Division of Nephrology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, New York.
Department of Mechanical Engineering, Columbia University, New York, New York.
J Am Soc Nephrol. 2020 Oct;31(10):2372-2391. doi: 10.1681/ASN.2019121261. Epub 2020 Jul 31.
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 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.
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.
维持足细胞错综复杂的指状形态对于肾小球滤过至关重要。足细胞特化形态的一个关键方面是将不同的细胞骨架丝分离并组织到不同的亚细胞成分中，其确切机制仍知之甚少。
使用来自大鼠、小鼠和人类的细胞来描述足细胞结构的细胞骨架结构。筛选嘌呤霉素氨基核苷诱导的大鼠肾病模型中的时间依赖性蛋白质组变化，强烈提示肌动蛋白结合蛋白 LIM-nebulette 与肾小球功能密切相关。单细胞 RNA 测序和免疫金标记用于确定 LIM-nebulette 在足细胞中的表达特异性。自动化高内涵成像、超分辨率显微镜、原子力显微镜 (AFM)、钙的活细胞成像以及运动性和粘附动力学的测量，表征了 LIM-nebulette 在足细胞中的生理作用。
敲除小鼠对阿霉素诱导的肾病更敏感，并表现出形态、细胞骨架和黏附异常，伴有钙动力学、运动性和 Rho GTPase 活性改变。糖尿病肾病和 FSGS 患者的 LIM-nebulette 表达在转录和蛋白水平均降低。在小鼠、大鼠和人类中，LIM-nebulette 表达定位于足细胞的初级、次级和三级突起，与黏附斑以及波形蛋白纤维共定位。在永生化的人足细胞中敲低 LIM-nebulette 会导致波形蛋白丝的组织紊乱和细胞弹性降低，这可通过 AFM 压痕测量来评估。
LIM-nebulette 是一种多功能细胞骨架蛋白，通过对黏附斑、肌动蛋白细胞骨架和中间丝的积极重排，在维持足细胞结构完整性方面至关重要。
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