NMY-2 maintains cellular asymmetry and cell boundaries, and promotes a SRC-dependent asymmetric cell division

Abstract

The nonmuscle myosin II NMY-2 is required for cytokinesis as well as for the establishment of zygote asymmetry during embryogenesis in Caenorhabditis elegans. Here we describe two conditional nmy-2 alleles that rapidly and reversibly inactivate the protein. We show that NMY-2 has late-cell-cycle roles in maintaining embryonic asymmetries and is also required for a surprisingly late step in the maintenance of the cytokinesis furrow. Finally, during a signaling-induced asymmetric cell division, NMY-2 is required for SRC-dependent phosphotyrosine signaling and acts in parallel with WNT-signaling to specify endoderm.

Figure 1

Partial amino acid sequence alignment for the NMY-2 orthologs from C. elegans (Ce), D. melanogaster (Dm) and H. sapiens (Hs).

Figure 2

Continuous 15°C imaging (Panel A) and temperature upshift from 15°C to 25°C (Panel B) of anaphase nmy-2(ne3409); PAR-6::mCherry; PAR-2::GFP zygotes. The anterior is to the left. Minutes: seconds were labeled. 0:00: upshift point. Each column consists of frames taken at the same time point: PAR-6::mCherry (first row), PAR-2::GFP (second row), and combined (third row). The bar indicates 10 um.

Figure 3

Temperature downshift from 25°C to 15°C of prometaphase nmy-2(ne3409); PAR-6::mCherry; PAR-2::GFP zygotes. The anterior is to the left. A: an embryo that developed complementary PAR-6 and PAR-2 cortical signals during downshift. B: an embryo that failed to develop an apical PAR-2 but later developed a PAR-2 signal at the cleavage furrow. Minutes: seconds were labeled. 0:00: downshift point. Each column consists of frames taken at the same time point: PAR-6::mCherry (first row), PAR-2::GFP (second row), and combined (third row). The bar indicates 10 um.

Figure 4

NMY-2 is required for the maintenance of nascent cell boundaries. (A–F) Diagrams and micrographs depicting the results of representative temperature-shift experiments performed on nmy-2(ne3409) embryos. Each row of three micrographs depicts a single embryo maintained at 15°C until cell cleavage was complete (time 0), and then shifted to 25°C at the time indicated in the first micrograph of each row (minutes:seconds). G: Summary of all 44 embryos assayed as described in A–F. Blank circles represent embryos whose furrows later fused back during the next mitosis, and grey circles represent embryos whose furrows never fused back. The position of each circle on the X-axis indicates the time at which the corresponding embryo was shifted up. The bar indicates 10 um.

Figure 5

ZEN-4 staining in an nmy-2(ne3409) (left) and a wildtype (right) embryos. Arrowheads demarcate the extent of the ZEN-4 signal. “*” denotes variable and probably nonspecific spindle pole and chromosome stainings.

Figure 6

NMY-2 is required for elevated phosphotyrosine staining at the P2/EMS junction. (A through E): Representative micrographs showing SRC_PY416 staining in Wildtype N2 (A); mes-1(bn74) (B); src-1 dsRNA-injected N2 (C); and nmy-2(ne3409) embryos shifted to 25c for 2 minutes (D) for an elevated SRC at P2/EMS junction similar to the wildtype (A); and (E) for an uniform SRC pattern similar to (B). Arrowheads indicate the P2/EMS junction. (F and G): Tabulation of the percentage of embryos analyzed with elevated or uniform SRC_PY416 signal (F), and PY99 signal (G), for each embryo type.