Supporting Information - PNAS · 2012. 11. 15. · Supporting Information Tanaka et al....

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Supporting Information Tanaka et al. 10.1073/pnas.1218017109 SI Materials and Methods siRNA Oligos. siRNA sequences for human calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) were described pre- viously (1). The following siRNAs were used to knock down CAMSAP2 (Invitrogen) and γ-tubulin 1 and 2 (Sigma): si- CAMSAP2-1, 5-UCUCGAAUCUGUUUCUGUGGAGAGG-3; siCAMSAP2-2, 5-UUUAACUGAGGUUCUCCAUCAUCCC- 3; si γ-tubulin 1-1 (#1), 5-CAUACUCAGUGUUUCCCAATT-3; si γ-tubulin 1-2 (#2), 5-GAACCUGUCGCCAGUAUGATT-3; si γ-tubulin 2-1 (#1), 5-CAUCUCCUCGCUCUUUGAATT-3; and si γ-tubulin 2-2 (#2), 5-GGUUCAGCCCUACAAUUCATT-3. Similar results were obtained with the two siRNAs for each mole- cule, and therefore only the results obtained with a single siRNA were shown, unless otherwise noted. We also used Stealth RNAi Negative Control Low or Medium GC Duplexes (Invitrogen) as a negative control for siRNA treatments. The effects of siRNA treatment of cells were analyzed 34 d after transfection. Commercial Antibodies and Reagents. The following primary anti- bodies were used: rat mAb (Nacalai), mouse mAb (Roche), and rabbit pAb (MBL, Chemicon, and Abmart) against GFP; mouse mAb (Sigma), rat mAb (Millipore), and rabbit pAb (Abcam) against α-tubulin; mouse mAb and rabbit pAb (Sigma) against γ-tubulin; rabbit pAbs against detyrosinated tubulin (Millipore) and giantin (Millipore); and mouse mAbs against end-binding protein 1 (EB1) and early endosome antigen 1 (EEA1) (BD), acetylated tubulin and tyrosinated tubulin (Sigma), GAPDH (Santa Cruz), Kusabira Orange (MBL), and Flag (Sigma). The following secondary anti- bodies were used: goat Alexa Fluor 488-, 555-, 594-, or 647-con- jugated anti-mouse or anti-rabbit IgG (Invitrogen); Cy3-conjugated anti-rat IgG (Millipore); CF488A/594-conjugated anti-rat IgG (Biotium); sheep HRP-conjugated anti-mouse or anti-rabbit IgG (GE Healthcare); peroxidase-conjugated anti-rat IgG (Jackson); and peroxidase-conjugated anti-mouse or anti-rabbit IgG (GE Healthcare). Immunostaining. Cells were xed, and then permeabilized with ice- cold methanol for 5 min at -20 °C, unless otherwise noted. For immunostaining for EEA1, cells were xed with 4% (wt/vol) PFA in PHM buffer [60 mM K1,4-piperazinediethanesulfonic acid (Pipes), pH 7.0, 25 mM Hepes, 2 mM MgCl 2 ] for 20 min at 37 °C. The xed cells were permeabilized with TBS containing 0.2% (vol/ vol) Triton X-100 for 5 min, and then incubated in blocking buffer [5% (wt/vol) skim milk or 5% (wt/vol) BSA in T-TBS] for 30 min. Cells were then incubated with primary antibodies for 2 h at room temperature or overnight at 4 °C, and subsequently with secondary antibodies for 1 h. Coverslips were mounted with FluorSave (EMD). Confocal images were acquired with a Zeiss LSM710 or LSM780 confocal microscope (Carl Zeiss) through a Plan-Apo- chromat 63×/1.40 N.A. oil-immersion objective lens (Carl Zeiss). Other images were analyzed as described previously (1). Images were processed by Photoshop (Adobe). Image Processing and Quantication. All images were modied by adjustments of levels and contrasts. Each set of data was acquired with the same microscope setting. For image analysis, we used Imaris 7.27.4 (Bitplane), Zen software (Zeiss), SoftWoRx (Ap- plied Precision), and ImageJ (NIH). Before image analysis, back- grounds were subtracted. Maximum-intensity projection of confocal stacks (0.5 μm sections) and time-lapse images were ac- quired using Zen and ImageJ, respectively. To measure the length of EB1, CAMSAPs, and microtubule images, we manually tracked them using Zen. EB1 comets tended to be longer at the peripheral than at the central regions of a cell; therefore, we analyzed 10 comets in each region and summed up the values, where the pe- ripheral region was dened as that spanning 10 μm from the cel- lular edge. The number of EB1 comets was quantied with the spot detection function of Imaris, with a setting of 0.3 μm as a minimal diameter and Gaussian Filter. If incorrect spots were detected, they were manually removed. The kymographs were created with the Multiple Kymograph plug-in for ImageJ. To estimate the velocity of growing microtubule plus ends, we calculated instantaneous displacement of each EB1 comet at growth phases over time by tracking them manually, using the spot detection function of Imaris. The band intensities in Western blots were measured by ImageJ software. For analysis of Golgi fragmentation, giantin- positive particles in 2D projection images were quantied by Im- ageJ software. Immunoprecipitation. For immunoprecipitation, HEK293T cells transiently transfected with CAMSAP2-Flag alone or both CAMSAP2-GFP and CAMSAP3-Flag were dissolved in lysis buffer [60 mM K-Pipes, pH 7.0, 25 mM Hepes, 10 mM EGTA, 2 mM MgCl 2 , 1% (vol/vol) Nonidet P-40, and protease and phosphatase inhibitor mixture (Sigma)]. After centrifugation, supernatants were precleared with protein G-conjugated Sepharose beads (GE Healthcare) for 1 h, incubated with appropriate antibodies for 1 h, and incubated with newly prepared protein G-conjugated Se- pharose beads for 1 h. After the beads were washed four times with the lysis buffer, precipitates were eluted with a sample buffer, separated by SDS/PAGE, and analyzed by immunoblotting. SDS/PAGE and Western Blotting. Samples were boiled with SDS sample buffer at 97 °C for 5 min, separated by SDS/PAGE, and transferred to Immobilon-P membranes (Millipore). Membranes were blocked with 5% (wt/vol) skim milk in T-TBS for 30 min and incubated with primary antibodies for 2 h at room temperature or overnight at 4 °C, and then with secondary antibodies for 1 h. The proteins were detected by using Western lightning plus ECL (PerkinElmer), Chemi-Lumi One Super (Nacalai), or Novex ECL Chemiluminescent Substrate Reagent Kit (Invitrogen). 1. Meng W, Mushika Y, Ichii T, Takeichi M (2008) Anchorage of microtubule minus ends to adherens junctions regulates epithelial cell-cell contacts. Cell 135(5):948959. Tanaka et al. www.pnas.org/cgi/content/short/1218017109 1 of 8

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  • Supporting InformationTanaka et al. 10.1073/pnas.1218017109SI Materials and MethodssiRNA Oligos. siRNA sequences for human calmodulin-regulatedspectrin-associated protein 3 (CAMSAP3) were described pre-viously (1). The following siRNAs were used to knock downCAMSAP2 (Invitrogen) and γ-tubulin 1 and 2 (Sigma): si-CAMSAP2-1, 5′-UCUCGAAUCUGUUUCUGUGGAGAGG-3′;siCAMSAP2-2, 5′-UUUAACUGAGGUUCUCCAUCAUCCC-3′; si γ-tubulin 1-1 (#1), 5′-CAUACUCAGUGUUUCCCAATT-3′;si γ-tubulin 1-2 (#2), 5′-GAACCUGUCGCCAGUAUGATT-3′; siγ-tubulin 2-1 (#1), 5′-CAUCUCCUCGCUCUUUGAATT-3′; andsi γ-tubulin 2-2 (#2), 5′-GGUUCAGCCCUACAAUUCATT-3′.Similar results were obtained with the two siRNAs for each mole-cule, and therefore only the results obtained with a single siRNAwere shown, unless otherwise noted. We also used Stealth RNAiNegative Control Low or Medium GC Duplexes (Invitrogen) as anegative control for siRNA treatments. The effects of siRNAtreatment of cells were analyzed 3–4 d after transfection.

