SUPPLEMENTARY INFORMATION

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1 SUPPLEENTRY INFORTION DOI: 1.138/ncb2577 Early Telophase Late Telophase B icrotubules within the ICB (percent of total cells in telophase) D G ultinucleate cells (% total) T without gaps n=351 n=12 inus-end retraction p<.1 n=293 CHP4B-KD T with gaps E icrotubules with gap (percent of total cells in telophase) C n=18 CHP4B localization within ICB (percent of total cells with T Gap) H n=167 CHP4B-KD idbody Only n=63 F idbody+t gap CHP4B-KD -CHP4B -FIP3 Figure S1 FIP3 and microtubule dynamics during late telophase. () Schematic representation of FIP3 dynamics during early and late telophase. (B) Quantification of telophase cells with microtubule (T) gaps and minusend retractions. The data shown are the means and standard deviation from three independent experiments. (C) Quantification of CHP4B localization within the ICB in telophase cells. The data shown are the means and standard deviation from three independent experiments. (D-E) Quantification of multinucleation (D) and microtubule gap formation (E) in cells treated with CHP4B sirn. The data shown are the means and standard deviation from three independent experiments. (F) Immunoblot analysis of lysates from mock or CHP4B sirn-treated HeLa cells. (G and H) Images of telophase Hela cells with CHP4B-YFP extending from the midbody to the abscission site in continuous manner (G) or forming a distinct pool (H). Scale bars 5 μm acmillan Publishers Limited. ll rights reserved.

2 SUPPLEENTRY INFORTION SCP1 B C erge D SCP1 E F FIP3-KD G SCP3 H E erge J SCP3 K L FIP3-KD Levels of SCP1 in the ICB (arbitrary fluorescence units/mm2) n=1 p<.5 n=1 FIP3-KD Levels of SCP3 in the ICB (arbitrary fluorescence units/mm2) n=1 p<.5 n=1 FIP3-KD N SCP1 SC1-KD SCP2 SC2-KD SCP3 SC3-KD Figure S2 Localization of SCP1 and SCP3 during telophase. (-L) (-C, G-E) or FIP3 sirn-treated (D-F, J-L) HeLa cells were fixed and stained with either anti-scp1 ( and D) or anti-scp3 (G-J) antibodies. sterisk marks midbodies. () Quantification of SCP1 or SCP3 immunofluorescence within the ICB. The data shown are the means and standard deviations of fluorescence from 1 randomly picked cells in telphase. (N) HeLa cells were treated with either SCP1, SCP2 or SCP3 sirns and stained with anti-scp1 (top panels), anti-scp2 (middle panels) or anti-scp3 (bottom panels) antibodies. Scale bars 5 μm acmillan Publishers Limited. ll rights reserved.

SUPPLEENTRY INFORTION HeLa- HeLa- B Synchronize (Thymidine/Nocodazole) # IgG icrosomes Immunoprecipitation anti-gfp ass-spec nalysis # 59% cells in telophase C Input α-igg Input α-gfp - -FIP1 D Total

3% ctin regulators 5.4% Kinases & phosphatases 3.9% Lipid regulators 2.7% Tubulin regulators 2.5% otor 1.8% Intermediate filament associated.

Cells were isolated by mitotic shake off, washed and incubated for 9 minutes. Cells were then lysed using glass-glass homogenizer and microsomes isolated.

3 SUPPLEENTRY INFORTION HeLa- HeLa- B Synchronize (Thymidine/Nocodazole) # IgG icrosomes Immunoprecipitation anti-gfp ass-spec nalysis # 59% cells in telophase C Input α-igg Input α-gfp - -FIP1 D Total Identified Proteins (441) DN/RN associated 35.1% isc 24.9% itochondria associated 7.7% ER proteins 5.7% Unknown 3.2% Candidate proteins 23.4% -FIP1 -Syntaxin6 -TfR embrane transport 6.3% ctin regulators 5.4% Kinases & phosphatases 3.9% Lipid regulators 2.7% Tubulin regulators 2.5% otor 1.8% Intermediate filament associated.7% Figure S3 Immunoisolation and proteomic analysis of endosomes. () HeLa cells were synchronized using thymidine/ nocodazole block. Cells were isolated by mitotic shake off, washed and incubated for 9 minutes. Cells were then lysed using glass-glass homogenizer and microsomes isolated. FIP3-endosomes were then immunoisolated using rabbit polyclonal anti-gfp antibody. Rabbit IgG was used as control. (B) HeLa cells were imaged 9 minutes after release from thymidine/nocodazole block. sterisks mark cells in late telophase. Pound sign marks cell in metaphase. (C) Western blot analysis enrichment within immunoisolated FIP3-endosome. icrosomes (input), IgG and anti-gfp immunoisolates were separated by SDS/PGE and immunoblotted with anti-gfp, anti-fip1, anti-tfr and anti-synatxin6 antibodies. TfR and FIP1 are the markers of recycling endosomes that are not enriched within the ICB. Syntaxin 6 is a marker for TGN. (D) Proteins identified in immunoisolated FIP3-endosomes were categorized based on their function. Scale bars 5 μm acmillan Publishers Limited. ll rights reserved.

