RESEARCH      PUBLICATIONS      MOVIE GALLERY      TEAM      CONTACT

Monitoring the cytoskeletal EGF response in live gastric carcinoma cells

Felkl M, Tomas K, Smid M, Mattes J, Windoffer R, Leube RE, 2012

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.

EGF does not affect microtubule plus-end-dynamics. Microtubule plus-ends were labeled by EB3-CFP in a MKN1 cell. The time-lapse sequence (10 frames/min) of fluorescence images (inverse presentation; left) and corresponding DIC images (right) was recorded first in the absence of EGF and then in the presence of 30 ng/ml EGF (corresponding Figure 3A, B). Note that EGF induces extensive ruffling and cell translocation that is coupled to retraction fiber formation at the rear. The associated substantial cell shape alterations necessitated adjustment of the focal plane resulting in a few out of focus images. Note also that plus-end-dynamics are not altered by EGF treatment.


Download: .mp4 / .wmv

EGF-induced restructuring of the actin and keratin cytoskeleton. The overlay images show a MKN1 cell that is labeled with mRFP-actin (red) and HK18-YFP (green) before and after addition of 5 ng/ml EGF (see also corresponding Fig. 2D, D'). Fluorescence images were recorded by confocal laser scanning microscopy (2 frames/min). Note the appearance of multiple actin-rich and keratin-free ruffles upon EGF treatment.


Download: .mp4 / .wmv

Ruffling activity is increased upon addition of EGF. MKN1 and Hs746T cells were transfected to produce enhanced yellow fluorescence protein (YFP) under CMV-promoter control. The fluorescence was recorded by confocal laser scanning microscopy three times per minute in the absence of EGF and after addition of 5 ng/ml EGF (corresponding Fig. 1A-B'). Note the EGF-induced cell spreading and the appearance of multiple ruffles and lamellipodia that leads to extensive lamella formation in MKN1 cells but not in Hs746T cells.


Download: .mp4 / .wmv

Microtubule exploration of EGF-induced cell extension. Fluorescence microscopy (inverse presentation) of a peripheral segment of a MKN1 cell synthesizing EB3-CFP after addition of 100 ng/ml EGF. The recording (6 frames/min) begins 1.5 min after EGF addition (see also corresponding Fig. 3E). Note the exploratory extension of microtubules into the newly-formed cell extension. Bar, 5 µm.


Download: .mp4 / .wmv

Triple labeling of MKN1 cells reveals sequential reorganization of cytoskeletal filaments in response to EGF. MKN1 were transfected with expression constructs for mRFP-actin, EB3-CFP and HK18-YFP. The composite Video displays images that were recorded every 38 s starting 10 min after addition of 30 ng/ml EGF. It depicts the individual fluorescence patterns of EB3-CFP (EB3), mRFP-actin (ACTB) and HK18-YFP (KRT18) first in inverse presentation separately (cell contours demarcated in orange), then as a merged overlays of EB3-CFP (false green color) and mRFP-actin (red), and finally as merged overlays of EB3-CFP (false green color), mRFP-actin (red) and HK18-YFP (false blue color) as indicated. Note that EGF-induced cell extensions are positive for actin and that dynamic microtubules enter these regions which contain very little keratin. Bar, 10 µm


Download: .mp4 / .wmv

EGF-induced restructuring of the actin (left) and keratin cytoskeleton (right) in relation to focal adhesion dynamics (middle). The images were recorded in a MKN1 cell (see corresponding Fig. 5) that was labeled with mRFP-actin (left), CFP-dSH2 (middle) and HK18-YFP (right). The images (inverse presentation) were collected at 2 frames/min before and after addition of 5 ng/ml EGF. Note the appearance of actin-rich ruffles that either lead to lamella formation (at left cell margin) with dSH2-labeled focal adhesions and extending keratin filaments or disappear (at right cell margin).


Download: .mp4 / .wmv

Lamella differ from ruffles and lamellipodia by the presence of focal adhesions. The time lapse series (25 s recording intervals) shows overlays of DIC recordings and CFP-dSH2 fluorescence (false red color) in MKN1 cells (see corresponding Fig. 6 A, A'). Note the induction of abundant, dSH2-negative ruffles and lamellipodia upon addition of 5 ng/ml EGF.


Download: .mp4 / .wmv

Focal adhesions are formed and turn over in lamella (corresponding Fig. 6 B, B'). The micrographs show overlays of YFP (white) and paxillin-dsRed fluorescence in a MKN1 cell before and after addition of EGF (5 ng/ml). Images were recorded every 28 s.


Download: .mp4 / .wmv

Paxillin is recruited to focal adhesions prior to tyrosine phosphorylation in newly-formed lamella upon EGF treatment (see also Fig. 7). The images are taken from a MKN1 cell simultaneously expressing paxillin-dsRed and CFP-dSH2 (false green color) immediately after addition of 5 ng/ml EGF. The composite Video presents the overlays of paxillin-dsRed and CFP-dSH2 (top) and the overlays in combination with DIC micrographs (bottom). Images were recorded every 25 s by confocal laser scanning microscopy. Note the appearance of paxillin-positive focal adhesion sites in the proceding lamellum prior to dSH2 recruitment.


Download: .mp4 / .wmv

Zyxin partially co-distributes with dSH2 in focal adhesions and is recruited from zyxin-enriched fibrillar adhesion upon EGF treatment. MKN1 were co-transfected with constructs encoding RFP-zyxin and CFP-dSH2 (depicted in false green color). Note that both co-localize in focal adhesions in the cell periphery but that the more centrally located focal adhesions and large fibrillar adhesions are predominantly positive for zyxin and contain very little dSH2 label. The video depicts a cell before and after addition of 5 ng/ml EGF (recording interval 50 s; see also corresponding Fig. 8D, D'). Note the reduction of the very large zyxin-positive plaque-like areas in the cell center and the appearance of novel contacts in newly-formed lamella (e.g., lower right). Bar, 5 µm.


Download: .mp4 / .wmv