Hypoxia alters the recruitment of tropomyosins into the actin stress fibres of neuroblastoma cells

Abstract

Background: Neuroblastoma is the most common extracranial solid tumor of childhood. The heterogeneous microenvironment of solid tumors contains hypoxic regions associated with poor prognosis and chemoresistance. Hypoxia implicates the actin cytoskeleton through its essential roles in motility, invasion and proliferation. However, hypoxia-induced changes in the actin cytoskeleton have only recently been observed in human cells. Tropomyosins are key regulators of the actin cytoskeleton and we hypothesized that tropomyosins may mediate hypoxic phenotypes.

Methods: Neuroblastoma (SH-EP) cells were incubated ± hypoxia (1 % O2, 5 % CO2) for up to 144 h, before examining the cytoskeleton by confocal microscopy and Western blotting.

Results: Hypoxic cells were characterized by a more organized actin cytoskeleton and a reduced ability to degrade gelatin substrates. Hypoxia significantly increased mean actin filament bundle width (72 h) and actin filament length (72-96 h). This correlated with increased hypoxic expression and filamentous organization of stabilizing tropomyosins Tm1 and Tm2. However, isoform specific changes in tropomyosin expression were more evident at 96 h.

Conclusions: This study demonstrates hypoxia-induced changes in the recruitment of high molecular weight tropomyosins into the actin stress fibres of a human cancer. While hypoxia induced clear changes in actin organization compared with parallel normoxic cultures of neuroblastoma, the precise role of tropomyosins in this hypoxic actin reorganization remains to be determined.

Fig. 1

Hypoxia alters the behavior of SH-EP neuroblastoma cells. a SH-EP cells were grown in normoxic (20 % O₂) and hypoxic conditions (1 % O₂) for 24–144 h. Hypoxic cell viability normalized to normoxic controls (dashed line through 1). b-i Representative images of a chamber slide (8-well) coated with an extracellular matrix mimic, GFP-tagged gelatin, seeded with 1.6 × 10⁴ SH-EP/well. After 72–96 h ± hypoxia, cells fixed in methanol and stained with DAPI and TRITC-phalloidin. Five fields of view (20× objective) were obtained for each well using a widefield microscope and images imported into Image J for analysis. DAPI nuclear counts (b, c) provided cell numbers. Thresholding on phalloidin (d, e) and the absence of GFP (f, g) provided cell and matrix degradation areas, respectively. Hypoxia resulted in decreased gelatin degradation per cell area at 72 h (h), although no significance was observed at 96 h (i). 1,000+ cells analysed per timepoint. Data is mean ± SEM; n = 4 (a), n = 3 (b-i). * P < 0.05, ** P <0.01 (t-test). Scale bar = 50 μm

Fig. 2

Hypoxia leads to a more ordered actin cytoskeleton. SH-EP cells were grown in normoxia (20 % O₂) or hypoxia (1 % O₂) for 72 – 96 h. Coverslips were fixed with 4 % PFA and stained with TRITC-phalloidin and DAPI. Single z-plane images were obtained by confocal microscopy. Linear feature detection software was used to quantitate actin cytoskeletal organization. a-d Representative linear detection after 72 h ± hypoxia. Hypoxia visibly increases the number of actin filament bundles known as stress fibres (b vs. d). Scale bar = 50 μm, or 5 μm in inset. e Hypoxia increases mean actin filament bundle width at 72 h. f Hypoxia increases total actin filament length per cell number (AFLC) and (g) per cell area (AFLA). Data is mean ± SEM (n = 3). 650+ cells analysed per timepoint. ** P <0.01, *** P <0.0001 (t-test). ^†AFLA is dimensionless, with [length (pixels)]/[area (pixels)]

Fig. 3

Hypoxia alters the expression and localization of HMW tropomyosins. a Normoxic (N) and hypoxic (H) SH-EP lysates were separated by SDS-PAGE. Representative Western blot stained with α9d antibody, which detects HMW tropomyosin isoforms Tm1, Tm2 and Tm3. Actin used loading control. Densitometry values were normalized to normoxic levels (dashed line) for Tm1 (b), Tm2 (c) and Tm3 (d) expression. Results are mean ± SEM; n = 3–4, * P <0.05, ** P <0.01 (t-test). e-l SH-EP cells were grown for 72 h ± hypoxia, fixed, permeabilized and incubated with Tm311 and CGβ6 antibodies to detect HMW isoforms Tm1/2/3 (e-h) and Tm2/3 (e-h), respectively. Cells then incubated with Alexa555-tagged goat anti-mouse and single z-plane images obtained by confocal microscopy. Hypoxia increased filamentous organization of Tm1/2/3 (f vs. h) and Tm2/3 (j vs. l) compared to normoxic control cells. Independent experiments (Tm311, n = 2; CGβ6, n = 5). Scale bar = 50 μm, 10 μm in inset

Fig. 4

LMW Tropomyosin expression and localization are unaltered by hypoxia. Normoxic (N) and hypoxic (H) SH-EP lysates were separated by SDS-PAGE and immunoblotted with a WD4/9d, b γ9d and c CG3 antibodies to examine expression of LMW isoforms Tm4, Tm5NM1/2 and Tm5NM1-11, respectively. Actin used as loading control (d-f) Densitometry was normalized to actin, before expressing hypoxic levels relative to normoxic values (dashed line). Hypoxia did not significantly alter the expression of these LMW isoforms. Results are mean ± SEM; n = 4. g-n SH-EP cells grown for 72 h ± hypoxia were fixed, permeabilized and incubated with γ9d to detect Tm5NM1/2 (g-j) and WD4/9d to detect Tm4 (k-n). Cells then incubated with Alexa555-tagged goat anti-mouse or Alexa488-tagged goat anti-rabbit, respectively. Hypoxia did not alter the intracellular organization of Tm5NM1/2 (h vs. j) nor Tm4 (l vs. n). Independent experiments (γ9d, n = 2; WD4/9d, n = 3). Scale bar = 50 μm, 10 μm in inset