RESUMO
The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell.
Assuntos
Células Acinares/ultraestrutura , Encéfalo/citologia , Retículo Endoplasmático/química , Retículo Endoplasmático/ultraestrutura , Neurônios/ultraestrutura , Glândula Parótida/citologia , Células Acinares/química , Células Acinares/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Modelos Biológicos , Neurônios/química , Neurônios/metabolismoRESUMO
This review highlights the complex membrane architectures and organelles observed along the renal tubular segments through careful review of ultrastructural and physiological studies published over the past several decades. We also showcase the vital role(s) played by the actin cytoskeleton and actin associated myosin motor proteins in regulating cell type-specific physiological functions within cells of the renal epithelium. The purpose of this review is to provide a fresh conceptual framework to explain the structure-function relationships that exist between the actin cytoskeleton, organelle structure, and cargo transport within the mammalian kidney. We believe that with recent advances in technologies to visualize the actin cytoskeleton and associated proteins within intact kidneys, it is imperative to reimagine the functional role(s) for these proteins in situ, which will provide a rationale for their unique, cell type specific function(s), necessary to build and maintain complex physiological processes.
RESUMO
Gap junctions have well-established roles in cell-cell communication by way of forming permeable intercellular channels. Less is understood about their internalization, which forms double membrane vesicles containing cytosol and membranes from another cell called connexosomes or annular gap junctions. Here, we systematically investigated the fate of connexosomes in intact ovarian follicles. High-pressure frozen, serial-sectioned tissue was immunogold labeled for connexin 43 (Cx43, also known as GJA1). Within a volume corresponding to â¼35 cells, every labeled structure was categorized and had its surface area measured. Measurements support the concept that multiple connexosomes form from larger invaginated gap junctions. Subsequently, the inner and outer membranes separate, Cx43 immunogenicity is lost from the outer membrane, and the inner membrane appears to undergo fission. One pathway for processing involves lysosomes, based on localization of cathepsin B to some processed connexosomes. In summary, this study demonstrates new technology for high-resolution analyses of gap junction processing.This article has an associated First Person interview with the first author of the paper.
Assuntos
Comunicação Celular , Junções Comunicantes , Feminino , Humanos , Lisossomos , Microscopia Eletrônica , Microscopia Imunoeletrônica , Folículo OvarianoRESUMO
Throughout the male reproductive lifespan, spermatogonial stem cells (SSCs) produce committed progenitors that proliferate and then remain physically connected in growing clones via short cylindrical intercellular bridges (ICBs). These ICBs, which enlarge in meiotic spermatocytes, have been demonstrated to provide a conduit for postmeiotic haploid spermatids to share sex chromosome-derived gene products. In addition to ICBs, spermatogonia exhibit multiple thin cytoplasmic projections. Here, we have explored the nature of these projections in mice and find that they are dynamic, span considerable distances from their cell body (≥25â µm), either terminate or physically connect multiple adjacent spermatogonia, and allow for sharing of macromolecules. Our results extend the current model that subsets of spermatogonia exist as isolated cells or clones, and support a model in which spermatogonia of similar developmental fates are functionally connected through a shared dynamic cytoplasm mediated by thin cytoplasmic projections.
Assuntos
Citoplasma/metabolismo , Mamíferos/metabolismo , Espermatogônias/metabolismo , Animais , Diferenciação Celular , Citoplasma/ultraestrutura , Difusão , Proteínas de Fluorescência Verde/metabolismo , Espaço Intracelular/metabolismo , Substâncias Macromoleculares/metabolismo , Masculino , Meiose , Camundongos Transgênicos , Papio , Ratos , Espermatócitos/citologia , Espermatócitos/metabolismo , Espermatogônias/citologia , Espermatogônias/ultraestruturaRESUMO
The Wnt family of secreted proteins has been proposed to play a conserved role in early specification of the bilaterian anteroposterior (A/P) axis. This hypothesis is based predominantly on data from vertebrate embryogenesis as well as planarian regeneration and homeostasis, indicating that canonical Wnt (cWnt) signaling endows cells with positional information along the A/P axis. Outside of these phyla, there is strong support for a conserved role of cWnt signaling in the repression of anterior fates, but little comparative support for a conserved role in promotion of posterior fates. We further test the hypothesis by investigating the role of cWnt signaling during early patterning along the A/P axis of the hemichordate Saccoglossus kowalevskii. We have cloned and investigated the expression of the complete Wnt ligand and Frizzled receptor complement of S. kowalevskii during early development along with many secreted Wnt modifiers. Eleven of the 13 Wnt ligands are ectodermally expressed in overlapping domains, predominantly in the posterior, and Wnt antagonists are localized predominantly to the anterior ectoderm in a pattern reminiscent of their distribution in vertebrate embryos. Overexpression and knockdown experiments, in combination with embryological manipulations, establish the importance of cWnt signaling for repression of anterior fates and activation of mid-axial ectodermal fates during the early development of S. kowalevskii. However, surprisingly, terminal posterior fates, defined by posterior Hox genes, are unresponsive to manipulation of cWnt levels during the early establishment of the A/P axis at late blastula and early gastrula. We establish experimental support for a conserved role of Wnt signaling in the early specification of the A/P axis during deuterostome body plan diversification, and further build support for an ancestral role of this pathway in early evolution of the bilaterian A/P axis. We find strong support for a role of cWnt in suppression of anterior fates and promotion of mid-axial fates, but we find no evidence that cWnt signaling plays a role in the early specification of the most posterior axial fates in S. kowalevskii. This posterior autonomy may be a conserved feature of early deuterostome axis specification.
