Abstract
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.
Highlights
Polarized epithelial cells have functionally distinct apical and basolateral membranes through which they communicate with external and internal bodily environments, respectively
To determine the functional diversity of the DENN domain– containing 1 (DENND1) family proteins in PODXL trafficking even in 2D monolayer formation, we further investigated whether DENN domain– containing 1A (DENND1A)–C are required for PODXL trafficking under 2D culture conditions
We presented new evidence indicating that distinct Rab35– guanine nucleotide exchange factors (GEFs) regulate PODXL trafficking under two culture conditions
Summary
Rab35–GEFs, DENND1A and folliculin differentially regulate podocalyxin trafficking in two- and three-dimensional epithelial cell cultures. We previously showed that Rab, 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. DENND1C has a unique actin-binding motif and is required for the regulation of actin dynamics [17] Another Rab35–GEF, FLCN [18], contains a DENN-like domain that harbors GEF activity toward Rab35 [19, 20], but FLCN is known to act as a GTPase-activating protein (GAP) for other small GTPases, such as RagC/D [21] and Rab7A [22]. Our findings suggest that Rab35-dependent PODXL transcytosis is differentially regulated by its upstream GEFs between 2D and 3D cell cultures
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