Cytoplasmic Rearrangements Research Articles

Functional topography of the fully grown human oocyte.

In vivo maturation (IVM) of human oocytes is a technique used to increase the number of usable oocytes for in vitro fertilization (IVF) and represents a necessity for women with different ovarian pathologies. During IVM the oocytes progress from the germinal vesicle stage (GV) through the metaphase II and during this journey both nuclear and cytoplasmic rearrangements must be obtained to increase the probability to get viable and healthy zygotes/embryos after IVF. As the successful clinical outcomes of this technique are a reality, we wanted to investigate the causes behind oocytes maturation arrest. For obvious ethical reasons, we were able to analyze only few human immature oocytes discarded and donated to research by transmission electron microscopy showing that, as in the mouse, they have different chromatin and cytoplasmic organizations both essential for further embryo development.

Ultrastructural characteristics of sheep oocytes during in vitro maturation (IVM)

The aim of this study was to describe the ultrastructure of sheep oocytes during 24h of in vitro maturation (IVM). Ovaries were collected from a slaughterhouse and the follicles aspirated. The selected cumulus oocyte complexes (COC's) were divided into groups and transferred to maturation medium, where they were maintained for 6, 12, 18 or 24h. Control immature oocytes were also collected (0h). After different maturation intervals the structures were removed and processed for transmission electron microscopy. Immature oocytes (0h) demonstrated pleomorphic mitochondria and presumptive lipid droplets as normally expected, and a moderate number of vesicles scattered throughout the cytoplasm. After 6h of IVM, there was a migration of the mitochondria and presumptive lipid droplets to the cytoplasm periphery and the onset of cumulus cell expansion. Between 12 and 18h of IVM, cytoplasmic rearrangements of the organelles were also observed e.g. aligning and increasing of cortical granules under the plasma membrane. Hooded mitochondria were observed during oocyte maturation. After 24h of IVM, the oocytes showed signs of degeneration, which can be related to cellular aging – because of the excessive time in the maturation medium, after acquisition of developmental competence. This work described the ultrastructural aspects of sheep oocytes during IVM and provided insight into oocyte maturation in the ovine species.

Short‐term weightlessness produced by parabolic flight maneuvers altered gene expression patterns in human endothelial cells

This study focused on the effects of short-term microgravity (22 s) on the gene expression and morphology of endothelial cells (ECs) and evaluated gravisensitive signaling elements. ECs were investigated during four German Space Agency (Deutsches Zentrum für Luft- und Raumfahrt) parabolic flight campaigns. Hoechst 33342 and acridine orange/ethidium bromide staining showed no signs of cell death in ECs after 31 parabolas (P31). Gene array analysis revealed 320 significantly regulated genes after the first parabola (P1) and P31. COL4A5, COL8A1, ITGA6, ITGA10, and ITGB3 mRNAs were down-regulated after P1. EDN1 and TNFRSF12A mRNAs were up-regulated. ADAM19, CARD8, CD40, GSN, PRKCA (all down-regulated after P1), and PRKAA1 (AMPKα1) mRNAs (up-regulated) provide a very early protective mechanism of cell survival induced by 22 s microgravity. The ABL2 gene was significantly up-regulated after P1 and P31, TUBB was slightly induced, but ACTA2 and VIM mRNAs were not changed. β-Tubulin immunofluorescence revealed a cytoplasmic rearrangement. Vibration had no effect. Hypergravity reduced CARD8, NOS3, VASH1, SERPINH1 (all P1), CAV2, ADAM19, TNFRSF12A, CD40, and ITGA6 (P31) mRNAs. These data suggest that microgravity alters the gene expression patterns and the cytoskeleton of ECs very early. Several gravisensitive signaling elements, such as AMPKα1 and integrins, are involved in the reaction of ECs to altered gravity.

Pollen vacuoles and their significance

Vacuoles of several types can be observed in pollen throughout its development. Their physiological significance reflects the complexity of the biological process leading to functional pollen grains. Vacuolisation always occurs during pollen development but when ripe pollen is shed the extensive translucent vacuoles present in the vegetative parts in previous stages are absent. Vacuole functions vary according to developmental stage but in ripe pollen they are mainly storage sites for reserves. Vacuoles cause pollen to increase in size by water accumulation and therefore confer some degree of resistance to water stress. Modalities of vacuolisation occur in pollen in the same manner as in other tissues. In most cases, autophagic vacuoles degrade organelles, as in the microspore after meiosis, and can be regarded as cytoplasm clean-up following the transition from the diploid sporophytic to the haploid gametophytic state. This also occurs in the generative cell but not in sperm cells. Finally, vacuoles have a function when microspores are used for pollen embryogenesis in biotechnology being targets for stress induction and afterwards contributing to cytoplasmic rearrangement in competent microspores.

β-Catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii

The canonical Wnt/β-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 β-catenin in metazoans is to define the posterior/vegetal part of the embryo. Although the specification of vegetal territories (endoderm) by β-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 β-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 β-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 β-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 β-catenin should be distinguished and carefully re-examined.

Cyclin B–Cdk1 activates its own pump to get into the nucleus

The transition to mitosis requires extensive nuclear and cytoplasmic rearrangements that must be spatially and temporally coordinated. In this issue, Gavet and Pines (2010a. J. Cell Biol. doi:10.1083/jcb.200909144) report on a simple yet elegant mechanism as to how this is achieved. By monitoring the activity of cyclin B–Cdk1 in real time, the authors show that concomitant with its activation in the cytoplasm, the kinase complex is rapidly imported into the nucleus by modifying the activity of the nucleocytoplasmic transport machinery. Thus, cyclin B–Cdk1 activates its own pump to get into the nucleus.

Signal transduction of fertilization in frog eggs and anti-apoptotic mechanism in human cancer cells: common and specific functions of membrane microdomains.

Membrane microdomains or lipid/membrane rafts are distinct areas on the plasma membranes, where a specific subset of lipids (e.g. cholesterol, sphingolipids) and proteins (e.g. glycosylphosphatidylinositol-anchored proteins, growth factor receptor/kinases) are getting together and functioning for several aspects of cellular functions. Our recent investigation has revealed that fertilization of African clawed frog, Xenopus laevis, requires cholesterol-dependent nature of egg membrane microdomains. Moreover, fertilization of Xenopus eggs involves proteolytic cleavage of the extracellular part and subsequent phosphorylation of a cytoplasmic tyrosine residue of uroplakin III, an egg membrane microdomain-associated protein. Protease activity toward uroplakin III seems to be derived from fertilizing sperm, while phosphorylation of uroplakin III seems to be catalyzed by the egg tyrosine kinase Src, whose activation is required for cytoplasmic rearrangement of fertilized eggs; so-called ‘egg activation’. Therefore, it is assumed that uroplakin III serves an integral part of signal transduction in fertilization of Xenopus. Our more recent study on human cancer cells has revealed that a similar but distinct scheme of signal transduction operates in anti-apoptotic growth of cells. Namely, in human bladder carcinoma cells, cooperation of uroplakin III and Src, both of which localize to the membrane microdomains, allows cells to escape from apoptotic cell death and proliferate under culture conditions deprived of serum. In this review, I briefly introduce about biology of fertilization and cancer, and then present and discuss our experimental data on general importance and specific features of membrane microdomains in Xenopus fertilization and anti-apoptosis in human bladder carcinoma cells.

Yeast shuttling SR proteins Npl3p, Gbp2p, and Hrb1p are part of the translating mRNPs, and Npl3p can function as a translational repressor.

A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.

MUCILAGE-MODIFIED4 encodes a putative pectin biosynthetic enzyme developmentally regulated by APETALA2, TRANSPARENT TESTA GLABRA1, and GLABRA2 in the Arabidopsis seed coat.

The Arabidopsis seed coat epidermis undergoes a complex process of differentiation that includes the biosynthesis and secretion of large quantities of pectinaceous mucilage, cytoplasmic rearrangement, and secondary cell wall biosynthesis. Mutations in MUM4 (MUCILAGE-MODIFIED4) lead to a decrease in seed coat mucilage and incomplete cytoplasmic rearrangement. We show that MUM4 encodes a putative NDP-l-rhamnose synthase, an enzyme required for the synthesis of the pectin rhamnogalacturonan I, the major component of Arabidopsis mucilage. This result suggests that the synthesis of monosaccharide substrates is a limiting factor in the biosynthesis of pectinaceous seed coat mucilage. In addition, the reduced cytoplasmic rearrangement observed in the absence of a key enzyme in pectin biosynthesis in mum4 mutants establishes a causal link between mucilage production and cellular morphogenesis. The cellular phenotype seen in mum4 mutants is similar to that of several transcription factors (AP2 [APETALA2], TTG1 [TRANSPARENT TESTA GLABRA1], TTG2 MYB61, and GL2 [GLABRA2]). Expression studies suggest that MUM4 is developmentally regulated in the seed coat by AP2, TTG1, and GL2, whereas TTG2 and MYB61 appear to be regulating mucilage production through alternate pathway(s). Our results provide a framework for the regulation of mucilage production and secretory cell differentiation.

