Both daughter cells traffic and exocytose membrane at the cleavage furrow during mammalian cytokinesis.

Cell culture and VSVG-YFP fluorescence labeling

BSC1 and Hela cells were grown in DME (American Type Culture Collection) supplemented with 10% FBS, 1% penicillin/streptomycin, 1% l -glutamine, and 1% nonessential amino acids (Invitrogen). Cells were cultured at 37°C (unless otherwise noted), supplemented with 5% CO 2 , and plated on dishes (Mattek) before imaging. For VSVG-YFP and YFP-GPI expression, BSC1 and Hela cells were infected with adenovirus as previously described ( Toomre et al., 2000 ). YFP-GPI–infected cells were incubated at 19°C for 2 h before imaging. Immunofluorescence and VSVG/transferrin labeling BSC1 and Hela cells expressing VSVG-YFP (ts045 with spacer; Toomre et al., 2000 ) were released from the 39°C temperature-sensitive block for 60 min, permeabilized with 0.1% Triton X-100 and 4% PFA for 30 s, and fixed in 4% PFA with 0.02% glutaraldehyde for 20 min at room temperature. Cells were washed with PBS and immunostained with mouse anti-tubulin (Sigma-Aldrich), mouse α-EEA1 (BD Biosciences), or mouse α-LAMP1 (BD Biosciences) antibodies, followed by donkey α-mouse Cy5 or donkey α-mouse Texas red (Jackson ImmunoResearch Laboratories) secondary antibodies, and imaged with SDCM. For midbody immunofluorescence, cells were fixed on ice with ethanol for 15 min, and immunostained with rabbit α-MKLP1 (Santa Cruz Biotechnology, Inc.) and HOECHST 15090 dye (Serva) followed by donkey α-rabbit Cy2 (Jackson ImmunoResearch Laboratories). All confocal images shown are 2D projections of confocal stacks (typically ∼10 sections; 0.5-μm intervals). For VSVG/transferrin colabeling, BSC1 cells were coinfected with adenoviruses separately expressing VSVG-YFP and transferrin receptor for 1 h at 37°C. Cells were washed with PBS and incubated in DME for 9–12 h at 39.5°C before imaging. Cells were transferred to 32°C for 15 min before adding a pulse of 5 mg/ml transferrin–Texas red (Invitrogen) for 5 min. Cells were washed with PBS and incubated in DME at 32°C for an additional 15 min before live imaging of both fluorophores with SDCM.

Microscopy

For ultra-sensitive 4D imaging (3D + time) a Yokagawa-type SDCM system equipped with a FRAP photokinesis unit was used (PerkinElmer). The system was mounted onto an inverted microscope (IX71; Olympus) equipped with a 1 Kb × 1 Kb electron-multiplying charge-coupled device camera (Hamamatsu Photonics) and a temperature-controlled stage set at 32°C, which was controlled by Ultraview ERS software (PerkinElmer). Samples were imaged in phenol red–free DME by a 60× 1.4 NA oil phase objective, yielding a spatial resolution of 0.14 μm per pixel. Excitation was achieved using 514-nm argon and 568-nm argon/krypton lasers (Melles Griot). Exposure times were 0.1–0.25 s. TIRFM images were acquired using an inverted microscope equipped with a 1.45 NA 60× TIRFM lens (Olympus), back-illuminated electron-multiplying charge-coupled device camera (512 × 512, 16-bit; iXon887; Andor Technologies), and controlled by Andor iQ software (Andor Technology). Excitation was achieved using a 514-nm line of argon laser, and exposure times were 0.1–0.2 s and acquired at 0.5–4 Hz. The calculated evanescent field depth was ∼100 nm. DIC images were acquired using either a 60× 1.2 NA water immersion objective or 60× 1.42 NA oil immersion objective, with an aluminum closed cell chamber that uses two 15-mm coverslips, and a BX-51 microscope (Olympus) with Nomarski optics, all enclosed within an Air-therm temperature-regulated environmental box at 37°C. Exposure times were 1 s, and two to four images were acquired per minute with an iXon887 electron-multiplying charge-coupled device camera. FRAP experiments A photokinesis unit mounted on the SDCM (described in Microscopy) was used for FRAP experimentation and controlled by Ultraview ERS software. Regions of interest (ROI) were identified and photobleached with a 514-nm argon laser at 100% power for 200 bleach cycles (∼3–5 s). Quantification of fluorescence recovery was obtained using Ultraview ERS software programs.

