Abstract
Rab5, EEA1 and APPL1 are frequently used in fluorescence microscopy to mark early endosomes, whereas Rab7 is used as marker for late endosomes and lysosomes. However, since these proteins localize poorly in immuno-electron microscopy, systematic studies on their ultrastructural distributions are lacking. Here we address this gap by presenting a quantitative, high-throughput, on-section correlative light-electron microscopy (CLEM) approach using the sensitivity of fluorescence microscopy to infer label to hundreds of organelles classified by ultrastructure. We show that Rab5 predominantly marks small, endocytic vesicles and early endosomes. EEA1 co-localizes with Rab5 on especially early endosomes, but unexpectedly also labels Rab5-negative late endosomes and even lysosomes. APPL1 is restricted to small Rab5-positive, vesicular profiles without any visible content or ultrastructural marks. Rab7 primarily labels late endosomes and lysosomes. Our studies reveal the first ultrastructural distribution of key endosomal proteins at their endogenous levels and introduce CLEM as sensitive alternative for quantitative immuno-EM.
Competing Interest Statement
The authors have declared no competing interest.
