Coupling of Posterior Cytoskeletal Morphogenesis to the G1/S Transition in the Trypanosoma brucei Cell Cycle

Xiaoming Tu and Ching C. Wang*

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-2280

Monitoring Editor: John Pringle

The expression levels of fourCdc2-related kinases (CRK1, 2, 4, and 6) in the procyclic formof Trypanosoma brucei were knocked down in pairs using the RNAinterference (RNAi) technique. A double knockdown of CRK1 andCRK2 resulted in arrested cell growth in the G1 phase accompaniedby an apparent cessation of nuclear DNA synthesis. The arrestedcells became elongated at the posterior end like the G1-phasecells generated by knockdown of CycE1/CYC2 in a previous study.However, $~$ 5% of the G1 cells in the current study also possessedmultiply branched posterior ends, which have not previouslybeen observed in T. brucei. DAPI and immunofluorescence stainingshowed a single nucleus, kinetoplast, basal body, and flagellumin the anterior portion of each G1 cell. The split and grosslyextended posterior ends were heavily stained with antibodiesto tyrosinated ${alpha}$ -tubulin, suggesting an accumulation of newlysynthesized microtubules. A significant population of anucleatecells (zoids), apparently derived from kinetoplast- dictatedcytokinesis and cell division of the G1 cells, also had extendedand branched posterior ends filled with newly synthesized microtubules.This continued posterior extension of microtubules in the G1cells and zoids suggests that CRK1 and CRK2 exert a coordinatedcontrol on G1/S passage and the limited growth of the microtubulecorset toward the posterior end. This connection may providea new insight into the mechanism of morphological maintenanceof an ancient protist during its cell-cycle progression.

^*Corresponding author. E-mail: ccwang{at}cgl.ucsf.edu