Assuntos
Linhagem da Célula/fisiologia , Desenvolvimento Embrionário/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Transporte Biológico , Padronização Corporal/fisiologia , Ectoderma , Receptores Frizzled/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Homeobox , Homeostase , Planárias , Poliquetos/embriologia , Poliquetos/fisiologiaRESUMO
The endoplasmic reticulum (ER) is an interconnected network of tubules and sheets. In most tissues of the body, ER tubules have a diameter of â¼60â nm. Using new methods for serial-section electron microscopy, a distinct class of very narrow, 20- to 30-nm-diameter tubules were found in neurons of both the central and peripheral nervous system. The narrow tubules appear to be the most abundant form of ER in axons, and are also found interspersed in the cell bodies and dendrites. At the site of branch points, there is a small sheet that has a similarly narrow lumen. The narrowness of the ER is likely to be important for the as yet poorly characterized functions of the axonal ER.
Assuntos
Axônios/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Neurônios/ultraestrutura , Animais , Dendritos/ultraestrutura , Camundongos , Microscopia EletrônicaRESUMO
Gap junction turnover occurs through the internalization of both of the plasma membranes of a gap junction plaque, forming a double membrane-enclosed vesicle, or connexosome. Phosphorylation has a key role in regulation, but further progress requires the ability to clearly distinguish gap junctions and connexosomes, and to precisely identify proteins associated with them. We examined, by using electron microscopy, serial sections of mouse preovulatory ovarian follicles that had been collected with an automated tape collecting ultramicrotome (ATUM). We found that connexosomes can form from adjacent cell bodies, from thin cell processes or from the same cell. By immunolabeling serial sections, we found that residue S368 of connexin 43 (also known as GJA1) is phosphorylated on gap junctions and connexosomes, whereas connexin 43 residue S262 is phosphorylated only on some connexosomes. These data suggest that phosphorylation at S262 contributes to connexosome formation or processing, and they provide more precise evidence that phosphorylation has a key role in gap junction internalization. Serial section electron microscopy of immunogold-labeled tissues offers a new way to investigate the three-dimensional organization of cells in their native environment.
Assuntos
Conexina 43/metabolismo , Microscopia Eletrônica/métodos , Animais , Feminino , Junções Comunicantes/metabolismo , Junções Comunicantes/ultraestrutura , Cavalos , Camundongos Endogâmicos C57BL , Fosforilação , Fosfosserina/metabolismo , Coloração e RotulagemRESUMO
FGFs act in vertebrate mesoderm induction and also play key roles in early mesoderm formation in ascidians and amphioxus. However, in sea urchins initial characterizations of FGF function do not support a role in early mesoderm induction, making the ancestral roles of FGF signaling and mechanisms of mesoderm specification in deuterostomes unclear. In order to better characterize the evolution of mesoderm formation, we have examined the role of FGF signaling during mesoderm development in Saccoglossus kowalevskii, an experimentally tractable representative of hemichordates. We report the expression of an FGF ligand, fgf8/17/18, in ectoderm overlying sites of mesoderm specification within the archenteron endomesoderm. Embryological experiments demonstrate that mesoderm induction in the archenteron requires contact with ectoderm, and loss-of-function experiments indicate that both FGF ligand and receptor are necessary for mesoderm specification. fgf8/17/18 gain-of-function experiments establish that FGF8/17/18 is sufficient to induce mesoderm in adjacent endomesoderm. These experiments suggest that FGF signaling is necessary from the earliest stages of mesoderm specification and is required for all mesoderm development. Furthermore, they suggest that the archenteron is competent to form mesoderm or endoderm, and that FGF signaling from the ectoderm defines the location and amount of mesoderm. When considered in a comparative context, these data support a phylogenetically broad requirement for FGF8/17/18 signaling in mesoderm specification and suggest that FGF signaling played an ancestral role in deuterostome mesoderm formation.
