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First published online October 24, 2007
doi: 10.1242/10.1242/jcs.011130

Journal of Cell Science 120, 3820-3829 (2007)
Published by The Company of Biologists 2007
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SH4-domain-induced plasma membrane dynamization promotes bleb-associated cell motility

Stella Tournaviti1,*, Sebastian Hannemann2,*, Stefan Terjung3, Thomas M. Kitzing4, Carolin Stegmayer1, Julia Ritzerfeld1, Paul Walther5, Robert Grosse4, Walter Nickel1,{ddagger} and Oliver T. Fackler2,{ddagger}

1 Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
2 Department of Virology, University of Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
3 Advanced Light Microscopy Facility, EMBL, Meyerhofstr. 1, Heidelberg, Germany
4 Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany
5 Electron Microscopy Facility, University Ulm, 89069 Ulm, Germany


Figure 1
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  		Fig. 1. The SH4 domain of Leishmania HASPB induces plasma membrane blebbing. (A) Confocal microscopy analysis of HASPB-expressing CHO cells. Stable CHO cell lines expressing the SH4 domain of Leishmania HASPB fused to GFP (N18-HASPB-GFP) (a-d) or expressing a HASPB mutant with the myristoyl acceptor glycin at position 2 replaced by alanine (N18-{Delta}myr-HASPB-GFP) (e-h) under control of a dox-dependent promoter were cultured in the presence of 1 µg/ml dox to induce transgene expression. 24 hours post-induction, cells were analyzed by confocal microscopy either live (a,e) or following fixation and staining with TRITC-labelled phalloidin to visualize cellular F-actin (fixed; b-d,f-h). Presented are representative mid-cell z-sections. Scale bars, 10 µm. (B) PM blebbing induced by non-fusion full-length HASPB. Shown are confocal micrographs of live cells expressing: full-length HASPB fused to GFP (a), full-length non-fusion HASPB (b), GFP (c) or a palmitoylation-deficient mutant of N18-HASPB fused to GFP (N18-{Delta}pal-HASPB-GFP; d). All GFP fusion proteins were expressed stably in CHO cell lines, whereas full-length non-fusion HASPB was transiently co-expressed in CHO cells with actin-RFP. Panels display the distribution of GFP fusion proteins with the exception of b, in which actin-RFP is shown. Scale bars, 10 µm. (C) PM blebbing as a function of N18-HASPB-GFP expression levels. N18-HASPB-GFP CHO cells were cultured for 24 hours with the indicated concentrations of dox, fixed and analyzed for PM blebbing by microscopy. Shown is the overall percentage of cells displaying PM blebs with the black and grey columns indicating the fraction of blebbing cells with high (more than five blebs per cell) or low (less than five blebs per cell) blebbing activity, respectively. Values are the arithmetic means of at least three independent experiments+s.d. In each experiment, over 100 cells were counted per condition. (D) Quantification of the blebbing efficiency of the cells depicted in selected panels in A and B. Values are the arithmetic means of at least three independent experiments experiments+s.d. In each experiment, over 100 cells were counted per condition.

 

Figure 2
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  		Fig. 2. HASPB-induced plasma membrane blebs are highly dynamic. (A-D) Representative images of N18-HASPB-GFP-induced PM blebs at four different time-points of a fast 2D time-lapse (10 minutes, 150 ms per frame, see also supplementary material Movie 1) show the dynamic appearance and retraction of blebs of different sizes. The kinetics of several blebs was analyzed using kymographs along the axis perpendicular to the bleb base. Boxed areas in C indicate the blebs analyzed in the kymographs shown in E,F. Scale bar, 10 µm. (E,F) Two kymographs (time-space plots; left) along the axis perpendicular to the bleb base and corresponding details of the blebs in the boxed areas in C during formation, retention and retraction (right). Notice the different time intervals between the stills during formation (1 second) and retention as well as retraction (3 seconds).

 

Figure 3
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  		Fig. 3. HASPB-induced plasma membrane blebs are non-apoptotic. (A) Confocal micrographs of GFP- or N18-HASPB-GFP-expressing CHO cells. Following dox induction, cells were treated with either medium or etoposide (ETO; 200 µg/ml) and cycloheximide (Chx; 200 µg/ml) overnight, fixed and subjected to TUNEL staining. Scale bar, 10 µm. (B) Quantification of TUNEL-positive blebbing cells. Values are the arithmetic mean of three independent experiments+s.d. In each experiment, over 100 cells were counted per condition. GFP-expressing cells were arbitrarily set to 100%.

