a Anti-Dab2 RGD Merge

Transcription

1 a Anti-Da Merge *** ** Percentage of cells adhered RGE RAD RGE RAD Supplementary Figure Da are localized at clusters. (a) Endogenous Da is localized at clusters. (), ut not RGE nor RAD peptides can trigger cell adheon. Peptides are pre-coated on the coverglass. After hr, majority of MEFs form spread morphology on -coated glass. On RGE and RAD coated glass, majority of MEFs do not spread. All data are the mean of three independent experiments (n > in each condition). P values y unpaired Kruskal Wallis test are: *** P <, and ** P < Error ars represent s.d., and all samples represent iological replicates. Detailed statistical test results are availale in Supplementary Tale Scale ar represents µm in panel (a) or µm in panel ().

2 a Anti-Beta3 (LIBS) Clathrin light chain Da Anti-Beta3 (LIBS) c Da CA-LRP6 Integrin-eta3 Da and CA-LRP6 Da and integrin-eta3 d Pearson's R value Colocalization with Da in MEF Integrin eta3 *** CA-LRP6 Supplementary Figure. Clathrin and Da are localized at activated -integrineta3 clusters. (a) Conformation-specific antiody LIBS confirms that integrin-eta3 is activated and localizes with clusters. Clathrin light chain-eyfp also localizes with activated integrin-eta3. () Da-mCherry localizes with activated integrineta3 clusters. (c) When adhered on -memranes, majority of Da-mCherry are not localized to CA-LRP6-GFP (constitutively-active LDL-receptor related protein 6, lacking its extracellular domain). Da-mCherry localizes with integrin-eta3-bfp clusters. (d) Pearson s correlation analys of Da/integrin-eta3 and Da/CA- LRP6. All data are the mean of three independent experiments (n > in each condition). P value y unpaired Kruskal Wallis test is: *** P < Error ars represent s.d., and all samples represent iological replicates. Detailed statistical test results are availale in Supplementary Tale Scale ar represents µm.

3 3 H46 Da UOS Da HeLa Da CHO a5 Da CHO a5 +3 Da Supplementary Figure 3. Da are localized at -integrin clusters in various cell types. Da-mCherry is found at clusters in various cell types on memranes, including H46, UOS, HeLa, and CHO cells. However, Da-mCherry is asent from clusters in the CHO cells lacking integrin-eta3. Scale ar 5µm.

4 4 a Da β3 Y77F Merge -glass -memrane CHO α5β + β3-y77f CHO α5β + β3-y784f Constitutively active Src-Y57F transfected Da Da Src-pY46 β3 Y784F Integrin-eta3 Merge Supplementary Figure 4. Da inding is not solely dependent on Integrin-eta3 tyrone phosphorylation. (a) Tyrone to phenylalanine mutation of integrin-eta3 fails to completely lock Da inding. Co-localization of Da-mCherry and integrineta3-y77f-gfp or Y784F can still e oserved on the -memranes. Scale ar represents µm. () Introduction of constitutive active Src kinase mutant does not promote Da inding to integrin-eta3 on -glass. Src activity, visualized y Src-pY46 staining is mainly at integrin-eta3 clusters that do not associate with Da. Scale ar represents 5µm.

5 5 a Normalized Intenty (a.u.) Da Talin Normalized Intenty (a.u.) Da Talin 5 5 Time (s) 5 5 Time (s) Supplementary Figure 5. Inversely correlated recruitment of Da and talin. (a) As Da is recruited over time, talin egins to dissociate. () When Da egins to dissociate, talin recruitment is increased. The intenty fluctuation of suggested that integrin-eta3 were dynamically reorganized during the Da/talin exchange. Error ars represent s.e.m (n=6).

6 6 a Da Rap GV Da and Da PIP5Kγ87 Da and c Da Talin L43G Da and d Pearson's R value Colocalization of Da and clusters Rap GV over-expreson PIP5Kγ87 over-expreson Talin L43G over-expreson Supplementary Figure 6. Da remains recruited to /integrin-eta3 clusters even with mutants to pro-activate integrins. (a-c) Overexpreson of GFP-Rap V, GFP-PIP5Kγ87, and calpain-cleavage restant mutant GFP-talin-L43G do not lock Da-mCherry recruitment at clusters. (d) Pearson s correlation analys of Da and clusters, with overexpreson of Rap V, PIP5Kγ87, and talin- L43G. All data are the mean of three independent experiments (n > in each condition). Error ars represent s.d., and all samples represent iological replicates. Detailed statistical results are availale in Supplementary Tale Scale ar represents µm.

7 7 a Da LDLRAP (ARH) Merge Da DOK Merge c Da Num Merge d Pearson's R value Colocalization with Num in MEF Integrin eta3 clusters Da Supplementary Figure 7. PTB domain containing protein Num is recruited at clusters. (a) The localization of PTB-containing adaptor proteins LDLRAP-GFP at clusters is low and limited. () DOK-GFP is not enriched at Da-ound clusters. (c) PTB-containing adaptor protein Num-GFP is colocalized with Da- mcherry and clusters. (d) Pearson s correlation analys of Num/integrineta3, Num/, and Num/Da. All data are the mean of three independent experiments (n > in each condition). Error ars represent s.d., and all samples represent iological replicates. Detailed statistical results are availale in Supplementary Tale Scale ar represents µm.

8 8 Pearson's R value Colocalization of Clathrin and clusters * * **** Control Da Num Da+Num Supplementary Figure 8. Colocalization analys of clathrin and. Single RNA knockdown of Da and Num causes a small decrease of colocalization of clathrin and. Doule knockdown of Da and Num results in further decline in colocalization. The knockdown efficiency is found to e 7-9% (Fig. 5c and 5d). All data are the mean of three independent experiments (n > in each condition). P values y unpaired Kruskal Wallis test are: **** P <, and * P < 5. Error ars represent s.d., and all samples represent iological replicates. Detailed statistical results are availale in Supplementary Tale

9 9 a Da + Num Control Anti-Da Integrin-eta3 Anti-Da Integrin-eta3 Talin Talin c Pearson's R value Colocalization of Talin and Integrin-eta3 clusters Control **** Da+Num Supplementary Figure 9. Increased colocalization of talin and integrin-eta3 in the Da/Num knockdown cells. (a) Anti-Da and GFP-talin are spatially separated at integrin-eta3-bfp clusters. Talin is enriched at integrin-eta3 clusters in the periphery of the adheon zone, ut asent at integrin-eta3 clusters in the center of the adheon zone. () Doule knockdown of Da and Num causes the increased colocalization of GTP-talin and integrin-eta3-bfp. (c) Pearson s correlation analys of talin and integrin-eta3. All data are the mean of three independent experiments (n > in each condition). Error ars represent s.d., and all samples represent iological replicates. Detailed statistical results are availale in Supplementary Tale Scale ar µm.

10 aa Da Num Da + Num C **** 3 **** **** 8 **** Da + Num N Speed (µm/min) + a D Control Da Num Da + Num D a + N N um um D a ol tr on C Num um um N D a ol tr on C Control Da c Speed (µm/min) **** 8 **** Relative frequency (fractions) Relative frequency (fractions) B Speed (µm/min) Control Speed (µm/min) Supplementary Figure Knockdown of Da and Num reduces cell migration speed. (a) Migration tracks of control, Da, Num, and Da+Num cells on compliant fironectin-coated PDMS gel (3kPa). Color-coded tracks represent migration speed within hrs of oservation. Also see Supplementary Movie 5. Scale ar µm. ( and c) Single knockdown of Da or Num causes a decrease in cell migration speed, and dual knockdown of oth Da and Num causes yet further decrease in migration speed. P values y unpaired Kruskal Wallis test are: **** P < All data are individual cell migration speed (n > in more than 3 cells in each condition) in two independent experiments. Error ars represent s.d., and all samples represent iological replicates. Detailed statistical results are availale in Supplementary Tale

11 a AP Merge Epn Merge 6 4 in am Merge D yn 8 A P Dynamin Colocalization with clusters Ep n c Pearson's R value d e Clathrin light chain Integrin-eta3 Merge -glass Supplementary Figure Clathrin-mediated endocytic machineries are recruited at clusters. (a-c) AP-mCherry, epn-cfp, and mcherry-dynamin in REF5 firolasts localize with clusters. (d) Pearson s correlation analys of /AP, /epn, and /dynamin. (e) mcherry-clathrin light chain was asent at integrin-eta3-gfp adheon plaques when the MEF adhered on glass. All data are the mean of three independent experiments (n > in each condition). Error ars represent s.d., and all samples represent iological replicates. Detailed statistical test results are availale in Supplementary Tale Scale ar represents µm.

