Figure 8.

Colocalization of CLN3 protein with lysosomal and early endosomal markers in transfected neurons. Postnatal rat hippocampal cells were transiently transfected with wt CLN3 or CLN3 carrying mutations in targeting motifs, fixed with PFA, permeabilized with Triton X-100 and analyzed by confocal microscopy after antibody labeling. The wt CLN3 protein (A and D) was found in vesicular structures in the soma and along neuronal processes. In the soma and proximal processes wt CLN3 (A) colocalized well with lgp120 (B) and to a lesser degree with EEA1 (our unpublished data); C, merge of A and B. The wt CLN3 found in vesicular structures in peripheral neuronal processes (D) was associated with EEA1-positive structures (E), indicating its presence in early endosomes; merge shown in F. A likely axon is marked by arrowheads, D-F. CLN3 carrying mutations in both targeting motifs (double mutant LI253-4A+M409A+G419A) was not found in lysosomes (our unpublished data), but at the plasma membrane (see cell bodies in G and Figure 6B). And also in vesicular structures in neuronal processes (G), which were positive for EEA1 (H); I, merge of G and H. Examples of colocalization are indicated by white arrows and arrowheads. Bar, 10 μm.