Figure 4.

Microneme Reduction and Mislocalization in Δshlp1 Ookinetes

(A) (i) Transmission electron micrograph (TEM) of a longitudinal section through a WT ookinete showing the conical apical end. The cytoplasm contains a number of apically located micronemes (M) and a more posteriorly located nucleus (N). Scale bar, 1 μm. (ii) Longitudinal TEM section through a Δshlp1 ookinete showing substantial reduction of micronemes and mislocalization of remaining micronemes at the posterior end. Bar = 1 μm. (iii) Enlargement of the anterior of a WT ookinete showing the complex nature of the apical end consisting of a conical shaped electron dense collar (C) with a central aperture and an underlying less electron dense ring (R). Micronemes (M) are located within the cytoplasm. Scale bar, 100 nm. (iv and v). Detail of the anterior of Δshlp1 ookinetes showing reduction in micronemes (M). Scale bar, 100 nm.

(B) Indirect immunofluorescence of a number of motor complex and micronemal proteins in WT and Δshlp1 ookinetes. Compared to WT, staining for micronemal proteins SOAP and CTRP was reduced in the Δshlp1 mutant and spread throughout the cell body as opposed to the normal concentration at the apical end (arrow). No difference was observed between WT and mutant ookinetes for glideosomal GAP45 and MTIP.

(C) Western blot analyses of ookinete micronemal proteins CTRP and SOAP. Expression levels were compared between WT and Δshlp1 ookinetes and normalized to the GFP control using ImageJ software. CTRP expression was unaltered in the mutant whereas SOAP expression was reduced to 40%.

(D) Relative RNA expression of microneme specific genes cht1, ctrp, and soap in Δshlp1 mutant parasites compared to WT controls. Each point is the mean of three biological replicates ±SD (normalized using qBasePlus). AG, activated gametocytes; OO, ookinete. ^∗p < 0.05; ^∗∗p < 0.01.

See also Figure S3.