Microtubule and Actin Cytoskeletons

Throughout oogenesis, different polarized microtubule networks are organized within the nurse cell-oocyte complex and mediate important processes related to oocyte determination and differentiation. One such microtubule network is organized in the germarium (see Subheading 1.3.). During stages 2-6, a common MTOC, situated behind the oocyte nucleus, organizes a microtubule network by focusing the minus ends of microtubules. The plus ends of the microtubules extend through the ring canals into the nurse cells (see Fig. 4C). No centrosomal proteins of this MTOC are known, except for Centrosomin (Cnn), which accumulates at the MTOC relatively late, at stages 5 and 6 (64). During stage 7, the terminal follicle cells adopt a posterior fate after receiving the Gurken signal, and their back-signal to the oocyte induces disassembly of the MTOC behind the oocyte nucleus (60,61). As a consequence, the microtu-bule network breaks down, and during stage 8, a new microtubule network is organized with microtubules nucleated all around the oocyte cortex, with the exception of the posterior pole (40). At this time, Cnn is seen relocalized to the anterior and lateral cortical regions of the oocyte (64). After this reorganization of the microtubule network has occurred, the oocyte nucleus moves along the microtubules, aided by the action of DLis-1 and dynein, from the posterior to a random anterior corner of the oocyte (65). This new position of the oocyte nucleus defines the dorsal side of the egg chamber through a second Gurken-signaling event (see Subheading 1.4.). During stages 8 and 9, the microtubule network becomes polarized in such a way that the plus ends of microtubules are enriched at the posterior pole of the oocyte, a process influenced by PAR-1 (66), Rab11 (67), and the actin cytoskeleton (68). This late reorganization of the microtubule network plays an essential role in the localization of anterior and posterior determinants within the Drosophila oocyte (69,70). For details on the localization of anterior and posterior determinants, see refs. 7,13,71, and 72.

The nurse cells and the oocyte are interconnected via ring canals, which are membrane-associated actin cytoskeletal elements. Together with the cortical actin, ring canals play an important role in intercellular cytoplasm transport during oogenesis (73) (see Figs. 4D,E). Ring canal assembly is initiated with the arrest of the cleavage furrows at a diameter of 0.5-1 ^m, followed by the recruitment of several proteins in a developmentally defined order (14,73-76). Thus, the ring canals develop an electron-dense outer rim and a proteinaceous inner rim, and their diameter increases to 10 ^m by the rapid phase of transport. The glycoprotein D-mucin, anillin, and the kinesin-like protein (KLP) Pavarotti are the first to be recruited next to the contractile actin ring of the cleavage furrows during the cystocyte divisions (76-78). At the time of the final cystocyte division, another protein (or proteins) that reacts with anti-

phosphotyrosine antibodies can be detected on the outer rim (14). As soon as the 16-cell cyst enters germarium region 2a, inner rim formation is initiated by the accumulation of Filamin, HtsRC, and actin, and a few hours later, in germarium region 3, the Kelch protein is recruited. The outer rim acquires Filamin at the same time as the inner rim, and as soon as the egg chamber buds off from the germarium, anillin disappears from the outer rim (76).

Cytoplasm is transported between nurse cells and oocyte in two phases, a slow-initial phase and a rapid-terminal phase (3,73,79). The slow initial phase occurs during stages 2-10a and seems to be highly regulated (80). The rapidterminal phase (also called "dumping") results in the regression of the nurse cells and doubling of the oocyte volume during stages 10b-12. In each nurse cell, a system of actin-based microfilaments forms, connecting the nuclei with the plasma membrane. These microfilaments may serve to tether the nuclei away from the ring canals, because they are too large to pass through (81). This phase of transport seems to be nonselective and the forces that drive egg chamber elongation during stages 10b-12 may be responsible for it (82).

The immunostaining methods described in Subheadings 3.1.-3.4. work well for visualizing microtubules (using the YL1/2 antibody) and ring canal components. Antibodies against ring canal proteins are described in refs. 74-78. Reference 14 describes a rhodamine-conjugated phalloidin-based protocol to visualize actin in egg chambers. To study the effect of cytoskeletal inhibitors, the drugs colchicine (microtubule-specific) and cytochalasin-D (actin-specific) can be employed as described in refs. 41 and 80, respectively. To study micro-tubule polarity, transgenes that express either the minus-end-directed motor Nod or a plus-end-directed kinesin fused to P-galactosidase can be used as described in ref. (70).

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