Abstract
Trypanosome brucei, the causative agent of African sleeping sickness, harbours a highly ordered, subpellicular microtubule cytoskeleton that defines many aspects of morphology, motility and virulence. This array of microtubules is associated with a large number of proteins involved in its regulation. Employing proximity-dependent biotinylation assay (BioID) using the well characterised cytoskeleton-associated protein CAP5.5 as a probe, we identified CAP50 (Tb927.11.2610). This protein colocalises with the subpellicular cytoskeleton microtubules but not with the flagellum. Depletion by RNAi results in defects in cytokinesis, morphology and partial disorganisation of microtubule arrays. Published proteomics data indicate a possible association of CAP50 with two other, yet uncharacterised, cytoskeletal proteins, CAP52 (Tb927.6.5070) and CAP42 (Tb927.4.1300), which were therefore included in our analysis. We show that their depletion causes phenotypes similar to those described for CAP50 and that they are essential for cellular integrity.
Highlights
The cytoskeletal network of eukaryotic organisms is involved in almost all aspects of cellular processes [1]
Procyclic PC449 cells were stably transfected with the pHD1800-CAP5.5-myc-BirA*-myc plasmid and the expression of the transgene after doxycycline induction was confirmed by Western blotting using an anti-myc antibody (Figure 1A)
An additional weak band at 70 kDa was detected in the soluble fraction, but not characterised further, assuming that binding partners of the abundant protein CAP5.5 are roughly present in equimolar amounts (Figure 1A)
Summary
The cytoskeletal network of eukaryotic organisms is involved in almost all aspects of cellular processes [1]. In protists, lacking intermediate filaments, microtubules are often essential for cell shape and morphological integrity This functional expansion, is unlikely due to, e.g., major differences in the way unit microtubules are built or assembled, as the sequences between tubulins of evolutionary highly diverse species and their in vitro properties are surprisingly conserved [2]. Key to these divergent functional trajectories is the emergence of specific sets of regulatory proteins [3,4,5,6]. Many CAPs have been identified in trypanosomes, their precise functions in the regulation and dynamics of the trypanosome cytoskeleton are largely unknown [4]
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