Supplementary Figures

Transcription

1 Electronic Supplementry Mteril (ESI) for Integrtie Biology. This journl is The Royl Society of Chemistry 214 Supplementry Figures

2 CWound Are µm2 A EGTA Low Clcium 8 1 min. fter wounding Wound + 1 min. 6 4 Wound + 1 min. 2 Low Clcium EGTA B 5 D 8 15 min. fter wounding *** Wound Are µm EGTA Low Clcium Time (min.) Wound Are µm *** Low Clcium EGTA Supplementry fig. 1 Extrcellulr Clcium is required for Xenopusleisoocyte cell membrne re-seling A. Representtie photogrphs of Xenopusleisoocyte cell membrne wounds t one nd ten minutes fter wounding, scle br is 5µm length. B. Digrm showing mens nd stndrd error of wound re t different times in control condition (blck squres, n=2), in presence of 5mM EGTA (red dots, n=21) or in presence of low extrcellulr clcium concentrtion (green tringles, n=17). C. Box Plot showing wound re distributions one minute fter wounding in the sme conditions s (B). D. Box Plot showing wound re distributions fifteen minutes fter wounding in the sme conditions s (B).

3 ¼ niml pole Mrginl zone ¼ egetl pole A B C Outwrd (positie) Inwrd (negtie) Outwrd (positie) Inwrd (negtie) Outwrd (positie) Inwrd (negtie) D Vibrting probe Vibrting probe Vibrting probe 1µm Vibrting probe Wound 1µm Wound 1µm Wound E D G H Electric current density (µa/cm 2 ) Electric current density (µa/cm 2 ) Electric current density (µa/cm 2 ) Intct membrne Intct membrne Intct membrne 1min. Post- Wounding 1min. Post- Wounding 1min. Post- Wounding egetl pole Outwrd (positie) Inwrd (negtie) Wound 1µm Electric current density (µa/cm 2 ) Intct membrne 1min. Post- Wounding Supplementry Fig. 2 Xenopusleisoocyte cell membrne wounding induces locl chnge of electric current density round the wound. A-D.Photogrphs illustrting electric current density mesurements t specific position long the niml egetl xis fter wounding of the cell membrne. Arrows indictes the direction of the electric current represented by positie lue when outwrd nd negtie lue when inwrd in (E-H). Positions were pproximtely 45 (A,E), 9 (B,F), 135 (C,G), or 18 (D,H) from the wound. E-H. Box Plots showing the electric current density distributions t intct membrnes nd t one minute fter wounding in control condition (n=15 for intct membrne mesurements nd 8-1 for wounded membrne mesurements).

4 A Animl Pole Ion selectie probe B 1µm 3 sec. Outwrd Flux (positie) InFlux (negtie) C Clcium Flux nm.cm.sec mM.5mM *** Low Clcium EGTA DCDCDCDCDCDCDCDCDCDCDCDCDCDCDCDCDCDCDCDCDCDCDC Supplementry Fig. 3: Extrcellulr clcium concentrtion ffects clcium flux t the intct cell membrne t the niml pole. A. Photogrph illustrting the clcium fluxes mesurements t the niml pole of Xenopusleisoocyte cell membrne. Arrows indicte the direction of the clcium flux representing outflow (positie lue) or influx (negtie lue) in (C). B. Exmple of ion selectie electrode mesurement trces of clcium concentrtion obtined t the niml pole of Xenopusleisoocyte cell membrne in control condition (blck), in presence of 5mM EGTA (red) or in presence of low extrcellulr clcium concentrtion (green). C. Box Plot showing clcium flux t the niml pole of Xenopusleisoocyte cell membrne wounds in control condition (n=21), in presence of 2mM EGTA (n=8) or in presence of low extrcellulr clcium concentrtion (n=12).

5 A Oscilltion Ion selectie Wound probe Wound Side Out Flux (positie) InFlux (negtie) 1µm B Clcium Flux nm.cm.sec C Clcium Flux nm.cm.sec Time (min.) Low Clcium *** *** Intct Membrne Wound side Intct Membrne Wound side Supplementry Fig. 4 Xenopusleisoocyte wounding does not ffect clcium flux t wound side. A. Photogrph illustrting the clcium flux mesurements t cell membrne wound side (ion selectie electrode is plced 1µm from the wound edge using micromnipultor). Arrows indicte the direction of the clcium flux representing outflow (positie lue) or influx (negtie lue) in (B), (C) nd (D). B. Digrm showing mens nd stndrd error of clcium flux t the wound side t different times in control condition (n=8). C. Box Plot showing the clcium flux t the intct membrne compre to wound side in control condition (n=8 nd 21) or in presence of low extrcellulr clcium concentrtion (n=8 nd 12). Asterisks represent the sttisticl differences between control intct membrnes ersus low clcium intct membrne or control wound side ersus low clcium wound side.

6 A Vibrting probe 1µm 1µm 1µm 1µm 1µm Outwrd (positie) Inwrd (negtie) niml pole ¼ niml pole Mrginl zone ¼ egetl pole egetl pole B C b c d e 3 sec. 1.5µA/cm 2 Electric current density (µa/cm 2 ) niml pole EGTA Low Clcium ¼ niml pole Mrginl zone ¼ egetl pole egetl pole Supplementry fig.5 The heterogeneous electric current density distribution t cell membrne long the niml-egetl xis is not ffected by low extrcellulr clcium concentrtion. A. Photogrph illustrting the electric current density mesurements t Xenopus leisoocyte cell membrne t different cell membrne domins long the niml-egetl xis. Arrows indicte the direction of the electric current represented by positie lue when outwrd nd negtie lue when inwrd in (C). B. Exmple of electric current mesurement trces obtined t different cell membrne domins long the niml-egetl xis in control condition. C. Digrm showing mens nd stndrd error of electric current density t different cell membrne domins long the niml-egetl xis in control condition (blck squres, n=15), in presence of 5mM EGTA (red dots, n=15) or in presence of low extrcellulr clcium concentrtion (green tringles, n=15).

7 A D E Membrne potentil (mv) B Membrne potentil (mv) C Membrne potentil (mv) EGTA Time (min.) Time (min.) Time (min.) Membrne potentil (mv) Membrne potentil (mv) D E *** Supplementry Figure 6: Extrcellulr clcium concentrtion ffects cell membrne potentil A. Digrm showing mens (solid lines) nd stndrd error (dshed lines) of Xenopus leisoocyte cell membrne potentil mesurements ersus time in control condition (blck, n=19) or fter ddition of 5mM EGTA t 3 seconds (red, n= 22). Letters show positions used to mesure dt shown in grphs (D) nd (E). B nd C. Exmples of Xenopusleisoocyte cell membrne potentil mesurements oer the time in control condition (B), nd in the presence of 5mM EGTA (C). D nd E. Box Plot showing the distribution of Xenopusleisoocyte cell membrne potentil mesurements oer 2 seconds period prior to ddition of EGTA (D) or eleen minutes fter (E).

8 Supplementry Tbles

9 Low C EGTA Wound center t 1min ± ±3 (***) -.8 ±.8 (***) Wound center t 3min ± ±1.7 (***).3 ±.8 (***) Wound center t 15min..1 ± ±1.2 (***).6 ±1 (NS) Wound side t 1min. 2.7 ± ±.3 (***) -.4 ±.1 (***) Wound side t 3min. 3.2 ± ±.3 (***) -1 ±.4 (***) Wound side t 9min..7 ± ±.1 (***).3 ±.1 (*) Supplementry tble 1: Extrcellulr clcium is required for electric current circuitry Vlues represent mens nd stndrd error of electric current density (in µa/cm 2 ) t different times in control condition (Center n=21; side n=18), in presence of 5mM EGTA (Center n=2; side n=19), or in presence of low extrcellulr clcium concentrtion (Center n=2; side n=19). Asterisks show sttisticl significnt differences between control ersus cow clcium nd control ersus EGTA; NS indictes no significnt difference.

10 prior wounding 1min. fter wounding 4min. fter wounding 15min. fter wounding -35+/ / / /-.5 EGTA /-.7 (NS) /-1.4 (NS) -6.5+/-1.4 (***) -2.7+/-1.8 (***) Supplementry Tble 2: Extrcellulr clcium is required for cell membrne repolriztion. Vlues represent mens nd stndrd error of Xenopusleisoocyte cell membrne potentil mesurements (in mv) before nd fter wounding t specific times in control condition (n=19) or in presence of 5mM EGTA (n= 22). Wound ws mde thirty seconds fter the mesurement strts nd EGTA dded thirty seconds fter wounding. Asterisks show significnt differences between control ersus EGTA; NS indictes no significnt difference.

11 Intct niml -5.2 ±.4 Wound center 1-2min ± 24.6(***) Wound Side 1-2min -5.8 ± 1.8 (NS) Wound center 3-4min ± 11.1 (***) Wound center 15-16min ± 4.7 (***) Supplementry tble 3: Dynmic of clcium flux round the wounds. Vlues represent mens nd stndrd error of clcium flux (in nm/cm 2 /sec) t different times in control condition (n=15). Asterisks show significnt differences between intct membrnes ersus the different conditions; NS indictes no significnt difference.

12 Low C EGTA Intct niml -5.2 ± ±.8 (***) -4 ±.4 (NS) Wound center 1-2min ± ±.3 (***) ±15.3 (*) Wound Side 1-2min ±1 -.1 ±.2 (***) ND Supplementry tble 4: Extrcellulr clcium is required for clcium influx through the wounds. Vlues represent mens nd stndrd error of clcium flux (in nm/cm 2 /sec) t different times in control condition (n=15), in presence of 5mM EGTA (n=8) or in presence of low extrcellulr clcium concentrtion (n=16).asterisks show significnt differences between control ersus low clcium nd control ersus EGTA; NS indictes no significnt difference; ND indictes not determined.

13 prior to +/- EGTA 2sec fter +/- EGTA 14 min fter+/- EGTA -34+/ / /-1.4 EGTA 34.2+/-1.1 (NS) /-3.2(***) -5.2+/-1.5(***) Supplementry Tble 5: Extrcellulr clcium concentrtion ffects cell membrne potentil. Vlues represent mens nd stndrd error of Xenopusleisoocyte cell membrne potentil mesurements (in mv) t specific times in control condition (n=19) or fter ddition of 5mM EGTA (n= 22). Asterisks show significnt differences between control ersus EGTA; NS indictes no significnt difference.

14 low clium EGTA Intct cell membrne electric current Intct cell membrne potentil ND - Intct cell membrne clcium flux -- Wound heling Wound center electric current ++ x -- Wound side electric current + x -- Wounded cell membrne potentil -/ ND -/-- Wound center clcium flux Wound side clcium flux -- Supplementry Tble 6: Summry of the effects of low extrcellulr clcium concentrtion on electric current, cell membrne potentil, clcium fluxes nd wound heling. (no chnge); -(decrese); + (increse), x (reersl), / (distinguish two time points); ND indictes not determined.