Ndel1 controls GluR1 localization in a similar way to Dyn2.
Fractionation of GluR1 in heavy membranes (HM) and light membranes (LM) from HeLa cells transfected with a combination of Dyn2, GluR1, Flag-Ndel1, Dyn2(K44A) constructs and/or treated with Ndel1 siRNA. (A) The HM fraction comprises several organelles, including the trans-Golgi network (TGN) and the endoplasmic reticulum (ER), as detected with p230 trans-Golgi/TGN38 and KDEL ER marker antibodies, respectively. The LM fraction includes cytosolic proteins and small organelles like early endosomes as indicated by Tubulin and EEA1 antibodies, respectively. (B) The framed Western blots depict the separation of GluR1 in HM vs GluR1 in LM in HeLa cells. Increasing the levels of Dyn2 or Ndel1 reduces the ratio GluR1 HM/GluR1 (HM+LM), indicating GluR1 is redistributed from the heavy membranes to the lighter membranes. The usage of a dominant negative mutant of Dyn2 (Dyn2(K44A)) or the treatment of cells with Ndel1 siRNA reverts the ratio, indicating accumulating GluR1 in the HM fraction. “C” corresponds to control (cells transfected with an empty vector). Error bars indicate s.d. (n = 3). One way ANOVA: ***, p<0.001; **, p<0.01; *, p<0.05. Note that the levels of stable (acetylated) Tubulin are similar in Ndel1 siRNA-treated cells vs control siRNA-treated cells. (C) In HeLa cells treated with control siRNA, the AMPA receptor GluR1 (red) is found at the cell periphery and up to the cell edge. In HeLa cells treated with Ndel1 siRNA most of GluR1 (red) is found close to the nucleus with very little amount at the cell periphery. Cells were double stained with a marker for the trans-Golgi network (p230 trans-Golgi, blue). Scale bar, 10 µm.