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. 2022 Oct 14;25(11):105357. doi: 10.1016/j.isci.2022.105357

Figure 4.

Figure 4

GMAP-1 carries phosphoglycerides and its deletion modifies the lipid composition of the epicuticle

(A) Purified GMAP-1-8xHis was incubated in solution with NBD-PC and NBD-PS. GMAP-1 complex with NBD-PG was visualized in a native gel following migration at 100V for 1h. Fluorescence imaging reveals that GMAP-1-8xHis binds and carries NBD-PC and NBD-PS.

(B) NBD-PC binding to purified GMAP-1 was confirmed by Flow Induced Dispersion Analysis (FIDA) which allows size-based characterization of biomolecules and their complexes in solution. The hydrodynamic radius of free NBD-PC was determined to 0.6 nm. The apparent size of NBD-PC increased steadily with GMAP-1 concentrations and was used for generating a binding curve. The complex size was determined to 3.14 nm, in line with the hydrodynamic radius of a 30 kDa protein with a flexible His tag. The dissociation constant (Kd) of the interaction was determined around 3 μM, assuming 1:1 binding stoichiometry.

(C) Purified GMAP-1 binds phosphoglycerides lipids on membrane strip, including phosphatidylserine (PS), phosphatidylinositols PI4P, PI3P, and PI5P.

(D and E). Surface lipids of the epicuticle were visualized by DiI staining of the cuticle for 12 N2 and 15 gmap-1 mutants. (D) A confocal plane at the surface of the cuticle highlights the furrows (blue arrowheads) that are less contrasted in gmap-1 than in N2. The bottom panel shows a zoom up of the area indicated by a square in the top panels. (E) A confocal plane at the level of hyp7 underneath the cuticle highlights the staining of hyp7 (green arrowhead) in gmap-1 but not in N2 and the reduced staining of the furrows in gmap-1. Scale bar = 20 μm.

(F) Analysis of the lipid composition of the epicuticle: PCA plot of the external cuticle layer extracted by lipodisq for N2 (black), gmap-1 mutants (red), and gmap-1 rescued in hypodermis (blue).

(G) Contribution of each lipid family (indicated by their color code on the right) on the total lipid extracted from the surface of the C. elegans cuticle from the N2, gmap-1, and gmap-1 rescued strains.

(H) Survival to hypotonicity was measured for adult worms after 15 min exposure to deionized water (N = 160 across 8 experimental replicates). Survival was measured for N2 controls, gmap-1(ulb13) and pld-1(ok2222) single mutants and gmap-1;pld-1 double mutants. One-way ANOVA followed by Kruskal-Wallis test. Data are represented as Mean +/− SEM.