Protein Kinase Involved in Flagellar-Length Control

Abstract

During its life cycle, the parasitic protozoon Leishmania mexicana differentiates from a flagellated form, the promastigote, to an amastigote form carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue designated LmxMKK was identified in a homology screening and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and is likely to be regulated by posttranslational mechanisms such as phosphorylation. A deletion mutant for the single-copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. We propose that LmxMKK influences anterograde intraflagellar transport to maintain flagellar length in Leishmania promastigotes; as such, it is the first protein kinase known to be involved in organellar assembly.Protein kinases are key regulatory molecules in the proliferation, differentiation, motility, and stress response of all eukaryotic cells. Together with their antagonists, the protein phosphatases, they are organized in complex networks to guarantee proper regulation of cellular processes according to environmental changes and intercellular communication. There is a wealth of information present on protein kinases in higher eukaryotes and Saccharomyces cerevisiae (18,45). Information on signal transduction processes for other unicellular organisms such as the sporozoa (Plasmodium, Toxoplasma, and Theileria) (22) and the kinetoplastida (Trypanosoma and Leishmania) (40, 50) as causative agents of infectious disease or the green alga Chlamydomonas (48) and the slime mold Dictyostelium discoideum (2) as model organisms for flagellar assembly and differentiation, respectively, is relatively scarce. It is likely that in Leishmania, as in other organisms, signal transduction pathways culminate in altered gene expression. As transcription factors and RNA polymerase II promoters are absent in these parasites, it is generally thought that regulation occurs posttranscriptionally on the level of mRNA maturation and turnover, efficiency of translation, and protein stability (10, 40). However, the regulatory molecules involved in these processes have not been identified.Leishmania parasites undergo a digenetic life cycle, differentiating from the promastigote form in the insect vector, the phlebotomine sand fly, to the amastigote form in the lysosomal compartment of the macrophages of mammals. Promastigotes are spindle-shaped cells, 11 to 20 m in length and 2 m in diameter, carrying a single flagellum of at least the length of the cell body at their anterior pole, which pulls the cell forward but also mediates...

Basal body and flagellum mutants reveal a rotational constraint of the central pair microtubules in the axonemes of trypanosomes

“…We reason that these reagents used to establish CP orientation in the latter two organisms were appropriate fixatives for our analysis in trypanosomes. Our conclusion of a fixed CP orientation is also compatible with data presented in previous studies on trypanosomes that involved other fixation techniques, such as high-pressure freezing and freeze substitution (Wiese et al, 2003), as well as detergent extraction followed by chemical fixation (Maga et al, 1999). (2) If the CP were falling back to a low-energy configuration within the axoneme, it might be expected that the precise orientation would be influenced by the degree of local strain as a result of bending.…”

SMP-1, a Member of a New Family of Small Myristoylated Proteins in Kinetoplastid Parasites, Is Targeted to the Flagellum Membrane inLeishmania

“…Axonemal truncation was associated with the loss of the paraflagellar rod and the degradation of PFR proteins. A similar loss of PFR proteins was observed in another L. mexicana mutant that exhibited a severely shortened flagellum (Wiese et al, 2003). Together these results suggest that perturbation of the lipid composition of the plasma membrane has a profound effect on axoneme extension.…”

Overexpression of S4D Mutant of Leishmania donovani ADF/Cofilin Impairs Flagellum Assembly by Affecting Actin Dynamics

“…In these cells the axoneme structure near the flagellar tip region also is missing, suggesting a role of the actin dynamics not only in the assembly of the PFR and the axoneme attachment fibers but also in the axoneme assembly. Defects in the axoneme assembly have also been observed in L. mexicana mitogen-activated protein (MAP) kinase (LmxMKK) gene deletion mutants (56). Further, LmxDH2.2 null cells have been reported to have nonemerging flagella with defective axoneme organization and no PFR (2).…”