Apical polarization during lumen formation
Upon plating into 3D culture, individual MDCK cells proliferate and assemble into cyst structures - a polarized spherical monolayer surrounding a central lumen. Lumenogenesis requires the apical membrane determinant gp135/podocalyxin5
(PCX in figures). Initially, MDCK aggregates have podocalyxin at the ECM-contacting surface (, 12 h; Fig. S1a
), before polarity inversion occurs, with β-catenin and Na/K-ATPase at cell-cell junctions and podocalyxin now at the lumen (, 24–48 h, arrows; Fig. S1d
. Early lumens occur at a site previously termed the “Pre-Apical Patch” (PAP), where opposing plasma membranes are separated, but the podocalyxin signal is non-resolvable by confocal microscopy7
; expansion enables luminal space visualization (). In contrast, apical proteins syntaxin-3 and GFP-CNT1 (Concentrative Nucleoside Transporter-1) label the entire surface before concentrating at the lumen (Fig. S1b–c, e–f
Characterization of MDCK cyst lumen initiation
Binding of antibodies to GFP-VSVG-podocalyxin at the periphery of cysts, then incubating further to allow lumenogenesis to occur, revealed that podocalyxin at the PAP (arrowheads) and in vesicles (arrows) is at least partially derived from transcytosed peripheral podocalyxin (Fig. S1g
Transcytosis of podocalyxin to the surface to establish the PAP represents formation of apical-basal polarization. We thus examined localization of select polarity (Par3/aPKC), trafficking (exocyst complex; Sec8/Sec10/Sec15A), and junctional (occludin) proteins during lumen initiation. Strikingly, although these proteins showed differing localizations before lumen formation, all converged transiently during lumen initiation (; ). When GFP-podocalyxin was peripheral, Par3 and Sec8 colocalized in puncta at the edge of cell-cell contacts (, arrowheads). When GFP-podocalyxin was internalized and transcytosed some Par3 and Sec8 concentrated at the first detectable site of GFP-podocalyxin delivery to the nascent apical surface (, arrow). We term this the A
ite (AMIS). Later, Par3 and Sec8 instead enriched at the tight junction (, arrowheads). We define early apical structures where several tight junction markers have become distinctly localized from podocalyxin as the PAP (see below)7
, rather than the earlier AMIS, where tight junction markers are not resolved from podocalyxin by confocal.
Distribution of trafficking and polarity proteins during lumen initiation and expansion.
Sec10 and occludin, in contrast, initially localized along the entire cell-cell contact in early aggregates with peripheral podocalyxin (Fig. S2a
; data not shown). Podocalyxin delivered to the AMIS partially overlapped with Sec10 and occludin (; Fig. S2b
; arrows). Though occludin remained along the entire contact, Sec10 condensed toward the AMIS. As the lumen expanded, Sec10 and occludin enriched at the tight junction (; Fig. S2c–f
), though some occludin remained along cell-cell contacts ().
aPKC follows yet a different pattern, with distinct pools initially localized with peripheral podocalyxin (arrowheads) and the AMIS (, arrows), before enriching at PAP edges and finally the tight junction and lumen (, arrowheads). Together, these data show the complex movement of trafficking and cortical polarity proteins, converging transiently at the AMIS.
The Rab8 and Rab11 GTPase families direct lumen initiation
We examined potential control of AMIS and lumen formation upon perturbation of select Rab GTPases involved in apical, basolateral, or junctional trafficking (; Fig. S3f–l
). In contrast to control cysts with a single lumen, apical podocalyxin and basolateral β-catenin, knockdown or dominant negatives of Rab8 (Rab8a/b) and Rab11 (Rab11a/25, but not Rab11b) family members significantly decreased single lumenogenesis (), instead displaying multi-lumens and accumulating podocalyxin in vesicles (, arrowheads; data not shown) close to the cell surface (marked by β-catenin). For single lumen-perturbing knockdowns, phenotypes were confirmed using additional shRNAs (Fig. S3l
), and additional cargos (Fig. S3a–c
). Rab10, Rab11b, Rab13, and Rab14 perturbation did not overtly perturb lumenogenesis and were not pursued ().
