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First published online 25 April 2006
doi: 10.1242/jcs.02938

Journal of Cell Science 119, 2107-2118 (2006)
Published by The Company of Biologists 2006
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Lgl mediates apical domain disassembly by suppressing the PAR-3-aPKC-PAR-6 complex to orient apical membrane polarity

Tomoyuki Yamanaka1,2,*,{ddagger}, Yosuke Horikoshi1,*, Natsuko Izumi1,2, Atsushi Suzuki1, Keiko Mizuno1 and Shigeo Ohno1,§

1 Department of Molecular Biology, Yokohama City University, Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
2 Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences, 1-7-29, Suehiro-cho, Turumi-ku, Yokohama 230-0045, Japan


Figure 1
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  		Fig. 1. Knockdown of mLgl retains apical proteins and F-actin at cell periphery in depolarized MDCK cells. (A) Western blot analysis of control cells (21-1 and 21-7) and mLgl-1-mLgl-2 double-kd MDCK cell clones (24-15 and 24-16) with antibodies against indicated proteins. mLgl-1 and mLgl-2 expression levels were specifically reduced in clones 24-15 and 24-16. The mLgl knockdown retains the apical proteins and F-actin at the cell periphery in depolarized MDCK cells. (B) Confluent monolayer of control cells or mLgl kd cells on permeable filter were incubated with LC medium for 20 hours, after which cells were fixed and co-stained with anti-gp135 antibody and Rhodamine-phalloidin. Arrowheads indicate cells with intracellular gp135 and F-actin staining. (C) Cells treated as in B, stained with antibodies against ezrin and ZO-1. Arrowheads indicate cells with intracellular ezrin staining. (D) Quantification of the cells with peripheral gp135 staining. The percentage of total cells is shown. Values are mean (±s.d.) of three independent experiments (21-1 and 24-15) or mean of two independent experiments (24-16). Each photograph represents the projected views of optical sections from the apical to the basal membranes of cells. Bars, 20 µm.

 

Figure 2
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  		Fig. 2. Knockdown of mLgl suppresses disassembly of apical proteins during the depolarization of MDCK cells. Confluent monolayers were incubated with mouse anti-gp135 antibody for 1 hour. After washing out the antibody, the cells were incubated with LC medium for the indicated time. The cells were fixed, permeabilized and stained with Alexa Fluor 488-conjugated anti-mouse IgG together with Rhodamine-phalloidin. (A) Projected views of optical sections from the apical to the basal membranes of cells. (B) Confocal z-sections. Arrowheads indicate cells with intracellular gp135 and F-actin staining. Bars, 20 µm.

 

Figure 3
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  		Fig. 3. Knockdown of mLgl increases interaction of aPKC-PAR-6 with PAR-3 or Cdc42. (A) Confluent monolayer of control cells (21-1 or 21-7) or mLgl kd cells (24-15 or 24-16) was lysed with lysis buffer and clarified by centrifugation. The supernatant fraction (Sup, lower panels) containing total 1 mg protein was subjected to immunoprecipitation with anti-PAR-6ß antibody and precipitated proteins were analyzed by western blot (IP, upper panels). Three independent experiments were performed for each clone and numbered as 1, 2 and 3. Notice that the co-precipitation of PAR-3 or Cdc42 with PAR-6 was increased in mLgl kd cells. (B-D) Quantification of the amount of PAR-3 (B), aPKC{lambda} (C) or PAR-6ß in IP fraction. The amount of PAR-3 increased significantly in both mLgl kd cells (*P<0.005, **P<0.001), whereas the amount of aPKC{lambda} or PAR-6ß was not changed in these cells.

 

Figure 4
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  		Fig. 4. PAR-3 knockdown decreases peripheral apical proteins in depolarized MDCK cells. (A) Western blot analysis of control cells (11-10 and 25-2) and PAR-3 kd cells (13-32 and 13-33) with antibodies of indicated proteins. Notice that the PAR-3 expression level was specifically reduced in clones 13-32 and 13-33. (B) A confluent monolayer of each cell on a permeable filter was incubated with LC medium for 20 hours, after which the cells were fixed and co-stained with anti-gp135 antibody and Rhodamine-phalloidin. (C) Cells treated as in B, stained with antibodies against ezrin and ZO-1. (D) Quantification of cells with cell peripheral gp135 staining. The percentage of total cells is shown. Values are mean (±s.d.) of three independent experiments (11-10 and 13-32) or mean of two independent experiments (13-33). Each photograph represents the projected views of optical sections from the apical to the basal membranes of cells. Bars, 20 µm.

 

Figure 5
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  		Fig. 5. Knockdown of PAR-3 accelerates disassembly of apical proteins during depolarization of MDCK cells. Confluent monolayers were incubated with gp135 mouse antibody for 1 hour. After washing out the antibody, the cells were incubated with LC medium for the indicated time. The cells were fixed, permeabilized and stained with Alexa Fluor 488-conjugated anti-mouse IgG together with rhodamine-phalloidin. (A) Projected views of optical sections from the apical to the basal membranes of cells. (B) Confocal z-sections. Arrowheads indicate the cells with intracellular gp135 and F-actin staining. (C) Quantification of cells with aggregated gp135 staining after incubation with LC medium for 5 hour. The percentage of total cells is shown. Values are mean (±s.d.) of three independent experiments. Bars, 20 µm.

 

Figure 6
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  		Fig. 6. Overexpression of a dominant-negative mutant of aPKC{lambda} enhances redistribution of gp135 and suppresses phenotype of mLgl knockdown. (A) mLgl kd cells (24-15) or control cells (21-1) on permeable filters were infected with adenovirus expression vectors encoding LacZ, wild-type aPKC{lambda} (aPKC{lambda} wt) or dominant-negative aPKC{lambda} (aPKC{lambda} dn) for 2 hours. The cells were then incubated with LC medium for 18 hours. (A) Cells were fixed and double stained with anti-aPKC{lambda} and anti-gp135 antibodies. Notice that the overexpression of aPKC{lambda} dn but not aPKC{lambda} wt in the control or the mLgl kd cells decreases the peripheral gp135 staining in depolarized cells. (B) Quantification of the cells with cell peripheral gp135 staining in A. Each photograph represents the projected views of confocal sections. Bars, 20 µm.

 

Figure 7
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  		Fig. 7. Knockdown of mLgl inhibits re-establishment of intercellular apical domains induced by collagen-gel overlay whereas knockdown of PAR-3 accelerates it. (A) mLgl kd cells (24-15 and 24-16) or control cells (21-1 or 21-7) were cultured on collagen-coated glass for 24 hours, after which the cells were overlaid with collagen gel for 48 hours. The cells were then fixed and triple stained with anti-gp135, anti-ZO-1 and anti-E-cadherin antibodies. (B) PAR-3 kd cells (13-32) or control cells (11-10) were cultured on collagen-coated glass for 24 hours, after which the cells were overlaid with collagen gel for 10.5 or 24 hours. The cells were fixed and double stained with anti-gp135 and anti-ZO-1 antibodies. (C) Quantification of colonies with concentrated gp135 staining at intercellular regions at indicated times after collagen-gel overlay. Scale bars represent 20 µm. Photographs represent the projected views of confocal sections. Bars, 20 µm (A) or 50 µm (B).

 

Figure 8
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  		Fig. 8. Knockdown of mLgl mislocalizes apical gp135 to outer surface of cyst where cells face collagen gel at initial stage of cystogenesis. mLgl kd cells (#1) or control cells were suspended in collagen gel and cultured for 2 days. Then, cysts were fixed and stained with anti-gp135 and anti-ß1 integrin antibodies. The respective major examples are shown. Notice that gp135 staining was detected at the outer surface of cell aggregates derived from Lgl kd cells, whereas gp135 staining was detected at the inner surface of cell aggregates from control cells. Each photograph represents the single x-y confocal section. Bars, 20 µm.

 

Figure 9
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  		Fig. 9. Knockdown of mLgl induces formation of abnormal cell aggregates without obvious lumens during MDCK cell cystogenesis. (A) mLgl kd cells (#1) or control cells were suspended in collagen gel and cultured for the indicated time. Then cysts were fixed and stained with rhodamine-phalloidin and anti-E-cadherin antibody. Cell nuclei were stained with TOPRO3. Notice that mLgl knockdown affected the actin distribution during cystogenesis, leading to the formation of cell aggregates without lumens. (B) Quantification of abnormal cell aggregates in total cysts at day 7 is shown. Values are the mean (±s.d.) of three independent experiments. Notice that about 80% of cysts of both mLgl kd cells (#1 and #2) formed abnormal cell aggregates. (C) mLgl kd (#1) or control cells were suspended in collagen gel and cultured for 7 days, and then were fixed and stained with anti-PAR-6ß and anti-E-cadherin antibodies. Notice that the distribution of PAR-6ß to the inner surfaces of the cysts was affected in cysts of mLgl kd cells. Each photograph represents the confocal single x-y section. Bars, 20 µm.

 

Figure 10
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  		Fig. 10. Schematic model of mechanism of mLgl-mediated disassembly of apical domain by suppressing PAR-3-aPKC-PAR-6 complex activity. mLgl suppresses the activity of PAR-3-aPKC-PAR-6 complex by the competitive interaction of aPKC-PAR-6 with PAR-3 and Cdc42. This leads to the disassembly of the apical membrane domain by internalization of apical proteins and orientation of apical membrane polarity. mLgl seems to also be involved in the regulation of tight junction and adherens junction disassembly.

 

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© The Company of Biologists Ltd 2006