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. 2010 Nov;12(11):1035-45.
doi: 10.1038/ncb2106. Epub 2010 Oct 3.

A molecular network for de novo generation of the apical surface and lumen

Affiliations

A molecular network for de novo generation of the apical surface and lumen

David M Bryant et al. Nat Cell Biol. 2010 Nov.

Abstract

To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is regulated by polarity complexes that are hypothesized to direct downstream events, such as polarized membrane traffic, although this interconnection is not well understood. We have found that Rab11a regulates apical traffic and lumen formation through the Rab guanine nucleotide exchange factor (GEF), Rabin8, and its target, Rab8a. Rab8a and Rab11a function through the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation through the Cdc42 GEF, Tuba. These components assemble at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical exocytosis during lumen formation, revealing an interaction between the machineries of vesicular transport and polarization.

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Conflict of interest statement

The authors declare that they have no competing financial interests

Figures

Figure 1
Figure 1. Characterization of MDCK cyst lumen initiation
(a–b) Development of polarity in MDCK cysts. Initially the apical marker podocalyxin (podocalyxin/gp135, red) is located at the extracellular matrix-contacting region of early aggregates (12 h after plating), while β-catenin (β-cat, green) is at cell-cell junctions. Polarity inversion then occurs (24 h) to form a preapical patch (PAP), with podocalyxin now at the cyst interior (arrow). The luminal space then opens (48 h). (b) Cartoon of cyst development from a. Black lines, plasma membrane; Red lines, apical surface; Blue, nuclei. (c–e) Transient localization of polarity and trafficking machinery to an Apical Membrane Initiation Site (AMIS). Sec8 (red) and Par3 (white) relocalize from puncta at the lateral most part of cell-cell contacts (arrowheads) in early aggregates with peripheral GFP-podocalyxin (green) (c), to sites of vesicle coalescence at the developing lumen (AMIS, d, arrow). Sec8 and Par3 then relocalizes to tight junctions (e, arrowheads) in cysts with an open lumen. Occludin initially localizes to the entire cell-cell contact (f), while aPKC localizes to the periphery with podocalyxin in early aggregates (i, arrowheads). Both Occludin and aPKC also converge at the AMIS (f, i), before redistributing to the edges of the lumen as the PAP forms (g, j, arrowheads). Both partially concentrate at the tight junctions in cysts with open lumens (h, k, arrowheads), though aPKC retains some luminal labeling. Blue, nuclei. Smaller panels in this and subsequent figures are higher magnifications of regions indicated. Bar, 20 μm.
Figure 2
Figure 2. The Rab8 and Rab11 GTPase families direct lumen initiation
Knockdown of select members of the Rab8 and Rab11 GTPase families (b, Rab8a; c, Rab8b; d, Rab11a; e, Rab25) revealed that in contrast to control RNAi cysts (a) with apical podocalyxin (red) and basolateral β-catenin (green), Rab knockdown resulted in the appearance of multi-lumens and vesicular podocalyxin accumulation (arrowheads). (f) Quantiation of single lumenogenesis at 48 h in cysts with either stable knockdown or overexpression of dominant-negative alleles (DN) of indicated Rab GTPases. Note requirement for Rab8 family (Rab8a, Rab8b), and select Rab11 family (Rab11a, Rab25), members in lumenogenesis. Line represents 0.75-fold single lumenogenesis, normalized to control levels. For all single lumenogenesis quantitation, values represent the average of ≥ three different experiments ± S.D., where *p <0.05, ***p<0.0001. Control, n = 868; Rab8a, n = 302; Rab8b, n = 312; Rab10, n = 336; Rab11a, n = 307; Rab11b, n = 1,528; Rab13, n = 315; Rab14, n = 331; Rab25, n = 310. (g–h) GFP-Rab11a was present on vesicles below the surface, initially very close to the AMIS marked by Par3 (g, arrow) in early aggregates, then underneath the apical surface, once lumens open and Par3 is at tight junctions (h, arrowheads). (i–m) Podocalyxin (red) and GFP-Rab11a (green) localization during cyst development. Initially podocalyxin (i, arrows) and a pool of GFP-Rab11a (i, yellow arrowheads) localized to the periphery, then internalized into GFP-Rab11a vesicles (j, k, white arrowheads), transiting to the nascent lumen (k–m, white arrows). The lumen then expands and GFP-Rab11a concentrates underneath the lumen (m, yellow arrowheads). Blue, nuclei. Smaller panels are higher magnifications of regions indicated. Bar, 20 μm.
Figure 3
