ten Brink, C. | Search bioRxiv

bioRxiv2021

Bimodal Endocytic Probe for Three-Dimensional Correlative Light and Electron Microscopy

Correlative light and electron microscopy (CLEM) can infer molecular, functional and dynamic information to ultrastructure by linking information of different imaging modalities. One of the main challenges, especially in 3D-CLEM, is the accurate registration of fluorescent signals to electron microscopy (EM). Here, we present fluorescent BSA-gold (fBSA-Au), a bimodal endocytic tracer as fiducial marker for 2D and 3D CLEM applications. fBSA-Au consists of colloidal gold (Au) particles stabilized with fluorescent bovine serum albumin (BSA). The conjugate is efficiently endocytosed and distributed throughout the 3D endo-lysosomal network of the cells, and has an excellent visibility both in fluorescence microscopy (FM) and EM. We demonstrate the use of fBSA-Au in several 2D and 3D CLEM applications using Tokuyasu cryosections, resin-embedded material, and cryo-EM. As a fiducial marker, fBSA-Au facilitates rapid registration of regions of interest between FM and EM modalities and enables accurate (50-150 nm) correlation of fluorescence to EM data. Endocytosed fBSA-Au benefits from a homogenous 3D distribution throughout the endosomal system within the cell, and does not obscure any cellular ultrastructure. The broad applicability and visibility in both modalities makes fBSA-Au an excellent endocytic fiducial marker for 2D and 3D (cryo-)CLEM applications.

cell biology↗

bioRxiv2019

VPS41 recessive mutation causes ataxia and dystonia with retinal dystrophy and mental retardation by inhibiting HOPS function and mTORC1 signaling

The vacuolar protein sorting protein 41 (VPS41) is a neuroprotective protein in models of Parkinsons disease (PD). As part of the HOPS (Homotypic fusion and Protein Sorting) complex, VPS41 regulates fusion of lysosomes with late endosomes and autophagosomes. Independent of HOPS, VPS41 regulates transport of newly synthesized lysosomal membrane proteins and secretory proteins. Here we report two brothers with compound heterozygous mutations in VPS41 (VPS41R662* and VPS41S285P), born to healthy and non-consanguineous parents. Both patients displayed transient retinal dystrophy, ataxia and dystonia, with brain MRI findings of cerebellar atrophy and a thin saber-shape corpus callosum. Patient-derived fibroblasts contained enzymatically active lysosomes that were poorly reached by endocytic cargo and failed to attract the mTORC1 complex. Consequently, transcription factor TFE3, a driver of autophagy and lysosomal genes, showed continuous nuclear localization which resulted in elevated LC3-II levels and an impaired response to nutrient starvation. CRISPR/CAS VPS41 HeLa knockout cells showed a similar phenotype that could be rescued by wildtype VPS41 but not by VPS41S285P or VPS41R662*. mTORC1 inhibition was also seen after knockout of HOPS subunits VPS11 or VPS18. Regulated neuropeptide secretion in PC12 VPS41 knockout cells was rescued by VPS41S285P expression, indicating that this HOPS-independent function was preserved. Co-expression of the VPS41S285P and VPS41R662* variants in a C. elegans model of PD abolished the protective effect of VPS41 against -synuclein-induced neurodegeneration. We conclude that both disease-associated VPS41 variants specifically abrogate HOPS function, which leads to a delay in endocytic cargo delivery to lysosomes, mTORC1 inhibition and irresponsiveness to autophagic clues. Our studies signify a link between HOPS function and mTORC1 signaling and imply that HOPS function is required for the neuroprotective effect of VPS41 in PD.

cell biology↗