Objective: 

Actin cytoskeleton supports the endosomal traffic in the cell. The actin also regulates signaling pathways by interacting with actin-binding proteins. It has been observed in our previous studies that the depolymerization of the filamentous actin occurs right after the interaction with fragment A (FA) of diphtheria toxin (DT) which is a well defined bacterial pathogen. The intracellular trajectory of DT has not been fully elucidated comparing to its inhibitory effect of protein synthesis which occurs following the ADP-ribosylation of eukaryotic elongasyon factor 2 by FA. In this study it is aimed to visualize the traffic of DT loaded endosomes and to show cytosolic and endosomal FA activity.

Methods: 

Human umbilical vein endothelial cells (HUVEC) were cultured and treated with DT. Filamentous actin, early endosomes and FA were detected by immunofluorescence microscopy. FA release into the cytosol from early endosomes was detected by Western blotting of affinity isolated protein complex. The activity of FA in fractions of cell lysates was checked by ADP-ribosylation assay.

Results: 

Immunostaining revealed that FA loaded endosomes were overlapped with actin and were accumulated around the nucleus. FA activity in nuclear cell fraction was found elevated.

Conclusions: 

FA release from toxin loaded endosomes was considered to be occurring due to the protein-protein interactions. Preliminary data presented here form the background for a new study with actin skeleton stabilizing drugs.