Protocol for monitoring fatty acid trafficking from lipid droplets to mitochondria in cultured cells.

Intracellular trafficking of fatty acids (FAs) between organelles is critical for cells to adjust their metabolism in response to stimuli such as exercise, fasting, and cold exposure. Here, we describe a protocol to monitor trafficking of FAs from lipid droplets to mitochondria. We describe the labeling of organelles in cultured C2C12 myoblasts with transfection and dyes.

Contact-FP: A Dimerization-Dependent Fluorescent Protein Toolkit for Visualizing Membrane Contact Site Dynamics.

Membrane contact sites (MCSs) are sites of close apposition between two organelles used to exchange ions, lipids, and information. Cells respond to changing environmental or developmental conditions by modulating the number, extent, or duration of MCSs. Because of their small size and dynamic nature, tools to study the dynamics of MCSs in live cells have been limited.

PLIN5 interacts with FATP4 at membrane contact sites to promote lipid droplet-to-mitochondria fatty acid transport.

Cells adjust their metabolism by remodeling membrane contact sites that channel metabolites to different fates. Lipid droplet (LD)-mitochondria contacts change in response to fasting, cold exposure, and exercise. However, their function and mechanism of formation have remained controversial.

Quantitative proteomic profiling identifies global protein network dynamics in murine embryonic heart development.

Defining the mechanisms that govern heart development is essential for identifying the etiology of congenital heart disease. Here, quantitative proteomics was used to measure temporal changes in the proteome at critical stages of murine embryonic heart development. Global temporal profiles of the over 7,300 proteins uncovered signature cardiac protein interaction networks that linked protein dynamics with molecular pathways.

The interdependent transport of yeast vacuole Ca and H and the role of phosphatidylinositol 3,5-bisphosphate.

Yeast vacuoles are acidified by the v-type H-ATPase (V-ATPase) that is comprised of the membrane embedded V complex and the soluble cytoplasmic V complex. The assembly of the V-V holoenzyme on the vacuole is stabilized in part through interactions between the V a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P). PI(3,5)P also affects vacuolar Ca release through the channel Yvc1 and uptake through the Ca pump Pmc1.

Phosphatidylinositol 3,5-bisphosphate regulates Ca transport during yeast vacuolar fusion through the Ca ATPase Pmc1.

The transport of Ca across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol-3-phosphate (PI3P) by the PI3P-5-kinase Fab1 to produce transient PI(3,5)P pools.

Copper blocks V-ATPase activity and SNARE complex formation to inhibit yeast vacuole fusion.

The accumulation of copper in organisms can lead to altered functions of various pathways and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg , Cd and Cu are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations.

Phosphatidylinositol 3,5-bisphosphate regulates the transition between trans-SNARE complex formation and vacuole membrane fusion.

Phosphoinositides (PIs) regulate a myriad of cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) on vacuole fusion remains unknown. PI(3,5)P is made by the PI3P 5-kinase Fab1 and has been characterized as a regulator of vacuole fission during hyperosmotic shock, where it interacts with the TRP Ca channel Yvc1.

Real-Time Fluorescence Detection of Calcium Efflux During Vacuolar Membrane Fusion.

During in vitro homotypic yeast vacuole fusion Ca is transported into and out of the organelle lumen. In vitro, Ca is taken up from the medium by vacuoles upon the addition of ATP. During the docking stage of vacuole fusion Ca is effluxed from the lumen upon the formation of trans-SNARE complexes between vesicles.

Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity.

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1.

Phosphatidylinositol (3,4,5)-trisphosphate binds to sortilin and competes with neurotensin: Implications for very low density lipoprotein binding.

Sortilin is a multi-ligand sorting receptor that interacts with B100-containing VLDL and LDL as well as other ligands including neurotensin (NT). The current study investigates the hypothesis that phosphatidylinositol (3,4,5)-trisphosphate (PIP3) generated downstream of insulin action can directly bind to sortilin. NT binds to sortilin at a well characterized site via its carboxy terminus (C-term).

The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.

The yeast vacuole requires four SNAREs to trigger membrane fusion including the soluble Qc-SNARE Vam7. The N-terminal PX domain of Vam7 binds to the lipid phosphatidylinositol 3-phosphate (PI3P) and the tethering complex HOPS (homotypic fusion and vacuole protein sorting complex), whereas the C-terminal SNARE motif forms SNARE complexes. Vam7 also contains an uncharacterized middle domain that is predicted to be a coiled-coil domain with multiple helices.

14-3-3 (Bmh) proteins regulate combinatorial transcription following RNA polymerase II recruitment by binding at Adr1-dependent promoters in Saccharomyces cerevisiae.

Adr1 and Cat8 are nutrient-regulated transcription factors in Saccharomyces cerevisiae that coactivate genes necessary for growth in the absence of a fermentable carbon source. Transcriptional activation by Adr1 is dependent on the AMP-activated protein kinase Snf1 and is inhibited by binding of Bmh, yeast 14-3-3 proteins, to the phosphorylated Adr1 regulatory domain. We show here that Bmh inhibits transcription by binding to Adr1 at promoters that contain a preinitiation complex, demonstrating that Bmh-mediated inhibition is not due to nuclear exclusion, inhibition of DNA binding, or RNA polymerase II (Pol II) recruitment.