The Rab8 and Rab11 GTPase families direct lumen initiation
The Rab11 family regulates transcytosis8
and lumenogenesis in diverse systems9–11
. GFP-Rab11a localized to vesicles underlying the AMIS (marked by Par3; arrow in ), then remained on subapical vesicles once lumens expanded (; ). Podocalyxin transcytosed to the AMIS via Rab11a vesicles. When podocalyxin was peripheral (arrow), GFP-Rab11a localized to juxtanuclear and peripheral vesicles (, white and yellow arrowheads, respectively). Upon internalization, podocalyxin localized to GFP-Rab11a vesicles (, arrowheads), then both were delivered to the cyst interior (, arrowheads). Here, regions of podocalyxin devoid of GFP-Rab11a began to emerge (), representing podocalyxin surface delivery (arrows). Similarly, IgA transcytosed to the PAP (Fig. S1h
). As the lumen expanded, GFP-Rab11a clustered underneath the apical surface (). Notably, overexpression of GFP-Rab11a (WT or activated Q70L) increased single lumenogenesis, while dominant negative GFP-Rab11aS25N
attenuated single lumens and accumulated podocalyxin intracellularly (Fig. S4a
; data not shown). Thus, Rab11a promotes transcytosis to the AMIS and single lumenogenesis.
Rab8 family GTPases were also required for single lumen formation (), and Rab8a localized to transcytosing podocalyxin vesicles and the AMIS7
(; data not shown). Knockdown of Rab11a caused upregulation of Rab8a, and vice versa (Fig. S3f–g
), suggesting compensation or co-operation between Rab11 and Rab8 families. To this end, we knocked down Rab8 and Rab11 family members, alone or in combination (Fig. S4b
). Of tested combinations, co-knockdown of Rab8a/b, with or without Rab11a knockdown, most severely reduced single lumenogenesis. This suggests that the Rab8 family may act downstream of Rab11a. Accordingly, Rab8a knockdown blocked GFP-Rab11aQ70L
-induced increased single lumenogenesis (Fig. S4c
). These data are consistent with hypothesis that the Rab8 family acts, at least in part, downstream of Rab11a during AP transport and lumenogenesis, though the precise interaction between these Rabs may be more complex.
Regulation of Rab8 during lumenogenesis
Rab11 binds the Rab GEF Rabin8 and stimulates its activity towards Rab812
. We reasoned that Rab11a may control subapical Rabin8/Rab8 targeting. In control cysts, a small pool of Rabin8, and to a lesser extent Rab8a localized to dispersed puncta, with some clustered subapically (, arrows). Expression of GFP-Rab11aWT
, but not GFP-Rab11aS25N
, strongly enhanced recruitment of Rabin8 and Rab8a to Rab11a-positive subapical vesicles (, arrowheads; see also for colocalization). Like endogenous Rab8a (), GFP-Rab8aWT
was cytoplasmic and in subapical vesicles, the latter of which was enhanced upon activated GFP-Rab8aQ67L
expression (Fig. S4f
). Thus, active Rab11a recruits active Rab8a to subapical vesicles, likely through Rabin8.
A Rab11-Rabin8-Rab8 module governs apical transport and single lumenogenesis
Tuba and Cdc42 regulate transport from Rab8a/11a vesicles
MDCK Rabin8 appeared as two bands, corresponding to its α and β isoforms: both possess the Rab11-binding region12
(; Fig. S5a
). Rabin8α knockdown accumulated some podocalyxin in vesicles (, arrowheads), and although only to a modest level, significantly decreased single lumenogenesis (). This modest effect is likely due to compensatory up-regulation of the Rabin8β isoform observed upon Rabin8α knockdown (). Dual α/β knockdown caused severe cell death, precluding further analysis (not shown). Expression of RNAi-resistant GFP-hRabin8αWT
, which localized to the luminal region (arrows), in endogenous Rabin8α knockdown cysts restored single lumenogenesis and podocalyxin localization (). In contrast Rabin8α GEF domain mutants (Fig. S5a–c
) further decreased single lumenogenesis and co-accumulated with podocalyxin on vesicles (, arrowheads) beneath the surface marked by F-actin. Similarly, overexpression of GFP-TBC1D30WT
, a GAP specific to the Rab8 family13
, but not GAP-deficient GFP-TBC1D30R140A
, perturbed single lumenogenesis (Fig. S5d–e
). These data suggest that a Rab11a-Rabin8α-Rab8a cascade, experimentally opposed by TBC1D30, is part of a regulatory module governing apical transport and lumenogenesis.
The exocyst and Par3/aPKC complexes regulate apical polarization
Rab8a and Rab11a associate with the Sec15 exocyst subunit14
, in turn linking to Sec10 and other subunits as part of a chain tethering vesicles to the basolateral3
and apical membranes15
. The exocyst also interacts with the Par3/aPKC complex16, 17
. As these factors converge at the AMIS, we examined their requirement in apical traffic and single lumenogenesis.
