Altered cell motility is considered to be a key factor in determining tumor 
invasion and metastasis. Epidermal growth factor (EGF) signaling has been 
implicated in this process by affecting cytoskeletal organization and dynamics 
in multiple ways. To sort the temporal and spatial regulation of EGF-dependent 
cytoskeletal re-organization in relation to a cell's motile behavior time-lapse 
microscopy was performed on EGF-responsive gastric carcinoma-derived MKN1 cells 
co-expressing different fluorescently labeled cytoskeletal filaments and focal 
adhesion components in various combinations. The experiments showed that EGF 
almost instantaneously induces a considerable increase in membrane ruffling and 
lamellipodial activity that can be inhibited by Cetuximab EGF receptor 
antibodies and is not elicited in non-responsive gastric carcinoma Hs746T cells. 
The transient cell extensions are rich in actin but lack microtubules and 
keratin intermediate filaments. We show that this EGF-induced increase in 
membrane motility can be measured by a simple image processing routine. 
Microtubule plus-ends subsequently invade growing cell extensions, which start 
to accumulate focal complexes at the lamellipodium-lamellum junction. Such 
paxillin-positive complexes mature into focal adhesions by tyrosine 
phosphorylation and recruitment of zyxin. These adhesions then serve as 
nucleation sites for keratin filaments which are used to enlarge the neighboring 
peripheral keratin network. Focal adhesions are either disassembled or give rise 
to stable zyxin-rich fibrillar adhesions which disassemble in the presence of 
EGF to support formation of new focal adhesion sites in the cell periphery. 
Taken together the results serve as a basis for modeling the early cytoskeletal 
EGF response as a tightly coordinated and step-wise process which is relevant 
for the prediction of the effectiveness of anti-EGF receptor-based tumor 
therapy.