RESUMO
Equal cell division relies upon astral microtubule-based centering mechanisms, yet how the interplay between mitotic entry, cortical force generation and long astral microtubules leads to symmetric cell division is not resolved. We report that a cortically located sperm aster displaying long astral microtubules that penetrate the whole zygote does not undergo centration until mitotic entry. At mitotic entry, we find that microtubule-based cortical pulling is lost. Quantitative measurements of cortical pulling and cytoplasmic pulling together with physical simulations suggested that a wavelike loss of cortical pulling at mitotic entry leads to aster centration based on cytoplasmic pulling. Cortical actin is lost from the cortex at mitotic entry coincident with a fall in cortical tension from â¼300pN/µm to â¼100pN/µm. Following the loss of cortical force generators at mitotic entry, long microtubule-based cytoplasmic pulling is sufficient to displace the aster towards the cell center. These data reveal how mitotic aster centration is coordinated with mitotic entry in chordate zygotes.
Assuntos
Sêmen , Fuso Acromático , Masculino , Humanos , Microtúbulos , Citoplasma , Divisão CelularRESUMO
The spindle assembly checkpoint (SAC) is a surveillance system that preserves genome integrity by delaying anaphase onset until all chromosomes are correctly attached to spindle microtubules. Recruitment of SAC proteins to unattached kinetochores generates an inhibitory signal that prolongs mitotic duration. Chordate embryos are atypical in that spindle defects do not delay mitotic progression during early development, implying that either the SAC is inactive or the cell-cycle target machinery is unresponsive. Here, we show that in embryos of the chordate Phallusia mammillata, the SAC delays mitotic progression from the 8th cleavage divisions. Unattached kinetochores are not recognized by the SAC machinery until the 7th cell cycle, when the SAC is acquired. After acquisition, SAC strength, which manifests as the degree of mitotic lengthening induced by spindle perturbations, is specific to different cell types and is modulated by cell size, showing similarity to SAC control in early Caenorhabditis elegans embryos. We conclude that SAC acquisition is a process that is likely specific to chordate embryos, while modulation of SAC efficiency in SAC proficient stages depends on cell fate and cell size, which is similar to non-chordate embryos.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Fuso Acromático , Animais , Fuso Acromático/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Caenorhabditis elegans/metabolismo , Tamanho Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Radial microtubule (MT) arrays or asters determine cell geometry in animal cells. Multiple asters interacting with motors, such as those in syncytia, form intracellular patterns, but the mechanical principles behind this are not clear. Here, we report that oocytes of the marine ascidian Phallusia mammillata treated with the drug BI-D1870 spontaneously form cytoplasmic MT asters, or cytasters. These asters form steady state segregation patterns in a shell just under the membrane. Cytaster centers tessellate the oocyte cytoplasm, that is divide it into polygonal structures, dominated by hexagons, in a kinesin-5-dependent manner, while inter-aster MTs form 'mini-spindles'. A computational model of multiple asters interacting with kinesin-5 can reproduce both tessellation patterns and mini-spindles in a manner specific to the number of MTs per aster, MT lengths and kinesin-5 density. Simulations predict that the hexagonal tessellation patterns scale with increasing cell size, when the packing fraction of asters in cells is â¼1.6. This self-organized in vivo tessellation by cytasters is comparable to the 'circle packing problem', suggesting that there is an intrinsic mechanical pattern-forming module that is potentially relevant to understanding the role of collective mechanics of cytoskeletal elements in embryogenesis. This article has an associated First Person interview with the first author of the paper.
