The Single Dynamin-like Protein of Trypanosoma brucei Regulates Mitochondrial Division and Is Not Required for Endocytosis

Abstract

Members of the evolutionarily conserved dynamin-related GTPase family mediate numerous cellular membrane remodeling events. Dynamin family functions include the scission of clathrin-coated pits from the plasma membrane, mitochondrial fission, and chloroplast division. Here we report that the divergent eukaryote Trypanosoma brucei possesses a single dynamin family gene, which we have designated TbDLP. Furthermore, a single dynamin family gene is also found in the Leishmania major and Trypanosoma vivax genomes, indicating that this is a conserved feature among the kinetoplastida. TbDLP is most homologous to the DMN/DRP family of dynamin-like proteins. Indirect immunofluorescence microscopy reveals that TbDLP is distributed in punctate structures within the cell that partially co-localize with the mitochondrion when labeled with MitoTracker. To define TbDLP function, we have used RNA interference to silence the TbDLP gene. Reduction of TbDLP protein levels causes a profound alteration in mitochondrial morphology without affecting the structure of other membrane-bound compartments, including the endocytic and exocytic apparatus. The mitochondrial profiles present in wild type trypanosomes fuse and collapse in the mutant cells, and by electron microscopy the mitochondria are found to contain an accumulation of constriction sites. These findings demonstrate TbDLP functions in division of the mitochondrial membrane. Most significantly, as TbDLP is the sole member of the dynamin family in this organism, scission of clathrin-coated pits involved in protein trafficking through the highly active endocytic system in trypanosomes must function in the absence of dynamin. The evolutionary implications of these findings are discussed.

Ablation of the single dynamin of T. brucei blocks mitochondrial fission and endocytosis and leads to a precise cytokinesis arrest

“…TbDLP is required for Bax-induced mitochondrial fission Ablation of TbDLP in procyclic T. brucei by RNAi, in agreement with previously published work (Morgan et al, 2004), resulted in a growth arrest within 24 hours. However, unlike previous reports based on Mitotracker-stained cells, the mitochondrial morphology as analyzed by immunofluorescence appeared not to be significantly affected (Fig.…”

“…In a wild-type population NKKN cells represent <0.1% indicating that this cell cycle stage is very short. NKKN cells in the induced RNAi cell line, in agreement with the previously reported Mitotracker analysis (Morgan et al, 2004), consistently have a single continuous mitochondrion showing a more collapsed morphology than in uninduced cells (Fig. 4B).…”

“…Indeed, analysis of TbDLPablated cells by light and electron microscopy shows an enlargement of the FP ( Fig. 2A,B) a phenotype that is very similar to that caused by the ablation of clathrin heavy chain (CLH) (Engstler et al, 2005;Morgan et al, 2004) and actin (Garcia-Salcedo et al, 2004), two proteins known to be required for endocytosis in T. brucei. However, in these cases the enlarged FPs were reported to be restricted to the bloodstream stage, whereas in TbDLP-RNAi cells they are detected in procyclic cells.…”

“…TbDLP is the only member of the dynamin family in T. brucei (Morgan et al, 2004). This raises the question whether it might also be required for endocytosis.…”

“…The genome of the parasitic protozoon Trypanosoma brucei, unlike most other eukaryotes, encodes only two tandemly linked, 97% identical DLPs (termed TbDLP), which are the sole members of the dynamin superfamily in this organism (Field and Carrington, 2004;Morgan et al, 2004). The two slightly different gene products are probably functionally equivalent, because the few amino acids that differ between them are spread over the entire length of the molecule and represent for the most part conservative substitutions.…”