12 Normalized Intenty (a.u.) Time (s) Clathrin Integrin-eta3 Supplementary Figure. Synchronized intenty decreases in integrin-eta3, clathrin, and. In the case of endocytic active puncta, the intenty profiles of integrin-eta3, clathrin, and decrease together. Also see Fig. 6. Error ars represent s.e.m. (n=6). Control Da Num Control on -glass Da+Num Chlorpromazine Supplementary Figure 3. Endocytos of ligands. Images represent inverted maximum projection of the confocal planes (-4 µm aove the sustrate) of endocytosed in control, Da, Num, Da+Num, and chlorpromazine treated cells on -memranes, as well as control cells on -coated glass. Also see Fig. 7. Scale ar µm.

13 3 Z= µm 5 µm µm 5 µm µm Dextran 3kD FN7- H Supplementary Figure 4. FN7- ligands on supported memranes are selectively endocytosed. Fironectin repeat III 7th to th domain monomer with ten histidines (FN7- H) contains the motif for integrin activation and is laeled y CF68R. When CF68R-FN7- H and micro-ruy dextran (3kD, with iotin and tetramethylrhodamine laels) are oth presented on supported memranes, endocytos of FN7-, not dextran is oserved. Scale ar represents µm. clustering With matrix-force development (fixed ligand) Adaptor proteins: Talin Actomyon Kindlin cytoskeleton assemly Activated Integrin-eta force-independent Activated integrin-eta3 force-dependent Without matrixforce development (moile ligand) Adaptor proteins: Da Num Clathrin coat assemly Other integrins? Supplementary Figure 5. Summary of the proposed model. Matrix-force development mediates adaptor protein inding to -integrin-eta3 clusters.

14 4 Marker anti-da anti-num Mol. wt. (kda) anti- -actin (loading control) Supplementary Figure 6. Full western lots of the Da and Num knockdown in MEF. The lots are stained and then recorded y a CCD camera.

15 5 Supplementary Tale : Detailed statistical test results Figure d Beta3 (LIBS) Beta (9EG7) Cell numers Mean SD P values > 5 < Figure e Beta3 (LIBS) Beta3-GFP Cell numers 3 4 Mean SD P values > 5 Figure j Moile zone Fixed zone Cell numers 4 3 Mean SD P values < Figure memrane glass Cell numers Mean SD 9 4 P values < Figure f MEF WT RhoA Q63L MRLC T8S9D Cell numers Mean 69 9 SD P values < < Figure 3 β3 β β3-y77f β3-y784f β3 + PP Cell numers Mean SD P values < < < < Figure 4i Talin Kindlin Tenn FilaminA Cell numers 4 7 Mean SD Figure 5 Da Integrin eta3 clusters Cell numers Mean SD Figure 6c, 6d Active Total Ratio Cell numers Mean SD Figure 7a α5β + αvβ3 α5β Cell numers 3 Mean SD P values < Figure 7 Wildtype Da Num Da+Num Chlorpromazine Wildtype of glass Cell numers Mean SD P values < < < < < Continue on the next page

16 6 Supplementary Tale : Detailed statistical test results Supplementary RGE RAD Figure Cell numers Mean SD P values < < Supplementary Beta3 CA-LRP6 Figure d Cell numers 5 Mean 6 37 SD P values < Supplementary Rap GV PIP5Kγ87 Talin L43G Figure 6d Cell numers 8 Mean SD Supplementary Beta3 Da Figure 7d Cell numers 9 6 Mean SD Supplementary Control Da Num Da+Num Figure 8 Cell numers Mean SD P values < 5 < 5 < Supplementary Control Da+Num Figure 9c Cell numers 5 Mean SD P values < Supplementary Control Da Num Da+Num Figure Speed dataset Mean SD P values < < < Supplementary AP Epn Dynamin Figure d Cell numers Mean SD