Burdyniuk M et al. (2018), F-Actin nucleated on chromosomes coordinates th...

ECB-ART-46408

J Cell Biol 2018 Aug 06;2178:2661-2674. doi: 10.1083/jcb.201802080.

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F-Actin nucleated on chromosomes coordinates their capture by microtubules in oocyte meiosis.

Burdyniuk M , Callegari A , Mori M , Nédélec F , Lénárt P .

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Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-µm starfish oocyte nucleus. How this F-actin-driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin "patches" form around chromosomes in a Ran-GTP-dependent manner, and we propose that these structures sterically block kinetochore-microtubule attachments. Once F-actin-driven chromosome transport is complete, coordinated disassembly of F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary because early capture of chromosomes by microtubules would interfere with F-actin-driven transport leading to chromosome loss and formation of aneuploid eggs.

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Genes referenced: actr2 dnah3 LOC115925415 LOC590297 ran tubgcp2

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	Figure 1. After F-actin–driven congression, chromosomes form lateral attachments and are transported along microtubules to the spindle poles. (A) Left: Scheme of an immature starfish oocyte with the nucleus anchored at the AP and with centrosomes nucleating astral microtubules. Right: Schematics of the nuclear region after NEBD. The F-actin network fills the nuclear region and, as it contracts, transports embedded chromosomes toward the AP. Chromosomes delivered within the capture range of astral microtubules are captured and transported on microtubules to the centrosomes at the AP. (B) Selected frames from a time series of single confocal sections through the nuclear region of an oocyte expressing EB3-3mCherry to visualize microtubule plus-tips (gray) and H2B-3mEGFP to label the chromosomes (cyan). See also Video 1. Chromosome trajectories are overlaid onto the images: green denotes actin-driven transport and red shows microtubule-driven transport. Lower panels: Zoom of the area marked with a dashed square, and the selected time points around microtubule capture are shown. Red arrowheads mark the contact between the microtubule and chromosome. Bars: (main images) 10 µm; (insets) 5 µm. (C) Plot of distance of chromosomes to the AP over time, calculated from the trajectories shown in B. Black dots mark capture events identified as the transition point between slow, F-actin–driven, and fast microtubule-driven transport. (D) Plot of chromosome distance to the AP over time in an oocyte treated with Ciliobrevin D to inhibit dynein (Fig. S1, G and H, for details). Time is given as minutes:seconds relative to NEBD.
	Figure 2. An F-actin–dependent mechanism delays chromosome capture. (A) Schematics of the experimental protocol for acute depolymerization of F-actin. (B) Selected maximum-intensity z-projections from a 3D confocal time series though the oocyte’s nuclear region during chromosome congression. Chromosomes (H2B-3mEGFP) are in cyan and F-actin (3mCherry-UtrCH) in gray. Bars, 10 µm. See also Video 2. (C) Left: Pseudo-colored time projection of a 3D confocal time series of chromosome congression (labeled with H2B-3mEGFP) in control or Latrunculin B–treated oocytes. See also Video 3. Right: Plot of chromosome distance to the AP over time, for the control and Latrunculin B–treated oocytes shown on the left. (D) Chromosome capture events identified for 13 pairs of control and Latrunculin B–treated oocytes (plotted in a different color for each oocyte). (E) Histograms of the data shown in D. Time is given relative to NEBD for all panels.
	Figure 3. The F-actin network does not prevent chromosome capture and transport by microtubules, but transport along microtubules interferes with F-actin network integrity. (A) A single selected slice from a deconvolved confocal stack of an oocyte fixed 5 min after NEBD and stained for tubulin (red), F-actin (green), and chromosomes by using Draq5 (cyan). (B) Microtubule length distribution measured in control and Latrunculin B–treated oocytes and plotted against each other. (C) Selected single confocal sections acquired over time showing the nuclear area of an oocyte expressing 3mEGFP-UtrCH (gray) and H2B-mCherry (cyan). See also Video 4. Right: Zoom-in on the region marked by a dashed rectangle of a chromosome transported along a microtubule (red arrowhead) causing local collapse of the F-actin network. (D) Maximum-intensity z-projections of a 3D confocal time series through the nuclear region of an oocyte expressing H2B-mCherry (cyan) and injected with UtrCH–Alexa Fluor 568 (gray). Single z-slice zooms of the regions marked by dashed rectangles are shown below visualizing the disruption of the F-actin network where chromosomes are pulled through. Bars: (main images) 10 µm; (C, inset) 5 µm. Time is given as minutes:seconds relative to NEBD for all panels.
	Figure 4. F-actin patches are nucleated on chromosomes by the Arp2/3 complex in a Ran-dependent manner. (A) Maximum projection of selected z-sections from a confocal z-stack of an oocyte expressing mEGFP-ArpC1 fixed 5 min after NEBD and immunostained. Anti-GFP antibody was used to enhance mEGFP-ArpC1, Phallodin-A568 to stain F-actin, and Draq5 for DNA. Below: Selected single z-slices zooming in on F-actin patches marked by dashed rectangles on the overview. Bars: (top images) 10 µm; (bottom images) 1 µm. (B) Single confocal slices selected from a time series of an oocyte injected with H1–Alexa Fluor 568 (cyan) and expressing either 3mEGFP-UtrCH (gray) to label F-actin or injected with Arp2/3–Alexa Fluor 488 protein (gray) to visualize the Arp2/3 complex. A region around a selected chromosome is shown. Oocytes were treated with CK-666 or with equal amount of DMSO 1 h before maturation. (C) Single confocal slices selected from a time series of an oocyte injected with H1–Alexa Fluor 568 (cyan) and either 3mEGFP-UtrCH mRNA to visualize F-actin or Arp2/3–Alexa Fluor 488 protein to visualize the Arp2/3 complex. A region around a selected chromosome is shown. Oocytes were injected with RanT24N or RanQ69L protein or equal amount of buffer as control. Time is given as minutes:seconds relative to NEBD. Bars, 5 µm.
	Figure 5. Disassembly kinetics of F-actin patches are tightly correlated with chromosome capture by microtubules. (A) Maximum-intensity z-projection of selected time points from a time series of the nuclear area of an oocyte expressing mEGFP-ArpC1 to label the Arp2/3 complex (gray), and H2B-mCherry to label chromosomes (cyan). Last frame: Chromosome tracks overlaid, color-coded as below. See also Video 5. Below: Single confocal slices of selected chromosomes marked by dashed rectangles on the overview. Bars: (top) 10 µm; (bottom) 5 µm. Time is given as minutes:seconds relative to NEBD. (B) Plot of chromosome distance to the AP over time, calculated from the trajectories shown in A. Trajectories are color-coded for actin- (green) and microtubule (red)-driven transport phases and arrival at the spindle (gray). Chromosome capture events are represented by black dots. (C) Normalized mEGFP-ArpC1 intensity profile for each chromosome tracked in B. Intensity is calculated in a 5-µm diameter sphere around the chromosome’s center of mass and normalized to the background level before NEBD onset (in gray). Plots are color-coded as in B. (D) Individual plots for chromosomes shown in A. (E) Oocytes expressing 3mCherry-UtrCH to label F-actin (gray) and H2B-3mEGFP to label chromosomes (cyan) were then treated with DMSO or Latrunculin B, respectively (left and right panels). Left side: Maximum z-projection of the first time point (00:30). Right side: Single confocal slices of selected chromosomes and an area sampling the F-actin network as marked by dashed rectangles on the overview. Below: Individual plots of mean intensities for chromosome patches (a and b) or network (c) shown above. Red bars indicate the chromosome capture event. Bars: (main images) 20 µm; (smaller images) 5 µm. Time is given as minutes:seconds relative to NEBD. (F) Plot of chromosome distance to the AP over time for the oocytes shown in E. Top right: Normalized 3mCherry-UtrCH intensity profiles for the 5-µm-diameter sphere surrounding each chromosome. Bottom right: Normalized mean F-actin network intensity profile.

References [+] :

Almonacid, Actin-based spindle positioning: new insights from female gametes. 2014, Pubmed, Echinobase