suppression of rac1 activity at the apical membrane of mdck cells is essential...

EMBO reports - Peer Review Process File - EMBOR-2011-35150

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Manuscript EMBOR-2011-35150 Suppression of Rac1 Activity at the Apical Membrane of MDCK cells is Essential for Cyst Structure Maintenance Shunsuke Yagi, Michiyuki Matsuda and Etsuko Kiyokawa Corresponding author: Etsuko Kiyokawa, Kyoto University Review timeline: Submission date: 17 June 2011 Editorial Decision: 14 July 2011 Correspondence: 25 July 2011 Revision received: 07 October 2011 Editorial Decision: 11 November 2011 Revision received: 30 November 2011 Accepted: 01 December 2011 Transaction Report: (Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity, letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this compilation.)

1st Editorial Decision 14 July 2011

Thank you for the submission of your research manuscript to EMBO reports. It has been sent to three referees, and so far we have received reports from two of them, which I copy below. As both referees feel that the manuscript is interesting and recommend that you should be given a chance to revise it, I would like to ask you to begin revising your manuscript according to the referees' comments. Please note that this is a preliminary decision made in the interest of time, and that it is subject to change should the third referee offer very strong and convincing reasons for this. As soon as we receive the third report it will be forwarded to you. As you will see, both referees are mainly concerned with the clarity and the presentation of the results. They also feel that some results deserve further discussion. Referee #1 has three concerns. First, s/he feels that the presentation of data shown in figure 1 is not clear enough. Second, he thinks the statistical analysis of the data presented in figure 2 is insufficient. Please remember that error bars, statistical significance and statistical tests used, must be included in the figure legends when data requiring them is presented. Finally, s/he thinks that the data concerning the comparison of the effects of TIAM1 vs. Beta2-Chimaerin is not properly explained and may need further experiments. Referee #2 main concern refers to FRET analysis. S/he thinks that more information on the procedures and controls should be provided.



Given the reviewers constructive comments and the potential interest of your study, I would like to give you the opportunity to revise your manuscript, with the understanding that the referees' concerns must be fully addressed and their suggestions (as detailed above and in their reports) taken on board. Acceptance of the manuscript will depend on a positive outcome of a second round of review and I should also remind you that it is EMBO reports policy to allow a single round of revision only and that, therefore, acceptance or rejection of the manuscript will depend on the completeness of your responses included in the next, final version of the manuscript. I look forward to seeing a revised version of your manuscript when it is ready. Yours sincerely, Editor EMBO reports REFEREE REPORTS: Referee #1: In this study Yagi et al. have used FRET in MDCK cells grown in matrigel to show that the activity of exogenous biosensors Raichu-Rac1, -RhoA, and -Cdc42 GTPases is different in the two distinct plasma membrane domains of these polarized cells. In particular, they find that the polarity of Raichiu-Rac1 is not evident at an early stage, while at a later stage of cystogenesis Raichiu-Rac1 activity appears higher at the lateral plasma membrane compared to the apical plasma membrane. The authors have then utilized a rapamycin-inducible Rac1 activity manipulating system based on the recruitment of fragments of either the Rac-GAP Beta2-chimaerin or the Rac GEF Tiam1 to affect Rac activation. From the data presented the authors conclude that Rac1 suppression at the apical membrane may be essential for the maintenance of cyst structure. Several points need to be addressed carefully to make the presentation of the data more clear: Figure 1G-I. The description of this part is fast and not clear. Authors should help the reader understand better why did they decide to show these data. They should also detail more what one should look at, and show or refer more precisely to proper controls. Figure 2C: the authors show that both GAP and GEF affect exogenous Rac activation. Is the 6% increase and decrease observed in Fig. 2C and 2D, respectively, statistically significant? If so, how has this been determined? The information is missing in the legend. Also, activation has been determined on cells plated on glass, while the rest of the analysis is performed on cysts. The authors should explain the reason for this choice in the text. Figure 2E and F: the way these figures are presented in the text is not very clear. The authors stress the stronger effects of the GEF Tiam1 on the maintenance of cysts, but the data shown indicate that also beta2-chimaerin affects the process, although differently. This part should be explained comprehensively and more clearly. In Fig.4D, staining for ZO1 is shown as an endogenous marker for tight junctions in Tiam1 cysts {plus minus} dimerizer; the same staining should be shown for tight junctions in bet2-chimaerin cysts {plus minus} dimerizer. Most results are based on overexpression experiments: does inhibition of endogenous Rac-GAPs (by RNAi) affect Rac activation and therefore cyst formation/maintenance? Referee #2: This is an interesting study in which the authors have used some innovative approaches to