 

Figure 4
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  		Fig. 4. Regulation of HASPB membrane blebbing by small Rho GTPases. N18-HASPB that was fused to mCherry and the indicated GTPase or C3 proteins fused to GFP were co-expressed in CHO cells and analyzed by live-cell microscopy. Shown is the percentage of cells displaying pronounced PM blebbing. Values are the arithmetic means of at least three independent experiments+s.d. In each experiment, over 100 cells were counted per condition.

 

Figure 5
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  		Fig. 5. Characterization of SH4-domain-mediated membrane blebbing. (A) PM blebs are dependent on Rock and myosin II activity as well as on an intact microfilament system. CHO cells expressing N18-HASPB-GFP were treated with solvent or the indicated drugs for 3 hours, fixed and stained for F-actin. Shown are representative confocal micrographs. Scale bar, 10 µm. (B) PM blebs depend on an intact microtubule architecture. Cells were treated and processed as described in A, but were additionally stained for {alpha}-tubulin. Scale bar, 10 µm. (C) Quantitative analysis of PM blebbing following drug treatment. CHO N18-HASPB-GFP cells were treated with medium or the indicated drugs for 3 hours (see Materials and Methods for details), fixed, stained for F-actin and evaluated for PM blebbing. Values are the arithmetic means of at least three independent experiments+s.d. In each experiment, over 100 cells were counted per condition. BS, blebbistatin.

 

Figure 6
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  		Fig. 6. N18-GFP-HASPB-induced plasma membrane blebbing correlates with enhanced cell invasion in 3D matrices. Cell migration across matrigel-coated transwells was assessed after 48 hours and invasiveness was calculated relative to GFP-expressing cells. Values are the arithmetic means of three independent experiments performed as duplicates with the indicated s.e.m. Statistical significance for the values obtained with HASPB-GFP-expressing cells was evaluated by Student's t-test relative to GFP-expressing cells (N18-HASPB-GFP P=0.019, N18-{Delta}myr-HASPB P=0.7).

 

Figure 7
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  		Fig. 7. Plasma membrane blebbing is a conserved activity of Src-kinase SH4 domains. (A) Confocal micrographs of live CHO cells stably expressing N18-HASPB-GFP (a), the indicated Src-kinase SH4 domains fused to GFP (b-e) or transiently full-length Lck fused to GFP (f). (B) Quantification of the blebbing efficiency of the cells depicted in A and in controls. Values are the arithmetic means of at least three independent experiments+s.d. In each experiment, over 100 cells were counted per condition. (C,D) SEM of CHO cells from cultures transfected with an expression construct for N18-Yes-GFP (C) or N18-Yes-{Delta}myr-GFP (D). Scale bars, 10 µm.

 

Figure 8
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  		Fig. 8. SH4-domain-mediated plasma membrane blebbing requires activity of endogenous Src. CHO N18-HASPB-GFP-expressing (A) or N18-Fyn-GFP-expressing (B) cells were treated with medium containing solvent or the indicated drugs for 3 hours (see Materials and Methods for details), fixed, stained for F-actin and evaluated for PM blebbing. Shown is the quantitative analysis of PM blebbing following drug treatment. Values are the arithmetic means of at least three independent experiments+s.d. In each experiment, over 100 cells were counted per condition.

 

Figure 9
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  		Fig. 9. Rho GTPases are selectively incorporated into Yes-SH4-domain plasma membrane blebs. Confocal micrographs of fixed and stained CHO-cells transiently co-expressing N18-Yes-GFP and the indicated Myc-tagged Rho GTPase. Arrows indicate individual blebs. `Zoom' pictures present higher-magnification pictures of the blebbing areas indicated by boxes in the merge panels. Scale bars, 10 µm.

 

Figure 10
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  		Fig. 10. Phosphorylated Src accumulates in Yes-SH4-domain plasma membrane blebs. Confocal micrographs of fixed and stained CHO-cells transiently expressing GFP, N18-Yes-{Delta}Myr or N18-Yes-GFP. (A) Localization of total Src. (B) Localization of active pSrc (Y418). Arrows indicate individual blebs. Insets present higher-magnification pictures of individual blebs. Scale bars, 10 µm.

 

Figure 11
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  		Fig. 11. Phosphorylated MLC accumulates in Yes-SH4-domain plasma membrane blebs. Confocal micrographs of fixed and stained CHO-cells transiently expressing GFP, N18-Yes-{Delta}Myr or N18-Yes-GFP. (A) Localization of total MLC. (B) Localization of pMLC (S19). Arrows indicate individual blebs. Scale bars, 10 µm.

 

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© The Company of Biologists Ltd 2007