Assuntos
Cinesinas , Microtúbulos , Animais , Tamanho Celular , Citoplasma , OócitosRESUMO
Spermatozoa of externally fertilizing freshwater fish possess several different modes of motility activation. Spermatozoa of common carp (Cyprinus carpio L.) are activated by hypoosmolality, whereas spermatozoa of sterlet (Acipenser ruthenus) require Ca2+ and low concentration of K+ for motility activation. Intracellular signaling differs between these two species as well, particularly in terms of utilization of secondary messengers (cAMP and Ca2+), and kinase activities. The current study was performed in order to determine the importance of protein phosphorylation and protein kinases for activation of sperm motility in carp and sterlet. Treatment with kinase inhibitors indicates that protein kinases A and C (PKA and PKC) participate in spermatozoa motility of both species. Immunodetection of phospho-(Ser/Thr) PKA substrates shows that phosphorylated proteins are localized differently in spermatozoa of carp and sterlet. Strong phosphorylation of PKC substrate was observed in flagella of sterlet spermatozoa, whereas in carp sperm, PKC substrates were lightly phosphorylated in the midpiece and flagella. Motility activation induced either phosphorylation or dephosphorylation on serine, threonine and tyrosine residues of numerous proteins in carp and sterlet spermatozoa. Proteomic methods were used to identify proteins whose phosphorylation state changes upon the initiation of sperm motility. Numerous mitochondrial and glycolytic enzymes were identified in spermatozoa of both species, as well as axonemal proteins, heat shock proteins, septins and calcium-binding proteins. Our results contribute to an understanding of the roles of signaling molecules, protein kinases and protein phosphorylation in motility activation and regulation of two valuable fish species, C. carpio and A. ruthenus.
Assuntos
Carpas , Proteínas de Peixes/metabolismo , Peixes , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismo , Animais , Carpas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peixes/metabolismo , Masculino , Fosforilação , Proteína Quinase C/metabolismo , Proteômica , Transdução de SinaisRESUMO
During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms.
Assuntos
Microtúbulos , Fuso Acromático , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Ciclo Celular , Divisão Celular , Cromossomos/metabolismo , Tubulina (Proteína)/metabolismo , MitoseRESUMO
Mitotic spindle orientation with respect to cortical polarity cues generates molecularly distinct daughter cells during asymmetric cell division (ACD). However, during ACD it remains unknown how the orientation of the mitotic spindle is regulated by cortical polarity cues until furrowing begins. In ascidians, the cortical centrosome-attracting body (CAB) generates three successive unequal cleavages and the asymmetric segregation of 40 localized postplasmic/PEM RNAs in germ cell precursors from the 8-64 cell stage. By combining fast 4D confocal fluorescence imaging with gene-silencing and classical blastomere isolation experiments, we show that spindle repositioning mechanisms are active from prometaphase until anaphase, when furrowing is initiated in B5.2 cells. We show that the vegetal-most spindle pole/centrosome is attracted towards the CAB during prometaphase, causing the spindle to position asymmetrically near the cortex. Next, during anaphase, the opposite spindle pole/centrosome is attracted towards the border with neighbouring B5.1 blastomeres, causing the spindle to rotate (10 degrees /minute) and migrate (3 microm/minute). Dynamic 4D fluorescence imaging of filamentous actin and plasma membrane shows that precise orientation of the cleavage furrow is determined by this second phase of rotational spindle displacement. Furthermore, in pairs of isolated B5.2 blastomeres, the second phase of rotational spindle displacement was lost. Finally, knockdown of PEM1, a protein localized in the CAB and required for unequal cleavage in B5.2 cells, completely randomizes spindle orientation. Together these data show that two separate mechanisms active during mitosis are responsible for spindle positioning, leading to precise orientation of the cleavage furrow during ACD in the cells that give rise to the germ lineage in ascidians.
