Figure 1.
Co-localization of FLRT1 and FGFR1.
A) Immunofluorescent staining of Cos-7 cells co-transfected with plasmids encoding FGFR1 and 3'HA-tagged FLRT1 Cells were stained with anti-FGFR1 (green) and anti-HA (FLRT1 -red) Merged images show areas of co-localisation in yellow Images (in section) were taken with a confocal microscope and are representative cells from 11 total fields of cells B) HEK 293T cells were co-transfected with FGFR1 and either control vector (pcDNA31) or FLRT1 (FLRT-HA) with or without stimulation with FGF2 (20ng/ml) in the presence of heparin (10mg/ml) for 30 min Anti-HA immunoprecipitation was performed on whole cell lysate which was subjected to western blot analysis with anti-phosphotyrosine (IP: HA, Blot: pY) to identify phosphorylated FLRT1 (pFLRT1) Phosphorylated FGFR1 (pFGFR1) was co-immunoprecipitated with FLRT1 The whole cell lysate (WCL) was probed for both FGFR1 (Blot: anti-FGFR1) and FLRT1 (Blot: anti-HA) expression Data is representative of at least 4 independent experiments.
Figure 2.
FLRT1 is not a SFK substrate but phosphorylation is FGFR-and SFK-dependent.
A) HEK 293T cells were transfected with control (pcDNA31) or FGFR1 and a panel of either full-length FLRT1-HA or tyrosine substitution contructs as indicated (see Materials & Methods) One sample was pre-treated with FGFR kinase inhibitor (SU5402, 50mM, 1 hr) where indicated Cell lysates were immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) or anti-HA (IP: HA, Blot: HA) to examine phosphorylated FLRT1HA levels (pFLRT1) or total immunoprecipitated FLRT1-HA levels (FLRT1), respectively Whole cell lysate (WCL) fractions were probed with anti-FGFR1 (Blot: FGFR) to control for protein expression B) HEK 293T cells were transfected with pcDNA31, FGFR1 and FLRT1-HA as indicated Cells were pre-incubated (1hr) with pharmacological inhibitors (SU5402, 50mM; SU6656, 20mM) Cell lysates were immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) for pFLRT1 and anti-HA (IP: HA, Blot: HA) for FLRT1 WCL fractions were probed with anti-FGFR1 (Blot: FGFR), anti-phospho-ERK (Blot: pERK) or anti-ERK (Blot: ERK) Data in A) and B) are representative of ≥3 independent experiments Densitometric analysis (mean ± sem, n = 3) is the ratio of pFLRT1:FLRT1 and normalised to FLRT1 phosphorylation in the absence of inhibitor in both cases (**p<001, *p<005 non-parametric one way ANOVA) C) HEK 293T cells were transfected with pcDNA31, FLRT1-HA alone or co-transfected with either a constitutively active (KA) or kinase dead (KD) c-Src construct Cells were serum starved for 1hr and cell lysates immunoprecipitated with anti-HA and subsequently blotted with anti-phosphotyrosine (IP: HA, Blot: pY) for pFLRT1 or anti-HA (IP: HA, Blot: HA) for FLRT1 WCL fractions were probed with anti-phospho-ERK (Blot: pERK) or anti-ERK (Blot: ERK) Data are representative of 3 independent experiments D) HEK 293T cells were transfected with either pcDNA31 vector, FLRT1-HA or Y3F-FLRT1-HA constructs Cells were co-stimulated with FGF2 (20ng/ml) and heparin (10mg/ml) for the indicated times Cell lysates were blotted for anti-phospho-ERK (WCL IB: pERK), membranes were stripped and re-probed for anti-ERK (WCL IB: ERK) Densitometric analysis has been adjusted for ERK loading and expressed as an arbitrary pERK:ERK ratio Data are representative of at least 4 independent experiments E) 293T cells transfected with Y3F-FLRT1-HA were serum-starved in the absence or presence of pharmacological inhibitors of FGFR1 (SU5402, 50mM) and SFKs (SU6656, 20mM) and whole cell lysates probed with antiphospho-ERK (IB: pERK), anti-ERK (IB: ERK) and anti-HA (Blot: HA) for Y3FFLRT1-HA.
Figure 3.
FLRT1 promotes neurite outgrowth in vitro.
SH-SY5Y cells 48 hrs after transfection of either FLRT1-HA or Y3F-FLRT1-HA alone or co-transfected with FGFR1, as indicated, were stained for FLRT1 (using anti-HA) prior to morphological analysis A) Cells were assigned a ‘morphology’ based on the number of large diameter processes (>5mm in length) and cell shape as typified by the examples shown and B) several characteristic neuronal parameters were determined, such as number of processes, length of the longest process, total dendritic length and maximum cell diameter (Scholl diameter) Data in A) and B) was derived from •100 cells from at least 3 independent experiments C) the complexity of the dendritic arbour (Table S1) in a radial profile from the soma, was measured in a variation of the Scholl analysis (Sholl, 1953; see Figure S2) Data are represented as mean ± sem, n • 50 cells from at least 3 independent experiments Statistical significance in all instances used non-parametric Kruskal-Wallis and post hoc Dunns test (***p<0001, **p<001, *p<005) Images were acquired on a Leica confocal microscope and Image J was used to process stacked confocal sections to allow data determination.
Figure 4.
FLRT1 and Y3F-FLRT1 increase the number of primary dendrites in rat hippocampal neurons.
(A–C) Representative confocal microscopic images of the number of primary dendrites in hippocampal neurons transfected with IRES GFP control vector (A), FLRT1 (B) and Y3F-FLRT1 (C) The number of primary dendrites increased when the neurons were transfected with FLRT1 and Y3F-FLRT1 compared to the control (D) Graphical representation of the average number of primary dendrites emerging from the soma under each condition There was a significant increase in primary dendrites in both the FLRT1-and Y3F-FLRT1transfected neurons compared to the control (P<00001, one-way analysis of variance and P = 00008, Kruskal-Wallis Test) Tukey's Multiple Comparison Test also revealed that there was a significant difference between control and FLRT1 (P<001, **), control and Y3F-FLRT1 (P<0001, ***) and FLRT1 and Y3F-FLRT1 (P<005, *) Data derived from several coverslips and two separate experiments.
Figure 5.
Differential trafficking and localization of activated FGFR1 by FLRT1 and Y3F-FLRT1.
A–F) Representative confocal microscope images of SH-SY5Y cells grown to confluency on coverslips and co-transfected with FGFR1, and either FLRT1-HA (A–C) or Y3F-FLRT1-HA (D–F) Cells were fixed with either no stimulation, A) and D), or after exposure to FGF2 (20ng/ml) in the presence of heparin (10mg/ml) for 5 min, B) and E), or 30 min, C) and F) Cells were stained with anti-pY766FGFR1 (pFGFR1, red), anti-HA (FLRT1 and Y3F-FLRT1, green) and anti-pY416Src (pSrc, blue) Merged images (and inset magnified images of cell body and neurites) demonstrated co-localization of FLRT1/FGFR1 (yellow), FLRT1/pSrc (cyan), pFGFR1/pSrc (magenta) and FLRT1/pFGFR1/pSrc (white, white arrows) Images were acquired on a Leica confocal microscope and processed using Image J and Adobe Photoshop 60 and represent at least 3 independent experiments• Scale bar: 20mm.