Wolbachia utilizes host microtubules and Dynein for anterior localization in the Drosophila oocyte

Abstract

To investigate the role of the host cytoskeleton in the maternal transmission of the endoparasitic bacteria Wolbachia, we have characterized their distribution in the female germ line of Drosophila melanogaster. In the germarium, Wolbachia are distributed to all germ cells of the cyst, establishing an early infection in the cell destined to become the oocyte. During mid-oogenesis, Wolbachia exhibit a distinct concentration between the anterior cortex and the nucleus in the oocyte, where many bacteria appear to contact the nuclear envelope. Following programmed rearrangement of the microtubule network, Wolbachia dissociate from this anterior position and become dispersed throughout the oocyte. This localization pattern is distinct from mitochondria and all known axis determinants. Manipulation of microtubules and cytoplasmic Dynein and Dynactin, but not Kinesin-1, disrupts anterior bacterial localization in the oocyte. In live egg chambers, Wolbachia exhibit movement in nurse cells but not in the oocyte, suggesting that the bacteria are anchored by host factors. In addition, we identify mid-oogenesis as a period in the life cycle of Wolbachia in which bacterial replication occurs. Total bacterial counts show that Wolbachia increase at a significantly higher rate in the oocyte than in the average nurse cell, and that normal Wolbachia levels in the oocyte depend on microtubules. These findings demonstrate that Wolbachia utilize the host microtubule network and associated proteins for their subcellular localization in the Drosophila oocyte. These interactions may also play a role in bacterial motility and replication, ultimately leading to the bacteria's efficient maternal transmission.

Figure 1. Wolbachia Distribution from Germ-Line Stem Cell Division through Mid-Oogenesis

In (B), (B′), (D), and (E), Wolbachia is green (hsp60), Vasa is blue, and DNA is red. In (F–H), Wolbachia is red (hsp60), α-Spectrin highlights the fusome (white), and microtubules are green. (A) The ovariole consists of the germarium (GER) at the anterior-most tip, and the vitellarium. Numbers represent the stages of oogenesis in the vitellarium (stages 2 through 14). Between stages 2 and 7, the cytoplasm of the nurse cells and the oocyte increases linearly. Following stage 7, the oocyte cytoplasm begins to grow disproportionately faster than nurse cell cytoplasm. Between stages 10 and 12, all nurse cell cytoplasm is transferred into the oocyte through “cytoplasmic dumping.” From stage 12 to 14, the nurse cell nuclei degenerate and dorsal filaments are formed. (B) Middle stage egg chambers infected with Wolbachia. White arrow indicates Wolbachia within follicle cells. (B′) High magnification of a stage 5 uninfected oocyte stained with anti-hsp60 antibody, showing that this antibody does not cross react with D. melanogaster proteins. (C) The germarium is divided into regions 1, 2, and 3. In the germarium, a germ-line stem cell (dark blue) divides to produce a daughter stem cell and a cystoblast. In region 1, the cystoblast is mitotically active; it undergoes four successive rounds of mitosis, resulting progressively in cysts of two, four, eight (brown), and sixteen (tan) cystocytes. Red shading represents the fusome. In region 2, the 16-cell cyst becomes surrounded by somatically derived follicle cells (green). At the most posterior position of the germarium (region 3), the stage 1 cyst buds off and enters the vitellarium. By this time the oocyte (white) has moved to the posterior side of the cyst. (D) High magnification of a stage 6 oocyte. Wolbachia (indicated by a white arrow) accumulate densely around the anterior of the oocyte. (E) High magnification of a stage 7 oocyte. Wolbachia begin to disperse from the anterior of the oocyte. (F) A germarium infected with Wolbachia: stage 1 egg chamber (white arrow) and stage 2 egg chamber (white arrow with asterisk). (G) High magnification of a germ-line stem cell (circumscribed by yellow line) undergoing asymmetric mitosis. The fusome material (indicated by a white arrow) is spherical in the stem cell. A cystoblast is visible in the upper right corner, identified by its shape and position in the germarium. A black arrow indicates Wolbachia. (H) A 16-cell cyst in region 2 of the germarium (circumscribed by yellow line) as it becomes surrounded by follicle cells. Wolbachia are present in all of the germ cells, but do not associate with the fusome. (A) adapted from [79]; (C) adapted from [39].

Figure 2. Wolbachia and Mitochondria Exhibit Different Localizations, and Wolbachia Anterior Localization Is Observed for Several Bacterial Strains and Drosophila Species

(A–C) A stage 5 egg chamber showing Wolbachia and mitochondria. (A) Host DNA and Wolbachia (greyscale-OliGreen). (B) Mitochondria (greyscale-MitoTracker). (C) Merged image of (A) and (B): Wolbachia and host DNA are green and Mitochondria are red. (D–F) Wolbachia and host DNA are red (propidium iodide), and cell cortex and ring canals are green. (D) An oocyte from D. melanogaster infected with the wMel Wolbachia strain. (E) An oocyte from D. melanogaster infected with the wMelpop (Popcorn) Wolbachia strain. (F) An oocyte from D. simulans infected with the wRiv Wolbachia strain. Scale bar = 10 μm. White arrows in (A) and (D) indicate Wolbachia concentrated at the anterior region of the oocyte.