Assuntos
Cordados/embriologia , Fatores de Crescimento de Fibroblastos/fisiologia , Mesoderma/embriologia , Animais , Cordados/genética , Cordados/metabolismo , Ectoderma/embriologia , Ectoderma/metabolismo , Embrião não Mamífero , Indução Embrionária/genética , Indução Embrionária/fisiologia , Endoderma/embriologia , Endoderma/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Gastrulação/genética , Gastrulação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Modelos Biológicos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The canonical Wnt/ß-catenin pathway is a key regulator of body plan organization and axis formation in metazoans, being involved in germ layer specification, posterior growth and patterning of the anteroposterior axis. Results from animals spanning a wide phylogenetic range suggest that a unifying function of ß-catenin in metazoans is to define the posterior/vegetal part of the embryo. Although the specification of vegetal territories (endoderm) by ß-catenin has been demonstrated in distantly related animals (cnidarians, a protostome, echinoderms and ascidians), the definition of the posterior part of the embryo is well supported only for vertebrates and planarians. To gain insights into ß-catenin functions during deuterostome evolution, we have studied the early development of the direct developing hemichordate Saccoglossus kowalevskii. We show that the zygote is polarized after fertilization along the animal-vegetal axis by cytoplasmic rearrangements resembling the ascidian vegetal contraction. This early asymmetry is translated into nuclear accumulation of ß-catenin at the vegetal pole, which is necessary and sufficient to specify endomesoderm. We show that endomesoderm specification is crucial for anteroposterior axis establishment in the ectoderm. The endomesoderm secretes as yet unidentified signals that posteriorize the ectoderm, which would otherwise adopt an anterior fate. Our results point to a conserved function at the base of deuterostomes for ß-catenin in germ layer specification and to a causal link in the definition of the posterior part of the embryonic ectoderm by way of activating posteriorizing endomesodermal factors. Consequently, the definition of the vegetal and the posterior regions of the embryo by ß-catenin should be distinguished and carefully re-examined.
Assuntos
Cordados/embriologia , Endoderma , Mesoderma , Organizadores Embrionários , beta Catenina/fisiologia , Animais , Padronização Corporal , Filogenia , ZigotoRESUMO
Due to its proximity to the axon initial segment (AIS), the paranode of the first myelin segment can influence the threshold for action potentials and how a neuron participates in a neuronal circuit. Using serial section electron microscopy, we examined its three-dimensional (3D) organization in the ventral horn of the mouse spinal cord. The myelin loops of postnatal day 18 mice resemble those at the node of Ranvier. However, in 3-month-old mice, 13 of 22 para-AIS showed 4 types of alteration: (A) A cytoplasmic foot process, with ultrastructural characteristics of an astrocyte, was interposed between the axolemma and the myelin loops. (B) A thin extension of the inner tongue was present between the foot process and axolemma. (C) The foot process was absent. The inner tongue extension was a broad lamella from which a thin extension reached beyond the loops and spiraled around axon. (D) One set of loops was adjacent to the axon, and another was further back and underlain by compact myelin. We suggest that (A)-(C) are steps in a progression toward (D). In this progression, a glial process displaces the original loops, the inner tongue reactivates and extends beneath the foot process, then wraps around the axon to form a new set of loops. This is the first study of the 3D organization of myelin at the AIS and provides evidence for glia-mediated age-dependent remodeling at this critical region.
Assuntos
Segmento Inicial do Axônio , Bainha de Mielina , Camundongos , Animais , Bainha de Mielina/ultraestrutura , Axônios/ultraestrutura , Neurônios , Microscopia EletrônicaRESUMO
Mitochondria-ER contact sites (MERCS) are involved in energy homeostasis, redox and Ca2+ signaling, and inflammation. MERCS are heavily studied; however, little is known about their regulation during mitosis. Here, we show that MERCS expand during mitosis in three cell types using various approaches, including transmission electron microscopy, serial EM coupled to 3D reconstruction, and a split GFP MERCS marker. We further show enhanced Ca2+ transfer between the ER and mitochondria using either direct Ca2+ measurements or by quantifying the activity of Ca2+-dependent mitochondrial dehydrogenases. Collectively, our results support a lengthening of MERCS in mitosis that is associated with improved Ca2+ coupling between the two organelles. This augmented Ca2+ coupling could be important to support the increased energy needs of the cell during mitosis.
