17 research outputs found
F-actin coordinates spindle morphology and function in Drosophila meiosis.
Meiosis is a highly conserved feature of sexual reproduction that ensures germ cells have the correct number of chromosomes prior to fertilization. A subset of microtubules, known as the spindle, are essential for accurate chromosome segregation during meiosis. Building evidence in mammalian systems has recently highlighted the unexpected requirement of the actin cytoskeleton in chromosome segregation; a network of spindle actin filaments appear to regulate many aspects of this process. Here we show that Drosophila oocytes also have a spindle population of actin that appears to regulate the formation of the microtubule spindle and chromosomal movements throughout meiosis. We demonstrate that genetic and pharmacological disruption of the actin cytoskeleton has a significant impact on spindle morphology, dynamics, and chromosome alignment and segregation during maturation and the metaphase-anaphase transition. We further reveal a role for calcium in maintaining the microtubule spindle and spindle actin. Together, our data highlights potential conservation of morphology and mechanism of the spindle actin during meiosis
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F-actin coordinates spindle morphology and function in Drosophila meiosis.
Acknowledgements: We are grateful to Jens Januschke, Torsten Krude, Jose Casal, and Paul Conduit for discussions and advice; Elise Wilby for detailed feedback on the manuscript; the Zoology Imaging Facility and Matt Wayland for assistance with microscopy; the Bloomington Drosophila Stock Center and Drosophila community for fly lines.Meiosis is a highly conserved feature of sexual reproduction that ensures germ cells have the correct number of chromosomes prior to fertilization. A subset of microtubules, known as the spindle, are essential for accurate chromosome segregation during meiosis. Building evidence in mammalian systems has recently highlighted the unexpected requirement of the actin cytoskeleton in chromosome segregation; a network of spindle actin filaments appear to regulate many aspects of this process. Here we show that Drosophila oocytes also have a spindle population of actin that appears to regulate the formation of the microtubule spindle and chromosomal movements throughout meiosis. We demonstrate that genetic and pharmacological disruption of the actin cytoskeleton has a significant impact on spindle morphology, dynamics, and chromosome alignment and segregation during maturation and the metaphase-anaphase transition. We further reveal a role for calcium in maintaining the microtubule spindle and spindle actin. Together, our data highlights potential conservation of morphology and mechanism of the spindle actin during meiosis
The spindle actin population is dynamic at egg activation.
A. Schematic and confocal Z-projection (10 μm) labeled for centromere (Cid-EGFP, green) and DNA (DAPI, cyan) (A′) or microtubules (Jup-GFP, green), actin (SiR-Actin, white), and DNA (DAPI, cyan) (A′′) in early (A′) and later (A′′) anaphase I (AI) oocytes. Post egg activation, the chromosomes start to separate to opposite poles while the spindle first increases in width (A′) and then in length (A′′). The completion of AI is marked by the spindle becoming perpendicular in orientation to the cortex. Insets show induvial channels. (A′) n = 34/34 early activated mature oocytes. (A′′) n = 20/20 late activated mature oocytes. B. Confocal Z-projection (10 μm) labeled for calcium (GCaMP3, green) and DNA (DAPI, cyan) of an AI oocyte. Calcium signal spread evenly across the elongated spindle with separating chromosomal mass. n = 10 activated mature oocytes. C. Confocal Z-projections (10 μm) labeled for actin (UtrCH-GFP) from live time series of MI oocytes (left, t = 0′) incubated in activation buffer (AB) (right, t = 5′). Similar changes in shape and rotation observed as with fixed samples. n = 25 activated mature oocytes. Scale bar: 5 μm (A-C).</p
The spindle actin is required for accurate segregation of chromosomes during anaphase I.
