural features that differed between neuronal and glial cilia influenced both cilium placement and shape and, thus, exposure to processes and synapses outside the cilium. Together, the ultrastructure both within and around neuronal and glial cilia suggest differences in cilia formation and function across cell types in the brain.
Asunto(s)
Cilios , Animales , Cilios/ultraestructura , Ratones , Microscopía Electrónica de Transmisión , Ratones Endogámicos C57BL , Neuronas/ultraestructura , Neuronas/fisiología , Corteza Visual/ultraestructura , Corteza Visual/fisiología , Neuroglía/ultraestructura , Neuroglía/fisiología , Femenino , Sinapsis/ultraestructura , Sinapsis/fisiología , MasculinoRESUMEN
Primary cilia on granule cell neuron progenitors in the developing cerebellum detect sonic hedgehog to facilitate proliferation. Following differentiation, cerebellar granule cells become the most abundant neuronal cell type in the brain. While essential during early developmental stages, the fate of granule cell cilia is unknown. Here, we provide nanoscopic resolution of ciliary dynamics in situ by studying developmental changes in granule cell cilia using large-scale electron microscopy volumes and immunostaining of mouse cerebella. We found that many granule cell primary cilia were intracellular and concealed from the external environment. Cilia were disassembed in differentiating granule cell neurons in a process we call cilia deconstruction that was distinct from pre-mitotic cilia resorption in proliferating progenitors. In differentiating granule cells, ciliary loss involved unique disassembly intermediates, and, as maturation progressed, mother centriolar docking at the plasma membrane. Cilia did not reform from the docked centrioles, rather, in adult mice granule cell neurons remained unciliated. Many neurons in other brain regions require cilia to regulate function and connectivity. In contrast, our results show that granule cell progenitors had concealed cilia that underwent deconstruction potentially to prevent mitogenic hedgehog responsiveness. The ciliary deconstruction mechanism we describe could be paradigmatic of cilia removal during differentiation in other tissues.
RESUMEN
A primary cilium is a thin membrane-bound extension off a cell surface that contains receptors for perceiving and transmitting signals that modulate cell state and activity. While many cell types have a primary cilium, little is known about primary cilia in the brain, where they are less accessible than cilia on cultured cells or epithelial tissues and protrude from cell bodies into a deep, dense network of glial and neuronal processes. Here, we investigated cilia frequency, internal structure, shape, and position in large, high-resolution transmission electron microscopy volumes of mouse primary visual cortex. Cilia extended from the cell bodies of nearly all excitatory and inhibitory neurons, astrocytes, and oligodendrocyte precursor cells (OPCs), but were absent from oligodendrocytes and microglia. Structural comparisons revealed that the membrane structure at the base of the cilium and the microtubule organization differed between neurons and glia. OPC cilia were distinct in that they were the shortest and contained pervasive internal vesicles only occasionally observed in neuron and astrocyte cilia. Investigating cilia-proximal features revealed that many cilia were directly adjacent to synapses, suggesting cilia are well poised to encounter locally released signaling molecules. Cilia proximity to synapses was random, not enriched, in the synapse-rich neuropil. The internal anatomy, including microtubule changes and centriole location, defined key structural features including cilium placement and shape. Together, the anatomical insights both within and around neuron and glia cilia provide new insights into cilia formation and function across cell types in the brain.
RESUMEN
Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.
Asunto(s)
Neocórtex , Células Precursoras de Oligodendrocitos , Animales , Ratones , Axones/metabolismo , Oligodendroglía/metabolismo , Neuronas/metabolismoRESUMEN
Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Transporte Biológico , Fusión Celular , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Ratones , RNA-Seq/métodos , Transducción de Señal/genética , Factores de Transcripción/genética , Proteínas del Envoltorio Viral/genética , Proteínas Señalizadoras YAPRESUMEN
Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.
