CRB3 navigates Rab11 trafficking vesicles to promote γTuRC assembly during ciliogenesis

Reviewer #1 (Public Review):

In this study the authors first perform global knockout of the gene coding for the polarity protein Crumbs 3 (CRB3) in the mouse and show that this leads to perinatal lethality and anopthalmia. Next, they create a conditional knockout mouse specifically lacking CRB3 in mammary gland epithelial cells and show that this leads to ductal epithelial hyperplasia, impaired branching morphogenesis and tumorigenesis. To study the mechanism by which CRB3 affects mammary epithelial development and morphogenesis the authors turn to MCF10A cells and find that CRB3 shRNA-mediated knockdown in these cells impairs their ability to form properly polarized acini in 3D cultures. Furthermore, they find that MCF10A cells lacking CRB3 display reduced primary ciliation frequency compared to control cells, which is in agreement with previous studies implicating CRB3 in primary cilia biogenesis. Using a combination of biochemical, molecular- and imaging approaches the authors then provide some evidence indicating that CRB3 promotes ciliogenesis by mediating Rab11-dependent recruitment of gamma-tubulin ring complex (gamma-TuRC) component GCP6 to the centrosome/ciliary base, and they also show that CRB3 itself is localized to the base of primary cilia. Finally, to assess the functional consequences of CRB3 loss on ciliary signaling function, the authors analyze the effect of CRB3 loss on Hedgehog and Wnt signaling using cell-based assays or a mouse model.

Overall, the described findings are interesting and in agreement with previous studies showing an involvement of CRB3 in epithelial cell biology, tumorigenesis and ciliogenesis. The results showing a role for CRB3 in mammary epithelial development and morphogenesis in vivo seem convincing. Although the authors provide evidence that CRB3 promotes ciliogenesis via (indirect) physical association with Rab11 and gamma-TuRC, the precise mechanism by which CRB3 promotes ciliogenesis remains to be clarified. Specific comments are as follows:

1. For all cell-based assays using shRNA to knock down CRB3, it would be desirable to perform rescue experiments to ensure that the observed phenotype of CRB3 depleted cells is specific and not due to off-target effects of the shRNA.
2. Figure 3G: it is very difficult to see that the red stained structures are primary cilia.
3. Figure 5A: it is unfortunate the authors chose not to show the original dataset (Excel file) used for generating this figure; this makes it difficult to interpret the data. It is general policy of the journal to make source data accessible to the scientific community.
4. The authors have a tendency to overinterpret their data, and not all claims put forth by the authors are fully supported by the data provided.

Reviewer #2 (Public Review):

In this work, the authors investigate the role of CRB3 in the formation of the primary cilium both in a mouse model and in human cells. They confirm in a conditional knock-out (KO) mouse model that Crb3 is necessary for the formation of the primary cilium in mammary and renal epithelial tissues and the new-born mice exhibit classical traits of ciliopathies. In the mouse mammary gland, the absence of Crb3 induces hyperplasia and tumorigenesis and in the human mammary tumor cells MCF10A the knock-down (KD) of CBR3 impairs ciliogenesis and the formation of a lumen in 3D-cultures with less apoptosis and spindle orientation defects during cell division.

To determine the subcellular localization of CRB3 the authors have expressed exogenously a GFP-CRB3 in MCF10A and found that this tagged protein localizes in cell-cell junctions and around pericentrin, a centrosome marker while endogenous CRB3 localizes at the basal body. To dissect the molecular role of CRB3 the authors have performed proteomic analyses after a pull-down assay with the exogenous tagged-CRB3 and found that CRB3 interacts with Rab11 and is present in the endosomal recycling pathway. CRB3 KD also decreases the interactions between components of the gamma-TuRC. In addition, the authors showed that CRB3 interacts with a tagged-Rab11 by its extracellular domain and that CRB3 promotes the interaction between Rab11 and CEP290 while CRB3 KD decreased the co-localization of GCP6 with Rab11 and gamma-Tub.

Finally, the authors showed that CRB3 depletion cannot activate the Hh pathway as opposed to the Wnt pathway.

Author Response

The following is the authors’ response to the original reviews.

Reviewer # 1

Specific comments

1. Figure 1: it is unclear how many mice were used for the described phenotypic analyses (panels D and E). Please clarify.

