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LET-99 determines spindle position and is asymmetrically enriched in response to PAR polarity cues in C. elegans embryos

Meng-Fu Bryan Tsou, Adam Hayashi, Leah R. DeBella, Garth McGrath^* and Lesilee S. Rose

Section of Molecular and Cellular Biology, University of California, Davis, CA 95616, USA
^* Present address: Exelixis, South San Francisco, CA 94080, USA

View larger version (58K): [in a new window]   Fig. 1. Analysis of nuclear and spindle pole movements. (A) DIC images of live let-99 (top row) and let-99; dhc-1(RNAi) (bottom row) embryos recorded by time-lapse video microscopy. Arrowheads mark the centrosomes and relative time points are indicated in the top left-hand corner of the images. Scale bar: 10 µm. (B) Traces of spindle pole position in representative one-cell embryos from metaphase to cytokinesis onset (time 0). 0% egg length indicates the anterior tip of the embryo; the posterior tip would be at 100%. Arrows in wild type (N2) and par-3 mark the time at which spindles began elongating and spindle poles started to oscillate; the most vigorous oscillations occurred in the middle of the elongation phase. Arrow in let-99 indicates the time at which nuclear/spindle rocking ceased.

View larger version (15K): [in a new window]   Fig. 2. Molecular identification of the let-99 gene. (A) Genetic and physical maps of the region containing let-99 on chromosome IV. (B) Restriction map and rescue data for cosmid C13H6 and fragments. (C) Restriction map and transcribed regions present in the smallest rescuing fragment. Northern blot analysis identified a 2.4 kb transcript that was expressed at high levels in the germline (let-99, open boxes) and two other transcripts (arrows), the positions of which were determined by the Genome Consortium (Genome Consortium, 1998). Injection of RNA corresponding to the 2.4 kb transcript into wild type resulted in a phenocopy of the let-99 mutant phenotype in progeny embryos. (D) The predicted LET-99 and LRG-1 proteins, showing the positions of let-99 mutations and the corresponding amino acids change. The DEP domain is boxed; shaded box indicates a block of amino acids (153-212) present in LET-99 but not LRG-1; LRG-1 is 86% identical to the N-terminal region of LET-99. The Genbank Accession Number for let-99 (K08E7.3) is Z77666 and for lrg-1(F55H2.4) is NP_499092. (E) Western blot of wild-type (N2) and let-99(or81) mutant embryos probed with affinity-purified anti-LET-99 antibodies and reprobed with anti-tubulin as a loading control.

View larger version (98K): [in a new window]   Fig. 3. Immunolocalization of the LET-99 protein in one-cell embryos. Confocal sections of mitotic stage wild-type embryos (A-J,M-N) and let-99 (or81) embryos (K,O) and meiotic stage wild-type (Q-V) and let-99 (or81) (W,X) embryos stained with affinity-purified LET-99 antibodies (A-D,I-K,Q,S,U,W) and DAPI (E-H,M-O,R,T,V,X). Anterior is towards the left in this and all subsequent figures unless indicated. (A,E) Early prophase embryo during pronuclear migration. (B,F) One-cell prophase embryo during centration, before nuclear rotation has occurred. (C,G) One-cell metaphase embryo. (D,H) One-cell anaphase embryo. (I,M) Two-cell embryo in which P1 is in prophase. (J,N) Six-cell embryo in which P2 is in prophase. (K,O) One-cell let-99 anaphase embryo. Arrowheads indicate polar bodies that are positive for LET-99 staining in wild-type embryos and negative for LET-99 in mutant embryos. Arrows in C,D indicate the metaphase plate- (C) and the spindle midzone- (D) associated staining of LET-99. Arrow in J indicates the LET-99 band in the P2 cell. (L,P) Schematic diagram of one-cell and two-cell embryos showing the three LET-99 domains in P lineage cells: anterior domain, posterior band and posterior domain. (Q,R) Wild-type embryo in anaphase of meiosis. (S,T) Wild-type embryo in metaphase of meiosis viewed from the side; the spindle axis is parallel to the edge of the embryo and tilted slightly towards viewer. arrows in T indicate faint gaps in DAPI staining between opposed chromatin masses to which the bars of LET-99 appear to localize. Such gaps and corresponding LET-99 bars were visible in meiotic prometaphase as well. (U,V) Wild-type embryo in meiosis viewed from one spindle pole. (W,X) let-99 (or81) embryo in anaphase of meiosis. Scale bars: in O, 10 µm for A-O; in X, 10 µm for Q-X.

View larger version (72K): [in a new window]   Fig. 4. Comparison of LET-99 and PAR-3 staining in wild-type embryos. Confocal micrographs of wild-type embryos triple labeled with DAPI (A, E, I), anti-PAR-3 (B, F, J) and anti-LET-99 antibodies (C, G, K). Merged images (D, H, L) show PAR-3 in red, LET-99 in green and DNA in blue. (A-D) 1-cell embryo during pronuclear migration. (E-H) 1-cell metaphase embryo. (I-L) 2-cell embryo in which P1 is in late prophase. The focal plane was chosen to show the P1 cell most clearly. Scale bar: 10 µm.

View larger version (61K): [in a new window]   Fig. 5. LET-99 localization depends on PAR-3 and PAR-2. Confocal images of par-3 embryos (A-H) and par-2 embryos (I-P) stained with anti-LET-99 antibodies (top panels) and DAPI (bottom panels). (A,E,I,M) One-cell prophase embryos. (B,F,J,N) One-cell metaphase embryos. (C,G,K,O) One-cell late anaphase embryos. (D,H,L,P) Two-cell prophase embryos. Arrows indicate the boundaries of the LET-99 central band in par-3 and of the posterior domain in par-2 embryos. Large arrowheads indicate polar bodies. Small arrowheads in B,J and C,K indicate the metaphase plate and spindle midzone-associated staining of LET-99, respectively; the intensity of these two patterns varies in both wild type and par mutants. Scale bar: 10 µm.

View larger version (50K): [in a new window]   Fig. 6. Nuclear rotation fails in spherical par-3 one-cell embryos. DIC images of live wild-type (A-D) and par-3 (E-H) embryos during chitinase treatment to remove the eggshell. Arrowheads mark the position of the sperm pronucleus before nuclear migration. Asterisks indicate the centrosomes. In the par-3 embryo, the two centrosomes are aligned into the plane of the image, transverse to the axis defined by the position of the sperm, and thus only one centrosome is visible. Scale bar: 10 µm.

View larger version (26K): [in a new window]   Fig. 7. Model for the role of LET-99 in nuclear rotation and anaphase spindle positioning in one-cell embryos. Anterior is towards the left. Centrosomes and microtubules are shown in green. Yellow arrows indicate forces from the cortex acting on subsets of astral microtubules. Broken lines indicate microtubules that contact regions enriched for LET-99 and experience less force (small yellow arrows; see text). Red arrows indicate the net force acting on the centrosomes or spindle poles, with size proportional to the magnitude of the force. (A) Prediction of forces during centration/nuclear rotation and anaphase spindle positioning in wild type. (B) Prediction of forces on spindle poles during anaphase in par-3 mutant embryos.