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  • LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments.

LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments.

Journal of the American Society of Nephrology : JASN (2020-08-02)
Xuhua Ge, Tao Zhang, Xiaoxia Yu, Alecia N Muwonge, Nanditha Anandakrishnan, Nicholas J Wong, Jonathan C Haydak, Jordan M Reid, Jia Fu, Jenny S Wong, Smiti Bhattacharya, Christina M Cuttitta, Fang Zhong, Ronald E Gordon, Fadi Salem, William Janssen, James C Hone, Aihua Zhang, Hong Li, John C He, G Luca Gusella, Kirk N Campbell, Evren U Azeloglu
ABSTRACT

Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood. Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes. Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation. LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, low endotoxin, pH 7, ≥98%
Sigma-Aldrich
Cytochalasin D, from Zygosporium mansonii, ≥98% (TLC and HPLC), powder
Sigma-Aldrich
Trisodium citrate dihydrate, meets USP testing specifications
Sigma-Aldrich
Retinoic acid, ≥98% (HPLC), powder
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, Type II-S, lyophilized powder, Protein ≥80 %, ≥2,000 units/mg protein
Sigma-Aldrich
Triton X-100, for molecular biology
Sigma-Aldrich
Puromycin aminonucleoside
Sigma-Aldrich
Anti-α-Tubulin antibody, Mouse monoclonal, clone DM1A, purified from hybridoma cell culture
Sigma-Aldrich
Iron(III) oxide, powder, <5 μm, ≥96%