DEV010983 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 - Adobe PDF

• Supplemental Table S2 - Adobe PDF

• Supplemental Models - Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. ARF6 is not required for myoblast fusion or midline crossing of axons. (A) Western blot to detect endogenous ARF6 using a rabbit polyclonal antibody against Drosophila ARF6, and rabbit anti-actin as a loading control. ARF6 protein is detected in WT adult flies. ARF6 protein could not be detected in arf6¹ zygotic mutant flies. The dashed line separates different regions of the same gel with no differences in image processing. (B-E) Stage 15 embryos derived from arf6¹/arf6¹ mothers. (B-C) Lateral view of control (B; arf6¹/CyO, hb-lacZ) and arf6¹/arf6¹ embryos (C) derived stained for the muscle marker MHC. Confocal z slices were average intensity projected in ImageJ. Dorsal is up and anterior to the left. (D-E) Dorsal view of control (D); arf6¹/CyO, hb-lacZ and (E) arf6¹/arf6¹ embryos, stained for the central nervous system marker BP102. Confocal z slices were maximum intensity projected in ImageJ. The CNS develops a normal morphology indicating that ARF6 is not required for midline crossing of axons.

• Supplemental Figure S2 -

  Fig. S2. ARF6 function during chorion formation. (A-D) Dorsal view of eggs laid by a wild type (A), homozygous arf6¹ mutant (B-C); (B) 68% incidence; (C) 32% incidence, and P(Ubi:arf6HAarf6¹/arf6¹ rescued female (D) 99.5% incidence. Anterior is to the left. In normal embryos, two dorsal appendages project from the chorion (arrowheads in A,B,D). In the mutants, various degrees of dorsal appendage fusion are observed (arrowhead in C).

• Supplemental Figure S3 -

  Fig. S3. Model of failure frequency during spermatocyte cytokinesis. (A) Possible lineages of a primary spermatocyte undergoing meiosis I and II to generate spermatids. p is the probability of cytokinesis failure. The probability of each lineage is given on the left. The proportion of spermatids containing one, two or four nuclei:Nebenkern is given on the right as a function of p. (B) Frequency of cells with one nucleus (blue), two nuclei (red) or four nuclei (black) after meiosis II. Circles, square and triangles correspond to published data. Theoretical curves are the frequency of the different cells (1:1, 2:1 and 4:1) as a function of p. The curves are derived from the equations given in A. Note that the published frequencies fit the theoretical curves well. (C) Values of p for mutants affecting cytokinesis. The error bars indicate the least sum of squares difference between the observed proportions of cells with one nucleus, two nuclei or four nuclei and those predicted by the model for the corresponding value of p. The alleles and source of frequency statistics (when not from this study) are indicated below. For sibling flies generated in this study in the genetic interaction test between pav and arf6, the pav allele of the siblings of Pr (control) flies is indicated in brackets, and the following abbreviations were used: arf6 Pr, w; FRTG13arf6³/FRTG13arf6³; Pr/+ and arf6 pav, w; FRTG13arf6³/FRTG13arf6³; pav/+.

• Supplemental Figure S4 -

  Fig. S4. Kinetics of furrow ingression and plasma membrane addition in control, arf6 and chic^13E mutant cells. (A) Kinetics of furrow diameter in control, arf6 early regressors and arf6 late regressor cells. Time zero is the appearance of an indentation as the cytokinesis furrow is initiated. arf6 early regressors have lower ingression rates than controls prior to furrow regression. (B) Kinetics of perimeter length in control, arf6 early regressors, arf6 late regressors and chic^13E cells. arf6 early regressors are impaired both in the rate of perimeter increase and the final perimeter attained. (C) Comparison of surface area with perimeter with the control (black) and arf6 (red) cells dividing in Schneider’s medium. Trendlines are the linear regression lines through the data points for each cell. The slope, intercept on the perimeter axis and R² value for each cell is shown in Table S2

• Supplemental Figure S5 -

  Fig. S5. Endosomal localization of ARF6 during interphase and cytokinesis. (A-D) Fixed primary spermatocytes, stained for ARF6HA (red) and expressing GFP-Rab4 (A), GFP-Rab5 (B,D), GFP-Rab11 (C). During interphase, ARF6HA colocalizes with Rab4 (arrowheads, A) and Rab5 (arrowheads, B) but shows only minimal colocalization with Rab11 (C). During cytokinesis ARF6HA partially colocalizes with GFP-Rab5 at the central spindle (arrowheads), stained with Pav (blue).

