Tuberous Sclerosis Complex Axis Controls Renal Extracellular Vesicle Production and Protein Content

International Journal of Molecular Sciences
Fahad ZadjaliJohn J Bissler

Abstract

The tuberous sclerosis complex (Tsc) proteins regulate the conserved mTORC1 growth regulation pathway. We identified that loss of the Tsc2 gene in mouse inner medullary collecting duct (mIMCD) cells induced a greater than two-fold increase in extracellular vesicle (EV) production compared to the same cells having an intact Tsc axis. We optimized EV isolation using a well-established size exclusion chromatography method to produce high purity EVs. Electron microscopy confirmed the purity and spherical shape of EVs. Both tunable resistive pulse sensing (TRPS) and dynamic light scattering (DLS) demonstrated that the isolated EVs possessed a heterogenous size distribution. Approximately 90% of the EVs were in the 100-250 nm size range, while approximately 10% had a size greater than 250 nm. Western blot analysis using proteins isolated from the EVs revealed the cellular proteins Alix and TSG101, the transmembrane proteins CD63, CD81, and CD9, and the primary cilia Hedgehog signaling-related protein Arl13b. Proteomic analysis of EVs identified a significant difference between the Tsc2-intact and Tsc2-deleted cell that correlated well with the increased production. The EVs may be involved in tissue homeostasis and cause disease by ov...Continue Reading

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Citations

Dec 15, 2020·Journal of Chromatography. a·Thanaporn LiangsupreeMarja-Liisa Riekkola
Jul 16, 2021·Frontiers in Physiology·Prashant KumarJohn J Bissler
Sep 10, 2021·Journal of Neuroimmune Pharmacology : the Official Journal of the Society on NeuroImmune Pharmacology·Santosh KumarJohn Bissler

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Methods Mentioned

BETA
size exclusion chromatography
dynamic
transition
transfection
electron microscopy
Size
dynamic light scattering
transfections
proteomics profiling

Software Mentioned

R
iPathway guide
limma
Izon Control Suite
Scaffold
Proteome
Mascot

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