Role of the p55-gamma subunit of PI3K in ALK-induced cell migration: RNAi-based selection of cell migration regulators

Abstract

Recently, unbiased functional genetic selection identified novel cell migration-regulating genes. This RNAi-based functional selection was performed using 63,996 pooled lentiviral shRNAs targeting 21,332 mouse genes. After five rounds of selection using cells with accelerated or impaired migration, shRNAs were retrieved and identified by half-hairpin barcode sequencing using cells with the selected phenotypes. This selection process led to the identification of 29 novel cell migration regulators. One of these candidates, anaplastic lymphoma kinase (ALK), was further investigated. Subsequent studies revealed that ALK promoted cell migration through the PI3K-AKT pathway via the p55γ regulatory subunit of PI3K, rather than more commonly used p85 subunit. Western blot and immunohistochemistry studies using mouse brain tissues revealed similar temporal expression patterns of ALK, phospho-p55γ, and phospho-AKT during different stages of development. These data support an important role for the p55γ subunit of PI3K in ALK-induced cell migration during brain development.

Keywords: ALK; PI3K; RNAi; brain development; cell migration.

Figure 1.

Schematic illustration of in vitro loss-of-function selection for cell migration-regulating genes. The production of a 63,996 pooled lentiviral shRNA library targeting 21,332 mouse genes was performed by the transient transfection of HEK293T cells with pHAGE-mir-30-RFP-shRNA, pVSV-G, pTat, pPM2, and pRev. For the introduction of the pooled lentiviral shRNA library, NIH3T3 fibroblast cells were seeded at a density of 1 × 10⁶ cells/100-mm culture plate and infected with the lentiviral shRNA library at an MOI of 1 in the presence of polybrene (8 µg/ml). Two days after infection, cells were selected with puromycin (10 ng/ml) for 7 d and then detached and seeded onto transwell culture inserts. After seeding, cells were allowed to migrate across a porous membrane at 37°C for 5 or 24 hr to determine if they had an increased or decreased migration phenotype, respectively. Migrated and non-migrated cells were collected by trypsin-EDTA treatment from the lower and upper faces of the inserts, respectively, and re-seeded onto transwell culture inserts for a second round of selection. This process was repeated 5 times. After the final round, genomic DNA was isolated from migrating and non-migrating cells in order to identify the shRNAs integrated into the combined cells. DNA was subjected to PCR and sequencing. shRNA segments were amplified using the following primer set: forward, AGTGAAGCCACAGATGTA; reverse, CCTCCCCTACCCGGTAGA. All clones were sequenced. The sequences of 22-27 nt, corresponding to the half-hairpins of shRNAs, were used to identify shRNAs.

Figure 2.

A similar temporal expression pattern of ALK, p55γ, and AKT during mouse brain development. Levels of ALK, phospho-p55γ, and phospho-AKT were assessed by Western blot analysis of whole brain lysates at different time points: embryonic stages (e.g., ED14.5, ED18.5), postnatal day (PD) 3, and adult brain. Tubulin acted as a loading control. Results of densitometric analysis are presented as means ± SDs (n = 3); * p values of < 0.05 indicate significance between the indicated conditions.

Figure 3.

Spatiotemporal expression pattern of ALK during mouse brain development. Sagittal sections were immunostained with anti-ALK antibody and visualized with DAB in the mouse brain at embryonic days (ED) 14.5, 18.5, at postnatal days (PD) 3, and in the adult. Scale bar = 2 mm (upper). Laminar patterns of ALK-immunoreactive cells are shown. Alternatively, for immunofluorescence analysis, brain tissue sections were immunostained with an anti-ALK antibody and Cy™3-conjugated secondary antibody (lower). The images represent the boxed region (upper panel) in the cerebral neocortical area. While ALK-positive cells were mainly seen in the migrating zone (CP of ED14.5 brain; SVZ of ED18.5 and PD3), no ALK-immunoreactive cells were observed in adult cortical layers (I – VI). P, pia meter; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone; WM, white matter. Scale bar = 100 μm.