Marie-Thérèse Leccia Journal of Investigative Dermatology

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Zyxin Redistributes Without Upregulation in Migrating Human Keratinocytes During Wound Healing Marie-Thérèse Leccia Journal of Investigative Dermatology Volume 113, Issue 4, Pages 651-657 (October 1999) DOI: 10.1046/j.1523-1747.1999.00726.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Migrating human keratinocytes cover in vitro wound after 24 h. Phase-contrast time-lapse images, at 0 h (A), 6 h (B), 12 h (C), and 24 h (D). By 24 h, the wound is 80% covered by migrating keratinocytes. Scale bar: 100 μm. Journal of Investigative Dermatology 1999 113, 651-657DOI: (10.1046/j.1523-1747.1999.00726.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Zyxin redistributes to focal contacts in human keratinocytes migrating into the in vitro wound. Double fluorescent confocal microscopic images of actin filaments (A) and zyxin (B) distribution in human keratinocytes next to the in vitro wound at low magnification. Migrating keratinocytes show developed actin stress fibers (A, F) and focal peripheral zyxin staining (B, H). Color overlay analysis (J) demonstrates colocalization of zyxin and ends of actin stress fibers or focal adhesions (yellow color). Non-migrating keratinocytes show peripheral actin staining (E), punctate zyxin staining centrally with marked peripheral localization (G), and marked colocalization with actin peripherally (I). Control preparation stained for actin (C) and the secondary fluorescein labeled antibody alone (D) revealed faint nonspecific fluorescein signal and absent “bleed-through”. Scale bar: 20 μm (A–D), 5 μm (E–J). Journal of Investigative Dermatology 1999 113, 651-657DOI: (10.1046/j.1523-1747.1999.00726.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Zyxin redistributes to focal contacts in migrating keratinocytes in the presence of the protein synthesis inhibitor cycloheximide. Migrating human keratinocytes show peripheral zyxin staining at focal contacts in the presence of 10 μg cycloheximide per ml (A) or 20 μg cycloheximide per ml (B). Scale bar: 5 μm. Journal of Investigative Dermatology 1999 113, 651-657DOI: (10.1046/j.1523-1747.1999.00726.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Zyxin upregulation is not detectable in migrating keratinocytes. (A) Western immunodetection of zyxin expression in high-density (HD) nonmigrating keratinocytes versus low-density (LD) migrating keratinocytes. Top panel: a single band at relative mobility of 84 kDa corresponds to zyxin. Bottom panel: Amido-black-stained bottom portion of nitrocellulose membrane shows protein loading of gel above. (B) Densitometry gel scan of zyxin immunoblots in (A); no significant difference was detectable between HD (nonmigrating) and LD (migrating) keratinocytes. (C) Immunoblot assay to determine sensitivity for detectable changes in zyxin protein levels. Zyxin immunoblot of increasing protein loading from lysates of confluent keratinocyte cultures. (D) Densitometry gel scan plot of zyxin immunoblot in (C) showing linear relationship of protein loading and densitometry gel scan values. Journal of Investigative Dermatology 1999 113, 651-657DOI: (10.1046/j.1523-1747.1999.00726.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Zyxin is localized in a punctate staining pattern throughout the keratinocyte cytoplasm in normal human epidermis and exhibits focal peripheral staining in wounds. (A) Confocal immunofluorescent microscopy reveals punctate zyxin staining throughout the keratinocyte cytoplasm in normal epidermis. Note increased staining in lower cell layers compared with upper layers. Sections incubated with the fluorescein-labeled goat anti-rabbit antibody alone exhibit faint nonspecific staining (B). (C) Hematoxylin and eosin stained section of wound site. (D) Migrating epidermal sheet shows peripheral actin localization (rhodamine-phalloidin staining). (E) The keratinocytes show zyxin dispersed throughout the cytoplasm with focal peripheral increased staining at the leading edge (arrows). In contrast, keratinocytes away from the leading edge show a punctate staining pattern (arrowheads), similar to normal skin zyxin distribution. (F) Magnification of leading edge of migrating keratinocytes in (E), showing focal peripheral increased staining adjacent to dermis. The dashed line indicates the dermoepidermal junction. Scale bar: 20 μm. Journal of Investigative Dermatology 1999 113, 651-657DOI: (10.1046/j.1523-1747.1999.00726.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Zyxin is present in human epidermis. Immunoblot analysis of lysate from whole isolated human epidermis. Single band at relative mobility of 84 kDa corresponds to zyxin. Journal of Investigative Dermatology 1999 113, 651-657DOI: (10.1046/j.1523-1747.1999.00726.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions