Cilia and flagella are complex, microtubule (MT)-filled cell organelles of which the structure is evolutionarily conserved from protistan cells to mammalian sperm and the size is regulated. The best-established model for flagellar length (FL) control is set by the balance of continuous MT assembly and disassembly occurring at the flagellar tip. Because steady-state assembly of tubulin onto the distal end of the flagellum requires intraflagellar transport (IFT)--a bidirectional movement of large protein complexes that occurs within the flagellum--FL control must rely upon the regulation of IFT. This does not preclude that other pathways might "directly" affect MT assembly and disassembly. Now, among the superfamily of kinesins, family-13 (MCAK/KIF2) members exhibit a MT-depolymerizing activity responsible for their essential functions in mitosis. Here we present a novel family-13 kinesin from the flagellated protozoan parasite Leishmania major, that localizes essentially to the flagellum, and whose overexpression produces flagellar shortening and knockdown yields long flagella. Using negative mutants, we demonstrate that this phenotype is linked with the MT-binding and -depolymerizing activity of this kinesin. This is the first report of an effector protein involved in FL control through a direct action in MT dynamics, thus this finding complements the assembly-disassembly model.

All the physical linkage groups constituting the genome of Leishmania infantum have been identified for the first time by hybridization of specific DNA probes to pulsed field gradient-separated chromosomes. The numerous co-migrating chromosomes were individualised using the distinctive size polymorphisms which occur among strains of the L. infantum/L. donovani complex as a tool. A total of 244 probes, consisting of 41 known genes, 66 expressed sequence tags (ESTs) and 137 anonymous DNA sequences, were assigned to a specific linkage group. We show that this genome comprises 36 chromosomes ranging in size from 0.35 to -3 Mb. This information enabled us to compare the genome structure of L. infantum with those of the three other main Leishmania species that infect man in the Old World, L. major, L. tropica and L. aethiopica. The linkage groups were consistently conserved in all species examined. This result is in striking contrast to the large genetic distances that separate these species and suggests that conservation of the chromosome structure may be critical for this human pathogen. Finally, the high density of markers obtained during the present study (with a mean of 1 marker/130 kb) will speed up the construction of a detailed physical map that would facilitate the genetic analysis of this parasite, for which no classical genetics is available.

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SummaryMicrotubules are key players in the biology of Trypanosomatid parasites, not only as classical components of the mitotic spindle, microtubule-organizing centres and flagellum but also as the essential constituent of the cytoskeleton. Their length dynamics are regulated by, among others, microtubule-severing proteins. Four and six genes encoding microtubulesevering proteins can be found bioinformatically in the Leishmania major and Trypanosoma brucei genome respectively. We investigated all these proteins in these organisms, which include the katanin, katanin-like, spastin and fidgetin, and looked at their subcellular localization as well as their putative function by examining 'loss-of-function' phenotypes. The katanin-like KAT60b was found implicated in flagellar length reduction, but not in its size increase, while the katanin p80 subunit appeared clearly involved in cytokinesis. Fidgetin and spastin homologues were both localized in the nucleus: the first as a discrete and variable number of dots during most of the cell cycle, redistributing to the spindle and midbody during mitosis; the second concentrated as Յ 5 perinucleolar punctuations, similar to the electron-dense plaques identified in T. brucei, which were assimilated to kinetochores. This first study of microtubulesevering proteins in 'divergent' eukaryotes gives further insight into the multiple functions of these proteins identified in the hitherto studied models.