Figure 7.
Downregulation of EHD1 affect L1/NgCAM trafficking through early endosomal compartments. A–C, Colocalization of endocytosed L1–myc/NgCAM (red) with endosomal markers (cyan) in cells expressing either shEHD1 #1–GFP or shRandom–GFP (colocalization appears white). The whole cell and piece of dendrite or cell soma are shown. A′–C′, Quantification of colocalization of endocytosed L1–myc/NgCAM with endosomal markers. Statistical significance, t test. Error bars indicate SEM; n = 10–25 cells per condition. A, Colocalization of L1–myc (20 min of anti-myc antibody uptake followed by 90 min chase) with endogenous EEA1. A′, Quantification of the extent of colocalization of endocytosed and chased L1–myc with endogenous EEA1 in cells expressing either shRandom–GFP or shEHD1 #1–GFP. B, Colocalization of NgCAM endocytosed for 20 min and fixed with endogenous NEEP21 in cells expressing either shRandom–GFP or shEHD1 #1–GFP. B′, Colocalization between endocytosed NgCAM and NEEP21 quantified as a proportion of NEEP21 endosomes containing NgCAM. C, Colocalization of NgCAM (endocytosed for 20 min and chased for 90 min) with lysosomal marker lgp120 in cell expressing either shRandom–GFP or shEHD1 #1–GFP. C′, Colocalization between endocytosed NgCAM and lysosomal marker lgp120 quantified as a proportion of colocalizing pixels of endocytosed NgCAM and lysosomes. D, Model of EHD1 localization and function in L1/NgCAM trafficking in neurons. L1/NgCAM (blue arrows) is endocytosed from the somatodendritic plasma membrane via an EHD1/EHD4-mediated pathway into EHD1-positive pre-early endosomal compartments (pre-EE) (green). These compartments then either mature or fuse into early endosomal (EE) compartments (yellow). The transition from pre-EE to EE is achieved through recruitment of EEA1 (yellow curved arrow) to EHD1 endosomal compartments. EHD1–EEA1-positive early endosomal compartments (yellow with green border) can either lose EHD1 (green curved arrow) or lose EEA1 (yellow curved arrow) and mature into EHD1-positive and/or NEEP21-positive endosomes (purple). L1/NgCAM is subsequently trafficked through NEEP21-positive neuronal early endosomes (purple). It is also possible that some L1/NgCAM enters NEEP21 compartments directly without passing through EEA1 compartments. From the NEEP21 compartment, L1/NgCAM is transported toward the axon in currently undefined endosomes (blue). Downregulation of EHD1 (red) leads to delay of L1/NgCAM exit from EEA1-positive compartments and delayed entrance into NEEP21-positive compartments, which in turn increases missorting of L1/NgCAM to the somatodendritic surface (red arrow) and therefore impairs transcytosis of L1/NgCAM to the axon.