investigate how a change in the localization of active Rac1 leads to dramatic perturbations of epithelial cyst morphology. They present a descriptive, yet interesting set of findings in this regard. A few clarifications are warranted and some questions/concerns should be addressed to fully justify the conclusions drawn. 1. The localization of Cdc42 to the apical membrane and Rac1 to the basolateral membrane of mature cysts or tubules is in accordance with previous reports (see Belmonte et al, Cell 2007 and Tushir et al, EMBO J. 2007, among others). It is unclear why the authors describe some of their findings in this regard as "unexpected". 2. It would be beneficial and greatly increase the clarity of the data if the researchers indicated the specific membrane locations they were using to quantify the FRET signal, particularly with regard to the point that there is essentially "uniform" activity over the membrane of immature cysts. Figure 1a shows relatively large signal variation over the region that would seem to be the apical patch. Given how weak FRET signals can be, the authors could show a magnified image of a region of a mature cyst to better indicate the differences in active Rac1-GTP levels in the various membrane domains. 3. It would also be helpful of the authors would present at least one complete set of images (supplemental would be fine) showing the CFP, YFP, and ratio image. 4. That spindle pole misalignment contributes to the altered morphology of the cyst is valid. However, these data could also easily be interpreted as the morphological drive to produce a filled cyst causes the angle of the spindle pole to be abnormal. Are Cdc42-GTP levels perturbed in these cysts? Also of note is that there is proliferation/invasion into the matrix (see Figure 2E and others). This latter point should also be addressed. The activation of Rac1 seems to lead to a significant overall increase in cell growth. 5. How does the GTP-pool of Rac1 in FKBP-beta2-chimaerin expressing cysts compare to untransfected cysts? The authors should discuss how their data compares with earlier findings of Rac1 inhibition on MDCK cyst development (O'Brien et al, Nat Cell Biol. 2001.) 6. While it seems clear that inducing activation of Rac1 at the apical membrane is accompanied by a disruption of the tight junction formation, the link between the two events is unclear from this study. den Elzen et al., MBC, 2009 report that it is more the positioning of the cadherin-based adhesion, or visa vie the neighboring cells, that dictates the position of the mitotic spindle than does the position of the tight junction. It is equally plausible that the redistribution of the Tiam1 is signaling both for a disruption of cell polarity and the hyperproliferative effects and that those events cause a disruption in tight junction integrity, spindle pole orientation, and cyst morphology. Minor Issues: ν Page 7, paragraph continuing from page 6, intersectin misspelled. ν Figure 4 D check image labels.

Correspondence 25 July 2011

Thank you again for the submission of your research manuscript to EMBO reports. As I mentioned in my previous letter, your manuscript was sent to three referees and we have just received the third report, which I copy below. As all three referees feel that the manuscript is interesting and recommend that you should be given a chance to revise it, I would like to ask you to continue revising your manuscript according to the referees' comments. As you can see below, referee #3 points out that the experimental evidence provided does not support the major claim of the manuscript. The referee remarks that explanations alternative to localized Rac1 activation in the apical domain could account for the phenotypes observed and that further experimental controls need to be added. S/he is also concerned with the interpretation of the data regarding luminal filling of the cysts.



