MBC Videos and Supplemental Data

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Supplemental Data PDF

Supplemental Figure 1. The coding region of the transgene sqh-GFP, encoding the RMLC-GFP chimera. The RMLC sequence is in black, the linker sequence in red, and the GFP sequence in green. The genomic fragment of sqh is shown from the NaeI site (-56 bases from the ATG) to the BspHI site (83 bases from the stop codon). A small intron is in lower case letters.

• Movie1Fig1A.mov RLC-GFP embryos during cellularization, as visualized by Time-Lapse Confocal Microscopy (TLCM). The furrow forms and invaginates over a period of 50 minutes. The slow phase (0-30 minutes) ingresses at approximately 0.2mu m/min, accelerating to about 1 mu m/min, once the furrow has passed the base of the nuclei (fast phase). Note the initial increase in fluorescence until the beginning of the fast phase. The subsequent disappearance of GFP signal as the furrow reaches its final depth is likely due to quenching by the yolk, as no loss of signal is seen in fixed and immunostained embryos. The biphasic rates of furrow formation are essentially identical in wild type and RLC-GFP embryos.
• Movie2Fig2.mov (5.18 MB) A cytoplasmic reservoir of myosin II-containing particles contributes to myosin in the advancing furrow. An RLC-GFP embryo, previously injected with Rhodamine-tubulin to label microtubules, was visualized by TLCM. The myosin II-containing particles (green), 1-2mu m in diameter, display a saltatory movement, but progress generally anteriorly at an average rate of 0.04mu m/min and fuse with the furrow (green), particularly during the slow phase. Particle movement is sensitive to colchicine (see Movie 3), and thus are probably moving along microtubules. Particle fusion with the furrow apparently contributes to the increase in myosin signal at the furrow front. Note the occasional reversal of direction of individual particles. The particles do not appear to be membrane vesicles (see text).
• Movie3Fig3A.mov (4.29 MB) The depolymerization of microtubules by colchicine blocks further furrow ingression, but does not block the rearrangement of the furrow into rings that pinch off basally at the end of cellularization. RLC-GFP Embryos were injected with colchicine 20 minutes after the onset of cellularization. Although furrowing ingression nearly stops, the subsequent rearrangement of the furrow into rings occurs “on time” (about 50 minutes after furrow onset), and the rings eventually close off basally. The fact that the transition from invagination to basal closure can occur in the absence of microtubules suggests that the transition is normally triggered by a clock marking time with respect to the onset of cellularization, independent of furrow depth. Note that myosin-containing particles stop moving following colchicine treatment.
• Movie4Fig3B.mov (3.76 MB) Depolymerization of actin does not fully block furrowing. RLC-GFP Embryos were injected with cytochalasin 20 minutes after the onset of cellularization. Furrowing continues, even though the myosin-rich front is profoundly disrupted. In contrast, there is no evidence of basal closure. Cytochalasin treatment has no effect on myosin particles, which continue to move apically, often passing right through the torn furrow front.
• Movie5Fig8A.mov (5.25 MB) Myosin II dynamics during the first divisions of the cellular blastoderm. A mitotic domain in the head region of an RLC-GFP embryo (previously injected with Rhodamine tubulin to visualize microtubules) was recorded at 5-second intervals by TLCM. In interphase myosin (green) is diffusely cytoplasmic. During mitosis, myosin begins to accumulate at the cortex as the cells round up. Soon after anaphase onset, cortical myosin declines at the poles and accumulates at the equator, suggesting cortical flow. The myosin ring then contracts, pinching the cell in two. Duration 3 min 20 sec.

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