Role of cytoplasmic dynein-2 in Leishmania mexicana
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Leishmania mexicana is a parasitic protozoan that exists in two distinct morphologic forms, the flagellated promastigote in the insect vector, and the non-flagellated amastigote in the vertebrate host. The promastigote flagellum is used for motility and attachment in the insect gut, and may also have a sensory role. Cytoplasmic dynein-2 is a retrograde motor involved in the assembly and maintenance of flagella in Chlamydomonas and the sensory cilia in Caenorhabditis elegans. Eukaryotic organisms with cilia or flagella typically express a single cytoplasmic dynein-2 heavy chain. In contrast, Leishmania and another kinetoplastid, Trypanosoma, have two cytoplasmic dynein-2s'. We have identified and sequenced the cytoplasmic dynein-2 heavy chain genes in Leishmania mexicana designated LmxDHC2.1 and LmxDHC2.2 . Quantitative RT-PCR showed that transcript of both genes is present in wild type promastigotes and amastigotes in equal abundance. To determine which of the two L. mexicana dynein-2 homologues is involved in flagella assembly both genes were independently disrupted by homologous gene replacement. I was unable to disrupt LmxDHC2.1 as all the transformants obtained were aneuploid and still contained a wild type copy of the gene, suggesting that LmxDHC2.1 is an essential gene. In contrast, disruption of LmxDHC2.2 resulted in immotile promastigotes that had a rounded cell body. Ultra-structural analysis revealed non-emergent flagella that lacked the paraflagellar rod and the axoneme was deficient in dynein arms, radial spokes, and the central pair microtubules. Western blot analysis confirmed the absence of paraflagellar rod proteins PFR1 and PFR2, and also confirmed that although they assume amastigote morphology, these cells express proteins consistent with the promastigote stage. These results show that LmxDHC2.2 is required for flagellar assembly and also participates in the maintenance of promastigote cell shape. Together, these findings demonstrate that the two dynein-2 heavy chain isoforms of Leishmania perform distinct functions.