We acknowledge that we made a mistake in failing to clearly describe the phenotypic analyses. In Figure 1D and E, we performed statistical analysis on the number of TEBs in whole mammary mounts. One mouse stained a mammary whole mount with Carmine-alum staining. Thus, “n” represents the 10 mice we analyzed. We have modified the legend of Figure 1 to " D, E. Quantification of the average number of TEBs and bifurcated TEBs in littermate Crb3fl/fl (n=10) and Crb3fl/fl;MMTV-Cre (n=10) mice at 8 weeks old" in lines 909-911.

1. Figure 2: in panels B and C it is unclear how the data was quantified; the legend states "n=10", does this mean the experiment in B was done 10 times? And that 10 acini per condition were measured in panel C? In panel D a difference in 0.3% between NC and shCRB3 seems miniscule; do the authors mean 30% instead? And how many acini were counted per condition per (how many) experiments? Same applies to panels G and H, it is unclear how many cells were analyzed per (how many) experiments.

Thanks for your suggestions. We failed to describe the details of the statistical analysis well in the experimental method. To provide a brief overview of our statistical analysis method, we took 3-4 random bright-field micrographs of each well in the chamber slide system and repeated the experiment three times. We then counted the number of acini in all micrographs (Figure 2B) and examined the diameter of all acini in each photograph, averaging the values as data (Figure 2C). We also determined the percentage of aberrant acini in each photograph, which was used as an analysis value (Figure 2D). We carefully confirmed that the vertical axis of Figure 3D was indeed mislabeled and should mean 30%, and revised the original figure. For IF analysis of the mitotic spindle orientation during lumen formation, we examined the division angle of one cell in one acinus that was mitotically dividing, 3-4 acini were randomly examined in each well in the chamber slide system, and this experiment was repeated three times (Figure 2G and H). Therefore, we have provided a detailed description of these issues in the Figure 2 legend. The revised parts are found in lines 922-924, lines 926-927, lines 929-930, and line 932.

1. Figure 2: it would be desirable if authors were able to quantify the data in panels E and I.

Thank you for your comments. According to your suggestions, we performed the quantitative analysis of Figure 2E and I, which is now presented in the new Figure 2D and H.

1. For all cell-based assays using shRNA to knock down CRB3 (Fig. 2A-H; Fig. 3A-F; Fig. 4C-E; Fig. 5G-J; Fig. 6C; Fig. 7C, D; Fig. 8E-G), it would be desirable to perform rescue experiments to ensure that the observed phenotype of CRB3 depleted cells is specific and not due to off-target effects of the shRNA.

Yes, rescue experiments involving overexpression of CRB3 in CRB3 depleted cells can accurately account for the specific phenotype as well as eliminate the off-target effects of shRNA. However, our group has long focused on the role of the cell polarity protein CRB3 in contact inhibition and tumorigenesis. Our previous studies have ruled out the off-target effects of shRNA and reported that CRB3 regulates contact inhibition and tumorigenesis through Hippo or Wnt signaling pathways (Cell Death Dis 2017;8(1):e2546, Oncogenesis 2017;6(4):e322, J Cell Mol Med 2018;22(7):3423-33). Therefore, we will pay close attention to rescue experiments to ensure experimental integrity and phenotypic specificity in our subsequent studies.

1. Figure 3: how many cells were counted/measured per condition (in how many experiments) in panels B, D, H, F, G and H? In panels C and D, what is the CRB3 protein level in these cells? This is of relevance as protein overexpression per se could impinge on ciliation frequency. This question could be addressed by performing a western blot analysis with CRB3 antibody.

We did not clearly describe the measurement and statistical analysis methods in the previous manuscript. Similarly, we took 3-4 random IF and SEM micrographs of each sample in one experiment, and this experiment was repeated three times. Subsequently, the number of ciliated cells and total cells were counted, and the proportion of ciliated cells was calculated (Figure 3B, D and F). In these figures, the cilium length of representative ciliated cells was measured in each photograph. In the knockout mouse model, we needed to find the intact mammary ductal lumen and renal tubule in IF staining of mouse mammary and renal tissue sections, with 5-6 random fields micrographs taken per slice, and the proportion of ciliated cell was measured by counting and taking the average. A total of ten mice were repeated in these experiments (Figure 3G and H). Therefore, the legend of Figure 3G and H has been partially modified and a detailed description has been added to the Figure 3 legend. The revised parts are in lines 945-946, lines 950-951, line 953.

