LRRK2 Transport Is Regulated by Its Novel Interacting Partner Rab32
Figure 4
Co-localization analysis of Rab32 wt and the constitutively active mutant Rab32 Q85L with different endosomal markers.
(A) NIH3T3 cells were co-transfected with plasmids encoding the recycling endosome marker GFP-Rab11B and DsRed-Monomer-Rab32 wt or DsRed-Monomer-Rab32 Q85L, fixed and analyzed by fluorescence microscopy. Scale bar = 10 µm. (B) Cells were transfected with plasmids encoding for GFP-Rab32 wt or GFP-Rab32 Q85L followed by fixation and subsequent immunofluorescence staining of Rab7. Scale bar = 10 µm. (C–E) NIH3T3 cells expressing either GFP-Rab32 wt or GFP-Rab32 Q85L were fixed and stained for Rab7. (C) Microscopic analysis of GFP-Rab32 Q85L that co-localized with endogenous Rab7 (arrows) in the perinuclear area. Non co-localizing Rab7 was indicated by arrowheads. The perinuclear area was defined by the red (outlines nucleus) and the yellow line (outer border for perinuclear area). The image illustrates the cellular area used for the following analysis. (D) Rab7 perinuclear aggregates co-localizing with GFP-Rab32 wt or GFP-Rab32 Q85L and non co-localizing ones. ctrl. = control (untransfected IHKE-1 cells) (E) Quantification of perinuclear Rab7-positive structures. ctrl. (control): perinuclear Rab7 in untransfected cells; wt/Q85L: perinuclear Rab7 co-localizing with GFP-Rab32 constructs. control/Rab32 wt/Rab32 Q85L: n = 1660/18/167 structures in 67/18/60 cells, 1/1/3 independent experiments. Statistical significance was tested by a Student's T-test: pcontrol-wt = 0.02; pcontrol-Q85L<0.005; pwt-Q85L = 0.02. Scale bar = 10 µm.