Intercellular protein movement in syncytial Drosophila follicle cells

Abstract

Ring canals connecting Drosophila germline, follicle and imaginal disc cells provide direct contact of cytoplasm between cells. To date, little is known about the formation, structure, or function of the somatic ring canals present in follicle and imaginal disc cells. Here, we show by confocal and electron microscopy that Pavarotti kinesin-like protein and Visgun are stable components of somatic ring canals. Using live-cell confocal microscopy, we show that somatic ring canals form from the stabilization of mitotic cleavage furrows. In contrast to germline cells, syncytial follicle cells do not divide synchronously, are not maximally branched and their ring canals do not increase in size during egg chamber development. We show for the first time that somatic ring canals permit exchange of cytoplasmic proteins between follicle cells. These results provide insight into the composition and function of ring canals in somatic cells, implying a broader functional significance for syncytial organization of cells outside the germline.

Fig. 1.

Proteins in somatic ring canals. (A–D) GFP::Pav-KLP (A,B) and GFP::Vsg (C,D) localize to ring canals in the germline (A,C yellow arrowheads) and somatic follicle cells from region 2a of the germarium (A,C) through stage-10 egg chambers (B,D) (white arrows). (E,F) ImmunoEM of post-mitotic follicle cells expressing GFP::Pav-KLP (E) and GFP::Vsg (F) shows specific localization of these proteins to follicle cell ring canals (circles). (G) GFP::Vsg localizes to ubiquitous puncta in imaginal wing discs. (H,I) Higher magnification confirms Vsg (H) and Pav-KLP (I) puncta on cell membranes of wing disc cells (arrows). Scale bars: 10 μm (A–D,H,I), 0.5 μm (E,F), 50 μm (G).

Fig. 2.

Localization of putative ring canal proteins and ring canal position. (A,B) Neither Nasrat-Flu puncta (A, red arrows) nor Polehole-Flu puncta (B, red arrows) colocalize with GFP::Pav-KLP-marked ring canals (green arrows). (C,D) GFP::Pav-KLP-marked ring canals (green arrows) are located basal to (above) adherens junctions (C, red arrows) and largely apical to (below) gap junctions (D). Scale bars: 10 μm.

Fig. 3.

Mitotic spindle orientation and cyst organization. (A,B) Schematic of branched and linear divisions in a syncytium. Asymmetric segregation of existing ring canals (green bars) in mitosis results in a branch in the syncytium (A), whereas a mitotic plane (dotted lines) that separates existing ring canals generates a linear cyst (B). (C–E) Egg chambers expressing GFP::Pav-KLP (green) were stained with DAPI (blue) and antibodies against centrosomin (Cnn) and Adducin (htsM) (red, marking centrosomes and membranes, respectively). Angles of ring canals relative to the cleavage plane were measured (illustrated in insets). (F) The positions of 79 ring canals (green) from 67 dividing cells do not show a significant bias relative to the cleavage plane. Measurements were grouped into 10°bins. (G) The observed distribution of ring canals in 796 follicle cells indicates an intermediate branching pattern. Models of linear and maximally branched 512-cell cysts (resulting from nine follicle cell cycles) and the 16-cell maximally branched pattern observed in the female germline are also shown. The unnumbered segments in the maximally branched 512-cell model represent cells having between five and nine ring canals and decrease geometrically in their proportion of the total. Scale bar: 5 μm.

Fig. 4.

Time-lapse images showing formation of follicle cell ring canals. Egg chambers expressing Cherry::Histone2Av and GFP::Pav-KLP were cultured in vitro and imaged to capture ring canal formation (movies are provided in the supplementary material). Diagrams below the images show the positions of chromosomes and cleavage furrows. White arrowheads denote GFP::Pav-KLP accumulation at the cleavage furrow and subsequent ring canal. Time is indicated as minutes:seconds. (A) The nascent ring canal remained centered between daughter nuclei. (B) The nascent ring canal moved off-center. (C) An existing ring canal (yellow arrowhead) was drawn in by the constricting cleavage furrow and resolved into one object (T=10:30).

Fig. 5.

Tripartite ring canals. (A) Confocal image of a stage-10 egg chamber expressing GFP::Pav-KLP and stained with htsM antibody to visualize membranes. Ring canals were located at two-cell or three-cell junctions (arrows). (B) ImmunoEM image of a tripartite ring canal that connects the cytoplasm of three cells (numbered). Membranes are indicated with dashed red lines. Scale bars: 10 μm (A), 0.5 μm (B).

Fig. 6.

Prevalence of ring canals in follicle cells. (A) A comparison of the total number of ring canals and cells in stage 2–9 egg chambers. The data support a linear trend (y=0.887x−11.3; R²=0.998), indicating that 88.7% of follicle cell divisions outside the germarium form a stable ring canal. Divisions within the germarium exhibit a larger deficit of ring canals resulting in a shift in the y-intercept. The dotted line (slope=1) represents the maximum possible number of ring canals. (B–D) Not all follicle cell subtypes have ring canals. Stalk cells (B,B′, arrowheads) and polar cells (C, highlighted) do not have ring canals. Polar cells were identified by elevated nuclear localization of GFP::Pav-KLP. Before their migration, presumptive border cells (C, adjacent to polar cells) do have ring canals. Once migration has begun, border cells contain fewer, smaller puncta that presumably represent ring canal remnants (D, arrowheads). Nurse cell nuclei and germline ring canals (arrows) are visible around the border cells. Scale bars: 10 μm.

Fig. 7.

Ring canals in follicle cell mitosis. (A) Progression through mitosis is revealed by PH3 staining. (B–D) Mitotic cells in GFP::Pav-KLP egg chambers were evaluated for the presence of connecting ring canals. Membranes are marked with htsM. (B) A single mitotic cell is connected by ring canals (arrows) to non-mitotic cells. Clusters of mitotic cells with two (C) or three (D) mitotic cells that are connected by a ring canals (arrowheads) are also connected by ring canals to other, non-mitotic cells. Pro, prophase; Prm, prometaphase; Met, metaphase; Ana, anaphase; Tel, telophase; Cyt, cytokinesis. Scale bar: 10 μm.

Fig. 8.

Movement through follicle cell ring canals. (A) Single-cell activation of PA-GFP reveals exchange of protein between follicle cells. (B–E) FLIP analysis demonstrates that some, but not all, proteins are exchanged between follicle cells. Multiple cells in each field were repeatedly bleached over 45–90 minutes. Red circles denote bleached cells; yellow flags indicate adjacent cells with significant loss of fluorescence. Oda, but not RpL30 or Yps, shows movement between cells. (C) Co-expression of GFP::Oda and GFP::Pav-KLP demonstrates that GFP movement occurs only between cells connected by ring canals (arrowheads). Asterisks highlight cells exhibiting measureable loss of fluorescence. Here, GFP::Oda is heterozygous and produces lower GFP expression compared with that in B. Minor differences in the position and intensity of ring canals between C and C′ are due to changes in the focal plane and are not significant. (F) UASp-Yps::mRFP displays mosaic expression in follicle cells and is expressed at different levels in cells connected by ring canals (arrows). Scale bars: 10 μm.