RESUMO
Craniometaphyseal dysplasia (CMD), a rare craniotubular disorder, occurs in an autosomal dominant (AD) or autosomal recessive (AR) form. CMD is characterized by hyperostosis of craniofacial bones and flaring metaphyses of long bones. Many patients with CMD suffer from neurological symptoms. To date, the pathogenesis of CMD is not fully understood. Treatment is limited to decompression surgery. Here, we report a knock in (KI) mouse model for AR CMD carrying a R239Q mutation in CX43. Cx43KI/KI mice replicate many features of AR CMD in craniofacial and long bones. In contrast to Cx43+/+ littermates, Cx43KI/KI mice exhibit periosteal bone deposition and increased osteoclast (OC) numbers in the endosteum of long bones, leading to an expanded bone marrow cavity and increased cortical bone thickness. Although formation of Cx43+/+ and Cx43KI/KI resting OCs are comparable, on bone chips the actively resorbing Cx43KI/KI OCs resorb less bone. Cortical bones of Cx43KI/KI mice have an increase in degenerating osteocytes and empty lacunae. Osteocyte dendrite formation is decreased with reduced expression levels of Fgf23, Sost, Tnf-α, IL-1ß, Esr1, Esr2, and a lower Rankl/Opg ratio. Female Cx43KI/KI mice display a more severe phenotype. Sexual dimorphism in bone becomes more evident as mice age. Our data show that the CX43R239Q mutation results in mislocalization of CX43 protein and impairment of gap junction and hemichannel activity. Different from CX43 ablation mouse models, the CX43R239Q mutation leads to the AR CMD-like phenotype in Cx43KI/KI mice not only by loss-of-function but also via a not yet revealed dominant function.
RESUMO
The maturation hormone 1-methyladenine (1-MA) causes meiotic resumption of prophase arrested immature starfish oocytes. Continuous exposure to > or = 0.5 microM 1-MA causes germinal vesicle breakdown (GVBD) in approximately 20 min, but oocytes pretreated for > 30 min with a subthreshold dose of 1-MA undergo GVBD much faster (approximately 10 min) when they are exposed to 1 microM 1-MA. Furthermore, a very low subthreshold 1-MA suffices to start the maturation process: oocytes exposed to 0.005 microM 1-MA for up to 10 min followed by 1 microM 1-MA is equivalent to continuous exposure to 1 microM 1-MA. These dose and timing relationships indicate that there is a two-stage dependence on 1-MA. A possible explanation for this dependence is that there are two processes involved: an initial process that is triggered by a low dose of 1-MA, and a second process that cannot start until the first process is completed and is stimulated by a higher dose of 1-MA. These subthreshold 1-MA effects on GVBD timing are not directly coupled to changes in calcium physiology that also occur during maturation. Subthreshold 1-MA was found to cause a transient accumulation of Cdc2/cyclin B into the nucleus. The two-stage dependence indicates that there are unsuspected features in this well-studied pathway leading to GVBD. In the animal, this hormone dependence may help to synchronize maturation throughout all parts of the ovary.
Assuntos
Adenina/análogos & derivados , Meiose/efeitos dos fármacos , Meiose/fisiologia , Oócitos/metabolismo , Estrelas-do-Mar/efeitos dos fármacos , Estrelas-do-Mar/metabolismo , Adenina/farmacologia , Animais , Proteína Quinase CDC2/metabolismo , Cálcio/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ciclina B/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Oócitos/citologiaRESUMO
Chromosome capture by microtubules is widely accepted as the universal mechanism of spindle assembly in dividing cells. However, the observed length of spindle microtubules and computer simulations of spindle assembly predict that chromosome capture is efficient in small cells, but may fail in cells with large nuclear volumes such as animal oocytes. Here we investigate chromosome congression during the first meiotic division in starfish oocytes. We show that microtubules are not sufficient for capturing chromosomes. Instead, chromosome congression requires actin polymerization. After nuclear envelope breakdown, we observe the formation of a filamentous actin mesh in the nuclear region, and find that contraction of this network delivers chromosomes to the microtubule spindle. We show that this mechanism is essential for preventing chromosome loss and aneuploidy of the egg--a leading cause of pregnancy loss and birth defects in humans.
