Hemidesmosome protein dynamics in live epithelial cells.
Hemidesmosomes mediate stable anchorage of epithelial cells to laminin-5 in the basement membrane zone and have been likened to spot-welds. Indeed, it has been assumed that hemidesmosomes are not dynamic, at least when compared to other matrix adhesion sites including focal contacts. We tested this notion by monitoring the fate of green fluorescent protein (GFP)-tagged human integrin beta4 subunit (GFP-hbeta4) and GFP-tagged 180-kD human bullous pemphigoid (BP) autoantigen (GFP-BP180) in live cultures of 804G cells that assemble numerous mature hemidesmosomes. In subconfluent 804G cells, both GFP-hbeta4 and GFP-BP180 protein clusters are not stable but assemble into and disassemble out of cat paw-like arrays at a relatively rapid rate. In confluent populations of 804G cells, although some cat paw-like clusters of both GFP-hbeta4 and GFP-BP180 are stable over periods of >60 min, other GFP-hbeta4 and GFP-BP180 protein arrays form and/or disappear during the same time period. Moreover, individual labeled particles show considerable motility in the plane of the membrane. Fluorescence recovery after photobleaching analyses provide a further indication of the dynamics of hemidesmosome proteins. In particular, bleached GFP-hbeta4 protein clusters in confluent cells recover signal within about 30 min, indicating that there is a relatively rapid turnover of hemidesmosome components in protein arrays clustered along the substratum attached surface of a cell. The rate of recovery is dependent on an intact microfilament system. In sharp contrast, bleached GFP-BP180 protein clusters in confluent cells fail to recover signal even when observed for longer than 60 min. To evaluate hemidesmosome protein dynamics in motile cells, we monitored GFP-hbeta4 and GFP-BP180 in 804G cells populating scrape wound sites in vitro. In these migratory cells, which lack mature hemidesmosomes, integrin beta4 subunit and BP180 protein clusters progressively assemble and disassemble into linear and cat-paw arrays. In summary, hemidesmosome protein clusters, like their counterparts in focal contacts, are dynamic. We discuss these results in relation to hemidesmosome functions.
Tsuruta
Daisuke
D
Department of Cell and Molecular Biology, The Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA.
Hopkinson
Susan B
SB
Jones
Jonathan C R
JC
D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins; D022002:Hemidesmosomes / Q000378:metabolism*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D039663:Integrin beta4 / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D024041:Non-Fibrillar Collagens; D000097805:Collagen Type XVII
D001324:Autoantigens; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D039663:Integrin beta4; D008164:Luminescent Proteins; D024041:Non-Fibrillar Collagens; D000097805:Collagen Type XVII