Study on interaction of autophagosome and microtubule during autophagy process in Cherax quadricarinatus

Insect research suggests that autophagy plays an important role in virus infection and proliferation, while shrimp autophagy research reports are rare. Understanding the autophagy of shrimp cells will open up new ideas for shrimp disease immunity research. Autophagy related protein LC3 is a marker protein of autophagy, existing in the membrane of autophagosome during the whole autophagy process. Our bioinformatics analysis found that Cherax quadricarinatus LC3 (CqLC3) and α-tubulin (Cqα-tubulin) had the interaction relations. To further reveal transport way of autophagosome during autophagy process, in this study, recombinant plasmids pET-HIS-CqLC3 and pET-GST-Cqα-tubulin were constructed and induced for protein expression in vitro. Then HIS and GST tagged fusion proteins HIS-CqLC3 and GST-Cqα-tubulin were purified by using affinity chromatography respectively. Recombinant proteins HIS-CqLC3 and GST-Cqα-tubulin were found binding each other by the GST pull-down assay. In order to further reveal the role of microtubules for autophagosome transport, C. quadricarinatus cell microtubules were depolymerized by vincristine which was a microtubule inhibitor, then the autophagosomes were detected by MDC staining. The results showed that autophagic reaction was incomplete, and autophagosomes increased after microtubule depolymerization. Therefore, the autophagosomes in C. quadricarinatus cells can interact with microtubules through CqLC3, and microtubules play an important role in autophagosome transport during autophagy process. This paper for the first time revealed the transport pathway of autophagosomes in shrimp autophagy, and the results laid the foundation for the study of autophagy in shrimp cells.