Mapping bilayer thickness in the ER membrane.

In the plasma membrane and in synthetic membranes, resident lipids may laterally unmix to form domains of distinct biophysical properties. Whether lipids also drive the lateral organization of intracellular membranes is largely unknown. Here, we describe genetically encoded fluorescent reporters visualizing local variations in bilayer thickness. Using them, we demonstrate that long-chained ceramides promote the formation of discrete domains of increased bilayer thickness in the yeast ER, particularly in the future plane of cleavage and at ER-trans-Golgi contact sites. Thickening of the ER membrane in the cleavage plane contributed to the formation of lateral diffusion barriers, which restricted the passage of short, but not long, protein transmembrane domains between the mother and bud ER compartments. Together, our data establish that the ER membrane is laterally organized and that ceramides drive this process, and provide insights into the physical nature and biophysical mechanisms of the lateral diffusion barriers that compartmentalize the ER.

Yeast ceramide synthases, Lag1 and Lac1, have distinct substrate specificity.

LAG1 was the first longevity assurance gene discovered in Saccharomyces cerevisiae The Lag1 protein is a ceramide synthase and its homolog, Lac1, has a similar enzymatic function but no role in aging. Lag1 and Lac1 lie in an enzymatic branch point of the sphingolipid pathway that is interconnected by the activity of the C4 hydroxylase, Sur2. By uncoupling the enzymatic branch point and using lipidomic mass spectrometry, metabolic labeling and in vitro assays we show that Lag1 preferentially synthesizes phyto-sphingolipids. Using photo-bleaching experiments we show that Lag1 is uniquely required for the establishment of a lateral diffusion barrier in the nuclear envelope, which depends on phytoceramide. Given the role of this diffusion barrier in the retention of aging factors in the mother cell, we suggest that the different specificities of the two ceramide synthases, and the specific effect of Lag1 on asymmetrical inheritance, may explain why Δlag1 cells have an increased lifespan while Δlac1 cells do not.

Fluorescence Recovery After Photo-Bleaching (FRAP) and Fluorescence Loss in Photo-Bleaching (FLIP) Experiments to Study Protein Dynamics During Budding Yeast Cell Division.

The easiness of tagging any protein of interest with a fluorescent marker together with the advance of fluorescence microscopy techniques enable researchers to study in great detail the dynamic behavior of proteins both in time and space in living cells. Two commonly used techniques are FRAP (Fluorescent Recovery After Photo-bleaching) and FLIP (Fluorescence Loss In Photo-bleaching). Upon single bleaching (FRAP) or constant bleaching (FLIP) of the fluorescent signal in a specific area of the cell, the intensity of the fluorophore is monitored over time in the bleached area and in surrounding regions; information is then derived about the diffusion speed of the tagged molecule, the amount of mobile versus immobile molecules as well as the kinetics with which they exchange between different parts of the cell. Thereby, FRAP and FLIP are very informative about the kinetics with which the different organelles of the cell separate into mother- and daughter-specific compartments during cell division. Here, we describe protocols for both FRAP and FLIP and explain how they can be used to study protein dynamics during cell division in the budding yeast Saccharomyces cerevisiae. These techniques are easily adaptable to other model organisms.