Asunto(s)
Encéfalo/diagnóstico por imagen , Nanotecnología , Neuroimagen/métodos , Imagen Óptica/métodos , Animales , Axones , Espinas Dendríticas , Drosophila , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Imagenología Tridimensional , Riñón/diagnóstico por imagen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Fantasmas de Imagen , Corteza Somatosensorial/diagnóstico por imagen , SinapsisRESUMEN
Type I collagen is the main component of bone matrix and other connective tissues. Rerouting of its procollagen precursor to a degradative pathway is crucial for osteoblast survival in pathologies involving excessive intracellular buildup of procollagen that is improperly folded and/or trafficked. What cellular mechanisms underlie this rerouting remains unclear. To study these mechanisms, we employed live-cell imaging and correlative light and electron microscopy (CLEM) to examine procollagen trafficking both in wild-type mouse osteoblasts and osteoblasts expressing a bone pathology-causing mutant procollagen. We found that although most procollagen molecules successfully trafficked through the secretory pathway in these cells, a subpopulation did not. The latter molecules appeared in numerous dispersed puncta colocalizing with COPII subunits, autophagy markers and ubiquitin machinery, with more puncta seen in mutant procollagen-expressing cells. Blocking endoplasmic reticulum exit site (ERES) formation suppressed the number of these puncta, suggesting they formed after procollagen entry into ERESs. The punctate structures containing procollagen, COPII, and autophagic markers did not move toward the Golgi but instead were relatively immobile. They appeared to be quickly engulfed by nearby lysosomes through a bafilomycin-insensitive pathway. CLEM and fluorescence recovery after photobleaching experiments suggested engulfment occurred through a noncanonical form of autophagy resembling microautophagy of ERESs. Overall, our findings reveal that a subset of procollagen molecules is directed toward lysosomal degradation through an autophagic pathway originating at ERESs, providing a mechanism to remove excess procollagen from cells.
Asunto(s)
Autofagia/fisiología , Retículo Endoplásmico/metabolismo , Procolágeno/metabolismo , Células 3T3 , Animales , Línea Celular , Colágeno Tipo I/metabolismo , Aparato de Golgi/metabolismo , Lisosomas/metabolismo , Ratones , Osteoblastos/metabolismo , Transporte de Proteínas/fisiologíaRESUMEN
[This corrects the article DOI: 10.1371/journal.pgen.1005810.].
RESUMEN
The hexameric AAA ATPase VPS4 facilitates ESCRT III filament disassembly on diverse intracellular membranes. ESCRT III components and VPS4 have been localized to the ciliary transition zone and spindle poles and reported to affect centrosome duplication and spindle pole stability. How the canonical ESCRT pathway could mediate these events is unclear. We studied the association of VPS4 with centrosomes and found that GFP-VPS4 was a dynamic component of both mother and daughter centrioles. A mutant, VPS4EQ, which can't hydrolyze ATP, was less dynamic and accumulated at centrosomes. Centrosome localization of the VPS4EQ mutant, caused reduced γ-tubulin levels at centrosomes and consequently decreased microtubule growth and altered centrosome positioning. In addition, preventing VPS4 ATP hydrolysis nearly eliminated centriolar satellites and paused ciliogensis after formation of the ciliary vesicle. Zebrafish embryos injected with GFP-VPS4EQ mRNA were less viable, exhibited developmental defects and had fewer cilia in Kupffer's vesicle. Surprisingly, ESCRT III proteins seldom localized to centrosomes and their depletion did not lead to these phenotypes. Our data support an ESCRT III-independent function for VPS4 at the centrosome and reveal that this evolutionary conserved AAA ATPase influences diverse centrosome functions and, as a result, global cellular architecture and development.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/análisis , Centrosoma/enzimología , Centrosoma/metabolismo , Cilios/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/análisis , Tubulina (Proteína)/metabolismo , ATPasas de Translocación de Protón Vacuolares/análisis , Células 3T3 , ATPasas Asociadas con Actividades Celulares Diversas/genética , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Pez CebraRESUMEN
Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid ß-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons.
Asunto(s)
Proteínas Hedgehog/metabolismo , Mitocondrias/metabolismo , Animales , Técnicas de Cultivo de Célula , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Hipocampo/metabolismo , Humanos , Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , Neuronas/metabolismo , Neuronas/fisiología , Ratas , Transducción de Señal/fisiologíaRESUMEN
Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model.
