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This file contains supplemental figures to the article entitled "A Genetic Titration of Membrane Composition in C. elegans Reveals its Importance for Multiple Cellular and Physiological Traits".

Biological membranes are primarily an assembly of lipids and proteins. Collectively, these constituents within a phospholipid bilayer determine the physical membrane properties such as fluidity, viscosity, thickness, packing and permeability. Maintenance of membrane properties within acceptable margins, i.e. membrane homeostasis, is fundamentally important for cellular processes. For example, it is a well-established phenomenon that poikilothermic organisms, that cannot control their body temperature, constantly adapt their membrane lipid composition in order to maintain optimal membrane fluidity for membrane functions in spite of variation in ambient temperatures. Regulatory mechanisms must also exist in mammals to maintain membrane lipid heterogeneity across the secretory pathway and to compensate for dietary lipid variation. However, the molecular mechanisms of such an adaptive response in mammals remain poorly understood. Here, using systematic genetics, lipidomics and membrane property assays, we have established that the PAQR-2/IGLR-2 complex in C. elegans and AdipoR2 (a PAQR-2 homolog) in mammalian cells specifically respond to the toxic membrane-rigidifying effects of dietary saturated fatty acids (SFAs) and promote fatty acid desaturation to restore membrane composition and fluidity. In an attempt to understand other mechanisms essential to prevent SFA-mediated cellular toxicity, we also performed an unbiased forward genetic screen in C. elegans. Strikingly, this screen for SFA-tolerance genes led only to the isolation of novel paqr-2 and iglr-2 alleles; this strongly indicates that paqr-2 and iglr-2 are important genes specifically essential to respond to toxic effects of dietary saturated fats. In particular, we noted that in worms and cells that lack PAQR-2/AdipoR2 function, exogenous SFAs becomes rapidly incorporated into membrane phospholipids, leading to membrane rigidification. This was accompanied by an abnormal transcriptional response, impaired mitochondrial respiration and increased ER-UPR as measured in HEK293 cells. Interestingly, we noticed that the toxic effects of exogenous SFAs can be completely mitigated by supplying the cultured cells with small amounts of membrane fluidizing unsaturated fatty acids (UFAs). Consistently, we also found that facilitating the accumulation of UFAs either with mutations in fld-1 in worms or silencing the fld-1 mammalian homologs TLCD1/2, which normally function to limit the incorporation of polyunsaturated fatty acids in membrane phospholipids, is protective and able to attenuate SFA-mediated cellular toxicity. Altogether, these results suggest that maintenance of an optimal SFA/UFA ratio is crucial for normal cellular function and that the PAQR-2/AdipoR2 proteins essentially act as “guardians of membrane homeostasis”.

Additional file 2. Clinical isolates used in this study and their sources.

The Caenorhabditis elegans ventral nerve cord (VNC) consists of two asymmetric bundles of neurons and axons that are separated by the midline. How the axons are guided to stay on the correct sides of the midline remains poorly understood. Here we provide evidence that the conserved Wnt signaling pathway along with the Netrin and Robo pathways constitute a combinatorial code for midline guidance of PVP and PVQ axons that extend into the VNC. Combined loss of the Wnts CWN-1, CWN-2, and EGL-20 or loss of the Wnt receptor CAM-1 caused >70% of PVP and PVQ axons to inappropriately cross over from the left side to the right side. Loss of the Frizzled receptor LIN-17 or the planar cell polarity (PCP) protein VANG-1 also caused cross over defects that did not enhance those in the cam-1 mutant, indicating that the proteins function together in midline guidance. Strong cam-1 expression can be detected in the PVQs and the guidepost cell PVT that is located on the midline. However, only when cam-1 is expressed in PVT are the crossover defects of PVP and PVQ rescued, showing that CAM-1 functions nonautonomously in PVT to prevent axons from crossing the midline.

In spite of the worldwide impact of diabetes on human health, the mechanisms behind glucose toxicity remain elusive. Here we show that C. elegans mutants lacking paqr-2, the worm homolog of the adiponectin receptors AdipoR1/2, or its newly identified functional partner iglr-2, are glucose intolerant and die in the presence of as little as 20 mM glucose. Using FRAP (Fluorescence Recovery After Photobleaching) on living worms, we found that cultivation in the presence of glucose causes a decrease in membrane fluidity in paqr-2 and iglr-2 mutants and that genetic suppressors of this sensitivity act to restore membrane fluidity by promoting fatty acid desaturation. The essential roles of paqr-2 and iglr-2 in the presence of glucose are completely independent from daf-2 and daf-16, the C. elegans homologs of the insulin receptor and its downstream target FoxO, respectively. Using bimolecular fluorescence complementation, we also show that PAQR-2 and IGLR-2 interact on plasma membranes and thus may act together as a fluidity sensor that controls membrane lipid composition., Author Summary Using the nematode C. elegans as a model, we show that glucose decreases the fluidity of cellular membranes, which suggests a novel mechanism for the glucose toxicity observed in diabetics. We also show that the membrane proteins PAQR-2 and IGLR-2 are located together on cellular membranes and regulate fluidity in the presence of glucose. This is an important finding because PAQR-2 is a worm homolog of AdipoR1 and AdipoR2, themselves important anti-diabetic proteins of which the cellular mechanism of action has been difficult to elucidate. Thus in one stroke our work may explain glucose toxicity, describes a cellular mechanism that counteracts it, helps clarify the anti-diabetic roles of AdipoR1/2, and identifies IGLR-2 as a PAQR-2 co-receptor.

Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism., Highlights: FALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effectsFALCON is applicable to many and diverse cell typesA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidityNew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for diseaseC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling.