Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells

Abstract

Mitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein. Soft X-ray tomography reveals the ultrastructure of mitochondria and discloses close contact to endosome-like organelles. Using fluorescence microscopy, we localize endo-lysosomes containing NPC2 relative to mitochondria based on the Euclidian distance transform and use statistical inference to show that about 30% of such LE/LYSs are in contact to mitochondria in human fibroblasts. Using Markov Chain Monte Carlo image simulations, we show that interaction between both organelle types, a defining feature of membrane contact sites (MCSs) can give rise to the observed spatial organelle distribution. We devise a protocol to determine the surface fraction of endo-lysosomes in contact with mitochondria and show that this fraction does not depend on functional NPC1 or NPC2 proteins. Finally, we localize MCSs between LE/LYSs containing NPC2 and mitochondria in time-lapse image sequences and show that they either form transiently or remain stable for tens of seconds. Lasting MCSs between endo-lysosomes containing NPC2 and mitochondria move by slow anomalous sub-diffusion, providing location and time for sterol transport between both organelles. Our quantitative imaging strategy will be of high value for characterizing the dynamics and function of MCSs between various organelles in living cells.

Influence of Nuclear Localization Sequences on the Intracellular Fate of Gold Nanoparticles

“…While SXT provides an excellent tool for screening of large cellular volumes for the gold nanostructures and for an observation of the cellular ultrastructure, − the single particles with a size of about 14 nm, or their dimers, remain unobserved within the resolution provided by the SXT setup used for these studies. , Further detailing the observations enabled by SXT, TEM micrographs clearly show that the incubation conditions have a strong influence on the localization of the particles. When the incubation time is only 1 h and is followed by a 24 h chase time (Figure A–C), small aggregates of individual Au_AV nanoparticles are found in endosomes (Figure A,B, green circles) but also in the cytosol during endosomal escape (Figure A, blue circles) and in the nucleus (Figure A,C, red circles).…”

RNA Sequencing Analysis Reveals Divergent Adaptive Response to Hypo- and Hyper-Salinity in Greater Amberjack (Seriola dumerili) Juveniles

“…Ebp (Emopamil binding protein), also known as EBP cholestenol delta-isomerase, is essential in the sterol biosynthesis pathway [59]. Sterols are essential cell membrane components and transporters in many biofilms [60]. SQLE (squalene epoxidase) is rate limiting and the first oxygenation enzyme in cholesterol synthesis [61].…”

The use of soft X-ray tomography to explore mitochondrial structure and function

“…Mitochondria have been explored by SXT in the context of healthy single cells [ 26 ] and cancer cells [ 37 , 38 ], and after isolation from cells to explore their internal structure [ 27 , 39 ]. For the purpose of this review, we will highlight four unique applications of SXT that have been used to investigate the internal structure of mitochondria [ 27 ], evaluate mitochondria-ER contacts [ 19 ], explore the interaction between mitochondria and endosome-like vesicles [ 40 ], and visualize the mitochondrial network structure during multiple phases of insulin secretion [ 16 ].…”

“…Biolipids such as cholesterol are important for maintaining several biophysical properties of membranes such as fluidity, curvature, permeability, and protein-lipid interactions [ 40 ]. Delivery of cholesterol to the mitochondria enables its conversion to sterol metabolites and steroid hormones.…”

“…Delivery of cholesterol to the mitochondria enables its conversion to sterol metabolites and steroid hormones. To investigate the mechanisms of cholesterol delivery to the mitochondria, SXT and cryo-fluorescence microscopy were used to reveal close contacts of mitochondria to endosome-like organelles in human fibroblasts [ 40 ]. The authors used these results to determine the surface fraction of endo-lysosomes in contact with mitochondria, providing clarity on how cholesterol can be delivered to the mitochondria.…”