Kono Yohei, Pack Chan-Gi, Ichikawa Takehiko, Komatsubara Arata, Adam Stephen A, Miyazawa Keisuke, Rolas Loïc, Nourshargh Sussan, Medalia Ohad, Goldman Robert D, Fukuma Takeshi, Kimura Hiroshi, Shimi Takeshi
Cell Biology Center, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama 226-8503, Japan.
Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa 920-1192, Japan.
PNAS Nexus. 2024 Nov 21;3(12):pgae527. doi: 10.1093/pnasnexus/pgae527. eCollection 2024 Dec.
The nuclear lamina (NL) lines the nuclear envelope (NE) to maintain nuclear structure in metazoan cells. The major NL components, the nuclear lamins contribute to the protection against NE rupture induced by mechanical stress. Lamin A (LA) and a short form of the splicing variant lamin C (LC) are diffused from the nucleoplasm to sites of NE rupture in immortalized mouse embryonic fibroblasts (MEFs). LA localization to the rupture sites is significantly slow and weak compared with LC, but the underlying mechanism remains unknown. In this study, wild-type (WT), Hutchinson-Gilford Progeria syndrome (HGPS) knock-in MEFs expressing progerin (PG, an LA mutant lacking the second proteolytic cleavage site), and LA/C-knockout MEFs transiently and heterogeneously expressing LA/C WTs and mutants fused to mEmerald are examined before and after NE rupture induced by single-cell compression and laser microirradiation. The farnesylation at the CaaX motif of unprocessed LA and the inhibition of the second proteolytic cleavage decrease the nucleoplasmic pool and slow the localization to the rupture sites in a long-time window (60-70 min) after the induction of NE rupture. Our data could explain the defective repair of NE rupture in HGPS through the farnesylation at the CaaX motif of unprocessed progerin. In addition, unique segments in LA-specific tail region cooperate with each other to inhibit the rapid accumulation within a short-time window (3 min) that is also observed with LC.
核纤层（NL）衬于核膜（NE）内侧，以维持后生动物细胞的核结构。核纤层的主要成分核纤层蛋白有助于抵御机械应力诱导的核膜破裂。在永生化小鼠胚胎成纤维细胞（MEF）中，核纤层蛋白A（LA）和剪接变体核纤层蛋白C（LC）的一种短形式会从核质扩散至核膜破裂部位。与LC相比，LA定位于破裂部位的速度明显较慢且定位较弱，但其潜在机制仍不清楚。在本研究中，对野生型（WT）、表达早老素（PG，一种缺乏第二个蛋白水解切割位点的LA突变体）的哈钦森 - 吉尔福德早衰综合征（HGPS）基因敲入MEF，以及瞬时且异源表达与mEmerald融合的LA/C野生型和突变体的LA/C基因敲除MEF，在单细胞压缩和激光微照射诱导核膜破裂前后进行了检测。未加工的LA的CaaX基序处的法尼基化以及第二个蛋白水解切割的抑制会减少核质池，并在核膜破裂诱导后的长时间窗口（60 - 70分钟）内减缓其向破裂部位的定位。我们的数据可以解释HGPS中核膜破裂修复缺陷是通过未加工的早老素的CaaX基序处的法尼基化导致的。此外，LA特异性尾部区域中的独特片段相互协作，以抑制在短时间窗口（3分钟）内的快速积累，这在LC中也有观察到。