I look forward to seeing a revised version of your manuscript when it is ready. Yours sincerely, Editor EMBO reports REFEREE REPORT: Referee #3: The manuscript by Yagi and colleagues proposes that activation of Rac selectively at the basolateral domain is necessary for appropriate epithelial morphogenesis during cyst formation. The authors design and optimize an interesting system to locally activate or inactivate Rac at the plasma membrane by recruiting Tiam1 or b2-chimerin using FKB2. The data is of high quality technically and figures are nicely done and structured, which makes it easier to understand the complexities of the system. The adaptation of this method may be of interest to different readers that face the challenge of understanding the spatial and temporal regulation of GTPase signalling. The main conclusion of the manuscript is that Rac activation has to be excluded from the apical domain to allow appropriate cyst morphogenesis. However, in this reviewer's opinion, this has not been conclusively demonstrated by the data. I would therefore not recommend this manuscript for publication in its current form. A number of other explanations could account for the loss of polarity and cyst morphogenesis upon aberrant Rac up-regulation. First, Rac activation all around cell surface (induced by Tiam-1) should be compared to Rac activation at the apical domain only or at the bottom of the cells only. It could well be that an increase in Rac activity levels is sufficient to induce the effects reported. Alternatively, GEFs are predicted to provide specificity of signalling downstream of small GTPases. Rac activation at the surface specifically by Tiam-1 may lead to cysts full of cells, while Rac activation at the surface by another GEF may disrupt polarity but induce a different phenotype. Another point is that the attempts to define the cellular processes responsible for cysts being filled with cells led to the identification of two processes that could participate: changes in spindle orientation and mislocalization of tight junction proteins. The manuscript does not demonstrate how these two distinct processes might be linked or a causal effect between their perturbation and abnormal cysts. Proliferation, another important Rac-dependent process, could also be enhanced when Rac is inappropriately activated and lead to the exclusion of cells from the epithelium towards the lumen. 1st Revision - authors' response 07 October 2011

Referee #1: In this study Yagi et al. have used FRET in MDCK cells grown in matrigel to show that

the activity of exogenous biosensors Raichu-Rac1, -RhoA, and -Cdc42 GTPases is

different in the two distinct plasma membrane domains of these polarized cells. In

particular, they find that the polarity of Raichiu-Rac1 is not evident at an early stage,

while at a later stage of cystogenesis Raichiu-Rac1 activity appears higher at the lateral

plasma membrane compared to the apical plasma membrane. The authors have then

utilized a rapamycin-inducible Rac1 activity manipulating system based on the

recruitment of fragments of either the Rac-GAP Beta2-chimaerin or the Rac GEF Tiam1

to affect Rac activation. From the data presented the authors conclude that Rac1

suppression at the apical membrane may be essential for the maintenance of cyst

structure.

Several points need to be addressed carefully to make the presentation of the data more

clear:

We thank the reviewer for helpful suggestions. Figure 1G-I. The description of this part is fast and not clear. Authors should help the

reader understand better why did they decide to show these data. They should also

detail more what one should look at, and show or refer more precisely to proper

controls.

According to the suggestion, we re-read the previous manuscript and found that we inappropriately emphasized HGF in this part. We wanted to show the small GTPase activities in the tubules, because cysts and tubules are representative structures in epithelial organs. We rewrote this part to explain our motivation (Page 4, line 17-18, page 5, line 1-8). The different activity distributions of the three members of the Rho- GTPase family in the tubular structure serve as excellent controls for each other, excluding intermolecular FRET. Figure 2C: the authors show that both GAP and GEF affect exogenous Rac activation.

Is the 6% increase and decrease observed in Fig. 2C and 2D, respectively, statistically

significant? If so, how has this been determined? The information is missing in the

legend.

According to this suggestion, we statistically analyzed the data to show the 6% difference is significant. The details of the method have been included in the legend (Page

15, line 26). Also, activation has been determined on cells plated on glass, while the rest of the

analysis is performed on cysts. The authors should explain the reason for this choice in

the text.

Since this is the first report of rapamycin-induced Rac1 activation, it is important to validate this system in tissue culture cells plated on plastic dishes. By bath application of rapamycin, the effect of dimerizer became evident within a few minutes. After validation in the two-dimensional situation, we proceed to show the data obtained in the Matrigel culture (Supplementary Fig S3, in the previous and present manuscripts). We have modified the text accordingly.