Assuntos
Actinas/metabolismo , Núcleo Celular/metabolismo , Segregação de Cromossomos , Cromossomos/fisiologia , Meiose , Oócitos/metabolismo , Actinas/química , Animais , Transporte Biológico/efeitos dos fármacos , Biopolímeros/química , Biopolímeros/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Núcleo Celular/genética , Segregação de Cromossomos/efeitos dos fármacos , Cromossomos/efeitos dos fármacos , Microscopia Confocal , Microtúbulos/metabolismo , Nocodazol/farmacologia , Oócitos/citologia , Especificidade da Espécie , Estrelas-do-Mar , Tiazóis/farmacologia , TiazolidinasRESUMO
The Drosophila ovary is an exceptional model for studying cell-cell interactions in vivo. Cells communicate with each other in a highly coordinated manner. Accurate spatiotemporal regulation of cell-cell interaction is critical for the development of eggs. Ultrastructural analysis using electron microscopy (EM) permits the visualization of both cells and subcellular signaling structures with high resolution. Here we describe a method for the processing of intact fly ovaries by scanning electron microscopy (SEM).
Assuntos
Ovário/ultraestrutura , Animais , Comunicação Celular , Drosophila , Feminino , Ovário/citologiaRESUMO
Odontoblast processes are thin cytoplasmic projections that extend from the cell body at the periphery of the pulp toward the dentin-enamel junction. The odontoblast processes function in the secretion, assembly and mineralization of dentin during development, participate in mechanosensation, and aid in dentin repair in mature teeth. Because they are small and densely arranged, their three-dimensional organization is not well documented. To gain further insight into how odontoblast processes contribute to odontogenesis, we used serial section electron microscopy and three-dimensional reconstructions to examine these processes in the predentin region of mouse molars and incisors. In molars, the odontoblast processes are tubular with a diameter of ~1.8 µm. The odontoblast processes near the incisor tip are similarly shaped, but those midway between the tip and apex are shaped like plates. The plates are radially aligned and longitudinally oriented with respect to the growth axis of the incisor. The thickness of the plates is approximately the same as the diameter of molar odontoblast processes. The plates have an irregular edge; the average ratio of width (midway in the predentin) to thickness is 2.3 on the labial side and 3.6 on the lingual side. The plate geometry seems likely to be related to the continuous growth of the incisor and may provide a clue as to the mechanisms by which the odontoblast processes are involved in tooth development.
Assuntos
Dentinogênese/fisiologia , Incisivo/crescimento & desenvolvimento , Animais , Camundongos , Odontoblastos/fisiologia , Odontogênese/fisiologiaRESUMO
Cytonemes are specialized filopodia that mediate paracrine signaling in Drosophila and other animals. Studies using fluorescence confocal microscopy (CM) established their general paths, cell targets, and essential roles in signaling. To investigate details unresolvable by CM, we used high-pressure freezing and EM to visualize cytoneme structures, paths, contents, and contacts. We observed cytonemes previously seen by CM in the Drosophila wing imaginal disc system, including disc, tracheal air sac primordium (ASP), and myoblast cytonemes, and identified cytonemes extending into invaginations of target cells, and cytonemes connecting ASP cells and connecting myoblasts. Diameters of cytoneme shafts vary between repeating wide (206 ± 51.8 nm) and thin (55.9 ± 16.2 nm) segments. Actin, ribosomes, and membranous compartments are present throughout; rough ER and mitochondria are in wider proximal sections. These results reveal novel structural features of filopodia and provide a basis for understanding cytoneme cell biology and function.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Actinas/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Mioblastos/metabolismo , Pseudópodes/metabolismo , Transdução de Sinais/fisiologia , Asas de Animais/metabolismoRESUMO
The capillary network of the kidney glomerulus filters small molecules from the blood. The glomerular 3D structure should help to understand its function, but it is poorly characterized. We therefore devised a new approach in which an automated tape collecting microtome (ATUM) was used to collect 0.5 µm thick serial sections from fixed mouse kidneys. The sections were imaged by scanning electron microscopy at ~ 50 nm/pixel resolution. With this approach, 12 glomeruli were reconstructed at an x-y-z resolution ~ 10 × higher than that of paraffin sections. We found a previously undescribed no-cross zone between afferent and efferent branches on the vascular pole side; connections here would allow blood to exit without being adequately filtered. The capillary diameters throughout the glomerulus appeared to correspond with the amount of blood flow within them. The shortest path (minimum number of branches to travel from afferent to efferent arterioles) is relatively independent of glomerular size and is present primarily on the vascular pole size. This suggests that new branches and longer paths form on the urinary pole side. Network analysis indicates that the glomerular network does not form by repetitive longitudinal splitting of capillaries. Thus the 3D structure of the glomerular capillary network provides useful information with which to understand glomerular function. Other tissue structures in the body may benefit from this new three dimensional approach.