    Commercial Antibodies and Reagents. The following primary anti-bodies were used: rat mAb (Nacalai), mouse mAb (Roche), andrabbit pAb (MBL, Chemicon, and Abmart) against GFP; mousemAb(Sigma), ratmAb(Millipore), andrabbit pAb (Abcam)againstα-tubulin; mouse mAb and rabbit pAb (Sigma) against γ-tubulin;rabbit pAbs against detyrosinated tubulin (Millipore) and giantin(Millipore); and mouse mAbs against end-binding protein 1 (EB1)and early endosome antigen 1 (EEA1) (BD), acetylated tubulinand tyrosinated tubulin (Sigma), GAPDH (Santa Cruz), KusabiraOrange (MBL), and Flag (Sigma). The following secondary anti-bodies were used: goat Alexa Fluor 488-, 555-, 594-, or 647-con-jugated anti-mouse or anti-rabbit IgG (Invitrogen); Cy3-conjugatedanti-rat IgG (Millipore); CF488A/594-conjugated anti-rat IgG(Biotium); sheep HRP-conjugated anti-mouse or anti-rabbit IgG(GE Healthcare); peroxidase-conjugated anti-rat IgG (Jackson);and peroxidase-conjugated anti-mouse or anti-rabbit IgG (GEHealthcare).

    Immunostaining. Cells were fixed, and then permeabilized with ice-cold methanol for 5 min at −20 °C, unless otherwise noted. Forimmunostaining forEEA1, cells werefixedwith 4% (wt/vol) PFA inPHM buffer [60 mM K–1,4-piperazinediethanesulfonic acid(Pipes), pH 7.0, 25 mM Hepes, 2 mMMgCl2] for 20 min at 37 °C.The fixed cells were permeabilized with TBS containing 0.2% (vol/vol) Triton X-100 for 5 min, and then incubated in blocking buffer[5% (wt/vol) skim milk or 5% (wt/vol) BSA in T-TBS] for 30 min.Cells were then incubated with primary antibodies for 2 h at roomtemperature or overnight at 4 °C, and subsequently with secondaryantibodies for 1 h. Coverslips were mounted with FluorSave(EMD). Confocal images were acquired with a Zeiss LSM710 orLSM780 confocal microscope (Carl Zeiss) through a Plan-Apo-chromat 63×/1.40 N.A. oil-immersion objective lens (Carl Zeiss).

    Other images were analyzed as described previously (1). Imageswere processed by Photoshop (Adobe).

    Image Processing and Quantification. All images were modified byadjustments of levels and contrasts. Each set of data was acquiredwith the same microscope setting. For image analysis, we usedImaris 7.2–7.4 (Bitplane), Zen software (Zeiss), SoftWoRx (Ap-plied Precision), and ImageJ (NIH). Before image analysis, back-grounds were subtracted. Maximum-intensity projection ofconfocal stacks (0.5 μm sections) and time-lapse images were ac-quired using Zen and ImageJ, respectively. To measure the lengthof EB1, CAMSAPs, and microtubule images, we manually trackedthem using Zen. EB1 comets tended to be longer at the peripheralthan at the central regions of a cell; therefore, we analyzed 10comets in each region and summed up the values, where the pe-ripheral region was defined as that spanning 10 μm from the cel-lular edge. The number of EB1 comets was quantified with the spotdetection function of Imaris, with a setting of 0.3 μm as a minimaldiameter andGaussian Filter. If incorrect spots were detected, theywere manually removed. The kymographs were created with theMultiple Kymograph plug-in for ImageJ. To estimate the velocityof growing microtubule plus ends, we calculated instantaneousdisplacement of each EB1 comet at growth phases over time bytracking them manually, using the spot detection function ofImaris. The band intensities in Western blots were measured byImageJ software. For analysis of Golgi fragmentation, giantin-positive particles in 2D projection images were quantified by Im-ageJ software.

    Immunoprecipitation. For immunoprecipitation, HEK293T cellstransiently transfected with CAMSAP2-Flag alone or bothCAMSAP2-GFPandCAMSAP3-Flagweredissolved in lysisbuffer[60 mM K-Pipes, pH 7.0, 25 mM Hepes, 10 mM EGTA, 2 mMMgCl2, 1% (vol/vol) Nonidet P-40, and protease and phosphataseinhibitor mixture (Sigma)]. After centrifugation, supernatantswere precleared with protein G-conjugated Sepharose beads (GEHealthcare) for 1 h, incubated with appropriate antibodies for 1 h,and incubated with newly prepared protein G-conjugated Se-pharose beads for 1 h. After the beads were washed four times withthe lysis buffer, precipitates were eluted with a sample buffer,separated by SDS/PAGE, and analyzed by immunoblotting.

