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 2H-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.

Hopanoid lipids: from membranes to plant–bacteria interactions

“…In the marine cyanobacterium Crocosphaera watsonii , hopanoids are present in insoluble, detergent-resistant membrane fractions, as is common for raft-forming lipids in eukaryotes 80 . Microanalysis of isotope-labelled hopanoids in the cyanobacterium Nostoc punctiforme and in R. palustris TIE-1 also showed that hopanoids preferentially localize to regions of high membrane curvature, where high degrees of membrane tension may require thicker, more condensed membranes 81 . Although these collective data are promising, further biophysical analyses of live membranes will be needed to determine whether hopanoids indeed form lipid rafts in bacterial cells.…”

Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains

“…Since sterols are absent from most bacterial membranes, domain organization should depend on sterol surrogates and, hence, the involvement of polyisoprenoid lipids has been proposed (reviewed in [161]). A recent study using nanoSIMS has suggested hopanoid (pentacyclic triterpenoids structurally similar to steroids)-enriched domains in cyanobacterium Nostoc punctiforme [163]. …”

Global Fecal and Plasma Metabolic Dynamics Related to Helicobacter pylori Eradication

“…Hopanoids are bacterial pentacyclic triterpenoids that are analogous to eukaryotic steroids structurally and biosynthetically (Ourisson et al, 1987; Doughty et al, 2011; Welander et al, 2012), but their cellular roles are poorly understood (Doughty et al, 2014). Hopanoids may play a role in the alteration of cell membrane permeability in adaptation to extreme environmental conditions in many bacteria.…”