Contrasting patterns of mitochondrial redistribution in the early lineages of Caenorhabditis elegans and Acrobeloides sp. PS1146

We compared the redistribution of mitochondria in the early embryos of Caenorhabditis elegans (C. elegans) and Acrobeloides sp. PS1146 (Acrobeloides)—two nematode species where the mechanisms for embryonic axis specification are different even though subsequent development is remarkably similar (Goldstein et al., 1998). During the first cell cycle of C. elegans, mitochondria move with the bulk cytoplasmic flows that are directed toward the sperm pronucleus and aggregate at the posterior cortex during the period known as “pseudocleavage.” In contrast, in Acrobeloides embryos, where prominent cytoplasmic rearrangements are absent, mitochondria that are initially distributed loosely around the pronuclei and the cytoplasm are relocated around the mitotic spindle prior to cell division. Interestingly, this rearrangement is reiterated only in the germline and not the somatic lineage. In both species, the location of the mitochondria immediately prior to cell division correlates with the known location of the germline determinants, P granules, leading us to speculate that they may be associated.

Isolation and Characterization of Mutants Defective in Seed Coat Mucilage Secretory Cell Development in Arabidopsis

In Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type producing extracellular pectinaceous mucilage and a volcano-shaped secondary cell wall. Differentiation involves a regulated series of cytological events including growth, cytoplasmic rearrangement, mucilage synthesis, and secondary cell wall production. We have tested the potential of Arabidopsis seed coat epidermal cells as a model system for the genetic analysis of these processes. A screen for mutants defective in seed mucilage identified five novel genes (MUCILAGE-MODIFIED [MUM]1-5). The seed coat development of these mutants, and that of three previously identified ones (TRANSPARENT TESTA GLABRA1, GLABRA2, and APETALA2) were characterized. Our results show that the genes identified define several events in seed coat differentiation. Although APETALA2 is needed for differentiation of both outer layers of the seed coat, TRANSPARENT TESTA GLABRA1, GLABRA2, and MUM4 are required for complete mucilage synthesis and cytoplasmic rearrangement. MUM3 and MUM5 may be involved in the regulation of mucilage composition, whereas MUM1 and MUM2 appear to play novel roles in post-synthesis cell wall modifications necessary for mucilage extrusion.

A cytoplasmic male sterility-associated rearrangement of the mitochondrial cob gene region in the sugar beet (Beta vulgaris L.) genome

Physical maps of the cob mtDNA region were constructed and compared between sugar beet Beta vulgaris L. plants with normal fertility and with cytoplasmic male sterility (CMS). A CMS-associated rearrangement did not affect the coding region of cob and combined two mtDNA regions which are normally about 150 kb apart. Two point substitutions were found in the 3'-untranslated region of cob.

Local mechanical stimulation induces components of the pathogen defense response in parsley.

Cell suspension cultures of parsley (Petroselinum crispum) have previously been used as a suitable system for studies of the nonhost resistance response to Phytophthora sojae. In this study, we replaced the penetrating fungus by local mechanical stimulation by using a needle of the same diameter as a fungal hypha, by local application of a structurally defined fungus-derived elicitor, or by a combination of the two stimuli. Similar to the fungal infection hypha, the local mechanical stimulus alone induced the translocation of cytoplasm and nucleus to the site of stimulation, the generation of intracellular reactive oxygen intermediates (ROI), and the expression of some, but not all, elicitor-responsive genes. When the elicitor was applied locally to the cell surface without mechanical stimulation, intracellular ROI also accumulated rapidly, but morphological changes were not detected. A combination of the mechanical stimulus with simultaneous application of low doses of elicitor closely simulated early reactions to fungal infection, including cytoplasmic aggregation, nuclear migration, and ROI accumulation. By contrast, cytoplasmic rearrangements were impaired at high elicitor concentrations. Neither papilla formation nor hypersensitive cell death occurred under the conditions tested. These results suggest that mechanical stimulation by the invading fungus is responsible for the observed intracellular rearrangements and may trigger some of the previously demonstrated changes in the activity of elicitor-responsive genes, whereas chemical stimulation is required for additional biochemical processes. As yet unidentified signals may be involved in papilla formation and hypersensitive cell death.