Assuntos
Blastômeros/metabolismo , Centrossomo/metabolismo , Citoesqueleto/metabolismo , Fuso Acromático/metabolismo , Urocordados/metabolismo , Actinas/genética , Actinas/metabolismo , Anáfase , Animais , Blastômeros/citologia , Ciclo Celular/genética , Divisão Celular , Citoesqueleto/genética , Células Germinativas/metabolismo , Mitose , Prometáfase , Proteínas/genética , Proteínas/metabolismo , Fuso Acromático/genética , Urocordados/citologiaRESUMO
Ascidian postplasmic/PEM RNAs constitute a large class of cortical maternal RNAs which include developmental determinants (macho-1 and pem-1). We have analyzed the localization, cortical anchorage and cell type segregation of postplasmic/PEM RNAs in Ciona intestinalis and Phallusia mammillata using very high-resolution fluorescent in situ hybridization. We also compared RNAs extracted from whole oocytes and from isolated cortices using microarrays and localized RNAs possessing clusters of xCACx motifs in their 3'UTRs. Based on these combined approaches we conclude that: (1) the vast majority of the 39 postplasmic/PEM RNAs (including vasa) are localized in the egg cortex. (2) Many postplasmic/PEM RNAs 3'UTR are enriched in xCACx motifs, allowing us to identify 2 novel postplasmic/PEM RNAs (PSD and MnK). (3) Postplasmic/PEM RNAs anchored to cortical Endoplasmic Reticulum (cER) and those associated with granules have different cell destinations. We propose that there are 2 distinct categories of postplasmic/PEM RNAs on the basis of their cortical anchorages and cell destinations: (1) macho-1-like postplasmic/PEM RNAs anchored to cER which segregate into somatic B8.11 cells. (2) vasa-like postplasmic/PEM RNAs associated with granules which in addition to B8.11 cells segregate into B8.12 germ cells.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oócitos/metabolismo , RNA Mensageiro/genética , Urocordados/genética , Regiões 3' não Traduzidas/genética , Animais , Linhagem da Célula/genética , Ciona intestinalis/citologia , Ciona intestinalis/embriologia , Ciona intestinalis/genética , Clonagem Molecular , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Perfilação da Expressão Gênica , Hibridização in Situ Fluorescente , Larva/citologia , Larva/genética , Microscopia Confocal , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Oócitos/citologia , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Urocordados/citologia , Urocordados/embriologiaRESUMO
Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. Using ascidians, we observed that PB1 becomes tethered to the fertilized egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small structure around 1 µm in diameter protruded from the cortical outpocket that will form the future PB2, which we define as the "polar corps". As emission of PB2 progressed, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 outpocket was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in fertilized eggs that had PB1 removed suggested that the midbody remnant remained within the fertilized egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 since our data are correlative in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in several species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.
Assuntos
Meiose/fisiologia , Corpos Polares/fisiologia , Actinas/metabolismo , Animais , Bivalves/metabolismo , Bivalves/fisiologia , Cromatina/metabolismo , Cromatina/fisiologia , Cromossomos/metabolismo , Cromossomos/fisiologia , Citocinese/fisiologia , Oócitos/metabolismo , Oócitos/fisiologia , Corpos Polares/metabolismo , Fuso Acromático/metabolismo , Fuso Acromático/fisiologia , Urocordados/metabolismo , Urocordados/fisiologia , Zigoto/metabolismo , Zigoto/fisiologiaRESUMO
Global tissue tension anisotropy has been shown to trigger stereotypical cell division orientation by elongating mitotic cells along the main tension axis. Yet, how tissue tension elongates mitotic cells despite those cells undergoing mitotic rounding (MR) by globally upregulating cortical actomyosin tension remains unclear. We addressed this question by taking advantage of ascidian embryos, consisting of a small number of interphasic and mitotic blastomeres and displaying an invariant division pattern. We found that blastomeres undergo MR by locally relaxing cortical tension at their apex, thereby allowing extrinsic pulling forces from neighboring interphasic blastomeres to polarize their shape and thus division orientation. Consistently, interfering with extrinsic forces by reducing the contractility of interphasic blastomeres or disrupting the establishment of asynchronous mitotic domains leads to aberrant mitotic cell division orientations. Thus, apical relaxation during MR constitutes a key mechanism by which tissue tension anisotropy controls stereotypical cell division orientation.
Assuntos
Blastômeros/citologia , Forma Celular , Mitose , Estresse Mecânico , Animais , Modelos Teóricos , UrocordadosRESUMO
In eukaryotic cells, a spindle assembly checkpoint (SAC) ensures accurate chromosome segregation, by monitoring proper attachment of chromosomes to spindle microtubules and delaying mitotic progression if connections are erroneous or absent. The SAC is thought to be relaxed during early embryonic development. Here, we evaluate the checkpoint response to lack of kinetochore-spindle microtubule interactions in early embryos of diverse animal species. Our analysis shows that there are two classes of embryos, either proficient or deficient for SAC activation during cleavage. Sea urchins, mussels, and jellyfish embryos show a prolonged delay in mitotic progression in the absence of spindle microtubules from the first cleavage division, while ascidian and amphioxus embryos, like those of Xenopus and zebrafish, continue mitotic cycling without delay. SAC competence during early development shows no correlation with cell size, chromosome number, or kinetochore to cell volume ratio. We show that SAC proteins Mad1, Mad2, and Mps1 lack the ability to recognize unattached kinetochores in ascidian embryos, indicating that SAC signaling is not diluted but rather actively silenced during early chordate development.