Image analysis Ultraview

ERS software was used to subtract background of images before analysis by Image J software (National Institutes of Health). No nonlinear algorithms were used to alter fluorescent signals. Colocalizations were quantified for numerous ROI that corresponded to EMMA on a pixel-per-pixel basis using Imaris (Biplane AG) automated software. Online supplemental material Fig. S1 shows that post-Golgi vesicles in the cleavage furrow are distinct from satellite Golgi. Fig. S2 shows that SDCM analysis of YFP-GPI–labeled post-Golgi vesicles indicates trafficking into the cleavage furrow from both daughter cells. Fig. S3 shows that quantitative imaging of VSVG-YFP in BSC1 cells by TIRFM differentiates between vesicles that leave the evanescent field and those that dock and fuse. Video 1 shows that SDCM analysis reveals that post-Golgi vesicles are directed to the cleavage furrow from both daughter cells (QuickTime video from Fig. 3 A). Video 2 shows manual tracking of post-Golgi vesicles that are directed to the cleavage furrow from both daughter cells (QuickTime video from Fig. 3 B). Video 3 shows that SDCM analysis reveals that YFP-GPI–labeled post-Golgi vesicles are directed to the cleavage furrow from both daughter cells. Video 4 shows that FRAP analysis suggests that post-Golgi membrane accumulations in the cleavage furrow consists of individual vesicles (QuickTime video from Fig. 3 E). Video 5 shows that TIRFM analysis reveals exocytosis of post-Golgi vesicles from both daughter cells at the cleavage furrow (QuickTime video from Fig. 4, A, D, and E). Video 6 shows that TIRFM analysis reveals exocytosis of post-Golgi vesicles from both daughter cells at the cleavage furrow. Video 7 shows that TIRFM analysis reveals exocytosis of post-Golgi vesicle at the cleavage furrow. Video 8 shows that DIC time-lapse imaging indicates asymmetrical midbody inheritance in BSC1 cells (QuickTime video from Fig. 5 D). Video 9 shows that DIC time-lapse imaging indicates asymmetrical midbody inheritance in Hela cells (QuickTime video from Fig. 5 E). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200712137/DC1 .

Online supplemental material Fig. S1 shows that post-Golgi vesicles in the cleavage furrow are distinct from satellite Golgi. Fig. S2 shows that SDCM analysis of YFP-GPI–labeled post-Golgi vesicles indicates trafficking into the cleavage furrow from both daughter cells. Fig. S3 shows that quantitative imaging of VSVG-YFP in BSC1 cells by TIRFM differentiates between vesicles that leave the evanescent field and those that dock and fuse. Video 1 shows that SDCM analysis reveals that post-Golgi vesicles are directed to the cleavage furrow from both daughter cells (QuickTime video from Fig. 3 A). Video 2 shows manual tracking of post-Golgi vesicles that are directed to the cleavage furrow from both daughter cells (QuickTime video from Fig. 3 B). Video 3 shows that SDCM analysis reveals that YFP-GPI–labeled post-Golgi vesicles are directed to the cleavage furrow from both daughter cells. Video 4 shows that FRAP analysis suggests that post-Golgi membrane accumulations in the cleavage furrow consists of individual vesicles (QuickTime video from Fig. 3 E). Video 5 shows that TIRFM analysis reveals exocytosis of post-Golgi vesicles from both daughter cells at the cleavage furrow (QuickTime video from Fig. 4, A, D, and E). Video 6 shows that TIRFM analysis reveals exocytosis of post-Golgi vesicles from both daughter cells at the cleavage furrow. Video 7 shows that TIRFM analysis reveals exocytosis of post-Golgi vesicle at the cleavage furrow. Video 8 shows that DIC time-lapse imaging indicates asymmetrical midbody inheritance in BSC1 cells (QuickTime video from Fig. 5 D). Video 9 shows that DIC time-lapse imaging indicates asymmetrical midbody inheritance in Hela cells (QuickTime video from Fig. 5 E). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200712137/DC1 .

Supplementary Material [Supplemental Material Index]