Figure 2E and F: the way these figures are presented in the text is not very clear. The

authors stress the stronger effects of the GEF Tiam1 on the maintenance of cysts, but the

data shown indicate that also beta2-chimaerin affects the process, although differently.

This part should be explained comprehensively and more clearly.

According to this comment, we rewrote the paragraphs related to this figure ( line 8 to page 7, line 12). In Fig.4D, staining for ZO1 is shown as an endogenous marker for tight junctions in

Tiam1 cysts {plus minus} dimerizer; the same staining should be shown for tight

junctions in bet2-chimaerin cysts {plus minus} dimerizer.

Following up on this comment, we performed ZO1 immunostaining in 2-chimaerin expressing cysts with or without dimerizer. As expected, ZO-1 localized to tight junctions, as in the parental MDCK cysts (Figure for the reviewer). Because this is expected from the modest effect of Rac1 inactivation and because of the space constraints, we refrained from referring to this data in the paper. Most results are based on overexpression experiments: does inhibition of endogenous

Rac-GAPs (by RNAi) affect Rac activation and therefore cyst formation/maintenance?

It has been reported that more than 30 GAP proteins for Rac1 exist (Tcherkezian and Lamarche-Vane 2007). Recent papers successfully identified the GEF responsible for a multilumen phenotype by RNAi screening (Rodriguez-Fraticelli et al. 2010;Qin et al. 2010). In this study, we focused on the role of Rac1 activity in the late stages of cystogenesis. Hence, we could not adopt RNAi-mediated screening, which affects the early stages of cystogenesis. One might perform tetracycline-mediated inducible shRNA

screening; however, we are afraid that it is going to require enormous work to cover all of the GAPs for Rac1. Since we still do not have enough information about GAP expression in MDCK cells, we believe our work will help other researchers to identify GEFs or GAPs for Rac1 to maintain the cyst. Ref: Qin Y, Meisen WH, Hao Y, Macara IG (2010) Tuba, a Cdc42 GEF, is required for

polarized spindle orientation during epithelial cyst formation. J Cell Biol, 189: 661-669.

Rodriguez-Fraticelli AE, Vergarajauregui S, Eastburn DJ, Datta A, Alonso MA, Mostov K, Martin-Belmonte F (2010) The Cdc42 GEF Intersectin 2 controls mitotic spindle orientation to form the lumen during epithelial morphogenesis. J Cell Biol, 189: 725-738.

Tcherkezian J, Lamarche-Vane N (2007) Current knowledge of the large RhoGAP family of proteins. Biol. Cell, 99: 67-86.

Referee #2: This is an interesting study in which the authors have used some innovative approaches

to investigate how a change in the localization of active Rac1 leads to dramatic

perturbations of epithelial cyst morphology. They present a descriptive, yet interesting

set of findings in this regard. A few clarifications are warranted and some

questions/concerns should be addressed to fully justify the conclusions drawn.

We thank the reviewer for the favorable comments and helpful suggestions. 1. The localization of Cdc42 to the apical membrane and Rac1 to the basolateral

membrane of mature cysts or tubules is in accordance with previous reports (see

Belmonte et al, Cell 2007 and Tushir et al, EMBO J. 2007, among others). It is

unclear why the authors describe some of their findings in this regard as "unexpected".

There is a famous dogma that Cdc42 activates Rac1 (Nobes & Hall, 1995). In agreement with this theory, we also found that Rac1 and Cdc42 are activated at lamellipodia in EGF- or NGF-stimulated cells (Itoh et al. 2008; Aoki et al. 2005). Furthermore, many Dbl-family GEFs activate both Rac1 and Cdc42. Therefore, it was “unexpected” for us to find that the activities of Rac1 and Cdc42 are spatially different in the three dimensional structure. However, we admit that this observation may not be surprising for those who know Mostov’s paper showing the different spatial activation pattern of Rac1 and Cdc42 in cysts. Therefore, we have deleted this sentence (Page 4, line 25). 2. It would be beneficial and greatly increase the clarity of the data if the researchers

indicated the specific membrane locations they were using to quantify the FRET signal,

particularly with regard to the point that there is essentially "uniform" activity over the

membrane of immature cysts. Figure 1a shows relatively large signal variation over

the region that would seem to be the apical patch. Given how weak FRET signals can be,

the authors could show a magnified image of a region of a mature cyst to better indicate

the differences in active Rac1-GTP levels in the various membrane domains.

