Mann Noah, Surabhi Keerthana, Sharp Josephine, Phipps Mary, Becton Maelee, Hill Jahiem, Roberts Davis, Szatmari Erzsebet M, Hughes Robert M
Department of Chemistry, East Carolina University, Greenville, NC, United States.
Department of Physical Therapy, East Carolina University, Greenville, NC, United States.
Front Cell Neurosci. 2025 Jun 4;19:1543199. doi: 10.3389/fncel.2025.1543199. eCollection 2025.
Cofilin-actin rods are a well-documented stress response in neuronal cells and their persistence is frequently associated with neurodegenerative disease. However, the role of specific actin residues in promoting the formation of cofilin-actin rods and other anomalous cytoskeletal structures is largely unknown. As it is increasingly suspected that specific mutations and post-translation modifications of actin may promote neurodegenerative disease, characterizing the role of these residues in cytoskeletal dysregulation is highly relevant. In this study, we focus on the actin-ATP interface, which has been proposed as a key mediator of cofilin-actin rod formation and the propensity of actin to respond to cellular stress. Using a light and stress-gated reporter of cofilin-actin cluster formation, we determine the impact of mutants associated with Actin-ATP binding on the propensity of actin to form anomalous structures in the presence and absence of applied cellular stress. This study identifies actin mutants that promote anomalous actin inclusions in HeLa cells and characterizes the manifestation of these phenotypes in cortical neurons. Mutations to the ATP phosphate tail-binding region of actin (K18A, D154A, G158L, K213A) were found to be particularly disruptive to actin phenotypes, and in several instances promote disease-associated actin-rich structures such as cofilin-actin rods and Hirano bodies. We find that these mutant phenotypes are largely consistent between cell types and display highly unusual inclusions in cultured cortical neurons, without leading to nuclear fragmentation and apoptotic death of the transfected cells. These mutants strengthen the association of residue-specific changes in actin with large-scale phenotypic and functional changes in the cytoskeleton, further implicating them in neurodegenerative disease progression.
丝切蛋白 - 肌动蛋白杆是神经元细胞中一种有充分文献记载的应激反应，其持续存在常与神经退行性疾病相关。然而，特定肌动蛋白残基在促进丝切蛋白 - 肌动蛋白杆及其他异常细胞骨架结构形成中的作用在很大程度上尚不清楚。由于越来越多的人怀疑肌动蛋白的特定突变和翻译后修饰可能促进神经退行性疾病，因此表征这些残基在细胞骨架失调中的作用具有高度相关性。在本研究中，我们聚焦于肌动蛋白 - ATP界面，该界面被认为是丝切蛋白 - 肌动蛋白杆形成以及肌动蛋白对细胞应激反应倾向的关键介质。使用丝切蛋白 - 肌动蛋白簇形成的光控和应激门控报告基因，我们确定了与肌动蛋白 - ATP结合相关的突变体在有无施加细胞应激情况下对肌动蛋白形成异常结构倾向的影响。本研究鉴定出了在HeLa细胞中促进异常肌动蛋白包涵体形成的肌动蛋白突变体，并表征了这些表型在皮质神经元中的表现。发现肌动蛋白ATP磷酸尾结合区域的突变（K18A、D154A、G158L、K213A）对肌动蛋白表型具有特别的破坏作用，在几种情况下会促进与疾病相关的富含肌动蛋白的结构，如丝切蛋白 - 肌动蛋白杆和平野小体。我们发现这些突变体表型在不同细胞类型之间基本一致，并且在培养的皮质神经元中显示出非常异常的包涵体，而不会导致转染细胞的核碎裂和凋亡死亡。这些突变体加强了肌动蛋白中特定残基变化与细胞骨架大规模表型和功能变化之间的关联，进一步表明它们与神经退行性疾病进展有关。
