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First published online January 10, 2008
doi: 10.1242/10.1242/jcs.015966

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Src and Wnt signaling regulate dynactin accumulation to the P2-EMS cell border in C. elegans embryos

¹ Laboratory of Genetics
² Laboratory of Molecular Biology
³ Department of Anatomy, University of Wisconsin, Madison, WI 53706, USA

View larger version (68K): [in this window] [in a new window]   Fig. 1. Dynactin complex is enriched at the P2-EMS border prior to and during EMS and P2 spindle rotation. Multi-photon time series of embryos expressing (A) DNC-1::GFP (supplementary material Movie 1); (B) DNC-2::GFP; dnc-2 3'UTR RNAi; and (C) ARP-1::GFP; arp-1 3'UTR RNAi show the accumulation of dynactin along the P2-EMS border (marked by white arrows). 3' UTR RNAi was used to improve the image contrast of DNC-2::GFP and ARP-1::GFP (see text). Notice that dynactin also localizes to centrosomes, serving as a marker for the spindle positioning. (A') Enlarged region between the EMS centrosome and the P2-EMS accumulation in a DNC-1::GFP embryo. Arrowhead points to a dynactin-labeled astral MT plus-end that terminates at the accumulation site. Scale bar: 10 µm.

View larger version (96K): [in this window] [in a new window]   Fig. 2. Dynactin::GFP enrichment at the P2-EMS border is regulated by both the Wnt and Src pathways, but not by G. P2 cell is to the right and EMS is at the bottom. Dynactin accumulation to the P2-EMS border is dynamic. In order to compare the fluorescence intensity among varied genetic backgrounds over a period of time, we selected ten adjacent frames (total 44 seconds) and projected these in ImageJ to produce an averaged image of this time interval. Intervals were chosen to show the best accumulation in (A) DNC-1::GFP wild type (WT); (B) DNC-1::GFP with mom-5 RNAi (supplementary material Movie 2); (C,C') DNC-1::GFP with src-1 RNAi (supplementary material Movie 3); and (D) DNC-1::GFP with mom-5; src-1 RNAi. In C, the spindle of P2 but not EMS rotates, whereas, in C', neither the P2 nor EMS spindle rotates. Arrows mark P2-EMS dynactin accumulation. Double-headed arrows show the misaligned spindles in src-1 RNAi embryos. (E) Quantitative analysis of the fluorescence intensity along the P2-EMS border using ImageJ. Two 25x7 pixel boxes were drawn in each of the generated averaged images, one along the P2-EMS border (box 1) and the other in the cytoplasm below this accumulation (box 2). A ratio of box1:box 2 fluorescence intensity is plotted for different genetic backgrounds. Error bars are s.d. Asterisks indicate a statistically significant difference (P<0.05) between wild-type and the RNAi-treated embryos (Student's t-test, two-tailed equal variance). (F-H) Multi-photon time series of embryos expressing DNC-1::GFP show the P2-EMS accumulation (white arrows), and P2 and EMS spindle positioning (double-headed arrows). In a wild-type embryo (F), DNC-1 accumulates at the P2-EMS border prior to and during EMS and P2 spindle rotations. The two spindles rotate and move such that one centrosome from each is adjacent to this accumulation site. In a mom-5; src-1 RNAi embryo (G), P2 centrosomes rotate partially but the final position of the spindle is away from the P2-EMS border (n=7); whereas, in another mom-5; src-1 RNAi embryo (G'; supplementary material Movie 4), P2 centrosomes fail to rotate and spindle forms along the D-V axis (n=3). In both cases, EMS spindle fails to align along the A-P axis, and DNC-1::GFP does not accumulate at the P2-EMS border. (H; supplementary material Movie 5) In gpa-16(it143ts);goa-1(RNAi) embryos, DNC-1::GFP still accumulates at the P2-EMS border, albeit weakly (see text). All the spindle orientations, including ABa, ABp, EMS and P2, are abnormal. (I) Quantitative analysis of the fluorescence intensity along the P2-EMS border between wild-type DNC-1::GFP and gpa-16(it143ts);goa-1(RNAi) embryos grown at permissive temperature. Measurements were taken as in E. There is no statistically significant difference (P=0.20) between wild-type and the G mutant embryos (Student's t-test, two-tailed equal variance). Scale bar: 10 µm.

View larger version (78K): [in this window] [in a new window]   Fig. 3. Dynactin activity is required at least for EMS spindle rotation. Multi-photon time series of embryos expressing tubulin::GFP in (A) wild type (WT) and (B) dnc-1(or404ts) embryos treated with partial dnc-1 RNAi (see Materials and Methods) and imaged at permissive temperature showed normal EMS spindle rotation; (C) dnc-1(or404ts) embryos treated with partial dnc-1 RNAi and imaged at restrictive temperature showed EMS set up its metaphase spindle perpendicular to the A-P axis (this embryo shows a ventral aspect). Scale bar: 10 µm.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Model for the role of dynactin in receiving polarized signals during nuclear rotation in P2 and EMS (see text).

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