Immunofluorescence staining is the key technique for visualizing organization of endogenous cellular structures in single cells. Labeling and imaging of blood stage Plasmodium falciparum has always been challenging since it is a small intracellular parasite. The gold standard for parasite immunofluorescence is fixation in suspension with addition of minute amounts of glutaraldehyde to the paraformaldehyde-based solution. While this maintains red blood cell integrity, it has been postulated that antigenicity of the parasite proteins was, if at all, only slightly reduced. Here we show the deleterious effect that even these small quantities of glutaraldehyde can have on immunofluorescence staining quality and present an alternative cell seeding protocol that allows fixation with only paraformaldehyde. The highly improved signal intensity and staining efficiency enabled us to carry out RescueSTED nanoscopy on microtubules and nuclear pores and describe their organization in greater detail throughout the blood stage cycle.

Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 and MRE11 are required for RAD51-dependent homologous recombination and phosphorylation of histone H2A following a DNA double strand break (DSB), but neither MRE11 nor RAD50 substantially influence DSB resection at a chromosome-internal locus. In addition, we reveal intrinsic separation-of-function between T. brucei RAD50 and MRE11, with only RAD50 suppressing DSB repair using donors with short stretches of homology at a subtelomeric locus, and only MRE11 directing DSB resection at the same locus. Finally, we show that loss of either MRE11 or RAD50 causes a greater diversity of expressed VSG variants following DSB repair. We conclude that MRN promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.

Highlights-Omitting glutaraldehyde from fixative improves immunofluorescence staining -STED nanoscopy is readily applicable to infected red blood cells -Intranuclear microtubules are nucleated from distinct sites AbstractImmunofluorescence staining is the key technique for visualizing organization of endogenous cellular structures in single cells. Labeling and imaging of blood stage Plasmodium falciparum has always been challenging since it is a small intracellular parasite. The gold standard for parasite immunofluorescence is fixation in suspension with addition of minute amounts of glutaraldehyde to the paraformaldehyde-based solution. While this maintains red blood cell integrity, it has been postulated that antigenicity of the parasite proteins was, if at all, only slightly reduced. Here we show the deleterious effect that even these small quantities of glutaraldehyde can have on immunofluorescence staining quality and present an alternative cell seeding protocol that allows fixation with only paraformaldehyde. The highly improved signal intensity and staining efficiency enabled us to carry out RescueSTED nanoscopy on microtubules and nuclear pores and describe their organization in greater detail throughout the blood stage cycle.

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. This process is facilitated by the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.