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First published online 28 March 2006
doi: 10.1242/jcs.02884

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Profilin-I-ligand interactions influence various aspects of neuronal differentiation

Anja Lambrechts^1,*, Veronique Jonckheere¹, Christa Peleman^1,, Debby Polet¹, Winnok De Vos², Joël Vandekerckhove¹ and Christophe Ampe¹

¹ Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, and Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology (VIB09), Albert Baertsoenkaai 3, 9000 Ghent, Belgium
² Department of Molecular Biotechnology, Faculty of Bio-engineer sciences, Ghent University, 9000 Ghent, Belgium

View larger version (41K): [in a new window]   Fig. 1. Subcellular localisation of EYFP-profilin IWT in PC12 cells. (A) Projection of a confocal image stack series. (B,C) Selected stacks at a position close to the substrate. EYFP-profilin IWT is seen enriched in growth cones (GC), neuronal sprouts (S), and branch points (B). Bars, 10 µm.

View larger version (20K): [in a new window]   Fig. 2. Dose-dependent effect of profilin IWT on PC12 cell differentiation. The percentage of differentiated cells 76 hours after the addition of NGF, is shown as a function of the overexpression level of human profilin IWT. The cells were scored as differentiated when they had at least one neurite that is longer than the cell diameter. Results are presented as means ± s.e.m. with n=177-278 cells.

View larger version (75K): [in a new window]   Fig. 3. Expression of WT and mutant human profilin I in PC12 cells: a quantitative analysis of neuronal differentiation. (A) Western blot showing the expression levels of human WT and mutant profilins in the stably transfected PC12 cells. The levels were quantified by densitometry of the blot. The apparent higher molecular weight of the profilin IW3A mutant is also observed for the purified recombinant protein and is probably due to the introduced substitution (data not shown). (B) Representative images of the cell lines used in this study. The cells were grown on collagen-coated plastic and stimulated with NGF/forskolin for 24 hours. The profilin IR136D-expressing cells are visibly more differentiated. Bars, 20 µm. (C) Percentage of differentiated cells at 24 and 96 hours after NGF/forskolin stimulation. Results of two independent experiments are presented as means ± s.e.m.; n=317-355 cells. (D) Percentage of differentiated cells with two or more neurites at different time points after the addition of NGF/forskolin. Results of three independent experiments are presented as means ± s.e.m.; n=355-404 cells. (E) Percentage of neurites with branches, 96 hours after the addition of NGF/forskolin. n ranges from 136 to 159 cells, covering two independent experiments. (F) Profilin IR136D and profilin IW3A cells have longer neurites and profilin IR74E cells have shorter neurites from profilin IWT. The length of the longest neurite per cell (n=50 cells) was measured 96 hours after the start of differentiation and these lengths were plotted from short to long. The neurites can be divided into two populations: short – newly formed neurites up to 80 µm; and long – established neurites longer than 80 µm. The difference between the cell lines is most obvious in the population of longer neurites. , profilin IWT; , profilin IW3A; , profilin IR74E; , profilin IR136D.

View larger version (104K): [in a new window]   Fig. 4. Profilin IR136D facilitates neurite outgrowth without NGF/forskolin stimulation. Profilin IWT (A) and profilin IR136D (B) cells were plated on collagen for 36 hours without adding NGF/forskolin to the medium. The profilin IWT cells are less spread and do not extend neurites. The profilin IR136D cells are well spread and several of them extend neurites. Bars, 25 µm.

View larger version (78K): [in a new window]   Fig. 5. Phalloidin staining of cells expressing WT and mutant profilin I. (A) PC12 cells were plated on poly-D-lysine-coated coverslips and F-actin was stained 60 hours after treatment with 20 ng/ml NGF and 10 µM forskolin. Note that neurite outgrowth on poly-D-lysine is slower than on collagen, resulting in fewer and shorter neurites (compare with Fig. 3A). (B) CAD cells were transiently transfected with IRES-GFP constructs of profilin I WT and mutants. All cells shown are transfected based on the GFP signal (not shown), except for one non-transfected cell in W3A(2), which is indicated with an arrowhead. The arrow in W3A(1) indicates a ridge of densely packed filopodia. Bars, 25 µm (A); 50 µm (B).

View larger version (78K): [in a new window]   Fig. 6. Modulation of neurite outgrowth by inhibition of ROCK. (A) Cells were grown on collagen-coated coverslips and stimulated with NGF/forskolin for 24 hours without (a-e) or with prior incubation of 20 µg/ml ROCK inhibitor Y27632 for 15 minutes (a'-e'). a,a', parental PC12 cells; b,b', profilin IWT; c,c', profilin IW3A; d,d', profilin IR74E; e,e', profilin IR136D. Bars, 20 µm. The percentage of differentiated cells (B), the neurite length (C) and the percentage of branched neurites (D) after 24 hours were determined for cells treated with NGF/forskolin with or without Y27632. (B) All cell lines, except profilin IR136D cells, have increased percentages of differentiated cells when treated with the ROCK inhibitor. Results of two independent experiments are presented as means ± s.e.m. (n=300 cells). (C) Treatment with the ROCK inhibitor enhances neurite elongation except for profilin IR136D cells. The lengths of the longest neurite per cell (n=60) are displayed in box-and-whisker plots. + and – indicate the presence or absence, respectively, of Y27632 in the medium. (D) There was a strong increase in the percentage of branched neurites in PC12, profilin IWT, profilin IW3A and profilin IR74E cells, but not in profilin IR136D, in the presence of Y27632 (n=65-70 neurites).

View larger version (29K): [in a new window]   Fig. 7. Model for the regulation of profilin-I-actin interaction by PtdIns(4,5)P2 during neurite formation and growth. NGF stimulation locally activates PLC1, causing PtdIns(4,5)P2 hydrolysis into inositol 3-phosphate (IP3) and diacylglycerol (DAG), and release of profilin I (and perhaps also profilin IIa), that can now interact with actin and stimulate neurite formation. In this early stage, RhoA is inactivated through Rac and phosphoinositide 3-kinase (PI3K). At later stages, RhoA becomes active again, thereby activating ROCK and forming a complex with profilin IIa, which is now inactivated. ROCK also activates PtdIns 5-kinase (PI5K), which again increases the local PtdIns(4,5)P2 levels, resulting in inactivation of profilin I. Continuous stimulation of the NGF-receptor starts the PtdIns(4,5)P2 hydrolysis cycle again, and creates a balanced level of free profilin I, which may then contribute to neurite elongation. Where inactive, proteins are shown in grey lettering.

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