Asunto(s)
Centrosoma/metabolismo , Cilios/metabolismo , Células Epiteliales/metabolismo , Animales , Citocinesis , Perros , Células de Riñón Canino Madin Darby , Modelos BiológicosRESUMEN
At the Drosophila NMJ, BMP signaling is critical for synapse growth and homeostasis. Signaling by the BMP7 homolog, Gbb, in motor neurons triggers a canonical pathway-which modulates transcription of BMP target genes, and a noncanonical pathway-which connects local BMP/BMP receptor complexes with the cytoskeleton. Here we describe a novel noncanonical BMP pathway characterized by the accumulation of the pathway effector, the phosphorylated Smad (pMad), at synaptic sites. Using genetic epistasis, histology, super resolution microscopy, and electrophysiology approaches we demonstrate that this novel pathway is genetically distinguishable from all other known BMP signaling cascades. This novel pathway does not require Gbb, but depends on presynaptic BMP receptors and specific postsynaptic glutamate receptor subtypes, the type-A receptors. Synaptic pMad is coordinated to BMP's role in the transcriptional control of target genes by shared pathway components, but it has no role in the regulation of NMJ growth. Instead, selective disruption of presynaptic pMad accumulation reduces the postsynaptic levels of type-A receptors, revealing a positive feedback loop which appears to function to stabilize active type-A receptors at synaptic sites. Thus, BMP pathway may monitor synapse activity then function to adjust synapse growth and maturation during development.
Asunto(s)
Unión Neuromuscular/genética , Organogénesis , Sinapsis/genética , Transmisión Sináptica/genética , Animales , Animales Modificados Genéticamente , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Neuronas Motoras/metabolismo , Unión Neuromuscular/crecimiento & desarrollo , Transducción de Señal/genética , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The presence of Sonic Hedgehog (Shh) and its signaling components in the neurons of the hippocampus raises a question about what role the Shh signaling pathway may play in these neurons. We show here that activation of the Shh signaling pathway stimulates axon elongation in rat hippocampal neurons. This Shh-induced effect depends on the pathway transducer Smoothened (Smo) and the transcription factor Gli1. The axon itself does not respond directly to Shh; instead, the Shh signal transduction originates from the somatodendritic region of the neurons and occurs in neurons with and without detectable primary cilia. Upon Shh stimulation, Smo localization to dendrites increases significantly. Shh pathway activation results in increased levels of profilin1 (Pfn1), an actin-binding protein. Mutations in Pfn1's actin-binding sites or reduction of Pfn1 eliminate the Shh-induced axon elongation. These findings indicate that Shh can regulate axon growth, which may be critical for development of hippocampal neurons. SIGNIFICANCE STATEMENT: Although numerous signaling mechanisms have been identified that act directly on axons to regulate their outgrowth, it is not known whether signals transduced in dendrites may also affect axon outgrowth. We describe here a transcellular signaling pathway in embryonic hippocampal neurons in which activation of Sonic Hedgehog (Shh) receptors in dendrites stimulates axon growth. The pathway involves the dendritic-membrane-associated Shh signal transducer Smoothened (Smo) and the transcription factor Gli, which induces the expression of the gene encoding the actin-binding protein profilin 1. Our findings suggest scenarios in which stimulation of Shh in dendrites results in accelerated outgrowth of the axon, which therefore reaches its presumptive postsynaptic target cell more quickly. By this mechanism, Shh may play critical roles in the development of hippocampal neuronal circuits.
Asunto(s)
Axones/fisiología , Dendritas/metabolismo , Proteínas Hedgehog/metabolismo , Hipocampo/citología , Neuronas/citología , Transducción de Señal/fisiología , Animales , Axones/ultraestructura , Células Cultivadas , Dendritas/efectos de los fármacos , Dendritas/ultraestructura , Embrión de Mamíferos , Regulación de la Expresión Génica/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/farmacología , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Neuronas/efectos de los fármacos , Profilinas/genética , Profilinas/metabolismo , Transporte de Proteínas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/genética , Receptor Smoothened , Factores de Tiempo , Proteína con Dedos de Zinc GLI1RESUMEN
New work identifies components of the abscission checkpoint that prevent premature severing of the bridge connecting cells at the end of cell division. Kinase activities allow the membrane remodeling machinery to take their mark, but prevent them from leaving the starting block.
Asunto(s)
Citocinesis , Proteínas Oncogénicas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , HumanosRESUMEN
Membrane association with mother centriole (M-centriole) distal appendages is critical for ciliogenesis initiation. How the Rab GTPase Rab11-Rab8 cascade functions in early ciliary membrane assembly is unknown. Here, we show that the membrane shaping proteins EHD1 and EHD3, in association with the Rab11-Rab8 cascade, function in early ciliogenesis. EHD1 and EHD3 localize to preciliary membranes and the ciliary pocket. EHD-dependent membrane tubulation is essential for ciliary vesicle formation from smaller distal appendage vesicles (DAVs). Importantly, this step functions in M-centriole to basal body transformation and recruitment of transition zone proteins and IFT20. SNAP29, a SNARE membrane fusion regulator and EHD1-binding protein, is also required for DAV-mediated ciliary vesicle assembly. Interestingly, only after ciliary vesicle assembly is Rab8 activated for ciliary growth. Our studies uncover molecular mechanisms informing a previously uncharacterized ciliogenesis step, whereby EHD1 and EHD3 reorganize the M-centriole and associated DAVs before coordinated ciliary membrane and axoneme growth.
Asunto(s)
Proteínas Portadoras/genética , Cilios/metabolismo , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular/genética , Animales , Axonema/metabolismo , Axonema/ultraestructura , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Línea Celular , Centriolos/metabolismo , Centriolos/ultraestructura , Cilios/ultraestructura , Embrión no Mamífero , Células Epiteliales/ultraestructura , Humanos , Túbulos Renales Colectores/metabolismo , Túbulos Renales Colectores/ultraestructura , Ratones , Morfogénesis/genética , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/ultraestructura , Transducción de Señal , Vesículas Transportadoras/ultraestructura , Proteínas de Transporte Vesicular/antagonistas & inhibidores , Proteínas de Transporte Vesicular/metabolismo , Pez Cebra , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.
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Cilios/fisiología , Movimiento , Animales , Centrosoma/fisiología , Perros , Células de Riñón Canino Madin Darby , Microscopía ElectrónicaRESUMEN
How adherent and contractile systems coordinate to promote cell shape changes is unclear. Here, we define a counterbalanced adhesion/contraction model for cell shape control. Live-cell microscopy data showed a crucial role for a contractile meshwork at the top of the cell, which is composed of actin arcs and myosin IIA filaments. The contractile actin meshwork is organized like muscle sarcomeres, with repeating myosin II filaments separated by the actin bundling protein α-actinin, and is mechanically coupled to noncontractile dorsal actin fibers that run from top to bottom in the cell. When the meshwork contracts, it pulls the dorsal fibers away from the substrate. This pulling force is counterbalanced by the dorsal fibers' attachment to focal adhesions, causing the fibers to bend downward and flattening the cell. This model is likely to be relevant for understanding how cells configure themselves to complex surfaces, protrude into tight spaces, and generate three-dimensional forces on the growth substrate under both healthy and diseased conditions.
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Citoesqueleto de Actina/metabolismo , Adhesión Celular , Movimiento Celular , Forma de la Célula , Adhesiones Focales/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Mecanotransducción Celular , Modelos Biológicos , Miosina Tipo IIA no Muscular/genética , Miosina Tipo IIA no Muscular/metabolismo , Presión , Factores de Tiempo , TransfecciónRESUMEN
The final cleavage event that terminates cell division, abscission of the small, dense intercellular bridge, has been particularly challenging to resolve. Here, we describe imaging innovations that helped answer long-standing questions about the mechanism of abscission. We further explain how computational modeling of high-resolution data was employed to test hypotheses and generate additional insights. We present the model that emerges from application of these complimentary approaches. Similar experimental strategies will undoubtedly reveal exciting details about other underresolved cellular structures.