Assuntos
Invertebrados/embriologia , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Fuso Acromático/metabolismo , Animais , Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Embrião não Mamífero/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/fisiologia , Nocodazol/farmacologia , Transdução de Sinais/fisiologiaRESUMO
Cells are arranged into species-specific patterns during early embryogenesis. Such cell division patterns are important since they often reflect the distribution of localized cortical factors from eggs/fertilized eggs to specific cells as well as the emergence of organismal form. However, it has proven difficult to reveal the mechanisms that underlie the emergence of cell positioning patterns that underlie embryonic shape, likely because a systems-level approach is required that integrates cell biological, genetic, developmental, and mechanical parameters. The choice of organism to address such questions is also important. Because ascidians display the most extreme form of invariant cleavage pattern among the metazoans, we have been analyzing the cell biological mechanisms that underpin three aspects of cell division (unequal cell division (UCD), oriented cell division (OCD), and asynchronous cell cycles) which affect the overall shape of the blastula-stage ascidian embryo composed of 64 cells. In ascidians, UCD creates two small cells at the 16-cell stage that in turn undergo two further successive rounds of UCD. Starting at the 16-cell stage, the cell cycle becomes asynchronous, whereby the vegetal half divides before the animal half, thus creating 24-, 32-, 44-, and then 64-cell stages. Perturbing either UCD or the alternate cell division rhythm perturbs cell position. We propose that dynamic cell shape changes propagate throughout the embryo via cell-cell contacts to create the ascidian-specific invariant cleavage pattern.
Assuntos
Padronização Corporal , Divisão Celular , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Urocordados/citologia , Urocordados/embriologia , Animais , FertilizaçãoRESUMO
Through their coordinated alignment and beating, motile cilia generate directional fluid flow and organismal movement. While the mechanisms used by multiciliated epithelial tissues to achieve this coordination have been widely studied, much less is known about regulation of monociliated tissues such as those found in the vertebrate node and swimming planktonic larvae. Here, we show that a calcium sensor protein associated with outer arm dynein, calaxin, is a critical regulator for the coordinated movements of monocilia. Knockdown of calaxin gene in sea urchin embryos results in uncoordinated ciliary beating and defective directional movement of the embryos, but no apparent abnormality in axoneme ultrastructure. Examination of the beating cycle of individual calaxin-deficient cilia revealed a marked effect on the waveform and spatial range of ciliary bending. These findings indicate that calaxin-mediated regulation of ciliary beating is responsible for proper basal body orientation and ciliary alignment in fields of monociliated cells.
Assuntos
Cílios/fisiologia , Dineínas/metabolismo , Ouriços-do-Mar/fisiologia , Animais , Axonema/ultraestrutura , Corpos Basais , Cílios/genética , Cílios/metabolismo , Dineínas/genética , Embrião não Mamífero/fisiologia , Movimento , Orientação Espacial , Ouriços-do-Mar/embriologia , Ouriços-do-Mar/genéticaRESUMO
Asymmetric positioning of the mitotic spindle is a fundamental process responsible for creating sibling cell size asymmetry; however, how the cortex causes the depolymerization of astral microtubules during asymmetric spindle positioning has remained elusive. Early ascidian embryos possess a large cortical subdomain of endoplasmic reticulum (ER) that causes asymmetric spindle positioning driving unequal cell division. Here we show that the microtubule depolymerase Kif2 localizes to this subdomain of cortical ER. Rapid live-cell imaging reveals that microtubules are less abundant in the subdomain of cortical ER. Inhibition of Kif2 function prevents the development of mitotic aster asymmetry and spindle pole movement towards the subdomain of cortical ER, whereas locally increasing microtubule depolymerization causes exaggerated asymmetric spindle positioning. This study shows that the microtubule depolymerase Kif2 is localized to a cortical subdomain of endoplasmic reticulum that is involved in asymmetric spindle positioning during unequal cell division.Early ascidian embryos have a cortical subdomain of endoplasmic reticulum (ER) that controls asymmetric spindle positioning driving unequal cell division. Here the authors show that the microtubule depolymerase Kif2 is localized to a cortical subdomain of the ER that is involved in asymmetric spindle positioning.