    SDS/PAGE and Western Blotting. Samples were boiled with SDSsample buffer at 97 °C for 5 min, separated by SDS/PAGE, andtransferred to Immobilon-P membranes (Millipore). Membraneswere blocked with 5% (wt/vol) skim milk in T-TBS for 30 min andincubated with primary antibodies for 2 h at room temperature orovernight at 4 °C, and then with secondary antibodies for 1 h. Theproteins were detected by using Western lightning plus ECL(PerkinElmer), Chemi-Lumi One Super (Nacalai), or Novex ECLChemiluminescent Substrate Reagent Kit (Invitrogen).

    1. Meng W, Mushika Y, Ichii T, Takeichi M (2008) Anchorage of microtubule minus endsto adherens junctions regulates epithelial cell-cell contacts. Cell 135(5):948–959.

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  • Fig. S1. (A) A Caco2 cell lysate was incubated in the absence (−) or presence (+) of Taxol-stabilized microtubules. These samples were fractionated into thesupernatant (S) and pellet (P), and analyzed by SDS/PAGE and Western blotting using antibodies against the indicated molecules. (B) Caco2 cells were tran-siently cotransfected with the plasmids for CAMSAP2-GFP and CAMSAP3-mKOR, and then double immunostained for GFP and mKOR (Top), or triple-im-munostained for GFP, mKOR and α-tubulin (Middle and Bottom). Cells with three different expression levels of CAMSAPs are shown, where the top sampleshows the lowest level, and the bottom one, the highest level. The boxed portion is enlarged in the Inset. Note that the overexpressed CAMSAPs overlap withmicrotubules. (Scale bar, 10 μm.) (C) Caco2 cells transiently transfected with the plasmids for CAMSAP2-GFP (Left) or CAMSAP3-GFP (Right) were treated withDMSO as a control for 1 h at 37 °C (Upper) or 10 μM nocodazole for 1 h at 4 °C to depolymerize microtubules (Lower), and then triple-immunostained for GFP,α-tubulin, and DAPI. The asterisks indicate untransfected cells. (Scale bar, 10 μm.) (D) HEK293T cells were transiently transfected with the plasmid for Flag-tagged CAMSAP2, and their lysates were subjected to immunoprecipitation (IP) with anti-flag or control (IgG) antibodies. The precipitates were thenanalyzed by SDS/PAGE and Western blotting with antibodies against the indicated molecules. Note the coprecipitation of endogenous CAMSAP3 with theFlag-tagged CAMSAP2.

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  • Fig. S2. (A) Western blot analysis of knockdown efficiency for CAMSAP2 and -3. The band intensity of CAMSAP2 was measured for the control and CAMSAP3-depleted samples, and normalized using the GAPDH band, as shown in the bar graph. Data represent the mean ± SEM from seven independent experiments.*P < 0.01. (B) Caco2 cells transfected with the indicated siRNAs were double immunostained for CAMSAP2 and CAMSAP3. The boxed area is enlarged in theInset. (Scale bar, 10 μm.) The length of CAMSAP clusters was measured and compared between the control and CAMSAP2- or -3–depleted cells. Data werecollected from three independent experiments, in which more than 80 clusters were analyzed per experiment. (C) Caco2 cells treated with CAMSAP3 siRNAwere double immunostained for CAMSAP2 and α-tubulin. Elongated CAMSAP2 clusters overlap with microtubules at their ends. (Scale bar, 10 μm.) (D) Time-lapse recording of DD-CAMSAP3-GFP (white arrowhead) and EB1-RFP introduced into a Caco2 cell. The GFP signal of DD-CAMSAP3-GFP disappeared at 12 s.Simultaneously, the minus end (marked with blue arrowheads) of the EB1-decorated microtubule began to retract. Kymograph of EB1-RFP and GFP signalscollected along the broken white line is shown at Right. Montage images of Movie S3 are shown. (Scale bars, 1 μm.)

