Super-resolution fluorescence-assisted diffraction computational tomography reveals the threedimensional landscape of the cellular organelle interactome

The emergence of super-resolution(SR)fluorescence microscopy has rejuvenated the search for new cellular substructures.However,SR fluorescence microscopy achieves high contrast at the expense of a holistic view of the interacting partners and surrounding environment.Thus,we developed SR fluorescence-assisted diffraction computational tomography(SR-FACT),which combines label-free three-dimensional optical diffraction tomography(ODT)with two-dimensional fluorescence Hessian structured illumination microscopy.The ODT module is capable of resolving the mitochondria,lipid droplets,the nuclear membrane,chromosomes,the tubular endoplasmic reticulum,and lysosomes.Using dual-mode correlated live-cell imaging for a prolonged period of time,we observed novel subcellular structures named dark-vacuole bodies,the majority of which originate from densely populated perinuclear regions,and intensively interact with organelles such as the mitochondria and the nuclear membrane before ultimately collapsing into the plasma membrane.This work demonstrates the unique capabilities of SR-FACT,which suggests its wide applicability in cell biology in general.

Superresolution live-cell imaging reveals that the localization of TMEM106B to filopodia in oligodendrocytes is compromised by the hypomyelination-related D252N mutation

Hypomyelination leukodystrophies constitute a group of heritable white matter disorders exhibiting defective myelin development.Initially identified as a lysosomal protein,the TMEM106B D252N mutant has recently been associated with hypomyelination.However,how lysosomal TMEM106B facilitates myelination and how the D252N mutation disrupts that process are poorly understood.We used superresolution Hessian structured illumination microscopy(Hessian-SIM)and spinning discconfocal structured illumination microscopy(SD-SIM)to find that the wild-type TMEM106B protein is targeted to the plasma membrane,filopodia,and lysosomes in human oligodendrocytes.The D252N mutation reduces the size of lysosomes in oligodendrocytes and compromises lysosome changes upon starvation stress.Most importantly,we detected reductions in the length and number of filopodia in cells expressing the D252N mutant.PLP1 is the most abundant myelin protein that almost entirely colocalizes with TMEM106B,and coexpressing PLP1 with the D252N mutant readily rescues the lysosome and filopodia phenotypes of cells.Therefore,interactions between TMEM106B and PLP1 on the plasma membrane are essential for filopodia formation and myelination in oligodendrocytes,which may be sustained by the delivery of these proteins from lysosomes via exocytosis.