In contrast to control cysts with apical podocalyxin and basolateral β-catenin (), Sec15A knockdown (Fig. S3m
) accumulated podocalyxin in prominent, GFP-Rab11a positive vesicles close to the surface (, arrows; 4c arrowheads); this Rab11a compartment seemed expanded relative to control cysts (compare ). Additionally, cysts were defective in apical polarization, mistargeting apical cargo to regions of cell-cell contact (; Fig. S3d
). Accordingly, Sec15A knockdown caused almost complete loss of single lumenogenesis (). Similarly, Sec10 knockdown decreased single lumenogenesis and caused vesicular accumulation of podocalyxin (Fig. S2g–i
). Thus, the exocyst regulates podocalyxin transport from Rab11a vesicles to the forming apical surface.
The exocyst and Par3/aPKC regulate lumenogenesis
We also examined the role of the exocyst on Par3 transport and AMIS formation. In control cysts, Par3 localized to tight junctions (, arrows). In Sec15A knockdown cysts, Par3 showed varying, though always abnormal, localization. In regions where a PAP formed, Par3 was recruited to the surface (). However, in regions of vesicular podocalyxin accumulation, Par3 failed to be recruited to the surface and an AMIS was undetectable (, arrowhead). Expression of RNAi-resistant GFP-Sec15AWT, which localized to subapical vesicles (), in cysts with endogenous Sec15A knockdown rescued single lumenogenesis, and surface delivery of podocalyxin and Par3 localization (i.e. at tight junctions once lumens had formed).
To test the role of exocyst coupling to Rabs, we used GFP-Sec15AN691A
, a Rab11-uncoupled mutant15
. This mutant was completely unable to rescue the trafficking and single lumenogenesis defects caused by knockdown of endogenous Sec15A (). Thus, coupling of exocyst to Rab8/11 is required for surface targeting of podocalyxin and Par3 to the AMIS.
Similar to exocyst knockdown, Par3 knockdown also resulted in intracellular podocalyxin accumulation close to the surface marked by β-catenin (, arrows), in vesicles co-labeled for GFP-Rab11a (, arrows), and a strong disruption of single lumenogenesis (). Par3 knockdown also mistargeted some GFP-CNT1 to cell-cell contacts (Fig. S3e
). Moreover, upon Par3 knockdown, Sec8 was not recruited to surface regions adjacent to vesicular podocalyxin (), representing a failure to form the AMIS.
Inhibition of aPKC, using its pseudosubstrate inhibitor (aPKC-PS; ) similarly perturbed AMIS and single lumenogenesis, causing accumulation of podocalyxin in Rab11a-positive vesicles close to the surface marked by β-catenin (, arrowheads). In addition, aPKC inhibition caused lack of podocalyxin internalization from the periphery in some cells (, arrows), likely representing an additional function of aPKC at this locale (see ). Together, these data demonstrate a crucial role for the exocyst/Par3/aPKC complex in podocalyxin delivery from Rab11a vesicles to form the lumen.
Annexin2-Cdc42 associate with Rab11a vesicles during lumeogenesis
Luminal targeting of aPKC in MDCK cysts requires interaction of GTP-Cdc42 with the PI(4,5)P2-binding protein Annexin2 (Anx2)6
. Anx2 both transits to the surface via, and regulates the function of, Rab11a recycling vesicles18, 19
. We thus examined interplay between Anx2, Cdc42 and the Rab11a-Rab8a module.
In early cysts with peripheral podocalyxin (arrow), and subperipheral Apple-Rab11a, GFP-Anx2 localized to the surface (Fig. S6a
). When podocalyxin was in condensed Rab11a vesicles beneath the AMIS, some GFP-Anx2 now also localized to these vesicles (Fig. S6b
, arrowheads). Once podocalyxin was at the open lumen (Fig. S6c
, arrow), GFP-Anx2 localized to both apical and basolateral surfaces, but no longer to subapical Rab11a vesicles. Thus, GFP-Anx2 transiently associates with Rab11a vesicles during lumen initiation.