Figure 3. A Rab11-Rabin8-Rab8 module governs apical transport and single lumenogenesis
(a) Rab11a recruits Rabin8 and Rab8a to subapical vesicles. Control MDCK, GFP-Rab11a (WT or S25N; green) cysts grown for 48 h were stained for either endogenous Rabin8 or Rab8a (both in red). Note diffuse vesicular labeling, with low-level subapical accumulation of Rabin8 and Rab8a (arrows) in control cysts. Arrowheads indicate strong co-recruitment and clustering of Rabin8 and Rab8a vesicles, co-labelled for GFP-Rab11aWT, to the subapical region, but not with the S25N mutant. (b) Rabin8 expression occurred as two bands, at the predicted MW of its α and β isoforms. Knockdown of the α isoform with two shRNAs (Rabin8_4, Rabin8_5) revealed reduction of Rabin8α was accompanied by upregulation of the β isoform. GAPDH was used as a loading control. (c–e) Lumogenesis and apical podocalyxin transport require Rabin8α. Rabin8α knockdown caused the appearance of multi-lumens, the intracellular accumulation of a pool of podocalyxin (c, red, arrows), and a significant decrease in single lumenogenesis (e) in cysts at 48 h. Expression of RNAi-resistant human GFP-Rabin8α WT (green) rescued luminal targeting of podocalyxin and GFP-Rabin8α (d, arrows), and restored single lumenogenesis (e). Two different GEF domain mutants (L196A, F201A) were unable to rescue single lumenogenesis (e) or podocalyxin surface targeting (d), instead coaccumulating with podocalyxin on vesicles (d, F201A, arrowheads) below the cell surface marked by F-actin (blue). Single lumenogenesis quantitation values represent the average of ≥ three different experiments ± S.D., where *p <0.05, **p<0.001. Control, n = 312; Rabin8α KD, n = 340; Rabin8α KD + GFP-hRabin8α WT, n = 316; Rabin8α KD + GFP-hRabin8α L196A, n = 317; Rabin8α KD + GFP-hRabin8α F201A, n = 328. Smaller panels are higher magnifications of regions indicated. Bar, 20 μm.
Figure 4
Figure 4. The exocyst and Par3/aPKC regulate lumenogenesis
(a–d) Sec15A is required for AMIS formation. In contrast to control cysts (a) with apical podocalyxin and basolateral β-catenin, knockdown of Sec15A via two separate shRNAs (Sec15A_2, Sec15A_5) revealed a marked decreased in single lumenogenesis at 48 h (d), and the striking intracellular accumulation of a pool of podocalyxin (b, arrows) in GFP-Rab11a vesicles (c, arrowheads), and mistargeting of podocalyxin to β-catenin-positive membranes (b, Sec15A_2 is depicted). Single lumenogenesis quantitation values represent the average of ≥ three different experiments ± S.D., where **p<0.001. Scramble, n = 334; Sec15A_2, n = 355; Sec15A_5, n = 345. (e–h) Par3 targeting to the AMIS requires the exocyst. Par3 is targeted to tight junctions in control cysts with open lumens (e), but is mostly lost at regions of podocalyxin vesicle coalescence upon Sec15A knockdown (f, arrowheads), though some Par3 can be recruited to abnormal lumens. Note that expression of RNAi-resistant GFP-Sec15AWT (g), but not a Rab-uncoupled mutant (h, N691A), in cysts with endogenous Sec15A knockdown rescued both single lumenogenesis and surface targeting of podocalyxin and Par3 (arrows). (i–n) The Par3/aPKC complex is required for lumen initiation. Knockdown of Par3 using two different shRNAs (l, Par3_3, Par3_4) resulted in the accumulation of podocalyxin in vesicles (i, red, arrows), co-labeled for GFP-Rab11a (j, green), beneath the surface (outlined by β-catenin in i, green), and a marked reduction in single lumenogenesis (l). Also upon Par3 knockdown, GFP-podocalyxin (k, pseudo-colored red) is at multi-lumens and intracellular vesicles near the surface (arrows), whereat Sec8 (green) failed to be recruited to form the AMIS. Similarly, inhibition of aPKCs (n, aPKC-PS) resulted in the accumulation of podocalyxin in Apple-Rab11a vesicles (arrowheads), beneath the surface marked by β-catenin. In contrast, control cysts (m) displayed apical podocalyxin, basolateral β-catenin and subapical Apple-Rab11a. In addition, aPKC-PS (n) also blocked internalization of peripheral podocalyxin in some cells (arrows). Single lumenogenesis quantitation values represent the average of ≥ three different experiments ± S.D., where *p <0.05, **p<0.001, and n ≥ 100 cysts/replicate. Scramble, n = 326; Par3_3, n = 348; Par3_4, n = 369. Smaller panels are higher magnifications of regions indicated. Bar, 20 μm.
Figure 5
Figure 5. Tuba and Cdc42 regulate transport from Rab8a/11a vesicles
