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1 DOI: 1.138/nc2975 GM13 / DNA F-ctin α shrna CLASP1 shrna shrna shrna CLASP1 shrna shrna e Tuulin CLASP1 CLASP1 shrna #32 shrna #33 #55 #58 Tuulin c Tuulin ppmlc E-Cdherin shrna CLASP1 shrna shrna f Golgi Frgmenttion Angle α ( ) g shrna #32 #33 #55 #58 CLASP1 shrna shrna d shrna CLASP1 shrna shrna Focl Adhesion Are (µm 2 ) F-ctin Pxillin #32 #33 #55 #58 shrna CLASP1 shrna shrna Supplementry Figure 1 Vlidtion nd phenotypes of CLASP depletion. () Representtive imges of Golgi frgmenttion in CLASP-depleted cells stined for GM13 to identify the Golgi pprtus, nd Sytox ornge s nucler stin. () Cytoskeleton phenotypes of CLASP depletion. Peripherl microtuules re sprser nd disorgnized. F-ctin stress fires nchoring into FAs re more predominnt in CLASP-depleted cells. (c) Immunofluorescence of phospho-myosin (ppmlc) nd E-cdherin indicte tht CLASP depletion increses cell contrctility. (d) Representtive imges of migrting HCT cells t the edge of cell monolyer stined for pxillin (mgent) nd F-ctin (green), expressing control (non-trgeting), CLASP1 or shrna. Only one of the two shrna sequences used for ech CLASP isoform is shown. Single chnnels of the indicted regions re displyed with inverted contrst. (e) Immunolot of lystes from cells expressing different shrna constructs fter 7 dys of puromycin selection. Tuulin ws used s loding control. Blots were proed with isoform-specific ntiodies to either CLASP1 or. Uncropped lots re shown in Supplementry Figure 8. (f) Quntifiction of the degree of Golgi frgmenttion round the nucleus in HCT cells migrting t the edge of cell monolyer. The Golgi frgmenttion ngle α ws defined s the ngle through the center of the nucleus tht encompsses ll Golgi structures. n = 73 (control shrna); 35 (CLASP1 shrna #32); 47 (CLASP1 shrna #33); 65 ( shrna #55); 43 ( shrna #58) cells. Representtive dt set of three experiments. (g) Quntifiction of FA size in CLASP-depleted cells. Ech dt point is the verge FA size from one imge which contined 3-5 cells. n = 28 (control shrna); 32 (CLASP1 shrna #32); 35 (CLASP1 shrna #33); 27 ( shrna #55); 34 ( shrna #58) imges. Representtive dt set of three experiments. Box-nd-whisker plots show medin, 1 st nd 3 rd qurtile (ox), nd 95% confidence intervls (notches) with whiskers extending to the furthest oservtions within ±1.5 times the interqurtile rnge. Dots re individul dt points. 1
2 shrna CLASP1 shrna shrna 2 µm Supplementry Figure 2 CLASPs re required for directionl migrtion. () Exmple phse imges of HCT cells migrting t the edge of cell monolyer expressing control, CLASP1 or shrnas. () Plots of representtive cell migrtion pths in control nd CLASP-depleted cells from 2 cells per condition. Dt is representtive of 3 independent experiments. Phse contrst time lpse sequences were rotted such tht the wound edge ws ligned with the verticl imge xis, nd cells re migrting to the right. The positions of drk fetures in the nucleus (nucleoli) were trcked over time with the Time Mesurements function in NIS Elements using Auto Detect ROI. Migrtion pths were normlized to the strting position. 2
3 Pxillin shrna #28 #39 c 1 µm Y µm LY 2942 shrna # µm Nocodzole GAPDH Tuulin Pxillin Pxillin shrna #39 Pxillin d shrna #39 2 µm e Pxillin Pxillin shrna #28 f g shrna #28 shrna #39 F-ctin 2 µm shrna F-ctin shrna #28 Supplementry Figure 3 Vlidtion nd phenotypes of depletion. () Locliztion of endogenous (green) round pxillin-leled FAs (mgent) in control HCT cells, nd cells treted with the indicted drugs. Insets show only the chnnel of the indicted regions with inverted contrst. () Immunolot of lystes from cells expressing different shrna constructs fter 7 dys of puromycin selection. Tuulin nd GAPDH were used s loding controls. Uncropped lots re shown in Supplementry Figure 8. (c) Immunofluorescence of nd pxillin, in wound-edge HCT cells expressing control (non-trgeting), or the indicted shrnas. (d) Immunofluorescence of, in HCT cells expressing control or the indicted shrnas. -depletion olishes peripherl CLASP ccumultions. (e) Immunofluorecence of pxillin nd in HCT cells expressing control or shrna. CLASP-depletion in HCT cells does not ffect locliztion to dhesion sites. (f) Immunofluorescence of tuulin or (g) F-ctin (phlloidin) in HCT cells expressing control or shrna. Cytoskeleton phenotypes of depletion re qulittively similr to CLASP-depletion. Peripherl microtuules re sprser nd disorgnized. F-ctin stress-fiers nchoring into FAs re more predominnt in depleted cells. 3
