ANALYSIS OF TRYPANOSOMAL ENDOCYTIC ORGANELLES USING PREPARATIVE FREE-FLOW ELECTROPHORESIS

In this paper we demonstrate the power of preparative free-flow electr ophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocke t. The uptake of fluid phase markers such as horseradish peroxidase (H RP) into the various compartments of the endocytic pathway of bloodstr eam forms of Trypanosoma brucei brucei was manipulated by regulating t he external environment (e.g., by altering the temperature of incubati on). The various subcellular compartments were then separated by free- flow electrophoresis (FFE) or isopycnic density gradient centrifugatio n and analyzed for marker uptake. At low temperatures, HRP was found p redominantly in the flagellar pocket. Increasing the temperature resul ted in a time-dependent uptake of HRP into more positively charged end osomal fractions. However, little HRP activity was detected in lysosom al compartments, suggesting that either HRP had not yet entered the ly sosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pat hway that were not possible to identify by density gradient centrifuga tion alone. Although the differences in FFE separation of the endocyti c compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conlusion, we show that preparative FFE is a pow erful technique for the analysis and separation of flagellar pocket-de rived membranes from other endosomal and lysosomal compartments of Afr ican trypanosomes.