19 research outputs found

    Cell cycle control of endoplasmic reticulum structure and Ca2+ -release in the mouse oocyte and early embryo

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    In mammals, fertilisation triggers a series of intracellular Ca2+ transients which are responsible for egg activation and completion of meiosis. These oscillations are generated by InsP3-induced release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ oscillations last for 3-4 hours in mouse, ceasing at the time of pronucleus formation. The subsequent breakdown of the pronuclei (NEBD) at mitosis entry is accompanied by the resumption of Ca2+ oscillations. The experiments presented in this thesis examine the relationship between ER structure and Ca2+ release in the mouse oocyte and early embryo, and investigate the role of Ca2+ release m mitosis. Using the ER-specific marker Dil, we report that germinal vesicle breakdown is associated with a dramatic microtubule-dependent redistribution of ER to the region surrounding the metaphase-I spindle. ER remains tightly packed around the spindle, during centro-cortical migration. The formation of clusters of ER in the oocyte cortex occurs around the time of polar body formation, and coincides with increased responsiveness of InsP3-mediated Ca2+ release. The decrease in cdkl-cyclin B activity which occurs following activation is both necessary and sufficient for the subsequent disappearance of ER clusters, and corresponds with diminished Ca2+ release in response to InsP3. Cortical ER clusters do not re-appear following NEBD, rather ER accumulates around the mitotic spindle. NEBD is associated with increased responsiveness of Ca2+ release both in fertilised and parthenogenetic embryos. The role of Ca2+ in mitosis was examined. Mitotic Ca2+ transients are dispensable since InsP3-receptor-downregulation and Ca2+ -chelators prohibit mitotic Ca2+ transients without affecting the first embryonic division. Microinjection of a fluorescent marker into one pronucleus reveals that nuclear membrane permeablisation begins prior to initiation of mitotic Ca2+ signals. The subsequent cessation of mitotic oscillations precedes the formation of nuclei in the two-cell embryo. No Ca transients are detected during the second mitotic division. These data demonstrate dynamic microtubule and cell cycle dependent ER reorganisations in meiosis and mitosis, in which clustering of ER in the cortex or around the spindle is associated with increased responsiveness of InsP3-releasable Ca2+ stores. Additionally, the results presented suggest that global mitotic Ca2+ transients are triggered by NEBD, rather than being the cause

    The roles of Ca2+, downstream protein kinases, and oscillatory signaling in regulating fertilization and the activation of development

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    AbstractReviews in Developmental Biology have covered the pathways that generate the all-important intracellular calcium (Ca2+) signal at fertilization [Miyazaki, S., Shirakawa, H., Nakada, K., Honda, Y., 1993a. Essential role of the inositol 1,4,5-trisphosphate receptor/Ca2+ release channel in Ca2+ waves and Ca2+ oscillations at fertilization of mammalian eggs. Dev. Biol. 158, 62–78; Runft, L., Jaffe, L., Mehlmann, L., 2002. Egg activation at fertilization: where it all begins. Dev. Biol. 245, 237–254] and the different temporal responses of Ca2+ in many organisms [Stricker, S., 1999. Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157–176]. Those reviews raise the importance of identifying how Ca2+ causes the events of egg activation (EEA) and to what extent these temporal Ca2+ responses encode developmental information. This review covers recent studies that have analyzed how these Ca2+ signals are interpreted by specific proteins, and how these proteins regulate various EEA responsible for the onset of development. Many of these proteins are protein kinases (CaMKII, PKC, MPF, MAPK, MLCK) whose activity is directly or indirectly regulated by Ca2+, and whose amount increases during late oocyte maturation. We cover biochemical progress in defining the signaling pathways between Ca2+ and the EEA, as well as discuss how oscillatory or multiple Ca2+ signals are likely to have specific advantages biochemically and/or developmentally. These emerging concepts are put into historical context, emphasizing that key contributions have come from many organisms. The intricate interdependence of Ca2+, Ca2+-dependent proteins, and the EEA raise many new questions for future investigations that will provide insight into the extent to which fertilization-associated signaling has long-range implications for development. In addition, answers to these questions should be beneficial to establishing parameters of egg quality for human and animal IVF, as well as improving egg activation protocols for somatic cell nuclear transfer to generate stem cells and save endangered species