Cellular and Transcriptional Changes during Excystation ofGiardia lamblia in Vitro

Hetsko, M. L., McCaffery, J. M., Svärd, S. G., Meng, T.-C., Que, X., and Gillin, F. D. 1998. Cellular and transcriptional changes during excystation ofGiardia lamblia in vitro. Experimental Parasitology88, 172–183. Excystation ofGiardia lambliaentails differentiation of dormant cysts into parasitic trophozoites. Despite its importance for infection, this transformation is not understood at the cellular or molecular levels. In these studies, we report that excystation entails detection of environmental stimuli across the tough extracellular cyst wall leading to highly coordinated physiological, structural, and molecular responses. We found that novel cytoplasmic rearrangements and changes in individual species of mRNA and in cytoplasmic pH occur within the cyst wall in the earliest stage of excystation, in response to conditions modeling cyst ingestion and passage into the human stomach. This suggests that cysts do not contain all the mRNA needed for excystation and emergence and supports ourhypothesisthat external stimuli, including hydrogen ions, may penetrate or be perceived across the cyst wall. In contrast, changes in cyst wall structure or proteins were detected only later in excystation, in the stage that models passage into the human small intestine, where trophozoites can emerge and survive. These findings show that excystation ofG. lambliais a highly complex and active process and provide important insights into its cellular and molecular components.

Synergistic Effects of Vg1 and Wnt Signals in the Specification of Dorsal Mesoderm and Endoderm

In amphibians, dorsoventral asymmetry is established by cortical rotation, a cytoplasmic rearrangement in the egg which activates a dorsal determinant on one side of the zygote. This determinant has been proposed to be either Vg1, an endodermally derived molecule that can directly induce ectoderm to form dorsal mesoderm, or a member of the Wnt family, which patterns the ectoderm such that it forms dorsal mesoderm in response to ventral inductive signals. In this study, we have investigated whether the endogenous dorsal determinant(s) functions as a direct inducer of dorsal mesoderm (Vg1-like) or whether it acts to pattern the response of ectoderm to inductive signals (Wnt-like). We report here that cortical rotation enhances both the dorsal-inductive activity of endodermal cells and the response of ectodermal cells to endogenous inductive signals and that both of these activities are required for notochord induction in ectoderm/endoderm recombinants. While ectopically expressed Xwnt-8b can substitute for the dorsalizing signals activated in either ectoderm or endoderm, and can allow notochord formation in recombinants, Vg1 alone is not sufficient to induce notochord in ectodermal explants in the absence of signals activated by cortical rotation. Coexpression of Xwnt-8b along with Vg1 restores ectodermal competence to form notochord. Finally, in endodermal explants, ectopically expressed Xwnt-8b, but not Vg1, can divert the fate of ventral endodermal cells along a dorsal pathway. Thus, while Vg1 is most likely required for induction of mesodermin vivo,our data suggest that a maternal Wnt-like signal acts synergistically with Vg1 to specify a dorsal fate not only in the mesoderm, but also in the endoderm.

Specification of the anteroposterior axis in Caenorhabditis elegans

Anteroposterior asymmetries are apparent in C elegans development before the first cell division. Here we identify the cue that specifies the anteroposterior axis, and investigate how this cue is interpreted to generate initial asymmetry. In C. elegans, the sperm normally enters the egg in an invariant position. We have found that causing fertilisation to occur in the abnormal end of the egg completely reverses the orientation of the anteroposterior axis, but gives otherwise normal development. This result suggests that a component of the sperm normally specifies the anteroposterior axis. We have found that a cytoplasmic rearrangement in the uncleaved zygote is directed by the sperm, suggesting a mechanism by which the sperm may specify the axis. The results additionally reveal that the C elegans oocyte is constructed with no axis prespecified in the form of asymmetrically localised cytoplasmic determinants.

Infection-induced rapid cell death in plants: a means of efficient pathogen defense

The hypersensitive reaction represents one of the major means by which plants actively defend themselves against infection by pathogenic bacteria, fungi, viruses, and nematodes. This complex defense reaction, often associated with the synthesis of phytoalexins (antimicrobial secondary metabolites), involves at the cellular level highly dynamic cytoplasmic rearrangements, rapid metabolic changes, and finally cell death. It also correlates with the rapid and transient activation of various defense-related genes in a region of tissue surrounding infection sites and later, with the systemic increase in expression of a number of other genes. Examination of the reactions of individual living cells of potato leaves infected with Phytophthora infestans enabled the comprehensive description of the dynamic aspects of all stages of the defense response. Cytochemical investigations, employing cultured cells of parsley infected with P. infestans as a versatile model system, have contributed to a better understanding of cytoplasmic and metabolic processes occurring during the defense response, and suggest that hypersensitive cell death requires the preceding activation of respiration and specific metabolic pathways. Key words: defense responses, defense-related genes, hypersensitive reaction, programmed cell death.