Assuntos
Retículo Endoplasmático/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Urocordados/metabolismo , Animais , Divisão Celular Assimétrica , Ciona intestinalis/citologia , Ciona intestinalis/embriologia , Ciona intestinalis/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Microscopia Confocal , Imagem com Lapso de Tempo/métodos , Urocordados/citologia , Urocordados/embriologiaRESUMO
Until recently the set of "model" species used commonly for cell biology was limited to a small number of well-understood organisms, and developing a new model was prohibitively expensive or time-consuming. With the current rapid advances in technology, in particular low-cost high-throughput sequencing, it is now possible to develop molecular resources fairly rapidly. Wider sampling of biological diversity can only accelerate progress in addressing cellular mechanisms and shed light on how they are adapted to varied physiological contexts. Here we illustrate how historical knowledge and new technologies can reveal the potential of nonconventional organisms, and we suggest guidelines for selecting new experimental models. We also present examples of nonstandard marine metazoan model species that have made important contributions to our understanding of biological processes.
Assuntos
Biologia Celular , Modelos Animais , AnimaisRESUMO
During embryonic development and maternal meiotic maturation, positioning of the mitotic/meiotic spindle is subject to control mechanisms that meet the needs of the particular cell type. Here we review the methods, molecular tools, and the ascidian model we use to study three different ways in which centrosomes or spindles are positioned in three different cellular contexts. First, we review unequal cleavage in the ascidian germ lineage. In the germ cell precursors, a large macromolecular structure termed the centrosome-attracting body causes three successive rounds of unequal cleavage from the 8- to the 64-cell stage. Next, we discuss spindle positioning underlying the invariant cleavage pattern. Ascidian embryos display an invariant cleavage pattern whereby the mitotic spindle aligns in a predetermined orientation in every blastomere up to the gastrula stage (composed of 112 cells). Finally, we review methods and approaches to study meiotic spindle positioning in eggs.
Assuntos
Centrossomo/ultraestrutura , Fuso Acromático/ultraestrutura , Animais , Embrião não Mamífero/ultraestrutura , Masculino , Microscopia de Fluorescência , Óvulo/ultraestrutura , Urocordados/ultraestruturaRESUMO
In an accompanying article (C. Sardet et al. m/s 2004; 20 : 414-423) we reviewed determinants of polarity in early development and the mechanisms which regulate their localization and expression. Such determinants have for the moment been identified in only a few species: the insect Drosophila melanogaster, the worm Caenorhabditis elegans, the frog Xenopus laevis and the ascidians Ciona intestinalis and Holocynthia roretzi. Although oogenesis, fertilization, and cell divisions in these embryos differ considerably, with respect to early polarities certain common themes emerge, such as the importance of cortical mRNAs, the PAR polarity proteins, and reorganizations mediated by the cytoskeleton. Here we highlight similarities and differences in axis establishment between these species, describing them in a chronological order from oocyte to gastrula, and add two more classical model organisms, sea urchin and mouse, to complete the comparisons depicted in the form of a Poster which can be downloaded from the site http://biodev.obs-vlfr.fr/biomarcell.
Assuntos
Padronização Corporal/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Gástrula/fisiologia , Anfíbios , Animais , Drosophila/embriologia , Ectoderma/fisiologia , Embrião não Mamífero , Endoderma/fisiologia , Camundongos , Modelos Animais , FilogeniaRESUMO
Embryonic development depends on the establishment of polarities which define the axial characteristics of the body. In a small number of cases such as the embryo of the fly drosophila, developmental axes are established well before fertilization while in other organisms such as the nematode worm C. elegans these axes are set up only after fertilization. In most organisms the egg posesses a primary (A-V, Animal-Vegetal) axis acquired during oogenesis which participates in the establishment of the embryonic axes. Such is the case for the eggs of ascidians or the frog Xenopus whose AV axes are remodelled by sperm entry to yield the embryonic axes. Embryos of different species thus acquire an anterior end and a posterior end (Antero-Posterior, A-P axis), dorsal and ventral sides (D-V axis) and then a left and a right side.