    Mechanisms of PLCζ induced Ca²⁺ oscillations in mouse eggs at fertilisation

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    All the events of egg activation in mammalian eggs are triggered physiologically by transient increases in cytosolic free Ca²⁺ referred to as Ca²⁺ioscillations. These oscillations are initiated by the sperm derived PLC isoform, PLCζ. PLCζ releases Ca²⁺ by hydrolysing its substrate PI(4,5)P₂ to produce IP₃, however, many of the mechanisms by which PLCζ elicits Ca²⁺release in eggs are poorly understood. The results of this thesis confirm that whilstPLCζ cRNA and recombinant protein is able to cause Ca²⁺ioscillations in mouse eggs the sperm derived protein PAWP does not cause any Ca²⁺ release in any circumstances. It is shown that EF hand domain and XY linker of PLCζ are important in determining its Ca²⁺i releasing ability by enabling PLCζ binding to its substrate PI(4,5)P₂ through electrostatic interactions. The C2 domain of PLCζ was also found to play a crucial role in the Ca²⁺ releasing ability of PLCζ, possibly by binding to lipids or proteins in the target membrane. The Ca²⁺releasing ability of eggs is acquired during oocyte maturation and a dramatic increase in PLCζ sensitivity of oocytes occurs after germinal vesicle breakdown.A variety of markers for PLCζ’s substrate PI(4,5)P₂ including fluorescent PI(4,5)P₂ and gelsolin based fluorescent probes suggests that this PI(4,5)P₂ is localised to intracellular vesicles that could derive from Golgi apparatus. Attempts are made to measure PI turnover in these intracellular compartments of eggs during PLCζ induced Ca²⁺i oscillations using several probes. The results of this thesis suggest that PLCζ releases Ca²⁺ by a novel IP₃ based signalling pathway that involves an intracellular source of PI(4,5)P₂

    Oscillatory fluid flow drives scaling of contraction wave with system size

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    Flows over remarkably long distances are crucial to the functioning of many organisms, across all kingdoms of life. Coordinated flows are fundamental to power deformations, required for migration or development, or to spread resources and signals. A ubiquitous mechanism to generate flows, particularly prominent in animals and amoeba, is acto-myosin cortex driven mechanical deformations that pump the fluid enclosed by the cortex. Yet, it is unclear how cortex dynamics can self-organize to give rise to coordinated flows across the largely varying scales of biological systems. Here, we develop a mechanochemical model of acto-myosin cortex mechanics coupled to a contraction-triggering, soluble chemical. The chemical itself is advected with the flows generated by the cortex driven deformations of the tubular-shaped cell. The theoretical model predicts a dynamic instability giving rise to stable patterns of cortex contraction waves and oscillatory flows. Surprisingly, simulated patterns extend beyond the intrinsic length scale of the dynamic instability - scaling with system size instead. Patterns appear randomly but can be robustly generated in a growing system or by flow-generating boundary conditions. We identify oscillatory flows as the key for the scaling of contraction waves with system size. Our work shows the importance of active flows in biophysical models of patterning, not only as a regulating input or an emergent output, but rather as a full part of a self-organized machinery. Contractions and fluid flows are observed in all kinds of organisms, so this concept is likely to be relevant for a broad class of systems.Comment: 7 pages, 5 figure

    The relationship between calcium and metabolism in mouse eggs at fertilisation

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    At fertilisation in mammals a series of Ca2+ oscillations are initiated that activate development. These Ca2+ oscillations cause the reduction of mitochondrial NAD+ and flavoproteins, suggesting that they might also stimulate changes in cytosolic ATP levels. Many events at fertilisation are triggered that require ATP; however, the changes in ATP during fertilisation are poorly defined. In this thesis intracellular C a2+ and ATP levels in individual m ouse eggs were m easured by monitoring the fluorescence of a C a 2+ dye (Oregon green bapta dextran) and lum inescence of firefly luciferase. During fertilisation of m ouse eggs it w as found that there are two phases of increase in ATP in both the cytosol and the mitochondria, during the series of sperm-induced Ca2+ oscillations. The increase in ATP is Ca2+ dependent since it did not occur when Ca2+ oscillations were prevented by BAPTA injection and, were abrogated by extracellular Ca2+ chelation. Additionally, it w as not seen when eggs were activated by cycloheximide, which does not cause a Ca2+ increase. The ATP increase is likely to be caused by oxidative phosphorylation by the mitochondria since the ATP levels in substrate free media are recovered by the addition of pyruvate. This recovery is blocked by the pyruvate uptake inhibitor ar-Cyano-4-hydroxycinnamic acid. T hese data suggest that mammalian fertilisation is associated with a sudden but transient increase in cytosolic ATP via oxidative phosphorylation, and that Ca2+ oscillations are both necessary and sufficient to cause this increase in ATP. Work in this thesis has also investigated the functionality of the sperm factor PLC?. Using luciferase tagged PLC constructs, the Ca2+ oscillation inducing ability of a series of PLC? truncated constructs, PLC5 and PLCy have been established. Results show that PLC? activation of m ouse eggs cannot be reproduced by other PLCs and that the C2, EF1 and catalytic site on the X domain are all essential for causing C a2+ oscillations

    Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

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    Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

    Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

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    Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin
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