Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
Laboratory of Membrane Trafficking Mechanisms, Department of Integrative Life Sciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, Miyagi 980-8578, Japan.
J Biol Chem. 2020 Mar 13;295(11):3652-3663. doi: 10.1074/jbc.RA119.011646. Epub 2020 Jan 28.
Polarized epithelial cells have functionally distinct apical and basolateral membranes through which they communicate with external and internal bodily environments, respectively. The establishment and maintenance of this asymmetric structure depend on polarized trafficking of specific cargos, but the precise molecular mechanism is incompletely understood. We previously showed that Rab35, a member of the Rab family small GTPases, differentially regulates the trafficking of an apical cargo, podocalyxin (PODXL), in two-dimensional (2D) and three-dimensional (3D) Madin-Darby canine kidney (MDCK) II cell cultures through specific interactions with two distinct effectors, OCRL inositol polyphosphate-5-phosphatase (OCRL) and ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 2 (ACAP2), respectively. However, whether the upstream regulators of Rab35 also differ depending on the culture conditions remains completely unknown. Here, we investigated four known guanine nucleotide exchange factors (GEFs) of Rab35, namely DENN domain-containing 1A (DENND1A), DENND1B, DENND1C, and folliculin (FLCN), and demonstrate that DENND1A and FLCN exhibit distinct requirements for Rab35-dependent PODXL trafficking under the two culture conditions. In 3D cell cultures, only DENDN1A-knockout cysts exhibited the inverted localization of PODXL similar to that of Rab35-knockout cysts. Moreover, the DENN domain, harboring GEF activity toward Rab35, was required for proper PODXL trafficking to the apical membrane. By contrast, FLCN-knockdown cells specifically accumulated PODXL in actin-rich structures similar to the Rab35-knockdown cells in 2D cell cultures. Our findings indicate that two distinct functional cascades of Rab35, the FLCN-Rab35-OCRL and the DENND1A-Rab35-ACAP2 axes, regulate PODXL trafficking in 2D and 3D MDCK II cell cultures, respectively.
极化上皮细胞通过具有不同功能的顶侧和基底外侧膜分别与外部和内部的身体环境进行通讯。这种不对称结构的建立和维持依赖于特定货物的极化运输，但精确的分子机制尚不完全清楚。我们之前表明，Rab35 是 Rab 家族小 GTPases 的成员，通过与两个不同的效应物 OCRL 肌醇多磷酸盐 5-磷酸酶 (OCRL) 和具有卷曲螺旋、锚重复和pleckstrin 同源结构域 2 (ACAP2) 的 ArfGAP 分别特异性相互作用，差异调节二维 (2D) 和三维 (3D) Madin-Darby 犬肾 (MDCK) II 细胞培养物中顶端货物足细胞蛋白聚糖 (PODXL) 的运输。然而，Rab35 的上游调节剂是否也因培养条件而异仍完全未知。在这里，我们研究了 Rab35 的四个已知鸟嘌呤核苷酸交换因子 (GEF)，即 DENN 结构域包含蛋白 1A (DENND1A)、DENND1B、DENND1C 和多囊蛋白 (FLCN)，并证明 DENND1A 和 FLCN 在两种培养条件下表现出对 Rab35 依赖性 PODXL 运输的不同要求。在 3D 细胞培养物中，只有 DENND1A 敲除小囊泡表现出与 Rab35 敲除小囊泡相似的 PODXL 定位倒置。此外，DENN 结构域，具有 Rab35 的 GEF 活性，是 PODXL 正确运输到顶膜所必需的。相比之下，FLCN 敲低细胞在 2D 细胞培养物中类似于 Rab35 敲低细胞的肌动蛋白丰富结构中特异性积累 PODXL。我们的研究结果表明，Rab35 的两个不同功能级联，FLCN-Rab35-OCRL 和 DENND1A-Rab35-ACAP2 轴，分别调节 2D 和 3D MDCK II 细胞培养物中 PODXL 的运输。