4 EGFP- / Clthrin HC EGFP- / Dynmin-II Clthrin HC / Pxilin 2 min wshout 9 min nocodzole Untreted 5 µm Supplementry Figure 4 CLASPs do not co-loclize with endocytic vesicles. () Nocodzole wshout experiment in cells expressing EGFP-. Cells were pre-treted for 9 minutes with 3.3 µm nocodzole prior to wshout, nd immunostined for endocytic mrkers clthrin hevy chin (HC) or dynmin II. Imges were cquired y spinning disc confocl microscopy. No ovious colocliztion is oserved etween nd either clthrin HC or dynmin II. () Nocodzole wshout experiment in cells immunostined for clthrin HC nd pxillin. Cells were pre-treted for 9 minutes with 3.3 µm nocodzole prior to wshout. Smples were imged using totl internl reflection microscopy (TIRF). 4
5 SP MT1-MMP-EGFP MMP HPX EGFP TM MT1-MMP-mCherry-TM MMP HPX mcherry min MT1-MMP-mCherry-TM c Pxillin-mCherry AcGFP-Sec61 min Supplementry Figure 5 MT1-MMP-mCherry-TM nd ER dynmics in migrting HCT cells. () Digrm of fluorescently tgged MT1-MMP constructs used. SP, signl peptide; TM, trnsmemrne domin; HPX, hemopexin domin () Spinning disk confocl microscopy of migrting HCT epithelil cell t the edge of cell monolyer expressing MT1-MMPmCherry-TM. Red rrowhed indictes the evolution of right, luminl lelled vesicle through mcropinocytosis indictive of clevge of the mcherry tg from the trnsmemrne domin. (c) Dynmics of AcGFPtgged Sec61 s n endoplsmic reticulum mrker 1 in migrting HCT cell expressing pxillin-mcherry. Although ER tuules re dynmiclly pulled forwrd into the leding lmell, there ws no ovious correltion etween ER dynmics nd FA turnover. Elpsed time is in minutes. 5
6 Normlized intensity ms 2 ms 4 ms Distnce (µm) 1.5 FWHM (µm) Totl intensity (.u.) Time (ms) Supplementry Figure 6 EGFP-R6A vesicle fusion with the plsm memrne. () High speed TIRF imging of EGFP-R6A vesicles. Red rrowheds indicte two independent fusion events ssocited with lterl spreding of EGFP-R6A signl in the plsm memrne. Elpsed time is in seconds. () Anlysis of fluorescence intensity cross EGFP-R6A vesicles t different time points during fusion normlized to vesicle intensity efore fusion (n = 11 vesicles). Solid lines re Gussin fits. The integrted intensity remins high while the signl spreds in the TIRF plne demonstrting lterl diffusion of EGFP-R6A indictive of fusion with the plsm memrne nd thus exocytosis. Error rs re 95% confidence intervls. 6
7 RAW Pre-filter Post filter Crop c Mx. Intensity Projection x-t Kymogrph series Supplementry Figure 7 Workflow of generting kymogrphs to quntify EGFP-R6A vesicle trnsport. () Rotted rw nd filtered dt, cropped region round FA (dshed ox), nd single x-t kymogrph t the ornge line. () Set of ll twenty x-t kymogrphs for ech horizontl row of pixels in the cropped imge. (c) Mximum intensity projection of ll twenty x-t kymogrphs. See methods for dditionl detils. 7
8 Supplementry Fig. 1e Short Exposure Medium Exposure Long Exposure CLASP1 Tuulin GAPDH Tuulin GAPDH Supplementry Fig. 3 Short exposure Long exposure Tuulin GAPDH Supplementry Figure 8 Uncropped immunolots shown in supplementry Figures 1 nd 3. Memrnes were cut long the dotted lines efore incution with primry ntiodies in order to proe shrna trgets nd loding controls (GAPDH nd tuulin) on the sme lots. 8
