TY - JOUR
T1 - LIM-Nebulette reinforces podocyte structural integrity by linking actin and vimentin filaments
AU - Ge, Xuhua
AU - Zhang, Tao
AU - Yu, Xiaoxia
AU - Muwonge, Alecia N.
AU - Anandakrishnan, Nanditha
AU - Wong, Nicholas J.
AU - Haydak, Jonathan C.
AU - Reid, Jordan M.
AU - Fu, Jia
AU - Wong, Jenny S.
AU - Bhattacharya, Smiti
AU - Cuttitta, Christina M.
AU - Zhong, Fang
AU - Gordon, Ronald E.
AU - Salem, Fadi
AU - Janssen, William
AU - Hone, James C.
AU - Zhang, Aihua
AU - Li, Hong
AU - He, John C.
AU - Luca Gusella, G.
AU - Campbell, Kirk N.
AU - Azeloglu, Evren U.
N1 - Funding Information:
The study was supported by National Institutes of Health (NIH) grants R01 DK118222 (to E.U. Azeloglu), R01 DK103022 (to K.N. Campbell), R01 DK106035 (to G.L. Gusella), and R01 DK78897 (to J.C. He). S. Bhat-tacharya was in part supported by grant F31 DK124135. Stimulated emission-depletion microscopy and immunogold images were performed in the Microscopy CoRE at the Icahn School of Medicine at Mount Sinai, supported in part with funding from NIH Shared Instrumentation grant FAIN: S10OD021838. The proteomics data were obtained from mass spectrometers funded in part by NIH grants NS046593 and 1S10OD025047 (to H. Li).
Publisher Copyright:
Copyright © 2020 by the American Society of Nephrology
PY - 2020/10
Y1 - 2020/10
N2 - Background 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. Methods 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. Results 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. Conclusions 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.
AB - Background 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. Methods 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. Results 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. Conclusions 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.
UR - http://www.scopus.com/inward/record.url?scp=85092220473&partnerID=8YFLogxK
U2 - 10.1681/ASN.2019121261
DO - 10.1681/ASN.2019121261
M3 - Article
C2 - 32737144
AN - SCOPUS:85092220473
SN - 1046-6673
VL - 31
SP - 2372
EP - 2391
JO - Journal of the American Society of Nephrology : JASN
JF - Journal of the American Society of Nephrology : JASN
IS - 10
ER -