Live imaging of calcium spikes during double fertilization in Arabidopsis.
Ontology highlight
ABSTRACT:
SUBMITTER: Hamamura Y
PROVIDER: S-EPMC4143913 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell-cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+]cyto) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+]cyto signals during the entire double fertilization process in Arabidopsis. [Ca2+]cyto oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+]cyto transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+]cyto transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+]cyto signature differing by timing and behaviour from [Ca2+]cyto waves reported in mammals.
| S-EPMC4143946 | biostudies-literature
Project description:Angiosperms have a unique sexual reproduction system called "double fertilization." One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.
| S-EPMC4194012 | biostudies-literature
Project description:Calcium imaging has led to discoveries about neural correlates of behavior in subcortical neurons, including dopamine (DA) neurons. However, spike inference methods have not been tested in most populations of subcortical neurons. To address this gap, we simultaneously performed calcium imaging and electrophysiology in DA neurons in brain slices and applied a recently developed spike inference algorithm to the GCaMP fluorescence. This revealed that individual spikes can be inferred accurately in this population. Next, we inferred spikes in vivo from calcium imaging from these neurons during Pavlovian conditioning, as well as during navigation in virtual reality. In both cases, we quantitatively recapitulated previous in vivo electrophysiological observations. Our work provides a validated approach to infer spikes from calcium imaging in DA neurons and implies that aspects of both tonic and phasic spike patterns can be recovered.
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Project description:During double fertilization in angiosperms, two male gametes (sperm cells), are released from a pollen tube into the receptive region between two female gametes; the egg cell and the central cell of the ovule. The sperm cells fertilize the egg cell and the central cell in a one-to-one manner to yield a zygote and an endosperm, respectively. The one-to-one distribution of the sperm cells to the two female gametes is strictly regulated, possibly via communication among the four gametes. Polyspermy block is the mechanism by which fertilized female gametes prevent fertilization by a secondary sperm cell, and has been suggested to operate in the egg cell rather than the central cell. However, whether the central cell also has the ability to avoid polyspermy during double fertilization remains unclear. Here, we assessed the one-to-one fertilization mechanism of the central cell by laser irradiation of the female gametes and live cell imaging of the fertilization process in Arabidopsis thaliana. We successfully disrupted an egg cell within the ovules by irradiation using a femtosecond pulse laser. In the egg-disrupted ovules, the central cell predominantly showed single fertilization by one sperm cell, suggesting that neither the egg cell nor its fusion with one sperm cell is necessary for one-to-one fertilization (i.e., monospermy) of the central cell. In addition, using tetraspore mutants possessing multiple sperm cell pairs in one pollen, we demonstrated that normal double fertilization was observed even when excess sperm cells were released into the receptive region between the female gametes. In ovules accepting four sperm cells, the egg cell never fused with more than one sperm cell, whereas half of the central cells fused with more than one sperm cell (i.e., polyspermy) even 1 h later. Our results suggest that the central cell can block polyspermy during double fertilization, although the central cell is more permissive to polyspermy than the egg cell. The potential contribution of polyspermy block by the central cell is discussed in terms of how it is involved in the one-to-one distribution of the sperm cells to two distinct female gametes.
| S-EPMC7835324 | biostudies-literature
Project description:Calcium is a key regulator of axon degeneration caused by trauma and disease, but its specific spatial and temporal dynamics in injured axons remain unclear. To clarify the function of calcium in axon degeneration, we observed calcium dynamics in single injured neurons in live zebrafish larvae and tested the temporal requirement for calcium in zebrafish neurons and cultured mouse DRG neurons. Using laser axotomy to induce Wallerian degeneration (WD) in zebrafish peripheral sensory axons, we monitored calcium dynamics from injury to fragmentation, revealing two stereotyped phases of axonal calcium influx. First, axotomy triggered a transient local calcium wave originating at the injury site. This initial calcium wave only disrupted mitochondria near the injury site and was not altered by expression of the protective WD slow (WldS) protein. Inducing multiple waves with additional axotomies did not change the kinetics of degeneration. In contrast, a second phase of calcium influx occurring minutes before fragmentation spread as a wave throughout the axon, entered mitochondria, and was abolished by WldS expression. In live zebrafish, chelating calcium after the first wave, but before the second wave, delayed the progress of fragmentation. In cultured DRG neurons, chelating calcium early in the process of WD did not alter degeneration, but chelating calcium late in WD delayed fragmentation. We propose that a terminal calcium wave is a key instructive component of the axon degeneration program.Significance statementAxon degeneration resulting from trauma or neurodegenerative disease can cause devastating deficits in neural function. Understanding the molecular and cellular events that execute axon degeneration is essential for developing treatments to address these conditions. Calcium is known to contribute to axon degeneration, but its temporal requirements in this process have been unclear. Live calcium imaging in severed zebrafish neurons and temporally controlled pharmacological treatments in both zebrafish and cultured mouse sensory neurons revealed that axonal calcium influx late in the degeneration process regulates axon fragmentation. These findings suggest that temporal considerations will be crucial for developing treatments for diseases associated with axon degeneration.