• Movie 1 -

  Movie 1. GFP-α-tubulin in control. The metaphase spindle, labelled with α-tubulin GFP, consists of two populations of microtubules: those in the centre of the cells inside the partially broken down nuclear envelope, and an outer population in close contact with the cortex. After anaphase onset, the central spindle consists of both inner and outer populations of non-kinetocore microtubules, which start to become bundled, appearing brighter. Bundles of the outer microtubule population concentrate at the position where the cleavage furrow will form. As the cytokinesis furrow invaginates, the populations appear to meet as a dense midbody is formed (arrowheads, time 20 min AA). Genotype P(Ubi:GFP-α-tubulin);arf6¹/CyO. All movies are time-lapse movie of Drosophila spermatocytes in meiosis I. The rate of image collection is not constant within each video, so in several movies, times are given in hours:mins:seconds and anaphase onset is indicated, and in other, times relative to anaphase onset (min AA) are indicated in mins:seconds. Frames are shown for 0.2 seconds except frames with annotations such as Anaphase onset or arrowheads, which are shown for 0.4 seconds.

• Movie 2 -

  Movie 2. GFP-α-tubulin in arf6¹. The spindle appears similar to the control throughout metaphase and anaphase. 20 min AA, some bundling of central spindle microtubules can be seen (arrowhead), but these microtubules are later lost from the central spindle and the furrow regresses. P(Ubi:GFP−α-tubulin);arf6¹/ arf6¹.

• Movie 3 -

  Movie 3. Pav-GFP in control. During metaphase, Pav-GFP is cytosolic. After anaphase onset, Pav-GFP accumulates on central spindle microtubules during anaphase B cell elongation, before the initiation of the cytokinesis cleavage furrow. Pav-GFP remains on the increasingly bundled central spindle microtubules during cleavage furrow invagination. Genotype w; arf6¹/CyO; P(UbiPav-GFP)/TM6B.

• Movie 4 -

  Movie 4. Sqh-GFP in control. During metaphase Sqh-GFP is localized in the cytosol and several punctae. After anaphase onset Sqh-GFP is transferred to the cortex, where it concentrates at the site of the future cleavage furrow. Sqh-GFP remains concentrated at the cleavage furrow during invagination. Genotype y w sqh^AX3; +; P(w+ sqh-gfp).

• Movie 5 -

  Movie 5. Pav-GFP in arf6¹. Pav-GFP is localized as in control cells until after cleavage furrow initiation. As the cleavage furrow stops invaginating, Pav-GFP signal fades from the central spindle microtubules, and eventually the cleavage furrow regresses. Genotype w; arf6¹/arf6¹; P(UbiPav-GFP)/TM6B

• Movie 6 -

  Movie 6. Sqh-GFP in arf6³. Sqh-GFP is localized as in the control until cytokinesis. As the cleavage furrow regresses, Sqh-GFP remains associated with it. Genotype y w sqh^AX3; FRTG13arf6³/FRTG13arf6³; P(w+ sqh-gfp)

• Movie 7 -

  Movie 7. GFP-Rab4 in control. GFP-Rab4 is localized to endosomes and cytosol. Rab4 endosomes can be seen concentrating at the central spindle as the cytokinesis furrow starts to ingress (arrowheads) and remains concentrated at the central spindle until furrow ingression is complete. Genotype w P(Ubi-GFP-Rab4); +/+

• Movie 8 -

  Movie 8. GFP-Rab4 in arf6³. Rab4 GFP is localized as in control cells, and concentrates at the central spindle during cytokinesis (arrowheads). Genotype w P(Ubi-GFP-Rab4); FRTG13arf6³/FRTG13arf6³

• Movie 9 -

  Movie 9. GFP-Rab11 in control. Rab11 is localized to endosomes and the cytosol. When the furrow is deeply invaginated, Rab11 endosomes concentrate at the central spindle (arrowheads). Genotype w; P(UbiGFP-Rab11)/SM6B.

• Movie 10 -

  Movie 10. GFP-Rab11 in arf6¹ late regressor. Prior to cytokinesis GFP-Rab11 is localized as in controls. Some accumulation of GFP-Rab11 at the central spindle can be seen (arrowhead) before the furrow regresses. Since the recording of this cell started after anaphase onset, the min AA shown were estimated on the basis of the furrow ingression. Genotype w; P(UbiGFP-Rab11)arf6¹/arf6¹.