In contrast to controls expressing Anx2 WT with luminal podocalyxin, subapical Apple-Rab11a, and apical and basolateral GFP-Anx2 (Fig. S6d, f
), expression of dominant negative Anx2 (Anx2 XM) perturbed lumenogenesis and caused the accumulation of GFP-Anx2 with podocalyxin in Rab11a-positive vesicles (Fig. S6e
, arrowheads). Conversely, knockdown of Rab8a or Rab11a caused intracellular accumulation of podocalyxin in structures co-labeled with GFP-Anx2 (Fig. S6g–h
, arrowheads). Thus, Anx2 and Rab8a/11a co-operate in the delivery of podocalyxin to the surface.
We next examined whether Cdc42 associated with Rab11a vesicles. Unlike Anx2, GFP-Cdc42, though possessing a large cytoplasmic pool, strongly overlapped with subapical Apple-Rab11a in cysts with open lumens (, arrowheads). Activated Cdc42 (GFP-Cdc42Q61L) localized to cell-cell contacts and the luminal region, marked by podocalyxin (; arrowheads). As GFP-Cdc42Q61L removed cytoplasmic background labeling, and expression did not perturb single lumens (), we used this allele to further examine Cdc42 localization. In early cysts with peripheral podocalyxin (arrows), GFP-Cdc42Q61L localized to the surface (). When podocalyxin was internalized into Rab11a vesicles and subsequently concentrated at the AMIS, GFP-Cdc42Q61L now extensively overlapped with these vesicles (, arrowheads). As the PAP () and open lumen () formed, podocalyxin and Rab11a largely no longer overlapped, whilst GFP-Cdc42Q61L maintained some overlap with both (arrows). Thus, active Cdc42 associates with Rab11a vesicles during lumenogenesis.
Rab8a/11a regulate Cdc42 during apical transport
Tuba-Cdc42 function in apical transport from Rab8a/Rab11a vesicles
We next determined whether Cdc42 is required for transport from Rab11a vesicles. As shown previously, Cdc42 knockdown perturbed lumenogenesis (), causing accumulation of podocalyxin in VACS (arrows) or vesicles (arrowheads) close to the surface marked by β-catenin ()6
. Notably, intracellular podocalyxin observed upon Cdc42 knockdown was localized to Rab8a/11a vesicles, suggesting Cdc42 regulates transport from these vesicles (, arrowheads).
and Qin et al21
identified Intersectin-2 and Tuba as the only Cdc42-specific GEFs essential for MDCK lumenogenesis. As Intersectin-2 knockdown did not disrupt transport of podocalyxin in cysts20
, we examined if Tuba regulates Cdc42-dependent podocalyxin transport. Tuba knockdown phenocopied Cdc42 knockdown, disrupted single lumenogenesis, and accumulated podocalyxin in Rab8a/11a vesicles (, arrowheads). Notably, Tuba, and to greater extent Cdc42, knockdown blocked GFP-Rab11a-induced increased single lumenogenesis (), suggesting that Rab11a operates upstream of both Tuba and Cdc42. Thus, Tuba-dependent Cdc42 activation is required for podocalyxin apical transport.
Tuba is required for Cdc42 apical targeting21
. We examined whether Rab8a/11a also influenced Cdc42 activation. Rab8a, but not Rab11a, knockdown strikingly decreased global GTP-Cdc42 levels (). Similarly, overexpression of GFP-Rab8aQ67L
, but not GFP-Rab11aQ70L
, robustly activated Cdc42 (), suggesting that Rab8a influences global Cdc42 activation.
We examined whether Rab8a/Rab11a regulate apical Cdc42 targeting. A PBD-YFP probe of activated Cdc426
labeled the luminal surface, along with podocalyxin (, arrowheads), and to a lesser extent cell-cell contacts, mirroring activated Cdc42 localization (). Rab8a knockdown abrogated PBD-YFP membrane association, despite retaining luminal podocalyxin labeling (, arrowhead). Strikingly, Rab11a knockdown resulted in a loss of apical (arrowhead), but not basolateral, PBD-YFP (arrows) (). Thus, Rab8a is required for global activation and surface targeting of Cdc42, while Rab11a controls apical Rab8a, and consequently, active Cdc42 targeting.
We reasoned that as Rab8a/11a influenced apical targeting of active Cdc42, overexpression of active Cdc42 may rescue single lumenogenesis upon Rab8a/11a knockdown. Indeed expression of active Cdc42 (GFP-Cdc42Q61L) rescued apical targeting of podocalyxin () and single lumenogenesis in cysts with Rab8a or Rab11a knockdown (). These data support the conclusion that Cdc42, regulated by Rab8a/11a, is required for apical transport of podocalyxin. Taken together, Rab11a regulates a molecular network directing the apical polarity and trafficking machineries to initiate de novo lumen formation.