Variant Surface Glycoprotein synthesis
and the trypanosome cell cycle

Molecular mechanisms mediating immune evasion
in African trypanosomes

Our main aims are to dissect the molecular mechanisms mediating trypanosome immune evasion, both from host antibodies and from complement.  First of all, we are investigating the role of the protective VSG coat in bloodstream form trypanosomes.

VSG is monitored in the cell cycle

The VSG coat is composed of a dense layer of VSG molecules that cover the entire bloodstream form trypanosome shielding invariant surface molecules like receptors.  This VSG coat can be considered analogous to a fur coat, with the individual VSG molecules attached to the cell surface analogous to hairs.  We attempted to make "naked" bloodstream form trypanosomes, by blocking VSG synthesis using tetracycline inducible VSG RNA interference (RNAi).  Surprisingly, there was no significant depletion of VSG within the coat.  Instead, blocking VSG synthesis triggers a rapid and specific cell-cycle arrest.


Inhibition of VSG synthesis triggers a cell cycle arrest

VSG graph

Above: Microscopic analysis of T. brucei cell cycle progression during the course of tetracycline inducible VSG RNAi (indicated in hours).  Cells have one nucleus (N) and kinetoplast (K) (1K1N).  As the cells undergo S phase, first the kinetoplast divides (2K1N) and then the nucleus undergoes mitosis (2K2N). Blocking VSG synthesis triggers a precytokinesis arrest.
From Sheader et al. (2005) PNAS  102: 8716-8721.


Cells stalled after VSG RNAi arrest at a very precise cell cycle stage

Using scanning electron microscopy in collaboration with the Gull laboratory, normal cells immediately before cell division were compared with those arrested before cell division after the induction of VSG RNAi.  Cells that have arrested after the induction of VSG RNAi are shorter and broader than normal precytokinesis cells, although they have the same volume.  This indicates that the cells have changed their shape in the face of a restriction on the protective VSG coat, possibly as a means of minimising their surface area to volume ratio.  Secondly, the cells appear to have arrested at a very precise stage of the cell cycle with parallel flagellar pockets (invagination where the flagellum exits the cell).

VSGRNAiVSGRNAi2

0h VSG RNAi (left) 24h VSG RNAi (right)
Above: Scanning electron microscopy of a normal bloodstream form trypanosome immediately before cell division. On the right is a trypanosome that has stalled immediately before cell division after the induction of tetracycline inducible VSG RNAi. Scanning electron microscopy performed by Sue Vaughan and Keith Gull (Dunn School of Pathology, Oxford). From Sheader et al. (2005) PNAS  102: 8716-8721.

All of these results are compatible with VSG being monitored during the cell cycle of African trypanosomes.  We propose that we have discovered a novel checkpoint that is triggered in the absence of VSG synthesis.  This results in a rapid and specific cell cycle block preventing what would otherwise be disastrous dilution of the protective VSG coat.  We are presently investigating the mechanism behind this cell cycle arrest.

 

A global translation arrest is triggered in cells
where VSG synthesis has been blocked

We have found that after the induction of VSG RNAi, protein synthesis stops. Metabolic labeling of the arrested cells showed that total protein synthesis was reduced to less than 1-4% normal levels after 24 hours induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. In addition, polysome analysis showed a rapid decrease in polysomes (transcripts associated with translating ribosomes) in the arrested cells.

vsg

Above: Blocking VSG synthesis gives rise to a global translation arrest in T. brucei. A) VSG synthesis was blocked for the time indicated above in hours prior to labeling with 35-S methionine for one hour. Proteins were separated on an SDS-PAGE gel. The left panel shows [35-S]-methionine labeled proteins detected by fluorography. On the right is the corresponding Coomassie stained gel. B) Triplicate samples of [35-S]-methionine labeled cells were analysed to determine the mean rate of uptake and incorporation of methionine into total protein after the induction of VSG RNAi for the time indicated in hours (h). From Smith et al. (2009) PLoS ONE  4 (10): e7532.

We are currently interested in determining how VSG synthesis and/ or trafficking is monitored during the trypanosome cell cycle. What is “sensed” during the trypanosome cell cycle to trigger this very precise cell cycle arrest? In addition, what is the significance of the observed global arrest in protein synthesis observed when VSG synthesis is blocked?