TrkAIII promotes microtubule nucleation and assembly at the centrosome in SH-SY5Y neuroblastoma cells, contributing to an undifferentiated anaplastic phenotype

Abstract

The alternative TrkAIII splice variant is expressed by advanced stage human neuroblastomas (NBs) and exhibits oncogenic activity in NB models. In the present study, employing stable transfected cell lines and assays of indirect immunofluorescence, immunoprecipitation, Western blotting, microtubule regrowth, tubulin kinase, and tubulin polymerisation, we report that TrkAIII binds α -tubulin and promotes MT nucleation and assembly at the centrosome. This effect depends upon spontaneous TrkAIII activity, TrkAIII localisation to the centrosome and pericentrosomal area, and the capacity of TrkAIII to bind, phosphorylate, and polymerise tubulin. We propose that this novel role for TrkAIII contributes to MT involvement in the promotion and maintenance of an undifferentiated anaplastic NB cell morphology by restricting and augmenting MT nucleation and assembly at the centrosomal MTOC.

Figure 1

Representative IF images illustrating the typical pattern of TrkAIII expression (green), α-tubulin positive MTs (red), and overlap (yellow/orange) in interphase (a) and mitotic (b) TrkAIII SH-SY5Y transfectants.

Figure 2

(a) Representative IF images comparing TrkA variant expression (green), α-tubulin positive MTs (red), and the overlap (yellow/orange) in untreated and CEP-701-treated (100 ng overnight) TrkAIII SH-SY5Y transfectants and in untreated kd-TrkAIII, TrkAI, and control pcDNA SH-SY5Y transfectants. (b) Representative IF images comparing the pattern of TrkAIII expression (green), TrkAIII-associated tyrosine phosphorylation (red), and overlap (yellow/orange) in untreated and CEP-701-treated (100 nM, overnight) TrkAIII SH-SY5Y transfectants.

Figure 3

Representative IF images and histograms of MT regrowth assays, demonstrating (a) indirect IF changes in α-tubulin positive MT regrowth (red) from the γ-tubulin positive centrosome (green) in untreated and CEP-701-treated (100 nM) TrkAIII SH-SY5Y transfectants and in untreated TrkAI SH-SY5Y transfectants at 0, 5, and 15 minutes after nocodazole washout. (b) Histogram demonstrating the differences in mean (±S.E.) α-tubulin IF intensity in untreated and CEP-701-treated (100 nM for 1 hour) TrkAIII SH-SY5Y transfectants and in untreated TrkAI and control pcDNA SH-SY5Y transfectants at 0, 5, and 15 minutes after nocodazole washout, normalised with respect to untreated TrkAIII transfectants (arbitrary value 100%; n = 50 per group; * = statistical significance). (c) Indirect IF demonstrating differences in α-tubulin positive MT regrowth area (red) in untreated and CEP-701-treated (100 nM) TrkAIII SH-SY5Y transfectants and in untreated TrkAI SH-SY5Y transfectants at 0, 5, and 15 minutes after nocodazole washout. (d) Histogram demonstrating differences in α-tubulin positive MT regrowth area in untreated and CEP-701-treated (100 nM) TrkAIII SH-SY5Y transfectants and in untreated TrkAI and control pcDNA SH-SY5Y transfectants at 0, 5, and 15 minutes after nocodazole washout, normalised with respect to untreated TrkAIII transfectants (arbitrary value of 100%; n = 50 per group; * = statistical significance). (e) Histogram demonstrating the differences in γ-tubulin positive centrosome size in untreated control, TrkAI, and TrkAIII SH-SY5Y transfectants, normalised with respect to untreated control transfectants given the arbitrary value of 1 (n = 50 per group; * = statistical significance).

Figure 4

(a) IP/Western blots demonstrating differences in α-tubulin levels pulled down by TrkAIII and TrkAI immunoprecipitates from respective SH-SY5Y transfectants, plus histograms displaying densitometric analysis of the adjacent blots, demonstrating the presence of similar levels of TrkAI and TrkAIII immunoprecipitates, normalised to input α-tubulin levels, plus the difference in α-tubulin levels pulled down as a densitometric ratio to TrkAI and TrkAIII. (b) IP/Western blots demonstrating the effect of CEP-701 (100 nM for 0–16 hours) on TrkAIII tyrosine phosphorylation (P-Tyr) and α-tubulin levels pulled down in coimmunoprecipitation assays, plus histograms displaying densitometric analysis of the adjacent blots, demonstrating CEP-701-induced loss of TrkAIII tyrosine phosphorylation from 3 hr onwards, associated with reduced α-tubulin binding at 16 hours only.

Figure 5

(a) Western blots demonstrating the relative levels of TrkAI, TrkAIII, and total tyrosine phosphorylated α-tubulins in a representative α-tubulin phosphorylation assay; (b) IF images demonstrating the difference in tubulin polymerisation induced by TrkAIII but not control or TrkAI immunoprecipitates in a representative tubulin polymerisation assay, in the presence but not in the absence of ATP.

Figure 6

(a) Representative IF images demonstrating the difference in the regular oval nuclear morphology exhibited by pcDNA control and TrkAI SH-SY5Y transfectants, compared to the lobulated nuclei in TrkAIII SH-SY5Y transfectants, plus the inhibition of nuclear lobulation in TrkAIII SH-Sy5Y transfectants following incubation with CEP-701 (100 nM for 16 hours) and nocodazole (10 μg/mL for 2 hours). (b) Representative IF images demonstrating the intracellular distribution of endogenous TrkAIII (green), α-tubulin positive MTs (red), TrkAIII/α-tubulin overlap (yellow/orange), and DAPI-stained nuclei (blue) in human U251 cells, plus IF images demonstrating the capacity of nocodazole (10 μg/mL for 2 hours) and CEP-701 (100 nM for 16 hours) to inhibit nuclear lobulation in U251 cells.