Probing the subcellular localization of hopanoid lipids in bacteria using NanoSIMS

Abstract

The organization of lipids within biological membranes is poorly understood. Some studies have suggested lipids group into microdomains within cells, but the evidence remains controversial due to non-native imaging techniques. A recently developed NanoSIMS technique indicated that sphingolipids group into microdomains within membranes of human fibroblast cells. We extended this NanoSIMS approach to study the localization of hopanoid lipids in bacterial cells by developing a stable isotope labeling method to directly detect subcellular localization of specific lipids in bacteria with ca. 60 nm resolution. Because of the relatively small size of bacterial cells and the relative abundance of hopanoid lipids in membranes, we employed a primary (2)H-label to maximize our limit of detection. This approach permitted the analysis of multiple stable isotope labels within the same sample, enabling visualization of subcellular lipid microdomains within different cell types using a secondary label to mark the growing end of the cell. Using this technique, we demonstrate subcellular localization of hopanoid lipids within alpha-proteobacterial and cyanobacterial cells. Further, we provide evidence of hopanoid lipid domains in between cells of the filamentous cyanobacterium Nostoc punctiforme. More broadly, our method provides a means to image lipid microdomains in a wide range of cell types and test hypotheses for their functions in membranes.

Figure 1. Chemical structures of hopanoid lipids used in this study.

Structures of the two hopene isomers (A) and bacteriohopanetetrol (B) that were isotopically labeled with ²H, purified, and imaged in this study.

Figure 2. Cell biology of R. palustris.

(A) Cell cycle of the budding mode of cell division displayed by R. palustris in which motile swarmer cells (S) lose their flagellum and develop into tubed mother cells (M) that give rise to new swarmer cells. (B–E) A pulse of ¹³C labeled acetate added 30 min prior to the fixation of the cells allows for the visualization of the R. palustris cell cycle. Quantitative raster ion images of ¹²C⁻ (B) and ¹³C⁻ (C) were used to calculate the δ¹³C image (D) that indicates the presence of ¹³C enrichment at one pole of the cells as indicated by the white arrow. (E) A bacterial flagellum is visible, indicated by the red arrow, within an early raster frame in the ¹²C⁻ ion image confirming that the ¹³C is preferentially associated with the growing bud. Scale bars are 2 µm. Images shown are representative of 3 fields of view and 3 independent biological experiments.

Figure 3. Detection of exogenously added ²H-labeled BHT in R. palustris.

NanoSIMS images showing the count rates of ¹H⁻ (A) and ²H⁻ (B) ions. Note that the ²H abundance is patchy within cells. (C) A non-quantitative overlay showing ²H⁻ on top of ¹²C⁻ counts to mark cells. δ²H values for regions of interest encompassing two different cells marked by arrows. Cells were defined by contiguous regions generating a ¹²C⁻ count rate greater than 300 cts s⁻¹ (shown as bright in the green image) and had a bulk δ²H of 638±10, indicating the broad incorporation of labeled hopanoids. The reported error reflects the standard error of total ion counts within each of the indicated cells. Images shown are representative of 20 fields of view and 3 independent biological experiments. All scale bars are 5 µm.

Figure 4. Depth profiles of R. palustris cells using NanoSIMS.

(A) Conceptual illustration of NanoSIMS analysis of a cell. As the beam is rastered over the surface of the sample in many cycles, more material is removed from the sample surface and thus time (i.e. cycle number) relates to depth. (B) A stack of ion images binned by cycle number shows how cells are degraded over time by the ion beam. Quantitative images of ¹²C⁻ (C), ²H⁻ frames 5 to 10 (D), and ²H⁻ frames 30–35 (E) are shown to illustrate changes that occur in the ²H⁻-labeling patterns as the primary beam burns through the cells. (F) A depth profile of the cell indicated by the white arrow in panel C provides a quantitative example of a cell initially appearing unlabeled but becoming positive for the label as the opposite side of the cell is sampled. All scale bars are 2 µm. Error bars reflect the standard error of the total number of ions. Images shown are representative of 5 fields of view and 3 independent biological experiments.

Figure 5. Analysis of BHT localization in N. punctiforme cells.

NanoSIMS images of ¹H⁻ (A) and ²H⁻ (B) ion count rate and a non-quantitative overlay (C) of the raster images depicting their relative localizations with ¹H⁻ ions shown in green and ²H⁻ ions shown in red. The thin white circles in C denote the regions of interest (numbered 1–8) selected for the pooled analysis of data in panel D. The white arrow indicates that region of interest 4 is within region of interest 2. (D) Plot depicting the δ¹³C and δ²H values for the regions of interest (numbered blue circles) and all image pixels containing greater than 100 cts s^{−1 1}H⁻ ions, which encompasses all biomass in the image (blue square). Additional regions of interest points shown as are data taken from other ion images (unnumbered blue circles). The line is an ordinary least squares regression of interest shown in C. (E) A white dashed line indicates the region enlarged in panel F. (F) The white dashed line indicates a region of interest along a filament for the isotopic profile shown in G. (G) δ²H values along the filament highlight a periodicity with labeled BHT concentrated at the junctions between cells. All scale bars are 5 µm. Error bars in (G) reflect the standard error of the total number of ions. Images shown are representative of 5 fields of view and 3 independent biological experiments.

Figure 6. Analysis of hopene localization in N. punctiforme cells.

(A) Phase contrast and (B) fluorescence images indicating the presence of a jettisoned akinete envelope (i), a heterocyst (ii), and vegetative cells (iii). NanoSIMS images of ¹H⁻ (C) and ²H⁻ (D) ion count rates and a non-quantitative overlay of the two ions depicting their relative localizations (E) with ¹H⁻ ions shown in green and ²H⁻ ions shown in red. Note the substantial concentrations of labeled BHT within the akinete envelope. (F) δ¹³C and δ²H values for the regions of interest denoted by solid white lines in E (numbered blue circles) and all image pixels containing greater than 100 ¹H⁻ ions (blue square). The dashed white lines in E depict the region selected for the isotopic profile shown in (G). A plot of δ²H values along the filament indicates the BHT label is localized near cell junctions. All scale bars are 5 µm. Error bars reflect the standard error of the total number of ions in the selected region. Images are representative of 5 fields of view and 3 independent biological experiments.