Assuntos
Padronização Corporal , Polaridade Celular , Embrião de Mamíferos/ultraestrutura , Oócitos/citologia , Animais , Padronização Corporal/fisiologia , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Sinalização do Cálcio , Proteínas de Ciclo Celular/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Morfogênese , Oogênese , RNA Mensageiro/fisiologia , Urocordados/citologia , Urocordados/embriologia , Xenopus laevis/embriologia , Zigoto/ultraestruturaRESUMO
Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates). The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.
Assuntos
Chaperonina 60/metabolismo , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Óvulo/metabolismo , Urocordados/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/metabolismo , Anticorpos Monoclonais/metabolismo , Chaperonina 60/genética , Chaperonina 60/isolamento & purificação , Cromatografia por Troca Iônica , Eletroforese em Gel Bidimensional , Embrião não Mamífero/citologia , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Immunoblotting , Hibridização In Situ , Masculino , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/isolamento & purificação , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/isolamento & purificação , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos , Urocordados/embriologia , Urocordados/genéticaRESUMO
The completely transparent eggs and embryos of the ascidian Phallusia mammillata are well suited for imaging-based studies of how cell cycle control mechanisms have been integrated into the processes of meiosis, fertilization, and embryonic development. Several cell cycle-related issues that pertain to reproduction and development have been addressed using the ascidian model. For example, how are sperm-triggered calcium oscillations controlled by cell cycle kinases? How is chromosome segregation during meiosis regulated? What processes does the Mos/MAPK signaling cascade control in eggs in addition to CSF-mediated cell cycle arrest? Following fertilization ascidians blastomeres display cell cycle asynchrony, oriented cell division, and unequal cleavage resulting in the formation of a distinctive gastrula composed of precisely 112 cells. Here, we shall summarize these topics and where possible show how the mechanisms identified in ascidians compare to those identified in other organisms.
Assuntos
Pontos de Checagem do Ciclo Celular , Fase de Clivagem do Zigoto/citologia , Embrião não Mamífero/citologia , Oócitos/citologia , Urocordados/citologia , Urocordados/embriologia , Animais , Fase de Clivagem do Zigoto/metabolismo , Embrião não Mamífero/metabolismo , Fuso Acromático/metabolismoRESUMO
In several species, axis formation and tissue differentiation are the result of developmental cascades which begin with the localization and translation of key maternal mRNAs in eggs. Localization and anchoring of mRNAs to cortical structures can be observed with high sensitivity and resolution by fluorescent in situ hybridization coupled with labeling of membranes and macromolecular complexes. Oocytes and embryos of ascidians--marine chordates closely related to vertebrates--are ideal models to understand how maternal mRNAs pattern the simple ascidian tadpole. More than three dozen cortically localized maternal mRNAs have been identified in ascidian eggs. They include germ cell markers such as vasa or pem-3 and determinants of axis (pem-1), unequal cleavage (pem-1), and muscle cells (macho-1). High resolution localization of mRNAs, proteins, and lipids in whole eggs and embryos and their cortical fragments shows that maternal mRNA determinants pem-1 and macho-1 are anchored to cortical endoplasmic reticulum and segregate with it into small posterior somatic cells. In contrast, mRNAs such as vasa are associated with granular structures which are inherited by the same somatic cells plus adjacent germ cell precursors. In this chapter, we provide detailed protocols for simultaneous localization of mRNAs and proteins to determine their association with cellular structures in eggs and embryos. Using preparations of isolated cortical fragments with intact membranous structures allows unprecedented high resolution analysis and identification of cellular anchoring sites for key mRNAs. This information is necessary for understanding the mechanisms for localizing mRNAs and partitioning them into daughter cells after cleavage.