Figure 3. Wolbachia Exhibit Movement in Nurse Cells but Not in the Oocyte In Vivo

(A) A still image taken from Video S1 showing bacterial motility in a live stage 5 egg chamber. Draq 5 stains Wolbachia and host DNA (both shown in green). As in fixed tissues, Wolbachia localize anteriorly (white arrow) in the oocyte and are distributed randomly in the nurse cells (red arrows) of live egg chambers. Wolbachia in nurse cells exhibit random movement while Wolbachia in the oocyte are relatively motionless (Video S1). White arrowheads indicate smaller groups of Wolbachia at the anterior region of the oocyte with lower bacterial densities, suggesting that their lack of movement is not due to bacterial overcrowding in the oocyte. Yellow arrowhead indicates the oocyte nucleus. (B) A still image taken from Video S2 showing a live stage 6 uninfected egg chamber. This image illustrates that Draq 5 stains Wolbachia (see [A]) but not mitochondria or other cytoplasmic organelles. Yellow arrowhead indicates the oocyte nucleus. Scale bar = 5 μm.

Figure 4. Manipulation of Specific Host Cytoskeletal Factors Affects Wolbachia Numbers

(A) Mean Wolbachia numbers for the average nurse cell and the oocyte through mid-oogenesis. (B) Mean Wolbachia numbers for the average nurse cell and the oocyte for colchicine-treated and control (untreated) egg chambers. (C) Mean Wolbachia numbers for the average nurse cell and the oocyte for maelstrom ^r20/+ and maelstrom ^r20/Df (3L) 79E-F egg chambers. (D) Mean Wolbachia numbers for the average nurse cell and the oocyte for Dhc64C ^6–6/+, Dhc64C ^6–12/+, and Dhc64C ^6–6/Dhc64C ^6–12 egg chambers. Error bars represent the standard error of the mean.

Figure 5. Depolymerization of Microtubules Causes Loss of Anterior Wolbachia Localization in the Oocyte

(A–C) Stage 5 and 7 infected egg chambers, untreated. (A) Microtubules (greyscale). (B) Wolbachia (greyscale). White arrow indicates Wolbachia concentrated at the anterior region of the oocyte. (C) Merged image of (A) and (B): microtubules are red, and Wolbachia are green (hsp60). (D–F) Stage 5 and 7 infected egg chambers treated with colchicine. (D) Microtubules (greyscale). (E) Wolbachia (greyscale). (F) Merged image of (D) and (E): microtubules are red, and Wolbachia are green (hsp60). White arrows indicate positions of the oocytes. Yellow asterisk indicates a nurse cell adjacent to the oocyte in which Wolbachia appear abnormally high.

Figure 6. Wolbachia Are Transferred into the Oocyte from Nurse Cells through Cytoplasmic Dumping

Wolbachia and host DNA are red (propidium iodide), and cell cortex and ring canals are green. (A) A stage 12 egg chamber nearing completion of cytoplasmic dumping. (B) Higher magnification of nurse cell remnants. Only a small number of Wolbachia are present in remaining nurse cell cytoplasm. White arrow indicates a few Wolbachia near a nurse cell nucleus. (C) Higher magnification of the anterior region of the oocyte. Nearly all Wolbachia have been “dumped” into the oocyte. White arrow indicates Wolbachia, and white arrowhead points to the oocyte nucleus.

Figure 7. Perturbation of Microtubules and Cytoplasmic Dynein Disrupts Anterior Localization of Wolbachia in the Oocyte

Wolbachia and host DNA are red (propidium iodide), and cell cortex and ring canals are green. (A–D) Stage 4–6 infected oocytes from colchicine-treated females. White arrows indicate Wolbachia. (E–H) Stage 4–6 infected oocytes from maelstrom ^r20/Df (3L) 79E-F females. (I–L) Stage 4–6 infected oocytes from Dhc64C ^6–6/Dhc64C ^6–12 females.

Figure 8. In the Oocyte, Wolbachia Localize along the Anterior Nuclear Envelope

In (A) and (B), Wolbachia and host DNA are red (propidium iodide), and cell cortex and ring canals are green. In (C), (D), and (F),) cytoplasmic Dynein is green, and Wolbachia and host DNA are red (OliGreen). In (E) and (G–I), nuclear envelope is blue, and Wolbachia and host DNA are red (OliGreen). White arrows indicate Wolbachia. (A) A stage 7 oocyte derived from a hs-Dmn/+ female, heat shocked. Wolbachia localize normally in the anterior region. (B) A stage 7 oocyte derived from a hs-Dmn/hs-Dmn female, heat shocked. Wolbachia become displaced from the anterior region. (C) A series of early-stage infected egg chambers. Dynein becomes enriched in the oocyte before stage 7. (D) A stage 5–6 infected oocyte. Within the oocyte, Dynein is enriched near the posterior pole and in a ring around the periphery of the oocyte nucleus. Many Wolbachia localize near nuclear-associated Dynein along the anterior periphery of the oocyte nucleus but not with the majority of Dynein concentrated at the posterior pole. Scale bar = 20 μm. (E) A stage 5–6 infected oocyte. In the oocyte, many Wolbachia maintain a close association with the nuclear envelope. (F) A stage 9 infected oocyte. Following the microtubule reorganization, Dynein localizes at the posterior pole of the oocyte and between the oocyte nucleus and the anterolateral cortex. At this time, Wolbachia become distributed throughout the oocyte cytoplasm. Scale bar = 20 μm. (G) A stage 9 infected oocyte. Following the microtubule reorganization, Wolbachia lose their association with the nuclear membrane. (H) A stage 6 Dhc64C ^6–6/Dhc64C ^6–12-derived oocyte. Wolbachia localize around the entire periphery of the oocyte nuclear envelope. (I) A stage 6 Dhc64C ^6–6/Dhc64C ^6–12-derived oocyte. Wolbachia dissociate from the oocyte nuclear envelope.