9 Supplementry Video Legends Supplementry Video 1 EGFP- nd pxillin-mcherry dynmics in migrting HCT cell. Spinning disk confocl microscopy time lpse sequence of migrting HCT epithelil cell t the edge of cell monolyer expressing EGFP- (lck) nd pxillin-mcherry (mgent). EGFP--decorted microtuules engulf FAs efore FA disssemly underneth the dvncing cell ody. Imges were cquired every 2 minutes. The video plys t 15 frmes s -1 nd is thus ccelerted 18 times. Supplementry Video 2 Focl dhesion turnover dynmics in control nd CLASP-depleted cells. Spinning disk confocl microscopy time lpse sequences of migrting HCT epithelil cells t the edge of cell monolyer expressing pxillin-mcherry. Left pnel: shrna; middle pnel: CLASP1 shrna; right pnel: shrna. FA disssemly underneth the dvncing cell ody is disrupted in CLASP-depleted cells. Imges were cquired every 3 minutes. The video plys t 15 frmes s -1 nd is thus ccelerted 27 times. Supplementry Video 3 Focl dhesion-ssocited CLASP clusters re microtuule independent. Spinning disk confocl microscopy time lpse sequence of HCT epithelil cell expressing EGFP- (lck) nd pxillin-mcherry (mgent). 3.3 µm nocodzole ws dded fter 2 minutes, when EGFP- -leled growing microtuule plus ends ruptly dispper. Imges were cquired every 2 minutes. The video plys t 15 frmes s -1 nd is thus ccelerted 18 times. Supplementry Video 4 Focl dhesion-ssocited EGFP- dynmics fter nocodzole wshout. Spinning disk confocl microscopy time lpse sequence of HCT epithelil cell expressing EGFP- (lck) nd pxillin-mcherry (mgent). 3.3 µm nocodzole ws wshed out t the eginning of the time lpse sequence. Re-growing microtuules ssocite with CLASP clusters prior to FA disssemly. Note tht other FAs tht did not hve ssocited CLASP clusters do not disssemle. Imges were cquired every 3 seconds. The video plys t 15 frmes s -1 nd is thus ccelerted 45 times. Supplementry Video 5 CLASP clusters depend on focl dhesions. Spinning disk confocl microscopy time lpse sequence of HCT epithelil cell expressing EGFP- (lck) nd pxillin-mcherry (mgent). 1 µm Y ws dded fter 2 minutes, which induces FA nd susequent EGFP- cluster disssemly. Imges were cquired every 2 minutes. The video plys t 15 frmes s -1 nd is thus ccelerted 18 times. Supplementry Video 6 EGFP- nd pxillin-mcherry dynmics in migrting HCT cell. Spinning disk confocl microscopy time lpse sequence of migrting HCT epithelil cell t the edge of cell monolyer expressing EGFP- (lck) nd pxillin-mcherry (mgent). Similr to CLASPs, EGFP- puncte surround FAs efore FA disssemly. Imges were cquired every 3 minutes. The video plys t 15 frmes s -1 nd is thus ccelerted 27 times. Supplementry Video 7 Focl dhesion-ssocited mtrix degrdtion in spreding HCT cell. Spinning disk confocl microscopy time lpse sequence of pxillin-mcherry (mgent) expressing HCT epithelil cell spreding on Alex488-leled geltin. Imges were cquired every 5 minutes. The video plys t 1 frmes s -1 nd is thus ccelerted 3 times. Supplementry Video 8 MT1-MMP-EGFP vesicle dynmics ner focl dhesions. Totl internl reflection microscopy time lpse sequence of HCT epithelil cells expressing MT1-MMP-EGFP (lck) nd pxillin-mcherry (mgent) fter initil photoleching of memrne-ound signl. The sme three fusion events s shown in Fig. 6c re highlighted y colored squres. Imges were cquired every.3 seconds. The video plys t 24 frmes s -1 nd is thus ccelerted 7 times. Supplementry Video 9 EGFP-R6A vesicle dynmics in migrting HCT cell. Totl internl reflection microscopy time lpse sequence of HCT epithelil cells expressing EGFP-R6A (lck) nd pxillin-mcherry (mgent). A numer of closely FA-ssocited memrne fusion events re highlighted in the mgnified region on the right. Imges were cquired every second. The video plys t 3 frmes s -1 nd is thus ccelerted 3 times. Supplementry Video 1 EGFP-R6A vesicle dynmics in CLASP-depleted cells. Totl internl reflection microscopy time lpse sequence of HCT epithelil cells expressing EGFP-R6A (lck) nd pxillin-mcherry (mgent). Left pnel: CLASP1 shrna; right pnel: shrna. Imges were cquired every second. The video plys t 3 frmes s -1 nd is thus ccelerted 3 times. Supplementry Tles Legends Supplementry Tle 1: CLASP nd shrna sequences. Supplementry Tle 2: List of primry ntiodies nd other regents used for immunofluorescence nd imunolotting. Supplementry Tle 3: Rw dt nd sttistics. 9
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