Dynamics of cell adhesion and motility in living cells is altered by a single amino acid change in E-cadherin fused to enhanced green fluorescent protein.
E-Cadherin regulates epithelial cell adhesion and is critical for the maintenance of tissue integrity. In sporadic diffuse-type gastric carcinoma, mutations of the E-cadherin gene are frequently observed that predominantly affect putative calcium binding motifs located in the linker region between the second and third extracellular domains. A single amino acid change (D370A) as found in a gastric carcinoma patient reduces cell adhesion and up-regulates cell motility. To study the effect of this mutation on the dynamics of cell adhesion and motility in living cells, enhanced green fluorescent protein (EGFP) was C-terminally fused to E-cadherin. The resulting mutant E-cadherin-EGFP fusion protein with a point mutation in exon 8 (p8-EcadEGFP) and a wild-type E-cadherin-EGFP fusion construct (wt-EcadEGFP) were expressed in human MDA-MB-435S cells. Fluorescent images were acquired by time-lapse laser scanning microscopy and E-cadherin was visualized during contact formation and in moving cells. Spatial and temporal localization of p8- and wt-EcadEGFP differed significantly. While wt-EcadEGFP was mainly localized at lateral membranes of contacting cells and formed E-cadherin puncta and plaques, p8-EcadEGFP-expressing cells frequently formed transient cell-cell contacts. During random cell migration, p8-EcadEGFP was found in lamellipodia. In contrast, wt-EcadEGFP localized at lateral cell-cell contact sites in low or non-motile cells. Inhibition of the epidermal growth factor (EGF) receptor, which plays a major role in lamellipodia formation and cell migration, reduced the motility of p8-EcadEGFP-expressing cells and caused lateral membrane staining of p8-EcadEGFP. Conversely, EGF induced cell motility and caused formation of lamellipodia that were E-cadherin positive. In conclusion, our data show that mutant E-cadherin significantly alters the dynamics of cell adhesion and motility in living cells and interferes with the formation of stable cell-cell contacts.
Fuchs
Margit
M
Technische Universität München, Klinikum rechts der Isar, Institut für Allgemeine Pathologie und Pathologische Anatomie, München, Germany.
Hutzler
Peter
P
Handschuh
Gabriele
G
Hermannstädter
Christine
C
Brunner
Ingrid
I
Höfler
Heinz
H
Luber
Birgit
B
D000199:Actins / Q000378:metabolism; D015820:Cadherins / Q000235:genetics* / Q000502:physiology; D002448:Cell Adhesion / Q000235:genetics / Q000502:physiology; D002465:Cell Movement / Q000235:genetics* / Q000502:physiology; D003599:Cytoskeleton / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D018613:Microscopy, Confocal; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000502:physiology; D013194:Staining and Labeling
D000199:Actins; D015820:Cadherins; D011993:Recombinant Fusion Proteins