Wühr M et al. (2010), A model for cleavage plane determination in ear...

XB-ART-43143

Curr Biol 2010 Nov 23;2022:2040-5. doi: 10.1016/j.cub.2010.10.024.

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A model for cleavage plane determination in early amphibian and fish embryos.

Wühr M , Tan ES , Parker SK , Detrich HW , Mitchison TJ .

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Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their astral microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, astral microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before astral microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of astral microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.

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Species referenced: Xenopus laevis
Genes referenced: trim9

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	Figure 1. Spindles Are Positioned and Oriented by Asters Prior to Mitosis Onset (A) Frog embryos were artificially elongated by compression and fixed at different time points. Immunofluorescence against Î±- (yellow) and Î³-tubulin (red) allows scoring of centrosome orientation and cell-cycle stage. Note centrosomes are already aligned in prophase before nuclear envelope breakdown. (B) Quantification of average centrosome orientation relative to longest cell axis (angle measured between 0Â° and 90Â°; random orientation would be 45Â°). Centrosomes are already well aligned (4.9Â° Â± 2.4Â° SD) before nuclear envelope breaks down, as soon as they can be visualized with Î³-tubulin staining. Alignment does not improve significantly in metaphase (4.2Â° Â± 3.7Â°) or anaphase-telophase (4.9Â° Â± 3.8Â°). Once the expanding telophase asters touch the cortex, just before cytokinesis, centrosome alignment is fine tuned significantly (1.4Â° Â± 1.1Â°). (C) Î±-tubulin (yellow) immunofluorescence in a frog embryo at anaphase-telophase of first mitosis, before cytokinesis. Duplicated centrosomes are already aligned parallel to the longest axes of the daughter cells. DNA (blue) follows centrosomes. The scale bars represent 500 Î¼m. See also Figure S1.
	Figure S1. Related to Figure 1. A) Cleavage plane orientation is mostly determined before anaphase/telophase: Xenopus laevis embryos were fertilized and compressed at various time points between glass plates 0.8 mm apart. Each point represents the start of deformation and the resulting average cleavage angle relative to the glass plates with standard error of the mean (angles were defined between 0Â° as parallel to and 90Â° as perpendicular to glass plates). Time was normalized relative to first cleavage (t=1). The embryos mostly lose their ability to reorient the directly following cleavage plane relative to the artificially introduced longest axis before anaphase onset which we determined by Immunofluorescence at t ~ 0.79 (blue line). B) The tendency of the first cleavage plane to cut through the sperm entry point can be explained with the initial long axis of the sperm aster. Immunofluorescence of a Xenopus laevis embryo against Î±- and Î³-tubulin of the sperm aster 35 min post fertilization at 18Â° C. The sperm entered from the periphery (approximated by arrow), the growth of the sperm aster is restricted by the close by cortex resulting in an elongated aster, with the longest axis parallel to the tangent of the sperm entry point. The centrosomes and the later first mitotic spindle orient with this long axis, resulting in a cleavage plane that cuts near the sperm entry point.
	Figure S2. Related to Figure 2. Length asymmetry in telophase asters at 1st mitosis in frog and fish embryos. A) Definition of axes plotted in B. Distance of Interaction-zone to centrosome (x-axis) and Centrosome to aster front in a direction normal to the interaction zone (y-axis) B) Green symbols show distances as defined in A were plotted for the first cell cycle in zebrafish from the movie S1. Red symbols show data from 29 frog embryos fixed during anaphase/telophase of the first cell cycle. The blue line would be equivalent to a symmetrical aster (slope=1). All data-points are above the blue line, showing the asters are always longer (from centrosome to periphery) on the freely expanding side compared to the side facing the interaction zone. This is consistent with our length dependent pulling model for aster movement.
	Figure S4. Related to Figure 4. A cartoon summarizing our understanding of microtubule organization in frog embryos based on published [3, 13] and unpublished micrographs. I) Fertilization triggers the continuation of the eggâs cell cycle, which was arrested at metaphase of meiosis 2. The small meiotic spindle at the top (animal pole) of the egg enters anaphase. The female pronucleus (blue) is deposited close to the animal pole. The sperm enters randomly at upper half of the egg (sperm entry point in yellow) delivering the centrosome (green) and male pronucleus. The centrosome initiates the outgrowth of microtubules (red) resulting in the sperm aster. II) The center of this aster moves towards the center of the cell. Male and female nuclei move along microtubules and meet at the center of the aster. Yolk content is higher at the bottom of the egg which biases centering towards the top III) At the onset of the first mitosis the sperm-aster breaks down and from the deposited centrosomes and DNA a relatively small first mitotic spindle forms (embryos from C onwards are rotated by 90Â° around the z-axis for ease of drawing). Typically the first mitotic spindle forms parallel to the tangent of the sperm entry point. IV, V) Upon anaphase the two small asters of the spindle start to grow out, but do not grow into each other due to a region of reduced microtubules density that forms where the asters overlap. We call these asters telophase-asters, and the region between them interaction zone. The centers of the asters move apart towards the center of the future daughter cells. VI) The cleavage furrow constricts where the interaction zone meets the cortex. Upon entry to second mitosis the telophase-asters break down and the second mitotic spindles form centered and oriented along the longest axis of the cells. B) Model how a pair of centrosomes could align with an asterâs long axis e.g. in a compressed embryo at the one cell stage (Fig. 1). We assume length dependent pulling forces on microtubules (blue and red). The centrosomes are held together with a spring (This spring might correspond to the mini-spindle observable in Fig. 1C and 2D or the nucleus in Fig. 1 A). It seems plausible that microtubules are more likely to transfer pulling forces towards the centrosome they are closest to (blue microtubules are more likely to interact with blue centrosome). The net force on the centrosomes generates a torque that aligns the pair with the asterâs long axis. The same model applies to the orientation of pairs of centrosomes within the dome-shaped telophase asters. Besides our arguments put forward above for length dependent pulling on microtubules this model is motivated by the following observations: In telophase a pair of centrosomes aligns with the asterâs longest axis (Fig. 1, S1 B, 2D). Changing the shape of the aster reorients centrosomes (Fig. 1 B). Centrosomes orientation is dynein dependent (Fig 3 C) but independent of aster-cortex contact (Fig 1 C, S1 B, 2 D).

References [+] :

Adames, Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae. 2000, Pubmed