Indian Institute of Science Education and Research (IISER) Pune, Dr Homi Bhabha Road, Pashan, Pune, Maharashtra 411008, India.
Biol Open. 2023 Apr 15;12(4). doi: 10.1242/bio.059669. Epub 2023 May 4.
Arf1 belongs to the Arf family of small GTPases that localise at the Golgi and plasma membrane. Active Arf1 plays a crucial role in regulating Golgi organisation and function. In mouse fibroblasts, loss of adhesion triggers a consistent drop (∼50%) in Arf1 activation that causes the Golgi to disorganise but not fragment. In suspended cells, the trans-Golgi (GalTase) disperses more prominently than cis-Golgi (Man II), accompanied by increased active Arf1 (detected using GFP-ABD: ARHGAP10 Arf1 binding domain) associated with the cis-Golgi compartment. Re-adhesion restores Arf1 activation at the trans-Golgi as it reorganises. Arf1 activation at the Golgi is regulated by Arf1 Guanine nucleotide exchange factors (GEFs), GBF1, and BIG1/2. In non-adherent fibroblasts, the cis-medial Golgi provides a unique setting to test and understand the role GEF-mediated Arf1 activation has in regulating Golgi organisation. Labelled with Man II-GFP, non-adherent fibroblasts treated with increasing concentrations of Brefeldin-A (BFA) (which inhibits BIG1/2 and GBF1) or Golgicide A (GCA) (which inhibits GBF1 only) comparably decrease active Arf1 levels. They, however, cause a concentration-dependent increase in cis-medial Golgi fragmentation and fusion with the endoplasmic reticulum (ER). Using selected BFA and GCA concentrations, we find a change in the kinetics of Arf1 inactivation could mediate this by regulating cis-medial Golgi localisation of GBF1. On loss of adhesion, a ∼50% drop in Arf1 activation over 120 min causes the Golgi to disorganise. The kinetics of this drop, when altered by BFA or GCA treatment causes a similar decline in Arf1 activation but over 10 min. This causes the Golgi to now fragment which affects cell surface glycosylation and re-adherent cell spreading. Using non-adherent fibroblasts this study reveals the kinetics of Arf1 inactivation, with active Arf1 levels, to be vital for Golgi organisation and function.
Arf1 属于 Arf 家族的小 GTP 酶，定位于高尔基体和质膜。活性 Arf1 在调节高尔基体组织和功能方面发挥着关键作用。在小鼠成纤维细胞中，失去黏附会触发 Arf1 活性的持续下降（约 50%），导致高尔基体解体但不碎裂。在悬浮细胞中，跨高尔基（GalTase）比顺高尔基（Man II）更明显地分散，伴随着与顺高尔基隔室相关的活性 Arf1（使用 GFP-ABD：ARHGAP10 Arf1 结合结构域检测到）增加。重新黏附会恢复跨高尔基的 Arf1 活性，因为它会重新组织。高尔基体的 Arf1 活性受 Arf1 鸟嘌呤核苷酸交换因子（GEFs）、GBF1 和 BIG1/2 调节。在非黏附的成纤维细胞中，顺间高尔基体提供了一个独特的环境，可以测试和理解 GEF 介导的 Arf1 激活在调节高尔基体组织中的作用。用 Man II-GFP 标记，用递增浓度的布雷菲德菌素 A（BFA）（抑制 BIG1/2 和 GBF1）或 Golgicide A（GCA）（仅抑制 GBF1）处理非黏附的成纤维细胞，均可使活性 Arf1 水平相应降低。然而，它们会导致顺间高尔基体的片段化和与内质网（ER）融合的浓度依赖性增加。使用选定的 BFA 和 GCA 浓度，我们发现 Arf1 失活的动力学变化可以通过调节 GBF1 在顺间高尔基体的定位来介导这一过程。在失去黏附后，120 分钟内 Arf1 活性下降约 50%会导致高尔基体解体。这种下降的动力学，当通过 BFA 或 GCA 处理改变时，会导致 Arf1 活性相似的下降，但持续 10 分钟。这会导致高尔基体现在碎片化，从而影响细胞表面糖基化和重新黏附的细胞铺展。本研究利用非黏附性成纤维细胞，揭示了 Arf1 失活的动力学，以及活性 Arf1 水平，对高尔基体的组织和功能至关重要。
