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First published online 16 May 2007
doi: 10.1242/dev.004952

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Actin-dependent cytoplasmic streaming in C. elegans oogenesis

¹ Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
² Molecular and Cellular Biology Program and Department of Biology, University of Washington, Seattle, WA 98195, USA.
³ Howard Hughes Medical Institute, Seattle, WA 98109, USA.

View larger version (80K): [in this window] [in a new window]   Fig. 1. Proximal streaming in the wild-type C. elegans gonad. (A) Diagram of one arm of an adult hermaphrodite gonad. Plasma membranes, red; nuclei, blue. Somatic sheath cells enclose most of the gonad, but are not shown for simplicity. (B) Single image from time-lapse movie. DIC particles are shown at high magnification in inset. (C) 2-minute particle tracks. Arrows indicate final positions of particles and dots indicate stationary particles. Most particles ceased movement in cellularized oocytes; the small arrows shown result primarily from a shift in oocyte position. (D-G) Movement of PGL-1::GFP particles. (D) Low magnification view of gonad indicating perinuclear P granules (arrow) and detached, cytoplasmic P granules (arrowhead). (E-G) Time-lapse series of boxed region in D. Particles tracked are a perinuclear P granule (orange circle) and cytoplasmic PGL-1::GFP particles (green square and yellow triangle). Movements are summarized in G. (H) Low magnification view of gonad showing PGL-1:: mRFP-1 (red) and mitochondria (green). For movements of mitochondria see Movie 1 in the supplementary material. (I-K) Time-lapse images of a single PGL-1::mRFP-1 particle (arrowhead). Time is shown in minutes. (L) Composite of particle positions at 15 time points in 30-second intervals. (M) Diagram of bulk particle movement (green). Scale bars: 30 µm in B; 3 µm in B inset; 10 µm in E,J.

View larger version (99K): [in this window] [in a new window]   Fig. 2. Injected oil droplets are transported by cytoplasmic streaming in C. elegans. (A-E) Time-lapse images showing movement of an oil droplet (false green color, white arrowhead) over 30 minutes. (F) Position of the oil droplet in 1-minute intervals. Images are from Movie 2-1 in the supplementary material. Scale bar: 30 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 3. Gonad microtubules are not required for proximal streaming in C. elegans. (A) Microtubules (red) and nuclei (blue) in pachytene region. Proximal is to the right. (B) High magnification image of microtubules around individual germ nuclei. (C) Microtubules extending between enlarging oocytes and gonad core. Oocyte membranes are indicated by dashed lines. (D) Summary diagram of gonad microtubules. (E,F) Gonads immunostained for -tubulin after treating worms with empty-vector control (E) or an -tubulin RNAi feeding strain (F) for 24 hours. (G,H) Normal particle movement in an -tubulin RNAi-treated gonad; 2-minute particle tracks. Scale bars: 10 µm in A-C; 30 µm in E-G.

View larger version (50K): [in this window] [in a new window]   Fig. 4. The actomyosin cytoskeleton is required for streaming in C. elegans. (A-C) Phalloidin-stained microfilaments (green). (A) Low magnification view of enlarging oocytes in loop; fixation method I. (B) High magnification view of enlarging oocyte; fixation method II. Note the long filaments (arrowheads) extending from the core plasma membrane into the oocyte. (C) Pachytene region. (D) Summary diagram of gonad microfilaments. (E,F) Cytoplasmic streaming in gonad extruded in culture medium with EGTA; 2-minute particle tracks. (G,H) Abnormal, reversed streaming in gonad extruded in culture medium with latrunculin A and EGTA; 2-minute particle tracks. Scale bars: 10 µm in A-C; 30 µm in E,G.

View larger version (68K): [in this window] [in a new window]   Fig. 5. The force driving proximal streaming appears to be generated close to or within the enlarging oocytes. Drops of mineral oil (yellow) were injected into different areas of the C. elegans gonad. (A,D,G) DIC image. (B,E,H) 2-minute particle tracks. (C,F,I) summary diagrams. (A-C) Blocking the early pachytene area results in normal proximal streaming. (D-F) Particle movement after injection of an oil drop between the late pachytene area and loop. (G-I) An oocyte was damaged by injecting a small mineral oil droplet directly into it (red arrowhead). Scale bars: 30 µm.

View larger version (18K): [in this window] [in a new window]   Fig. 6. Models for oocyte-driven actin-dependent proximal streaming in C. elegans. Gray arrows indicate direction of cytoplasmic streaming into enlarging oocytes (blue outline). (A) Cortical contraction (green arrows) generates reciprocal cytoplasmic flow. (B) Particle transport along oriented actin cables (green). (C) Expansion of cortical surface. (D) Contractile actomyosin network at the entrance to an oocyte linked to a stable interior meshwork.

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