| S-EPMC4642236 | biostudies-literature
Project description:BACKGROUND:Of the animals typically used to study fertilization-induced calcium dynamics, none is as accessible to genetics and molecular biology as the model organism Caenorhabditis elegans. Motivated by the experimental possibilities inherent in using such a well-established model organism, we have characterized fertilization-induced calcium dynamics in C. elegans. RESULTS:Owing to the transparency of the nematode, we have been able to study the calcium signal in C. elegans fertilization in vivo by monitoring the fluorescence of calcium indicator dyes that we introduce into the cytosol of oocytes. In C. elegans, fertilization induces a single calcium transient that is initiated soon after oocyte entry into the spermatheca, the compartment that contains sperm. Therefore, it is likely that the calcium transient is initiated by contact with sperm. This calcium elevation spreads throughout the oocyte, and decays monotonically after which the cytosolic calcium concentration returns to that preceding fertilization. Only this single calcium transient is observed. CONCLUSION:Development of a technique to study fertilization induced calcium transients opens several experimental possibilities, e.g., identification of the signaling events intervening sperm binding and calcium elevation, identifying the possible roles of the calcium elevation such as the completion of meiosis, the formation of the eggshell, and the establishing of the embryo's axis of symmetry.
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Project description:To follow the dynamics of meiosis in the model plant Arabidopsis, we have established a live cell imaging setup to observe male meiocytes. Our method is based on the concomitant visualization of microtubules (MTs) and a meiotic cohesin subunit that allows following five cellular parameters: cell shape, MT array, nucleus position, nucleolus position, and chromatin condensation. We find that the states of these parameters are not randomly associated and identify 11 cellular states, referred to as landmarks, which occur much more frequently than closely related ones, indicating that they are convergence points during meiotic progression. As a first application of our system, we revisited a previously identified mutant in the meiotic A-type cyclin TARDY ASYNCHRONOUS MEIOSIS (TAM). Our imaging system enabled us to reveal both qualitatively and quantitatively altered landmarks in tam, foremost the formation of previously not recognized ectopic spindle- or phragmoplast-like structures that arise without attachment to chromosomes.
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Project description:The role of calcium signaling in cytokinesis has long remained ambiguous. Past studies of embryonic cell division discovered that calcium concentration increases transiently at the division plane just before cleavage furrow ingression, suggesting that these calcium transients could trigger contractile ring constriction. However, such calcium transients have only been found in animal embryos and their function remains controversial. We explored cytokinetic calcium transients in the fission yeast Schizosaccharomyces pombe by adopting GCaMP, a genetically encoded calcium indicator, to determine the intracellular calcium level of this model organism. We validated GCaMP as a highly sensitive calcium reporter in fission yeast, allowing us to capture calcium transients triggered by osmotic shocks. We identified a correlation between the intracellular calcium level and cell division, consistent with the existence of calcium transients during cytokinesis. Using time-lapse microscopy and quantitative image analysis, we discovered calcium spikes both at the start of cleavage furrow ingression and the end of cell separation. Inhibition of these calcium spikes slowed the furrow ingression and led to frequent lysis of daughter cells. We conclude that like the larger animal embryos, fission yeast triggers calcium transients that may play an important role in cytokinesis (197).
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Project description:The microenvironment has been shown to regulate cellular functions including cell growth, differentiation, proliferation, migration, cancer development and metastasis. However, the underlying molecular mechanism remains largely unclear. We have integrated micro-pattern technology and molecular biosensors based on fluorescence resonance energy transfer (FRET) to visualize calcium responses in cells constrained to grow on a micro-patterned surface. Upon ATP stimulation, human umbilical vein endothelial cells (HUVECs) cultured on different surface micro-patterns had a shorter decay time and a reduced peak of a transient intracellular calcium rise compared to control cells without constraints. The decay time is regulated by the plasma membrane and the membrane calcium channels, while the peak by endoplasmic reticulum (ER) calcium release. Further results revealed that voltage operated channels (VOCs), coupling the plasma membrane and ER, can affect both the decay time and the peak of calcium response. The inhibition of VOCs can eliminate the effect of different micro-patterns on calcium signals. When two connected HUVECs were constrained to grow on a micro-pattern, drastically distinct calcium responses upon ATP stimulation can be observed, in contrast to the similar responses of two connected cells cultured without patterns. Interestingly, the inhibition of VOCs also blocked this difference of calcium responses between two connected cells on micro-patterns. These results indicate that a micro-patterned surface can have a profound effect on the calcium responses of HUVECs under ATP stimulation, largely mediated by VOCs. Therefore, our results shed new light on the molecular mechanism by which HUVECs perceive the microenvironment and regulate intracellular calcium signals.
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Project description:The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.
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