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. Couturier, fused to the PH domain of the human PLC-g gene using overlapping PCR synthesis and cloned into the Stinger-attB-pneur-GFP plasmid (Aerts et al., 2010) where it replaced GFP. After sequencing, the resulting transgene was, PH-iRFP670: the codon-optimized sequence of the iRFP670 gene (Shcherbakova and Verkhusha, 2013) was PCR-amplified from neur-nlsFP670, 2014.

. Aerts, 2010) where it replaced GFP. After sequencing, the resulting transgene was inserted at position 99F8 (PB, Gal80: the sequence of the Gal80 gene was PCR-amplified from pCaspeR4-Gal80 and inserted into the Stinger-attBneur-GFP plasmid

. Couturier, For each pupa, the raw data were normalized by the mean value of nuclear NiGFP in pIIa and pIIb at t10. The LifeAct-GFP and Utrn-GFP signals were quantified on 2-image projections (Dz = 1mm) corresponding to the region just below the midbody. Intensity profiles were obtained using a manually defined ROI spanning the pIIa-pIIb interface. Images of the same pIIa-pIIb pairs were acquired at t10, t20 and t30. Lateral surfaces were quantified in SOPs surrounded by epidermal cells expressing a membrane marker. First, the membranes of the neighboring epidermal cells which locate between pIIa and pIIb were segmented automatically in each z section using the PH-ChFP, mCherry-CAAX or Resille-GFP markers. The lateral interface was next semi-automatically and independently segmented using the DlgGFP, PH-iRFP670 or PH-ChFP markers. Segmented interfaces were subsequently processed using the ''3D ImageJ Suite'' on Fiji to reconstruct a 3D object and determine its surface area. Images of the same pIIa-pIIb pairs were acquired at t10, t20, t30 and t60. In Rac1 DN cells, edges of interfaces were determined using the weak actin patches detected at lateral tricellular junctions. Contact width corresponds here to the distance between these two vertices. Movies were acquired with a 1-minute (movie 1) or a 10 s frame rate (movies 2-4) and a Dz = 0.5mm step. Images were furthered time window (t0-t20) were selected. After an additional 5 min, these pupae were dissected and fixed. This dissection step took $5 minutes, so that pIIa-pIIb pairs were t10-t30. Nota were then processed for antibody staining using goat anti-GFP (1:1000, Abcam) and guinea-pig anti-Spdo (1:1000, Live-imaging was performed at 20 ± 2 C with a scanning confocal microscope (LSM780, Zeiss) with a 63x Plan APO N.A. 1.4 DIC M27 objective. Images were acquired using z stacks (Dz = 0.5mm) in time-series, 2012.

. Couturier, Dl endocytosis assay SOPs undergoing the metaphase-anaphase transition were first selected in living pupae within a 10-min window to stay in the timing of cytokinesis (t0-t10), 2014.

, DHSB) in Schneider medium at 25 C (pIIa-pIIb cells were t20-t30), then fixed (paraformaldehyde 4%). Nota were then immunostained using goat anti-GFP (1:1000, Abcam) to detect Dlg-GFP. Secondary antibodies (1:1000) were from Jackson ImmunoResearch Laboratories. Two categories of SOPs were studied. First, those that had divided during the 10 min of live imaging produced the t20-t30 pIIa-pIIb pairs. In these cells, endocytosis took place between t5 and t30. Second, SOPs that had divided prior to live imaging were used to study endocytosis after t30, This dissection step took $5 min; hence pIIa-pIIb cells were t5-t15. Dissected and living nota were incubated 15 min with the anti-Dl antibody (mouse, 594.9B concentrate, vol.1, pp.1-10, 2019.