According to this comment, we prepared magnified images (Figure 1A the lower panels, in the revised manuscript). 3. It would also be helpful of the authors would present at least one complete set of

images (supplemental would be fine) showing the CFP, YFP, and ratio image.

In accordance with this suggestion, we added images of CFP and YFP (Supplemental Figure S1 B-D, in the revised manuscript). In Supplementary Figure S1E, we also added the method for apical and lateral FRET ratio measurement, hoping for better understanding of our analysis.

4. That spindle pole misalignment contributes to the altered morphology of the cyst is

valid. However, these data could also easily be interpreted as the morphological drive

to produce a filled cyst causes the angle of the spindle pole to be abnormal.

We observed the misalignment of the spindle pole before the abnormal monolayer becomes evident (Figure 3B and Supplemental Movie 2). We therefore conclude that spindle pole misalignment caused luminal cell filling. We have included this comment (Page 7 line 23-25).

Are Cdc42-GTP levels perturbed in these cysts? Using the Cdc42 FRET biosensor, we found that expression of FKBP-Tiam1 per se induced Cdc42 activation at the lateral membrane. However, upon dimerizer treatment, we could not observe further activation of Cdc42. We therefore excluded the possibility that Cdc42 is involved in the Rac1-induced phenotype. We have also included this note in the revised manuscript (page6, line 18-22). Also of note is that there is proliferation/invasion into the matrix (see Figure 2E and

others). This latter point should also be addressed. The activation of Rac1 seems to

lead to a significant overall increase in cell growth.

We could see cells invading into the Matrigel in Figure 2E, but not in Figure 3B, 3E, 4B, or 4D. Thus, this finding is not reproducible. It is possible that Rac1 activation forced cells to invade; however at this moment, we would like to refrain from referring to this observation.

To examine the relationship between Rac1 activation and proliferation, we counted cell number upon dimerizer-induced Rac1 activation and inactivation (Page 6 line 25 to page 7 line 3, and Supplementary Figure S4). Results indicated that Rac1 activation did not accelerate cell proliferation, while Rac1 inactivation significantly decreased it. We also confirmed that dimerizer treatment alone did not affect cell proliferation. From these results we discount the possibility that Tiam1-induced Rac1 activation induced the morphological disorder by upregulating cell proliferation.

5. How does the GTP-pool of Rac1 in FKBP-beta2-chimaerin expressing cysts

compare to untransfected cysts?

In the cysts, we could not observe a remarkable decrease in Rac1 activity upon 2-chimaerin translocation. This is probably due to the sensitivity of our current biosensor. The authors should discuss how their data compares with earlier findings of Rac1

inhibition on MDCK cyst development (O'Brien et al, Nat Cell Biol. 2001.)

We appreciate that the reviewer is interested in the phenotype of Rac1-inactivation. In the previous report (O'Brien et al. 2001), expression of dominant-negative Rac1 caused a selective inversion of the apical pole to the cyst periphery, accompanied by a failure of lumen formation. In agreement with this previous paper, we found that Rac1 inactivation by FKBP-chimaerin resulted in spheroid formation without lumen formation (Supplemental Figure S5 and Page 7, line13-16). Please note that O'Brien et

al. did not examine the role of Rac inhibition after cyst formation, even though they utilized a tetracycline-regulatable system. Thus, Rac1 inactivation perturbs cyst formation but does not have a significant effect once the cysts are matured. 6. While it seems clear that inducing activation of Rac1 at the apical membrane is

accompanied by a disruption of the tight junction formation, the link between the two

events is unclear from this study. den Elzen et al., MBC, 2009 report that it is more the

positioning of the cadherin-based adhesion, or visa vie the neighboring cells, that

dictates the position of the mitotic spindle than does the position of the tight junction.

