Abstract: FR-PO324
High-Resolution Imaging of Inter-Organelle Interaction and Vesicle Trafficking in Live-Cells Expressing the INF2 Variants
Session Information
- Genetic Diseases: Models, Mechanisms, Treatments
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1102 Genetic Diseases of the Kidneys: Non-Cystic
Authors
- Tran Thuy, Huong Quynh, Kansai Medical University Second Department of Internal Medicine, Hirakata, Osaka, Japan
- Ueda, Hiroko, Kansai Medical University Second Department of Internal Medicine, Hirakata, Osaka, Japan
- Tran, Linh Nguyen Truc, Kansai Medical University Second Department of Internal Medicine, Hirakata, Osaka, Japan
- Kondo, Naoyuki, Kansai Medical University Department of Molecular Genetics, Hirakata, Osaka, Japan
- Tsukaguchi, Hiroyasu, Kansai Medical University Second Department of Internal Medicine, Hirakata, Osaka, Japan
Background
Focal segmental glomerulosclerosis (FSGS) is primarily caused by podocyte deletion. Regulation of cytoskeleton plays a key role in maintaining the slit diaphragm integrity. INF2, an actin regulator, is one of the most prevalent genes in monogenic FSGS. The DID domain INF2 mutations cause two subtypes of disorders; one is FSGS alone and FSGS/CMT dual phenotypes. However, the detail mechanisms by which the DID mutations exert differential tissue effect remain unclear.
Methods
We characterized the cytoskeletal organization and inter-organelle interaction in Hela/COS7 cells expressing pathogenic INF2 variants identified in our cohort. Cellular effects are compared between FSGS only and CMT/FSGS variants. To clarify the actin dynamics and vesicle trafficking, we took advantage of the Spinning Disk Microscope DragonFly, which allows real-time imaging with 100nm resolution.
Results
When expressed in Hela and COS7 cells, INF2 variants causing FSGS alone (T161N, N202S) distributed a diffuse cytoplasmic ER pattern with Golgi enrichment similar to wild-type. In contrast, CMT/FSGS variants (G73D, V108D) exhibited a coarse granular distribution with aberrant accumulation at cell periphery. In fixed cells, actin stress fibers were more remarkably reduced in cells expressing CMT/FSGS variants than in FSGS variants. The extent of stress fiber reduction correlated with the severity of intracellular INF2 misdistribution.
Spinning Disk Microscopy with time-lapse imaging of living COS7 cells revealed that T161N variant increases the frequency of mitochondrial fragmentation as well as the number of mitochondria-ER interfaces than the wild-type. Moreover, trajectory analysis of lysotracker-labeled vesicles in living COS7 disclosed that in T161N variant reduces the motility of cytoplasmic vesicles than the wild-type (n=100 for each group, P<0.0001), suggesting that disorganized actin network may perturb the intracellular vesicular trafficking.
Conclusion
Our expression study indicates that the INF2 variants cause severer actin network disarrangement and thus more deteriorating effects on the ER-mitochondria interaction and endosomal trafficking than wild-type. Such mechanisms may represent a common, global feature that underlies the pathogenesis of INF2-related disorders, affecting podocyte and/or Schwann cells.
Funding
- Government Support – Non-U.S.