Immunolocalization Of Clathrin Heavy Chain (CHC) And APs ?-class Adapter Protein In Laticifers Of Euphorbia Helioscopia L.

Euphorbia helioscopia L. is a herb which has broad application values. Studies before were mainly focused on its biology characteristics, chemical constituents, pharmacological effects, vegetative growth, the development of the laticifers, the laticifer ultrastructure and latex proteomics, etc., while, there were no systematic researches on the mechanism of protoplasmic degeneration in laticifers. Lysosomal autophagy pathway is an important way for the metabolism of protein and aging, damaged organelles in the eukaryotic cells, it has a very important role in cell immunity and development, tissue remodeling and adaptation to the environment. Clathrin Heavy Chain and APs ?-class adapter protein were closely related with lysosomal autophagy pathway. Clathrin Heavy Chain is primarily responsible for the formation of clathrin-coated, dicytosome budding vesicles which is called the primary lysosomes, APs are responsible for recruiting clathrin to form clathrin-coated primary lysosomes, which would be fused with autophagosomes containing organelles or protein to form the secondary lysosomes, also known as autophagy lysosome. Lysosomes could release a variety of acid hydrolases making the organelles or proteins degrade away. However, if the protoplasmic degeneration in E. helioscopia laticifers are correlated with lysosomal autophagy pathway? If Clathrin Heavy Chain and APs ?-class adapter protein involve in the protoplasmic degeneration of E. helioscopia laticifers? As a result, based on above questions, present study was focused on E. helioscopia laticifers and analysized the immunolocalization of Clathrin Heavy Chain and APs ?-class adapter protein in tissue, molecular and cellular level with techniques of immunohistochemistry, immunoblotting and colloidal gold immunoelectronic microscopy. The results lay a foundation for exploring their relationship with laticifers growth. The results were as follows:1. The immunohistochemical analyses confirmed that laticifers contain Clathrin Heavy Chain. In the laticifers at early development stage, there were no distribution of Clathrin Heavy Chain, or less content, and then following the laticifer development, protoplasm began to degrade, the amount of Clathrin Heavy Chain increased along the entire process of protoplasm degradation. It also indicated that the protoplasm degradation of E. helioscopia laticifers and clathrin protein were closely linked.2. Through Western Blotting, it could be concluded that Clathrin Heavy Chain and APs ?-class adapter protein were expressed in E. helioscopia laticifers. Their relative molecular mass could be about 170 kDa and 100 kDa respectively, these results were similar to our expectations.3. In E. helioscopia laticifers at the early development stage, the cell protoplasm was rich, and the number of organelle was abundant, there contained a large number of mitochondria, endoplasmic reticulum and dicytosomes and so on. As the development, autophagy precursor increased gradually, some organelles including mitochondria and some protoplasm were wrapped to form autophagosomes, and then fused with lysosomes vesicles to form autolysosome, lysosomal enzymes in it made organelles degrade. Therefore, a large number of vesicles arounded these organelles, those vesicles were lysosomal vesicles. By colloidal gold immunoelectronic microscopy technique, it was found that Clathrin Heavy Chain and APs ?-class adapter protein were located in these lysosomal vesicles or on their membranes. In addition, two types of proteins were also distributed in autolysosomes. Therefore, Clathrin Heavy Chain and APs ?-class adapter proteins has a very close relationship with lysosomal autophagy pathway, they were involved in the protoplasm degradation of E. helioscopia laticifers by sorting and assembly lysosomal enzymes in the lysosomes which fused with autophagosomes.