We thank the reviewer for introducing the reference. In that paper (den Elzen et al. 2009), they observed that expression of dominant negative E-Cadherin (DN-E-Cad) disrupted adhesion-complex distribution and spindle orientation in a MDCK sheet (Figure 4). In the same situation, tight junctions and polarity were not affected (Figure 5b). They suggested that DN-E-Cad sequestered APC in the cytosol.

In the same paper, they found DN-E-Cad induced no obvious change of the mitotic spindle regulators LGN and NuMA, nor Dynein localization (Figure 8). Interestingly, it has been recently reported that LGN is necessary for proper spindle orientation in cysts (Zheng et al. 2010). This discrepancy might be brought about by a difference of the polarity formed in the 2- or 3-dimensions, or redundant pathways to maintain the spindle orientation. Since Cadherin localization was not examined in the 3-D cysts in this paper, we could not deny the possibility that tight junction disruption induced Cadherin-mediated cell-cell adhesion. Nevertheless, we assume that Cadherin, which localizes to the basolateral membrane, is not directly involved in the downstream

of Rac1-induced activation at the apical membrane. We tried to stain E-Cadherin using an E-Cadherin antibody (BD Transduction Laboratories, #610182); unfortunately, we could not observe positive staining, both in parent and FKBP-Tiam1 expressing cysts, while we could stain cysts with ZO-1 antibody (Figure 4D). Further experiments, such as identification of molecules connecting Rac1 activation at the apical membrane and tight junction disruption, will answer this question. We have included this in the revised manuscript (Page 9 line 2-8). It is equally plausible that the redistribution of the Tiam1 is signaling both for a

disruption of cell polarity and the hyperproliferative effects and that those events cause

a disruption in tight junction integrity, spindle pole orientation, and cyst morphology.

We answered this question above (question #4 from the same reviewer). Minor Issues:

Page 7, paragraph continuing from page 6, intersectin misspelled.

We thank the reviewer for careful reading of the manuscript. We corrected the word (Page 8, line 4). Figure 4 D check image labels.

We apologize for our mislabeling of the cell staining.

Ref:

Aoki K, Nakamura T, Fujikawa K, Matsuda M (2005) Local Phosphatidylinositol 3,4,5-Trisphosphate Accumulation Recruits Vav2 and Vav3 to Activate Rac1/Cdc42 and Initiate Neurite Outgrowth in Nerve Growth Factor-stimulated PC12 Cells. Mol. Biol. Cell, 16: 2207-2217.

den Elzen N, Buttery CV, Maddugoda MP, Ren G, Yap AS (2009) Cadherin adhesion receptors orient the mitotic spindle during symmetric cell division in mammalian epithelia. Mol. Biol Cell, 20: 3740-3750.

Itoh RE, Kiyokawa E, Aoki K, Nishioka T, Akiyama T, Matsuda M (2008) Phosphorylation and activation of the Rac1 and Cdc42 GEF Asef in A431 cells stimulated by EGF. Journal of Cell Science, 121: 2635-2642.

Nobes CD & Hall A (1995) Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 81:53-62.

O'Brien LE, Jou TS, Pollack AL, Zhang Q, Hansen SH, Yurchenco P, Mostov KE (2001) Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly. Nat Cell Biol, 3: 831-838.

Zheng Z, Zhu H, Wan Q, Liu J, Xiao Z, Siderovski DP, Du Q (2010) LGN regulates mitotic spindle orientation during epithelial morphogenesis. J Cell Biol, 189: 275-288.

Referee #3: The manuscript by Yagi and colleagues proposes that activation of Rac selectively at the

basolateral domain is necessary for appropriate epithelial morphogenesis during cyst

formation. The authors design and optimize an interesting system to locally activate or

inactivate Rac at the plasma membrane by recruiting Tiam1 or b2-chimerin using FKB2.

