Tetraspanins are Localized at Motility-Related Structures and Involved in Normal Human Keratinocyte Wound Healing Migration  Pablo F. Peñas, Amaro García-Díez, Francisco Sánchez-Madrid, María Yáñez-Mó  Journal of Investigative Dermatology  Volume 114, Issue 6, Pages 1126-1135 (June 2000) DOI: 10.1046/j.1523-1747.2000.00998.x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of tetraspanin proteins on keratinocytes. (A) Flow cytometry study. All tetraspanin molecules studied are expressed by keratinocytes with different intensities: (a) CD9 (VJ1/20) (b) CD81 (I.33.22) (c) CD151 (LIA1/1), and (d) CD63 (TEA3/18). (B) Immunofluorescence of isolated keratinocytes. CD9 (VJ1/20) and CD81 (I.33.22) are expressed in the filopodia of cells. CD151 (LIA1/1) showed a weak stain, mainly in an intracellular pattern. CD63 (TEA3/18) is expressed mainly intracellularly but some filopodial expression was also found. Scale bar: 30 μm. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression of tetraspanin proteins on keratinocytes. (A) Flow cytometry study. All tetraspanin molecules studied are expressed by keratinocytes with different intensities: (a) CD9 (VJ1/20) (b) CD81 (I.33.22) (c) CD151 (LIA1/1), and (d) CD63 (TEA3/18). (B) Immunofluorescence of isolated keratinocytes. CD9 (VJ1/20) and CD81 (I.33.22) are expressed in the filopodia of cells. CD151 (LIA1/1) showed a weak stain, mainly in an intracellular pattern. CD63 (TEA3/18) is expressed mainly intracellularly but some filopodial expression was also found. Scale bar: 30 μm. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Tetraspanins accumulate in keratinocyte cell-to-cell contacts. (A) When cells are grown in high calcium medium (1 mM) intercellular junctions are very tight. β1 integrin (TS2/16), CD9 (VJ1/20), CD81 (5A6), and CD151 (LIA1/1) show enhanced intercellular staining. Photographs of couples of cells were taken to avoid the artifact produced by the stratification of keratinocytes. Scale bar: 30 μm.B) When cells are grown in low calcium medium (< 0.1 mM) to avoid the effect of cadherins, filopodia of keratinocytes connecting neighboring keratinocytes show accumulation of CD9 (VJ1/20), CD81 (5A6), CD151 (LIA1/1), and α3β1 integrins. Bridges between cells are marked by arrows. CD63 (TEA3/18) only stains the intracellular compartment. Scale bar: 30 μm. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Tetraspanins accumulate in keratinocyte cell-to-cell contacts. (A) When cells are grown in high calcium medium (1 mM) intercellular junctions are very tight. β1 integrin (TS2/16), CD9 (VJ1/20), CD81 (5A6), and CD151 (LIA1/1) show enhanced intercellular staining. Photographs of couples of cells were taken to avoid the artifact produced by the stratification of keratinocytes. Scale bar: 30 μm.B) When cells are grown in low calcium medium (< 0.1 mM) to avoid the effect of cadherins, filopodia of keratinocytes connecting neighboring keratinocytes show accumulation of CD9 (VJ1/20), CD81 (5A6), CD151 (LIA1/1), and α3β1 integrins. Bridges between cells are marked by arrows. CD63 (TEA3/18) only stains the intracellular compartment. Scale bar: 30 μm. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Expression of tetraspanins in normal skin. CD9 (VJ1/20), CD81 (I.33.22), and CD151 (LIA1/1) stain the basal and spinous layers whereas β1 integrin (TS2/16) only stains the basal cell layer. Both groups of molecules colocalized at the intercellular junctions of basal cell keratinocytes (white arrows). Arrows are displayed in parallel on both panels to identify the same structures. Staining was visualized with Alexa Fluor 488 goat antimouse (VJ1/20, I.33.22, and LIA1/1) and Cy3-labeled avidin (TS2/16). Scale bar: 30 μm. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Tetraspanins and α3β1 integrins are found in the rips of motile keratinocytes. In low calcium medium, the staining obtained with β1 integrin (TS2/16), α3 integrin (P1B5), CD9 (VJ1/20), CD81 (I.33.22), and CD63 (TEA3/18) is very conspicuous compared with that in high calcium medium. Keratinocytes were grown in low calcium and high calcium media. Scale bar: 30 μm. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Tetraspanin molecules colocalize with β1 integrins in the footprints, filopodia, and intercellular junctions but not in focal adhesions. (A) β1 integrin (TS2/16), CD9 (VJ1/20), and CD81 (I.33.22) show coincident fluorescence stainings in rips (white arrows), filopodia (white arrowheads), and intercellular junctions (black arrows). Staining was visualized with FITC-conjugated goat antimouse (TS2/16) or Cy3-labeled avidin (VJ1/20 and I.33.22). Arrows are displayed in parallel on both panels to identify the same structures. Scale bar: 30 μm. (B) Both the anti-β1 integrin activation reporter (HUTS-21) and anti-talin (8D4) MoAbs clearly delineate the focal adhesions of keratinocytes (white arrowheads), with no CD9 (VJ1/20) and CD81 (I.33.22) expression. Although some HUTS-21 expression was found in the footprints of keratinocytes (white arrows), it was weaker than tetraspanin expression. When anti-talin MoAb was used, no expression on the footprints was found. Staining was visualized with FITC-conjugated goat antimouse (HUTS-21 and 8D4) or Cy3-labeled avidin (VJ1/20 and I.33.22). Arrows are displayed in parallel on both panels to identify the same structures. Scale bar: (β1) 20 μm, (talin) 30 μm. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Tetraspanin molecules colocalize with β1 integrins in the footprints, filopodia, and intercellular junctions but not in focal adhesions. (A) β1 integrin (TS2/16), CD9 (VJ1/20), and CD81 (I.33.22) show coincident fluorescence stainings in rips (white arrows), filopodia (white arrowheads), and intercellular junctions (black arrows). Staining was visualized with FITC-conjugated goat antimouse (TS2/16) or Cy3-labeled avidin (VJ1/20 and I.33.22). Arrows are displayed in parallel on both panels to identify the same structures. Scale bar: 30 μm. (B) Both the anti-β1 integrin activation reporter (HUTS-21) and anti-talin (8D4) MoAbs clearly delineate the focal adhesions of keratinocytes (white arrowheads), with no CD9 (VJ1/20) and CD81 (I.33.22) expression. Although some HUTS-21 expression was found in the footprints of keratinocytes (white arrows), it was weaker than tetraspanin expression. When anti-talin MoAb was used, no expression on the footprints was found. Staining was visualized with FITC-conjugated goat antimouse (HUTS-21 and 8D4) or Cy3-labeled avidin (VJ1/20 and I.33.22). Arrows are displayed in parallel on both panels to identify the same structures. Scale bar: (β1) 20 μm, (talin) 30 μm. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Inhibitory effect of antitetraspanin MoAbs on keratinocyte motility in a wound healing model. (A)(a) TS2/16 (anti-β1 integrin) and P1B5 (anti-α3 integrin) almost totally inhibited the motility of keratinocytes. (b) VJ1/20, VJ1/10 (anti-CD9) and (c) 5A6 (anti-CD81) showed a strong inhibition that was not attained with (c) I.33.22 (anti-CD81), (d) VJ1/16 or LIA1/1 (both anti-CD151). Units are square micrometers of wound covered by the keratinocytes at different times in a 1000 μm wide field. The mean ± SE of two experiments performed in duplicate are shown. (B) Keratinocyte cell monolayer in the wound healing model after 28 h of incubation with no MoAb, P1B5 (anti-α3 integrin), 5A6 (anti-CD81), and LIA1/1 (anti-CD151). Plates were fixed and stained with violet-crystal. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Inhibitory effect of antitetraspanin MoAbs on keratinocyte motility in a wound healing model. (A)(a) TS2/16 (anti-β1 integrin) and P1B5 (anti-α3 integrin) almost totally inhibited the motility of keratinocytes. (b) VJ1/20, VJ1/10 (anti-CD9) and (c) 5A6 (anti-CD81) showed a strong inhibition that was not attained with (c) I.33.22 (anti-CD81), (d) VJ1/16 or LIA1/1 (both anti-CD151). Units are square micrometers of wound covered by the keratinocytes at different times in a 1000 μm wide field. The mean ± SE of two experiments performed in duplicate are shown. (B) Keratinocyte cell monolayer in the wound healing model after 28 h of incubation with no MoAb, P1B5 (anti-α3 integrin), 5A6 (anti-CD81), and LIA1/1 (anti-CD151). Plates were fixed and stained with violet-crystal. Journal of Investigative Dermatology 2000 114, 1126-1135DOI: (10.1046/j.1523-1747.2000.00998.x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions