This transport is to a big degree dependent on intact actin filaments and has been shown to occur along focal adhesion-anchored stress fibers [24], [54]. Later steps of the migratory response were not investigated in this study as they are far downstream of EGF signaling and involve a complex series of further alterations such as microtubule-dependent focal adhesion turnover, activation of the actin-myosin system (white arrows in cell 3 of Fig. corresponding DIC images (right) was recorded first in the absence of EGF and then in the presence of 30 ng/ml EGF (corresponding Physique 3A, B). Note that EGF induces considerable 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. Notice also that plus-end-dynamics are not altered by EGF treatment.(MPG) pone.0045280.s003.mpg (4.9M) GUID:?823BEE45-B34D-454C-867B-D06749AFEEBC Video S4: 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 m.(MPG) pone.0045280.s004.mpg (7.0M) GUID:?0F308090-3814-49CA-BC4F-43EA93AB3209 Video S5: 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 (reddish), and finally as merged overlays of EB3-CFP (false green color), mRFP-actin (reddish) 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 m.(MPG) pone.0045280.s005.mpg (9.4M) GUID:?BF0A139E-4A75-4D92-A7B0-22B01A2D6B46 Video S6: 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 (observe 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 LSHR antibody cell margin) with dSH2-labeled focal adhesions and extending keratin filaments or disappear (at right cell margin).(MPG) pone.0045280.s006.mpg (6.0M) GUID:?A0196213-8DE7-4DD2-9B28-F1136834D482 Video S7: 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 (observe corresponding Fig. 6 A, A). Note the induction of abundant, dSH2-unfavorable ruffles and lamellipodia upon addition of 5 ng/ml EGF.(MPG) pone.0045280.s007.mpg (3.8M) GUID:?2941FF92-AA60-4B63-AC3D-93BF2D5AC0C2 Video S8: 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.(MPG) pone.0045280.s008.mpg (8.8M) GUID:?C357839B-F27C-4EB5-87FF-0EDBA5B52B3C Video S9: Paxillin is usually 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.(MPG) pone.0045280.s009.mpg (9.4M) GUID:?2C90EAAF-0FC3-4FB4-9D91-74B5D855DDF5 Video S10: Zyxin partially co-distributes with dSH2 SAFit2 in focal SAFit2 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 SAFit2 periphery but that this 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; observe 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., lesser right). Bar, 5 m.(MPG) pone.0045280.s010.mpg (8.8M) GUID:?51B9971C-48A5-45E7-B59E-DC5290C631D2 Abstract.