The data is of high quality technically and figures are nicely done and structured, which

makes it easier to understand the complexities of the system. The adaptation of this

method may be of interest to different readers that face the challenge of understanding

the spatial and temporal regulation of GTPase signalling. The main conclusion of the

manuscript is that Rac activation has to be excluded from the apical domain to allow

appropriate cyst morphogenesis. However, in this reviewer's opinion, this has not been

conclusively demonstrated by the data. I would therefore not recommend this

manuscript for publication in its current form.

We thank the reviewer for the favorable comments and helpful suggestions. A number of other explanations could account for the loss of polarity and cyst

morphogenesis upon aberrant Rac up-regulation. First, Rac activation all around cell

surface (induced by Tiam-1) should be compared to Rac activation at the apical domain

only or at the bottom of the cells only.

We agree that experiments with spatially restricted localization of FKBP proteins will bring us clear and definitive results. We have tried to do so by constructing syntaxin3- and syntaxin4-FRB. Even though syntaxin-tagged with fluorescent proteins properly located in the cyst (see Figure 2 E and F for syntaxin 4, data not shown for syntaxin3), FRB-tagged protein did not.

When we quantified Rac1 activation by FRET imaging, we found that Lyn-FRB-mediated Tiam1 recruitment to the plasma membrane up-regulated only Rac1 at the apical membrane (Supplementary Fig S3). We therefore concluded that inactivation of Rac1 at the apical membrane is responsible for cyst maintenance. It could well be that an increase in Rac activity levels is sufficient to induce the effects

reported. Alternatively, GEFs are predicted to provide specificity of signalling

downstream of small GTPases. Rac activation at the surface specifically by Tiam-1 may

lead to cysts full of cells, while Rac activation at the surface by another GEF may

disrupt polarity but induce a different phenotype.

We agree with the reviewer. We have added this possibility in the revised manuscript (Page 6 line 18-21). Another point is that the attempts to define the cellular processes responsible for cysts

being filled with cells led to the identification of two processes that could participate:

changes in spindle orientation and mislocalization of tight junction proteins. The

manuscript does not demonstrate how these two distinct processes might be linked or a

causal effect between their perturbation and abnormal cysts.

We agree that we could not show direct evidence to connect the two observations of changes in spindle orientation and mislocalization of tight junction proteins. We therefore softened the sentence in the abstract (Page 2, line 6). We observed a merging of apical and basolateral protein prior to the luminal cell filling (Figure 2E), and it has been established that the tight junction functions as a fence between the apical and lateral domains. From these, we assume it is most likely at this moment that a tight junction abnormality results in a change in spindle orientation. To probe our hypothesis that tight junction disruption causes the spindle misorientation, we have to examine the polarity marker upon specifically disrupting spindle formation, but not tight junction formation. To our knowledge, however, there is no molecule that has such a specific function. Even though we could not show the direct evidence, our speculation might be supported by the observation that the dynein-dynactin complex, which is involved in spindle positioning, is found in a circumferential ring below the tight junction (Ahringer 2003), and that tight junctions are retained during mitosis to separate the apical and basolateral membrane domains for epithelial integrity (Baker and Garrod 1993). Proliferation, another important Rac-dependent process, could also be enhanced when

Rac is inappropriately activated and lead to the exclusion of cells from the epithelium

towards the lumen.

This comment is the same as the comment from Reviewer#2. We reiterate our answer below.

To examine the relationship between Rac1 activation and proliferation, we counted cell number upon dimerizer-induced Rac1 activation and inactivation (Page 6 line 25 to page 7 line 3, and Supplementary Figure S4). Results indicated that Rac1 activation did not accelerate cell proliferation, while Rac1 inactivation significantly decreased it. We also confirmed that dimerizer treatment alone did not affect cell proliferation. From these results we discount the possibility that Tiam1-induced Rac1

activation induced the morphological disorder by upregulating cell proliferation. Ref Ahringer J (2003) Control of cell polarity and mitotic spindle positioning in animal cells.

Curr. Opin. Cell Biol, 15: 73-81. Baker J, Garrod D (1993) Epithelial cells retain junctions during mitosis. J Cell Sci, 104

( Pt 2): 415-425.

