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First published online 26 March 2003
doi: 10.1242/jcs.00401

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Rit promotes MEK-independent neurite branching in human neuroblastoma cells

¹ Department of Anatomy and Neurobiology, University of Kentucky Medical Center, Lexington, KY 40536-0298, USA
² Department of Molecular and Cellular Biochemistry, University of Kentucky Medical Center, Lexington, KY 40536-0298, USA

View larger version (69K): [in a new window]   Fig. 1. Rit supports neurite outgrowth in SH-SY5Y cells on endogenous matrix. SH-SY5Y cells were either not exposed to adenovirus or infected with adenovirus expressing only green fluorescent protein (GFP) or co-expressing GFP and wild-type (WT-Rit) or constitutively active Rit (CA-Rit). Expression of the transgene was confirmed by western blotting for Rit (A). Images of uninfected cells (B) or cells infected with GFP alone (C, and corresponding fluorescent image D) displayed basal levels of neurite outgrowth. Cells co-expressing GFP and either WT-Rit (E, and corresponding fluorescent image F) or CA-Rit (G, and corresponding fluorescent image H) displayed a qualitative increase in neurite outgrowth from SH-SY5Y cells. Bar, 50 µm.

View larger version (15K): [in a new window]   Fig. 2. Quantification of the number of neurites per cell (A, excluding cells not bearing neurites), the percent of neurite-bearing cells (B), the total neurite length per cell (C) and the number of branch points per neurite (D) from SH-SY5Y cells cultured on endogenous matrix. Wild-type (WT-Rit) and/or constitutively active (CA-Rit) Rit increases neurite initiation (A,B), elongation (C) and branching (D) in comparison with both uninfected cells (Control) and cells infected with adenovirus expressing only green fluorescent protein (GFP). Results are means±s.e.m. across experiments for four separate experiments. Asterisks (*) indicate significant difference compared with Control or GFP only cultures at P<0.05 (ANOVA with Least Significant Difference post hoc test).

View larger version (70K): [in a new window]   Fig. 3. Qualitative assessment of neurite outgrowth from SH-SY5Y cells plated on purified laminin-1. Cells were either not exposed to adenovirus (A) or exposed to adenovirus expressing only green fluorescent protein (GFP, B, and corresponding fluorescent image C), co-expressing GFP and wildtype (WT-Rit, D, and corresponding fluorescent image E) or co-expressing GFP and constitutively active Rit (CA-Rit, F, and corresponding fluorescent image G). SH-SY5Y cells expressing WT-Rit or CA-Rit have well developed growth cones (arrowheads in D,F) and display a qualitative increase in neurite outgrowth including marked increases in branching (arrows in F). Bar, 50 µm.

View larger version (15K): [in a new window]   Fig. 4. Quantification of the number of neurites per cell (A, excluding cells not bearing neurites), the percentage of neurite-bearing cells (B), the total neurite length per cell (C) and the number of branch points per neurite (D) from SH-SY5Y cells cultured on laminin-1. Both wild-type (WT-Rit) and constitutively active (CA-Rit) Rit increase neurite initiation (A,B), elongation (C) and branching (D, CA-Rit only) compared with control cultures not infected with adenovirus (Control) or cultures infected with adenovirus expressing only green fluorescent protein (GFP). Results are means±s.e.m. from four separate experiments. Asterisks (*) indicate significant difference compared with Control cultures at P<0.05 (ANOVA with Least Significant Difference post hoc test).

View larger version (64K): [in a new window]   Fig. 5. Digital images of SH-SY5Y cells three days (3d, A-E) and two weeks (2w, F-J) after no infection (A,F) or initial infection with adenovirus expressing green fluorescent protein (GFP) only (B,G) or constitutively active (CA)-Rit (C,H), CA-Ras (D,I) or CA-Raf (E,J). Qualitatively, Rit induces greater outgrowth than Ras or Raf, and its effects on elongation and branching increase over two weeks.

View larger version (21K): [in a new window]   Fig. 6. Quantification of neurite outgrowth from SH-SY5Y cells expressing CA-Rit, CA-Ras or CA-Raf. CA-Rit significantly increases the number of neurites per cell (A, including cells not bearing neurites), the percentage of neurite-bearing cells (B), the length of the longest neurite per cell (C) and the number of branch points per neurite at three days (open bars) or two weeks (filled bars) after initial infection. In contrast, CA-Ras or CA-Raf increase neurite initiation (the number of neurites per cell, A; the percent of neurite-bearing cells, B) or elongation (the total neurite length per cell, C) over two weeks (CA-Ras also increased the total neurite length per cell at three days, C). Results are means±s.e.m. of pooled data from three separate experiments. Asterisks (*) indicate significant difference compared with Control or GFP only cultures at P<0.05 (ANOVA with Least Significant Difference post hoc test).

View larger version (30K): [in a new window]   Fig. 7. Western blots for phosphorylated ERK (A) and total ERK (B) from SH-SH5Y cells not infected (Con) or infected with adenovirus to express green fluorescent protein (GFP) or constitutively active (CA) Rit, Ras or Raf, quantified in arbitrary units compared with uninfected cells (C), demonstrate that CA-Rit, CA-Ras, and CA-Raf all increased ERK phosphorylation. Western blots for phosphorylated Akt (D), and total Akt (E), quantified in F, demonstrate that CA-Ras, but not CA-Rit, increased Akt phosphorylation. Treatment of CA-Rit infected cells with the MEK inhibitor PD 098059 (Rit, PD) inhibits Rit-induced ERK phosphorylation (A,C) but not Akt phosphorylation (F). Data shown are representative blots from an experiment performed three times (quantification includes all three experiments).

View larger version (17K): [in a new window]   Fig. 8. Quantification of the number of neurites per cell (A, including cells not bearing neurites), the percent of neurite-bearing cells (B), the total neurite length per cell (C) and the number of branch points per neurite (D) from SH-SY5Y cells either not infected (Control) or infected with adenovirus to express green fluorescent protein (GFP) alone or co-express GFP and constitutively active (CA-) Rit or CA-Raf. Cells were also subjected to treatment with the MEK inhibitor PD 098059 (PD, CA-Rit + PD, CA-Raf+PD). CA-Rit increases neurite initiation (A,B), elongation (C, P=0.056) and branching (D) over Control or cultures infected with adenovirus expressing only GFP. PD 098059 blocks the CA-Rit-induced increase in the number of neurites per cell (A) and the percentage of neurite-bearing cells (B) but not the total neurite length (C) or the number of branch points per neurite (D). PD 098059 decreased basal or CA-Raf-induced neurite initiation (the number of neurites per cell, A; the percentage of neurite-bearing cells, B), but not elongation (C) or branching (D). Results are means±s.e.m. of pooled data from two separate experiments. Asterisks (*) indicate significant difference compared with Control or GFP only cultures (not bracketed), and bracketed asterisks indicate significant differences between those respective groups at P<0.05 (ANOVA with Least Significant Difference post-hoc test).

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