Supplementary Figure s1. M23-AQP4 C-terminus analysis for the ability to induce morphological changes in glioma cells. a) Schematic representation of the untagged and mCherry-tagged M23-AQP4 isoforms used for the analysis in (b). b) Immuno fluorescence analysis of U87 and U251 cells transfected with tagged and untagged M23-AQP4 isoforms. AQP4 (red) and Phallodin (green) to visualize F-actin. No morphological changes were induced by M23-AQP4 when M23-AQP4 with mCherry tagged C-terminus was used. Scale bar 50 µm. c) Quantification analysis of morphological alterations observed in the experiments described in (a). The histobar shows the percentage of cells with altered morphology. Values are expressed as mean {plus minus} SEM of % of cells with altered cell morphology on the total number of transfected cells per field (**** P<0.0001; *** P<0.0005; n=3).
ARTICLE ABSTRACT
The glial water channel protein aquaporin-4 (AQP4) forms heterotetramers in the plasma membrane made of the M23-AQP4 and M1-AQP4 isoforms. The isoform ratio controls AQP4 aggregation into supramolecular structures called orthogonal arrays of particles (AQP4-OAP). The role of AQP4 aggregation into OAP in malignant gliomas is still unclear. In this study, we demonstrate that AQP4 aggregation/disaggregation into OAP influences the biology of glioma cells. Selective expression of the OAP-forming isoform M23-AQP4 (AQP4-OAP) triggered cell shape changes in glioma cells associated with alterations to the F-actin cytoskeleton that affected apoptosis. By contrast, expression of M1-AQP4 (AQP4-tetramers), which is unable to aggregate into OAP, ameliorated glioma cell invasiveness, improved cell migration, and increased methalloproteinase-9 activity. Two prolines (254 and 296) at the C-terminus tail were shown to be important in mediating the relationship between the actin cytoskeleton and AQP4-OAP and AQP4-tetramers. In conclusion, this study demonstrates that AQP4 aggregation state might be an important determinant in orienting glioma cells to persist or perish. AQP4 disaggregation may potentiate invasiveness potential, whereas AQP4 aggregation may activate the apoptotic path. This study shows a new perspective on the role of AQP4 in brain tumors not necessarily associated with edema formation but with AQP4 aggregation/disaggregation dynamics and their link with the actin cytoskeleton.
This study demonstrates how AQP4 aggregation influences plasma membrane dynamics to alter cell proliferation, invasiveness, migration, and apoptotic potential in glioma cells.