Han J, Luby-Phelps K, Das B, Sgu X, Xia Y, Mostelle R, Krishna M, Falck J, White M, Broek D. Role of Substrates and Products of PI 3-kinase in Regulating Activation of Rac-Related Guanosine Triphosphatases by Vav. Science 279[5350], 558-560. 1998.

Rossman KL, Der CJ, Sondek J (2005) GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors. Nat Rev Mol Cell Biol, 6: 167-180.



2nd Editorial Decision 11 November 2011

Thank you for the submission of your revised manuscript to our offices. We have now received the enclosed reports from the two referees that were asked to assess it. Your manuscript still has one minor suggestion that I would like you to incorporate before we can proceed with the official acceptance of your manuscript. As you will se below, both referees are reasonably content with your revision of the manuscript but show some concerns that according to them should be addressed before acceptance. As you know, it is our policy to ask for only one round of revision. In order to make sure that your main claims are fully supported by the experimental evidence but, at the same time, not ask you to perform additional experiments that might not be critical and would only add to an unnecessary waste of time, I have contacted an expert advisor to specifically comment on the need for these additional experiments. Our advisor is very satisfied with your revision and your point by point response. Although s/he considers that the experiments suggested by the referees might be out of the scope of this report, particularly the biochemical analysis of Rac1 activity proposed by referee #2, s/he also thinks that this point should be addressed using the FRET probes in cysts (3D), as Rac1 activity has only been measured in 2D cell culture conditions. This is the only assay considered essential by our advisor and thus I do not consider necessary for you to perform any additional experiments besides this one. Do not hesitate to contact me in case you need further clarifications. I look forward to seeing a new revised version of your manuscript as soon as possible. Editor EMBO Reports REFEREE REPORTS: Referee #1: The authors have answered satisfactorily to most comments. Concerning the following point: In Fig.4D, staining for ZO1 is shown as an endogenous marker for tight junctions in Tiam1 cysts {plus minus} dimerizer; the same staining should be shown for tight junctions in bet2-chimaerin cysts {plus minus} dimerizer. The data presented by the authors in response to this comment should be at least mentioned in the text, and possibly included as supplementary figure. Referee #2: The authors have attempted to address the issues raised by the referees and their explanations for most points are reasonable. The following points should be addressed in the final version of the manuscript. It is important to correlate the translocation of FKB-B2-chimaerin with down regulation of Rac1 activity. The authors could attempt this with well established biochemical assays.



It is also important that they localize E-cadherin (point 6 reviewer 2) . Many studies have attempted this labeling successfully. Especially given a reported link between Rac1 and E-cadherin the conclusions drawn can change if a mislocalization of E-cadherin is indeed observed. 2nd Revision - authors' response 30 November 2011

Reply to the editor; I am very glad to hear that our revised manuscript can be accepted with a minor revision. According to Referee #1’s suggestion, we have included the Figure for the reviewer as Supplementary Figure S6 in the revised manuscript. The text has been modified accordingly (page 9, line 2-3, in the revised manuscript). Following your advisor’s comment, Rac1 inactivation was validated in cysts expressing Raichu-Rac1, Lyn-FRB, and FKBP-β2-chimaerin. FRET images were obtained before and after rapamycin-induced β2-chimaerin recruitment to the plasma membrane. When we performed unpaired t-test, we failed to detect the difference of Rac1 activity before and after β2-chimaerin recruitment to the plasma membrane. However, by paired t-test, the difference was significant; i.e., β2-chimaerin recruitment to the plasma membrane decreased Rac1 activity at both apical and lateral plasma membranes (p = 0.002 and p = 0.016, respectively). The data have been included as Supplementary Figure S3 F&G. The result section was modified accordingly (page 7, line 9-11). I hope this revision satisfies your requirement. 3rd Editorial Decision 01 December 2011

I am very pleased to accept your manuscript for publication in the next available issue of EMBO reports. Thank you for your contribution to EMBO reports and congratulations on a successful publication. Please consider us again in the future for your most exciting work. Yours sincerely, Editor EMBO Reports

suppression of rac1 activity at the apical membrane of mdck cells is essential...

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