Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

Eisenberg-Bord et al. describe a lipid droplet (LD) subpopulation with a unique proteome, which is adjacent to the nucleus–vacuole junction contact site. They identify the LD machinery, which cooperates with the lipodystrophy factor seipin as a key determinant of LD identity and suggest a mechanism for functional organelle diversification.

The Drosophila heart tube represents a structure that similarly to vertebrates' primary heart tube exhibits a large lumen; the mechanisms promoting heart tube morphology in both Drosophila and vertebrates are poorly understood. We identified Multiplexin (Mp), the Drosophila orthologue of mammalian Collagen-XV/XVIII, and the only structural heart-specific protein described so far in Drosophila, as necessary and sufficient for shaping the heart tube lumen, but not that of the aorta. Mp is expressed specifically at the stage of heart tube closure, in a polarized fashion, uniquely along the cardioblasts luminal membrane, and its absence results in an extremely small heart tube lumen. Importantly, Mp forms a protein complex with Slit, and interacts genetically with both slit and robo in the formation of the heart tube. Overexpression of Mp in cardioblasts promotes a large heart lumen in a Slit-dependent manner. Moreover, Mp alters Slit distribution, and promotes the formation of multiple Slit endocytic vesicles, similarly to the effect of overexpression of Robo in these cells. Our data are consistent with Mp-dependent enhancement of Slit/Robo activity and signaling, presumably by affecting Slit protein stabilization, specifically at the lumen side of the heart tube. This activity results with a Slit-dependent, local reduction of F-actin levels at the heart luminal membrane, necessary for forming the large heart tube lumen. Consequently, lack of Mp results in decreased diastolic capacity, leading to reduced heart contractility, as measured in live fly hearts. In summary, these findings show that the polarized localization of Mp controls the direction, timing, and presumably the extent of Slit/Robo activity and signaling at the luminal membrane of the heart cardioblasts. This regulation is essential for the morphogenetic changes that sculpt the heart tube in Drosophila, and possibly in forming the vertebrates primary heart tube.

Eukaryotic cells evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge on molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore critical for understanding eukaryotic metabolism. Here we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. The dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.

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Cancer cells are highly heterogeneous, both at the transcriptional level and in their epigenetic state. Methods to study epigenetic heterogeneity are limited in their throughput and the information obtained per cell. Here, we adapted Cytometry by Time of Flight (CyTOF) to analyze a wide panel of histone modifications and chromatin regulators in primary tumor-derived lines of Diffused Intrinsic Pontine Glioma (DIPG). DIPG is a lethal pediatric brain cancer, driven by a mutation in histone H3 leading to substitution of lysine 27 with methionine (H3-K27M mutation). We identified two epigenetically distinct subpopulations in DIGP, reflecting inherent heterogeneity in the expression levels of the mutant histone. These two epigenetic subpopulations are robust across tumor lines derived from different patients and show differential proliferation capacity as well as expression of stem-cell and differentiation markers. Moreover, we demonstrate the use of this single-cell high-dimensional data to elucidate potential interactions between histone modifications and epigenetic alterations during the cell-cycle. Our work establishes new concepts for the analysis of epigenetic heterogeneity and cross-talk in cancer that could be applied to diverse biological systems.

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