A-B. Confocal Z-projections (10 μm) labeled for actin (Lifeact-GFP, green) and DNA (DAPI, cyan) of AI oocytes incubated in control media (A) and cytoD (B). Spindle actin surrounds the segregating AI chromosomes in control media (A), but appears less defined than in MI oocytes, and is not detected after cytoD treatment (B). Chromosome segregation phenotype is disrupted in cytoD treated oocytes, many showing chromosomal units separated from the main mass (B′-B′′) compared to untreated controls (A′-A′′). Insets show induvial channels and additional chromosomal examples. n = 30 activated mature oocytes per treatment. C-D. Confocal Z-projections (10 μm) labeled for microtubules (Jup-GFP, green) and DNA (DAPI, cyan) of capuEY12344/+ (C) and capuEY12344/spire2F (D) AI oocytes. Microtubule spindle are detected in both backgrounds, but chromosome segregation is misregulated. Examples of the variety of disrupted chromosome segregation phenotypes indicate chromosomal units separated from the main mass (C′-C′′, D′-D′′). Insets show induvial channels and additional chromosomal examples. n = 17 activated mature oocytes per genotype. E-G. Graphs summarising the number of aberrant chromosomal masses (E), maximum chromosomal distance (F), and microtubule spindle length (G) for WT, cytoD treated, capuEY12344/+, and capuEY12344/spire2F AI oocytes. To quantify the aberrant chromosomal masses, we first defined the main chromosomal mass as the aligned materials in close association ( 1 μm or when present > 1 μm outside of the spindle shape observed in wild type segregation. We also completed a 3D analysis to check the Z distance of > 1 μm from the main mass. Comparison of the number of aberrant chromosomal masses shows a significant increase in cytoD treated and mutant backgrounds compared to the WT. (E) n > 20 mature oocytes per treatment or genotype, student’s t-test, * WT. (F-G) n > 30 mature oocytes per treatment or genotype, student’s t-test, not significant (ns). H. Graph summarising the angle of spindles from cortex before (left) and after (right) egg activation in WT and cytoD treated oocytes. Comparison of the angle between the spindle and cortex shows cytoD only has a significant effect on MI oocytes. There are no significant differences between the WT AI oocyte, and the cytoD treated MI and AI oocytes. n > 20 mature oocytes per treatment and stage, student’s t-test, not significant (ns). Scale bar: 5 μm (A-D).</p
A spindle actin population is present in the metaphase-arrested <i>Drosophila</i> mature oocyte.
A-D. Schematic and confocal Z-projection (10 μm) labeled for microtubules (Jup-GFP, green), actin (SiR-Actin, white), and DNA (DAPI, cyan). In stage 12 oocytes prior to egg maturation (A,A′) the DNA is condensed (white arrowhead, pointing right) and actin appears to enrich around the nucleus (white arrowhead, pointing left). (A′) n = 24 stage 12 oocytes and the depicted features were observed in 24 out of 24 samples (24/24). In stage 13 at maturation (B-B′′) actin spikes penetrate the nucleus (B′, white arrowhead) and then surround the DNA (B′′, white arrowhead actin inset) with the microtubule material appearing diffuse (B′′). (B′) n = 12/15 early stage 13 oocytes (B′′) n = 3/3 late stage 13 oocytes. In stage 14 metaphase I arrested oocytes (mature oocytes) (C,C′) the microtubule spindle forms and a population of actin, spindle actin, appears to associate with microtubules. Insets show induvial channels. (C′) n = 125/130 mature oocytes. Higher resolution image of early metaphase I arrest nucleus (D), actin appears to form a cage around the microtubules and DNA. In some cases, the 4th non-exchange chromosomes are visible as a smaller mass at each tip of the main body of chromosomes. (D) n = 56/60 mature oocytes. E-F. Confocal Z-projections (10 μm) labeled for actin (Act5C-GFP, green) (E), (Lifeact-GFP, green) (F), and DNA (DAPI, cyan) of a mature oocytes. The MI stage is marked by the spindle becoming parallel in orientation to the cortex. The cortical actin is visualized here as vertical green band of fluorescence between the oocyte nucleus and the follicle cell nuclei (far left) (E). The spindle actin is visible surrounding a central chromosomal mass using both markers (E, F). Insets show induvial channels, n > 15 mature oocytes per genetic actin marker. G. Confocal Z-projection (10 μm) labeled for Ca2+ (GCaMP3, green) and DNA (DAPI, cyan) of a MI oocyte. Higher calcium signal detected at the tip of the spindle pole, n = 15 mature oocytes. H-I. Confocal Z-projections (10 μm) from live time series labeled for actin (UtrCH-GFP) (H) or microtubules (Jup-GFP) (I) of MI oocytes. Similar structures and orientations are observed for actin and microtubule as compared to fixed samples. n > 37 mature oocytes per genetically encoded marker. Scale bar: 10 μm (A-C, H-I), 5 μm (D-G).</p
The spindle actin is required for recruitment and the position of the metaphase I spindle and to maintain chromosomal integrity.
A-B. Confocal Z-projections (10 μm) from live time series of WT, capuEY12344/+, and capuEY12344/spire1 or capuEY12344/spire2F in MI oocytes, labelled for actin (UtrCH-GFP) (A) or microtubules (Jup-GFP) (B). While the cortical actin (A, right panel, top right corner) could still be observed, spindle actin is not detected in the heterozygous or trans-heterozygous mutants. (A) n = 30 mature oocytes per genotype. Microtubules are elongated (B) and no longer parallel to the cortex in both the heterozygous and the trans-heterozygous mutants. (B) n = 30 mature oocytes per genotype. (Note: capuEY12344 referred to as capuEY in panels in all figures). C. Confocal Z-projections (10 μm) labeled for microtubules (Jup-GFP, green) and DNA (DAPI, cyan) of capuEY12344/+ (left) and capuEY12344/spire2F (right) in MI oocytes. DNA stains in both mutants reveal a separation of the chromosomal mass into two units that begin to migrate along the spindle axis. Insets show induvial channels. n = 20 mature oocytes per genotype. D. Confocal Z-projection (10 μm) labeled for calcium (GCaMP3, green) and DNA (DAPI, cyan) of a capuEY12344/+ MI oocyte. A loss of the calcium signal in a spindle shape and separation of the chromosomes into two separate masses are detected. Insets show induvial channels. n = 13 mature oocytes. E-G. Graphs summarising the proportion of microtubule spindle phenotype (E), maximum chromosomal distance and microtubule spindle length (F), and the angle of spindles from cortex (G), for WT, post-colchicine, post- cytoD, capuEY12344/+ and capuEY12344/spire2F MI oocytes. The proportion of oocytes with a WT microtubule spindle phenotype are significantly decreased after drug treatment and in mutant backgrounds (E). Most microtubule spindles found in the experimental groups are either disrupted (elongated in cytoD and mutants or shortened in colchicine) or not detected. We do see a proportion of WT oocytes with no spindle detected, which is likely a technical issue due to the orientation of the oocyte or labelling efficiency. (E) n > 16 mature oocytes per treatment or genotype, Fishers Exact Test, P value WT (F). In the few mature oocytes where microtubule spindle where still detected 15 minutes after colchicine addition (n = 11 mature oocytes), the spindle length is reduced without any significant impact on the maximum chromosomal distance. (F) n >16 mature oocytes per treatment or genotype, student’s t-test, ***WT. (G) n >16 mature oocytes per treatment or genotype, student’s t-test, ***H. Recovery of fluorescence intensity following photobleaching of microtubules in WT and capuEY12344/spire2F mutant. WT and mutant oocytes initially show similar recovery dynamics, however, a significant difference between the mutant and WT is observed over time (after 270 s). n = 6 mature oocytes per genotype, student’s t-test, *A-D).</p
The actin cytoskeleton promotes maintenance of spindle microtubules and regulation of chromosomal alignment.
A-F. Confocal Z-projections (10 μm) from live time series labeled for actin (UtrCH-GFP) or microtubules (Jup-GFP) of MI oocytes treated with colchicine (A-B), cytoD (C-D) and BAPTA-AM (E-F). Microtubules (A) first appear as a typical spindle structure (t = 0′) and depolymerize post colchicine treatment (t = 30′), whereas spindle actin (B, white arrowhead) is not affected. (A-B) n = 10 mature oocytes per genetically encoded marker treated. Before addition of cytoD (t = 0′), the microtubule spindle appears as a typical elliptical structure of approximately 10 μM (C). Post-cytoD treatment (t = 10′) the spindle has undergone a distinct morphological change as it elongates (C). Spindle actin (D, white arrowhead) appears parallel to the cortex before the addition of cytoD (t = 0′) and becomes depolymerized post treatment (t = 10′). (C-D) n = 9 mature oocytes per genetically encoded marker treated. Both microtubule spindle (E) and spindle actin (F, white arrowhead) depolymerize post BAPTA-AM treatment (t = 15′), suggesting calcium is required to maintain the metaphase spindles. (E,F) n = 8 mature oocytes per genetically encoded marker treated. G-J. Confocal Z-projections (10 μm) labeled for microtubules (Jup-GFP, green), actin (SiR-Actin, white), and DNA (DAPI, cyan) (G,I) or centromere (Cid-EGFP, green) and DNA (DAPI, cyan) (H,J) of MI oocytes incubated in control media (G-H) or cytoD (I-J). Spindle actin and the microtubule spindle appear to surround the chromosomes in MI oocytes incubated in control media (G). Centromeres are detected on each aligned chromosome (H, white arrowhead). (G-H) n = 40 mature oocytes per genetically encoded marker. Spindle actin depolymerizes post-cytoD treatment and the microtubule spindle is significantly elongated (I). Distance between centromeres also increases and the number of centromeres is unequally distributed between the two poles (J). (I,J) n = 50 mature oocytes per genetically encoded marker treated, (I) unpaired t-test, P value G,H,K). K. Confocal Z-projections (10 μm) labeled for microtubules (Jup-GFP, green), actin (SiR-Actin, white), and DNA (DAPI, cyan) of MI oocytes incubated in BAPTA-AM. A loss of the spindle and a round chromosomal mass in close proximity of the cortex are detected post-treatment. n = 10 mature oocytes. Insets show induvial channels. Scale bar: 10 μm (A-K).</p
Phosphorus Composition and Phosphatase Activities in Soils Affected by Long-Term Application of Pig Manure and Inorganic Fertilizers
Self-Template Synthesis of Porous Perovskite Titanate Solid and Hollow Submicrospheres for Photocatalytic Oxygen Evolution and Mesoscopic Solar Cells
We
describe a general synthesis strategy, which combines sol–gel
and hydrothermal processes, for the large-scale synthesis of porous
perovskite titanates spheres with tunable particle size and inner
structures. Amorphous hydrous TiO<sub>2</sub> solid spheres (AHTSS)
are first synthesized by a sol–gel method and are then used
as precursor and template for the subsequent hydrothermal reaction
with alkaline earth metal ions in an alkaline medium. This strategy
can be generalized to synthesize porous spheres of various perovskite
titanates (i.e., SrTiO<sub>3</sub>, BaTiO<sub>3</sub>, and CaTiO<sub>3</sub>) consisting of single-crystalline nanocubes. By controlling
the textural properties (i.e., size, porosity, and structure) of AHTSS,
perovskite titanates with tunable size and inner structures are selectively
synthesized. The underlying formation mechanism is manifested by XRD
and TEM to involve in situ crystallization or Ostwald ripening during
the hydrothermal process. The obtained porous SrTiO<sub>3</sub> spheres
present superior performance in photocatalytic oxygen evolution and
CdSe-sensitized mesoscopic solar cells