Discussion

Variant surface glycoprotein (VSG) is the most highly expressed protein in bloodstream form trypanosomes (10% total). Blocking VSG221 synthesis using RNAi or morpholino oligos triggers a precise pre-cytokinesis arrest which is rescued by ectopic VSG117 expressed from the VSG221 expression site.  However, we find that VSG117 expressed from the rDNA array does not rescue this arrest.  High levels of VSG117 are expressed, but the precise cell-cycle checkpoint is not triggered.  Here, VSG221 RNAi results in multi-nucleated cells that do not divide but re-enter S-phase and re-initiate nuclear division.  This less severe reduction in VSG levels results in cells that presumably do not have adequate VSG to form a cleavage furrow, but still reinitiate S-phase.  This indicates that a precise cell-cycle checkpoint is only triggered when VSG synthesis drops below a critical level.  We find that levels of the ER chaperone BiP increase when VSG synthesis is blocked and RNAseq analysis of these stalled cells has revealed additional up-regulated ER-associated genes following VSG RNAi.  Epitope tagging has confirmed ER localisation of these genes and we are currently characterising them to dissect the signalling pathway between the ER stress response induced by a VSG synthesis block and progression through the trypanosome cell-cycle.