p5rhogp-rfp F G erge H p5rhogp-rfp Figure S4

levels in cells treated with either p5rhogp or

Transferrin receptor and FIP1 are used as

(B-D) (B and C) or p5rhogp sirn-treated (D)

anti-p5rhogp antibody in the presence (C) or

4 SUPPLEENTRY INFORTION pkd#1 pkd#2 pkd#3 FIP3-KD#1 FIP3-KD#2 -p5rhogp -FIP3 -Transferrin Receptor -FIP % 93.11% 94.34% KD levels 62.9% 56.9% B C D KD levels p5rhogp b p5rhogp b GST-p5RhoGP 5 um p5rhogp b 5 um pkd#2 E p5rhogp-rfp F G erge H p5rhogp-rfp Figure S4 Time-lapse analysis of p5rhogp-rfp dynamics during late telophase. () Western blot analysis of p5rhogp and FIP3 levels in cells treated with either p5rhogp or FIP3 sirns. Transferrin receptor and FIP1 are used as loading controls. (B-D) (B and C) or p5rhogp sirn-treated (D) expressing HeLa cells were stained using rabbit anti-p5rhogp antibody in the presence (C) or absence (B and D) of 1- fold excess of purified recombinant GST-p5RhoGP protein. was also imaged to identify cell in late telophase. (E-H) HeLa cells co-expressing and p5rhogp-rfp were analyzed by time lapse microscopy. Square box in (E) marks the region that is enlarged in (H). In (H) panels asterisks mark the organelles that contain both, and p5rhogp- RFP. Scale bars 5 μm (B-D), 1 μm (E-G), and 1 μm (H) acmillan Publishers Limited. ll rights reserved.

5 SUPPLEENTRY INFORTION Ut-GFP min 4 min 13 min 1 hr 55 min B Ut-GFP D yoii-gfp C Furrow actin/body actin (arbitrary values) min 8 min 11 min 1 hr 24 min Figure S5 Filamentous actin is depolymerised in the ICB prior to abscission. Time-lapse images of HeLa cells expressing utrophin-gfp ( and B) or myosin II (D) were taken at the indicated time points. Lines in mark the 5μm region used for quantification of f-actin levels (C). rrows mark the secondary ingression sites. sterisks mark the midbody. Scale bars 5 μm acmillan Publishers Limited. ll rights reserved.

SUPPLEENTRY INFORTION Ut-RFP Ut Fluorescence (arbitrary units) B

D Furrow actin/body actin (arbitrary units) 3. 2.5 2. 1.5 1.

1 Ut Fluorescence (arbitrary units) Furrow actin Body actin.

required for actin depolymerization during late telophase.

cells expressing utrophin-rfp were analyzed by live cell imaging.

within the ICB) were chosen for analysis.

Line through the ICB indicates where line intensity scans were

(D) Ratiometric quantification of f-actin intensity within the ICB.