(a–g) Cdc42 associates with Rab11a vesicles. In cysts with an open lumen (a), whilst the majority of GFP-Cdc42 was cytoplasmic, a pool of GFP-Cdc42 (green) localized to Apple-Rab11a-positive (red) subapical vesicles (yellow, see arrowheads). Expression of activated Cdc42 (GFP-Cdc42Q61L; b, green) in cysts with an expanded lumen revealed GFP-Cdc42Q61L localization at cell-cell contacts, and the luminal region marked by podocalyxin (red, arrowheads). In early cysts with peripheral podocalyxin (red) (c), GFP-Cdc42Q61L localized all over the surface. When podocalyxin was internalized into Rab11a vesicles (d, vesicular podocalyxin) and concentrated at the AMIS (e), GFP-Cdc42Q61L now extensively overlapped with these vesicles (arrowheads). As the PAP (f) and lumen (g) formed, podocalyxin and Rab11a no longer overlapped, whilst GFP-Cdc42Q61L maintained some overlap with both (arrows). (h–n) Tuba and Cdc42 are required for transport from Rab8a/11a vesicles. In contrast to control cysts (h) with apical podocalyxin (red) and either basolateral β-catenin (h–j, green), or subapical GFP-Rab11a and Rab8a (k–m, green and blue, respectively), Cdc42 (i,l) or Tuba (j,m) knockdown resulted in the appearance of multi-lumens and the accumulation of podocalyxin in Rab8a/Rab11a-positive vesicles (l–m, arrowheads). Quantitation of Cdc42 and Tuba knockdown (n; verified by two different shRNAs each) revealed strongly perturbed single lumenogenesis both in MDCK or MDCK stably overexpressing GFP-Rab11a cysts at 48 h. Single lumenogenesis quantitation values represent the average of ≥ three different experiments ± S.D., where **p<0.001, ***p <0.0001. Control, n = 311; Cdc42_2, n = 321; Cdc42_3, n = 307; Tuba_1, n = 319; Tuba_2, n = 319; GFP-Rab11a Control, n = 600; GFP-Rab11a + Cdc42_2, n = 316; GFP-Rab11a + Tuba_1, n = 308. Blue in a–b, h–j, nuclei (nuc); c–g, Rab11a; k–m, Rab8a. Smaller panels are higher magnifications of regions indicated. Bar, 20 μm.
Figure 6
Figure 6. Rab8a/11a regulate Cdc42 during apical transport
(a–e) Rab8a-Rab11a control Cdc42 activation and targeting. Global Cdc42-GTP levels upon either Rab knockdown (a) or stable expression of activated Rabs (b) were determined as described in Methods. Values represent the average of three different experiments ± S.D., where *p<0.05. Notably knockdown of Rab8a, but not Rab11a, decreased global GTP levels of Cdc42 (a), while Rab8aQ67L, but not Rab11aQ70L, strongly increased GTP-Cdc42 levels (b). Thus Rab8a modulates global Cdc42 activation. Examination of localization of a PBD-YFP probe to detect activated Cdc42 (and possibly also Rac) revealed that PBD-YFP targeted to surface domains in control cells (c), but particularly the luminal surface (marked by podocalyxin in red; luminal localization denoted by yellow arrowhead). Rab8a knockdown abrogated global PBD-YFP surface labeling (d). Notably, Rab11a depletion blocked luminal, but not lateral, targeting of PBD-YFP (e), suggesting that Rab11a targets luminal Cdc42 via Rab8a. (f–i) Correction of Rab8a/Rab11a knockdown by active Cdc42 overexpression. In MDCK cysts, Rab8a or Rab11a knockdown decreased single lumenogenesis (i). In contrast, over-expression of active Cdc42 (GFP-Cdc42Q61L), which localized to the plasma membrane and was enriched at the lumen (f), in Rab8a (g) or Rab11a (h) knockdown cysts restored apical targeting of podocalyxin (f–g) and single lumenogenesis to levels resembling control cells (f, i). Blue, nuclei (nuc). Lumenogenesis values represent the average of ≥ three different experiments ± S.D., where *p<0.05. Control, n = 868; Rab8a KD, n = 302; Rab11a KD, n = 307; GFP-Cdc42Q61L Control, n = 343; GFP-Cdc42Q61L + Rab8a KD, n = 319; GFP-Cdc42Q61L + Rab11a KD, n = 308. Smaller panels are higher magnifications of regions indicated. Bar, 20 μm.
Figure 7
Figure 7. A molecular network for de novo lumen generation
(a) Cartoon diagram of the different stages of lumenogenesis and apical polarization in MDCK cysts. Initially podocalyxin is localized to the periphery of cysts (Early Aggregate), before internalization into Rab8a/11a-positive vesicles, and delivery to the AMIS (Apical Membrane Initiation). As podocalyxin at the apical domain and tight junctions become separately localized, the AMIS progresses to a PAP (Pre-Apical Patch), representing the early stages of apical-basal polarization. Expansion then allows opening of the luminal space (Open Lumen). Note the co-accumulation of polarization and trafficking machinery at the AMIS, despite varying localization during other stages of lumenogenesis. Red lines, podocalyxin; black lines, plasma membrane; grey ovals, nuclei; brown ovals, tight junctions; brown rectangle, AMIS; L, lumen. (b) A model cartoon diagram of the molecular network involved in delivery of apical vesicles (podocalyxin) to the AMIS during apical membrane initiation.

Comment in

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