Zeger Matthias, Stanisławczyk Lena Sarah, Bulić Marija, Binder Andrea Maria, Huber Armin
Department of Biochemistry, Institute of Biology, University of Hohenheim, Stuttgart, Germany.
Front Cell Dev Biol. 2024 Sep 20;12:1444953. doi: 10.3389/fcell.2024.1444953. eCollection 2024.
In polarized cells, the precise regulation of protein transport to and from the plasma membrane is crucial to maintain cellular function. Dysregulation of intracellular protein transport in neurons can lead to neurodegenerative diseases such as Retinitis Pigmentosa, Alzheimer's and Parkinson's disease. Here we used the light-dependent transport of the TRPL (transient receptor potential-like) ion channel in photoreceptor cells to study the role of Rab proteins in TRPL recycling. TRPL is located in the rhabdomeric membrane of dark-adapted flies, but it is transported out of the rhabdomere upon light exposure and localizes at the Endoplasmatic Reticulum within 12 h. Upon subsequent dark adaptation, TRPL is recycled back to the rhabdomeric membrane within 90 min. To screen for Rab proteins involved in TRPL recycling, we established a tissue specific (ts) CRISPR/Cas9-mediated knock-out of individual genes in photoreceptors and assessed TRPL localization using an eGFP tagged TRPL protein in the intact eyes of these mutants. We observed severe TRPL recycling defects in the knockouts of , , , , . Using immunohistochemistry, we further showed that Rab3 and RabX2 each play a significant role in TRPL recycling and also influence TRPL transport. We localized Rab3 to the late endosome in photoreceptors and observed disruption of TRPL transport to the ER in knock-out mutants. TRPL transport from the ER to the rhabdomere ensues from the trans-Golgi where RabX2 is located. We observed accumulated TRPL at the trans-Golgi in knock-out mutants. In summary, our study reveals the requirement of specific Rab proteins for different steps of TRPL transport in photoreceptor cells and provides evidence for a unique retrograde recycling pathway of TRPL from the ER via the trans-Golgi.
在极化细胞中，蛋白质往返于质膜的精确运输调控对于维持细胞功能至关重要。神经元内蛋白质运输失调可导致神经退行性疾病，如色素性视网膜炎、阿尔茨海默病和帕金森病。在此，我们利用光感受器细胞中TRPL（类瞬时受体电位）离子通道的光依赖性运输来研究Rab蛋白在TRPL循环中的作用。TRPL位于暗适应果蝇的微绒毛膜中，但在光照下会从微绒毛中运输出来，并在12小时内定位于内质网。随后在暗适应过程中，TRPL会在90分钟内循环回到微绒毛膜。为了筛选参与TRPL循环的Rab蛋白，我们建立了组织特异性（ts）CRISPR/Cas9介导的光感受器中单个基因敲除，并在这些突变体的完整眼睛中使用eGFP标记的TRPL蛋白评估TRPL定位。我们在敲除 、 、 、 、 后观察到严重的TRPL循环缺陷。通过免疫组织化学，我们进一步表明Rab3和RabX2在TRPL循环中均发挥重要作用，并且还影响TRPL运输。我们将Rab3定位于光感受器中的晚期内体，并在敲除突变体中观察到TRPL向内质网的运输中断。TRPL从内质网到微绒毛的运输发生在RabX2所在的反式高尔基体。我们在敲除突变体的反式高尔基体中观察到TRPL积累。总之，我们的研究揭示了光感受器细胞中TRPL运输不同步骤对特定Rab蛋白的需求，并为TRPL从内质网经反式高尔基体的独特逆行循环途径提供了证据。