quantification; black lines (furrow actin intensity) and grey

6 SUPPLEENTRY INFORTION Ut-RFP Ut Fluorescence (arbitrary units) B SC2/3-KD#1 Ut-RFP Ut Fluorescence (arbitrary units) C Ut-RFP pkd#2 D Furrow actin/body actin (arbitrary units) n=22 n=12 n=6 n=1 n=9 p<.5 p<.1 Ut Fluorescence (arbitrary units) Furrow actin Body actin. SC2/3-KD#1 SC2/3-KD#2 pkd#2 pkd#3 Figure S6 p5rhogp and SCP2/3 are required for actin depolymerization during late telophase. (), SCP2/3 sirn-treated (B), or p5rhogp sirn-treated (C) HeLa cells expressing utrophin-rfp were analyzed by live cell imaging. Only cells in late cytokinesis (as determined by localization within the ICB) were chosen for analysis. sterisks mark the midbodies. Line through the ICB indicates where line intensity scans were obtained (graphs on the right). (D) Ratiometric quantification of f-actin intensity within the ICB. (Inset) Graphic representation of the regions used for ratiometric quantification; black lines (furrow actin intensity) and grey lines (cell body actin). Data shown in D are the means and standard deviations derived from 8-2 randomly chosen cells in late telophase. Scale bars 5 μm acmillan Publishers Limited. ll rights reserved.

7 SUPPLEENTRY INFORTION Figure 5-Left anti-scp1 2 1 anti-scp2 anti-scp3 2 1 anti-tfr Figure 5-Right Figure 6J anti-scp1 anti-scp2 anti-scp3 anti-fip3 anti-fip1 anti-p5rhogp Supplemental Figure 1F anti-chp4b anti-fip Supplemental Figure 3C anti-gfp () anti-fip1 anti-syntaxin6 anti-tfr Supplemental Figure 4-Left anti-p5rhogp anti-tfr Supplemental Figure 4-Right anti-gfp () anti-fip Figure S7 Full scans of all immunoblots shown in this study acmillan Publishers Limited. ll rights reserved.

8 SUPPLEENTRY INFORTION Supplementary Tables Supplemental Table 1 Putative endosome cargo proteins. Proteins were categorized based upon protein function analysis. Only proteins identified in immunoisolated FIP3-endosomes but not IgG controls are listed in this table. The shown list includes proteins identified in three different experiments. Supplemental Table 2 List of all proteins identified in IgG control and anti-gfp sample from experimental run #1. Supplemental Table 3 List of all proteins identified in IgG control and anti-gfp sample from experimental run #2. Supplemental Table 4 List of all proteins identified in IgG control and anti-gfp sample from experimental run #3. Supplemental ovie 1 endosome movement during mid-telophase. expressing HeLa cell at mid-telophase was imaged by time-lapse microscopy. Shown move consists of 5 sequential images. Exposure 2 ms, time-lapse 5 ms. Cells were determined to be in mid-telophase based on the fact that has started to accumulate at the ICB, with the large pool of FIP3-endosomes still present at the cell bodies. Shown cell is a representative of 25 imaged cells. Supplementary ovies Supplemental ovie 2 endosome movement during late-telophase. expressing HeLa cell at late-telophase was imaged by time-lapse microscopy. Shown move consists of 5 sequential images. Exposure 2 ms, time-lapse 5 ms. Cells were determined to be in late-telophase based on the fact that endosomes are now present only in the ICB. Shown cell is a representative of 25 imaged cells. Supplemental ovie 3 Formation of the secondary ingression in mock-treated HeLa cell. Late telophase HeLa cell with secondary ingression and expressing was imaged by bright-field time-lapse microscopy. Shown move consists of 5 sequential images. Exposure 3 ms, time-lapse 5 sec. Supplemental ovie 4 Formation of the secondary ingression in CHP4B sirn-treated HeLa cell. CHP4B sirn-treated late telophase HeLa cell with secondary ingression and expressing was imaged by bright-field time-lapse microscopy. Shown move consists of 5 sequential images. Exposure 3 ms, time-lapse 5 sec. time-lapse sequence of the same cell is shown in supplemental movie 5. Supplemental ovie 5 dynamics during the formation of the secondary ingression in CHP4B sirn-treated HeLa cell. CHP4B sirn-treated late telophase HeLa cell with secondary ingression and expressing was imaged by time-lapse microscopy. Shown move consists of 5 sequential images. Exposure 2 ms, time-lapse 5 sec. Bright-field time-lapse sequence of the same cell is shown in supplemental movie acmillan Publishers Limited. ll rights reserved.

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION DOI: 10.1038/ncb3076 Supplementary Figure 1 btrcp targets Cep68 for degradation during mitosis. a) Cep68 immunofluorescence in interphase and metaphase. U-2OS cells were transfected with control sirna