Localization of polyadenylated RNAs during teloplasm formation and cleavage in leech embryos.

In the embryos of glossiphoniid leeches, as in many annelids, cytoplasmic reorganization prior to first cleavage generates domains of yolk-deficient cytoplasm (called teloplasm) that are sequestered during the first three cell divisions to the D' macromere. Subsequently, the D' macromere generates a set of embryonic stem cells (teloblasts) that are the progenitors of the definitive segmental tissues. The hypothesis that fate-determining substances are localized within the teloplasm and segregated to the D' macromere during cleavage is supported by experiments in which a redistribution of yolk-defcient cytoplasm changes the fate of blastomeres that inherit it (Astrow et al. 1987; Devries 1973; Nelson and Weisblat 1992). As a step toward identifying fate-determining factors in teloplasm, we describe the distribution of polyadenylated RNAs (polyA+ RNA) in the early embryo of the leech, Helobdella triserialis, as inferred from in situ hybridization using tritiated polyuridylic acid (3H-polyU). Our results indicate that polyA+ RNA colocalizes with teloplasm during cytoplasmic rearrangements resulting in teloplasm formation, and that it remains concentrated in the teloplasm during the cell divisions and a second cytoplasmic rearrangement during early embryogenesis. Lesser amounts of polyA+ RNA appear to be localized in cortical cytoplasm at most stages.

Rhizobium nod factors reactivate the cell cycle during infection and nodule primordium formation, but the cycle is only completed in primordium formation.

Rhizobia induce the formation of root nodules on the roots of leguminous plants. In temperate legumes, nodule organogenesis starts with the induction of cell divisions in regions of the root inner cortex opposite protoxylem poles, resulting in the formation of nodule primordia. It has been postulated that the susceptibility of these inner cortical cells to Rhizobium nodulation (Nod) factors is conferred by an arrest at a specific stage of the cell cycle. Concomitantly with the formation of nodule primordia, cytoplasmic rearrangement occurs in the outer cortex. Radially aligned cytoplasmic strands form bridges, and these have been called preinfection threads. It has been proposed that the cytoplasmic bridges are related to phragmosomes. By studying the in situ expression of the cell cycle genes cyc2, H4, and cdc2 in pea and alfalfa root cortical cells after inoculation with Rhizobium or purified Nod factors, we show that the susceptibility of inner cortical cells to Rhizobium is not conferred by an arrest at the G2 phase and that the majority of the dividing cells are arrested at the G0/G1 phase. Furthermore, the outer cortical cells forming a preinfection thread enter the cell cycle although they do not divide.

Slow cellular dynamics in MDCK and R5 cells monitored by time-lapse atomic force microscopy

We have examined dynamic events that occur on a time scale of minutes in an epithelial monolayer of Madine-Darby Canine Kidney (MDCK) cells and in ras-transformed MDCK cells by atomic force microscopy (AFM). Cells were imaged under physiological conditions, and time-lapse movies representing approximately 60 s real time per frame were assembled. In normal MDCK cells, two types of protrusions in the apical plasma membrane exhibit dynamic behavior. First, smooth bulges formed transiently over the time scale of minutes to tens of minutes. Second, spike-like protrusions appear initially as bulges, extend well above the apical surface and, finally, seem to detach. R5, an oncogenic transformant derived from MDCK cells, grows very flat on glass. During AFM imaging, these cells sometimes round up and detach from the substrate. In light microscopic observations of parallel preparations, cells rarely detach, suggesting that this is an active response of these cells to irritation by the AFM tip. R5 cells often extend processes that are supported by actin stress fibers. During imaging with the AFM, these processes withdraw at a rate of 1-5 microns/min, similar to that observed by light microscopy. During the withdrawal, movement of the stress fibers can be clearly seen. In the flat periphery of these cells, the transport of intracellular particles along cytoskeletal elements was seen. In addition, we have observed two types of wave-like movements through the cell, which appear to be an organized rearrangement of cytoplasm. One type of wave moves radially out from center of the cell while the other moves circularly along the cell periphery.