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  • Fig. S3. Caco2 cells were transfected with CAMSAP2 and -3 siRNAs, together with the mouse CAMSAP2-GFP and CAMSAP3-HA cDNAs (Top), mouse CAMSAP2-GFP cDNA (Middle), or mouse CAMSAP3-GFP (Bottom). Cells were immunostained for GFP and α-tubulin. The arrowheads point the centrosomal regions.Centrosomal microtubules, generated by CAMSAP deletion, are no longer detectable in the cells expressing the mouse CAMSAPs. However, some condensationof microtubules is detected near the nucleus in the cells singly transfected with mouse CAMSAP2 or -3, as indicated by arrows. (Scale bar, 10 μm.)

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  • Fig. S4. (A) Double immunostaining for γ-tubulin and CAMSAP2 or CAMSAP3 in Caco2 cells. In the paired centrosomes marked by γ-tubulin signals, onecentrosome overlaps CAMSAP2 or -3 signals more extensively than the other. (Scale bar, 10 μm.) (B) Western blot analysis of γ-tubulin knockdown efficiency.Two mixtures of siRNAs specific for γ-tubulin 1 and 2 were tested. See SI Materials and Methods for the sequences of the combined siRNAs. (C) Caco2 cellstransfected with si γ-tubulin1+2 #1 siRNAs were double immunostained for α-tubulin and γ-tubulin. Note that γ-tubulin signals were greatly reduced in thecytoplasm, although their centrosomal signals are still detectable. (Scale bar, 10 μm.)

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  • Fig. S5. (A) Double immunostaining for α-tubulin and EEA1 in Caco2 cells transfected with the indicated siRNAs. The arrowheads indicate centrosomal po-sitions. (Scale bars, 10 μm.) (B) Immunostaining for giantin, γ-tubulin (arrowheads), and DAPI in Caco2 cells treated with the indicated siRNAs. (Scale bars, 10μm.) (C) Quantification of the total number (white box) and area (gray box) of Golgi vesicles identified by immunostaining for giantin. Data represent themean ± SEM from four independent experiments, in which more than 10 cells were analyzed per experiment. **P < 0.001, ***P < 0.0001. (D) Double im-munostaining for giantin and α-tubulin in Caco2 cells transfected with the indicated siRNAs. The arrowheads show the centrosomal areas. (Scale bars, 10 μm.)(E) Golgi fragmentation induced by CAMSAP deletion was abolished by cotransfection of CAMSAP2 and -3 siRNAs with the plasmids for the mouse CAMSAP2-GFP and CAMSAP3-HA cDNAs. Cells were immunostained for GFP and giantin. The original clustering profile of Golgi membranes was restored in the cellsexpressing exogenous CAMSAPs. (Scale bars, 10 μm.)

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  • Movie S1. Time-lapse images of CAMSAP2 and CAMSAP3. CAMSAP2-GFP and CAMSAP3-mKOR were expressed in a Caco2 cell. Images were acquired at 2-sintervals for 1 min. (Scale bar, 1 μm.)

    Movie S1

    Movie S2. Time-lapse images of CAMSAPs and EB1. EB1-RFP and CAMSAP2-GFP (Left) or CAMSAP3-GFP (Right) were coexpressed in Caco2 cells, which weretransfected with control (Upper), CAMSAP2 siRNA (Lower Right), or CAMSAP3 siRNA (Lower Left). The arrows indicate CAMSAP2 or -3 signals. Images wereacquired at 2-s intervals for 1 min. (Scale bar, 1 μm.)

    Movie S2

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  • Movie S3. Time-lapse images of DD-tagged CAMSAP3-GFP and EB1-RFP expressed in a Caco2 cell. The white arrowhead indicates DD-tagged CAMSAP3/Nezhaand simultaneously the minus end of a noncentrosomal microtubule decorated with EB1-RFP. The blue arrowhead points to the retracting end of the samemicrotubule after degradation of DD-tagged CAMSAP3/Nezha. Images were acquired at 2-s intervals for 50 s. (Scale bar, 1 μm.)

    Movie S3

    Movie S4. Time-lapse images of EB1-GFP expressed in a Caco2 cell, which was treated with control (Left) or CAMSAP2+3 siRNAs (Right). The arrows in theright image indicate the putative centrosomal positions. Images were acquired at 1-s intervals for 2 min. (Scale bar, 5 μm.)

    Movie S4

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    http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218017109/-/DCSupplemental/sm03.movhttp://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1218017109/-/DCSupplemental/sm04.movwww.pnas.org/cgi/content/short/1218017109