Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation

Abstract

Mass spectrometry imaging has been used here to suggest that changes in membrane structure drive lipid domain formation in mating single-cell organisms. Chemical studies of lipid bilayers in both living and model systems have revealed that chemical composition is coupled to localized membrane structure. However, it is not clear if the lipids that compose the membrane actively modify membrane structure or if structural changes cause heterogeneity in the surface chemistry of the lipid bilayer. We report that time-of-flight secondary ion mass spectrometry images of mating Tetrahymena thermophila acquired at various stages during mating demonstrate that lipid domain formation, identified as a decrease in the lamellar lipid phosphatidylcholine, follows rather than precedes structural changes in the membrane. Domains are formed in response to structural changes that occur during cell-to-cell conjugation. This observation has wide implications in all membrane processes.

Fig. 1.

Schematic of two domain formation scenarios for mated T. thermophila. Heterogeneously distributed lipids in the plasma membranes of T. thermophila might form as a result of (Red Arrow) or in anticipation of pore formation (Blue Arrows). Black lipids are cylindrical, blue are H II, and green are lysolipids.

Fig. 2.

SIMS analysis of a strongly paired mated T. thermophila. (A) Differential interference contrast microscopy image of a mating cell pair. (Scale bar: 25 μm.) (B–D) SIMS images of a triturated pair of mating T. thermophila. C₅H₉ is mapped in (B), PC is mapped in (C), and 2-AEP is mapped in (D) (the intensity in the 2-AEP image has been multiplied by 3). (E) Region of interest analysis of the same cells. The red mass spectrum is from the cell bodies, and the green mass spectrum is from the junction. The Inset highlights the regions on the SIMS image, red for the cell bodies and green for the junction.

Fig. 3.

Pair formation and strong pair formation. The percent of cells paired plotted over time under quiescent conditions and the percent of cells paired following trituration. Pairs resisting trituration are strongly paired.

Fig. 4.

SIMS images show the temporal evolution of the observed lipid domain formed during cell conjugation. First row: Total ion images of cells imaged at 1 (A), 2 (B and C), and 3 h (D). Second row: Phosphatidylcholine ion images (m/z 184) of cells imaged at 1 (E), 2 (F and G), and 3 h (H). Third row: C₅H₉ ion images (m/z 69) of cells imaged at 1 (I), 2 (J and K), and 3 h (L). Fourth row: Line scans of the ion intensities of phosphatidylcholine and C₅H₉ across the cell–cell junctions for cells imaged at 1 (M), 2 (N and O), and 3 h (P). Fifth row: Normalized phosphatidylcholine ion images (m/z 184 divided by total ion) of cells imaged at 1 (Q), 2 (R and S), and 3 h (T). The color-coded bars indicate the counts per pixel observed in A–L, and in Q–T these show the counts for phosphatidylcholine divided by total counts in each pixel.

Fig. 5.

The percent of paired cells displaying lipid domains over time vs. the percent of paired cells that resisted separation following trituration. Three time points were investigated: 60 (n = 6), 120 (n = 9), and 180 min (n = 4). Domains were defined as a decrease in phosphocholine at the junction between cells (4). Line scans of PC intensity were used to measure this decrease. The appearance of a trough was used to determine that a pair had formed a lipid domain.