Thank you for your suggestions that we perform a western blot analysis with CRB3 antibody in Figure 3C and D. And we have added the western blotting with CRB3 analysis in the new Supplementary Figure 3A.

1. Figure 3G: it is very difficult to see that the red stained structures are primary cilia.

Yes, the staining structure of primary cilia in mammary ductal lumen are less clear than that of individual cells and in renal tubule in Figure 3G. We used recognized acetylated tubulin and γ-tubulin to stain the primary cilia, which were clearly labeled in individual cells. However, the labeled primary cilia in renal tubule were longer length and demonstrated a more pronounced structure than those in the mammary ductal lumen. In the mammary ductal lumen of the 10 mice we analyzed, the primary cilia showed shorter length and staining structure than the others shown in Figure 3G. This difference may be due to the distinct characteristics of primary cilia in different tissues.

1. Figure 4B: how many cells were analyzed in how many experiments?

Our statistical methods for analyzing cellular experiments using IF were essentially the same. We randomly selected 3-4 IF micrographs of each sample in one experiment, and this experiment was repeated three times. Subsequently, the number of colocalization cells and total cells were counted, and the proportion of cells with pericentrin and CRB3 colocalization was calculated (Figure 4B). The detailed description has been added to the Figure 4 legend. The revised part is in lines 962-963.

1. Lines 217-219: since the cells were not stained with a cilia marker, only a centrosome marker, the claim that CRB3 localizes to the base of cilia is unsubstantiated.

Thank you for your comments. The base of cilia is the basal body, which develops from the mother centriole of the centrosome (Cancer Res. 2006;66(13): 6463-7). Firstly, we found colocalization of CRB3 and pericentrin, a centrosome marker, in MCF10A cells (Figure 4A and B). Secondly, we verified the colocalization of CRB3 with γ-tubulin, a marker of basal body in primary cilia, in confluent quiescence cells (Figure 4C and D). In addition, we found that CRB3 was localized at the base of primary cilia labeled with acetylated tubulin (Figure 4E and F). Due to the species of commercialized CRB3 antibody, we were able to indirectly claim that CRB3 localizes to the base of cilia through these experiments.

1. Figure 3 and Figure 4: is it problematic to use gamma tubulin as centrosome marker if CRB3 depletion causes reduced centrosomal recruitment of gamma tubulin ring complex components? Also, in Figure S3A no gamma tubulin staining can be seen in the lower panel, why?

Thank you for your positive comments. As is well known, γ-tubulin is a marker of the centrosome, and we found that CRB3 depletion causes reduced centrosomal recruitment of gamma tubulin ring complex components. However, Our Figure 3 was illustrated the effect of CRB3 on ciliary assembly, and Figure 4 was analyzed the localization of CRB3 in primary cilia. In some reports on ciliary assembly, the fluorescent double staining of acetylated tubulin and γ-tubulin have been used to label primary cilia, and the effect of target genes on ciliary number and assembly were analyzed by these markers (Nature. 2013;502(7470): 254-7, Cell. 2007;130(4): 678-90 and so on). Although CRB3 affects the recruitment of gamma tubulin ring complex components, it does not affect the analysis of ciliary number and localization in Figures 3 and 4.

In Figure S3A, green staining labeled with γ-tubulin could be clearly found in the lower left panel. The representative area from the left amplification may have been poorly selected, resulting in no γ-tubulin staining on the right side. We have updated the lower right panel in the new Supplementary Figure 3B.

1. Figure S4A: the grouping of indicated proteins is factually wrong. For example, FBF1, SCLT1 and ODF2 are not IFT-B components, and several of the proteins indicated as localizing to the basal body also localize to (unciliated) centrioles. In contrast, CP110 is usually only found on unciliated centrioles and not mature basal bodies. Authors should consult the relevant literature and correct the figure accordingly. Alternatively, this misleading text/grouping could be removed from the figure. Furthermore, in the legend to Figure S4 there is no information provided about this quantitative analysis (how many independent experiments, which cells were analyzed etc.).

Thank you for your helpful suggestions. We have taken your advice and removed this misleading information from the manuscript, Supplementary Figure 4A and its corresponding legend. In the legend to Supplementary Figure 4A, we have added the detailed information for this quantitative analysis in the legend. The revised legend is shown in lines 1098-1100.

1. Figure S4B: how do authors know which of the bands correspond to CRB3 fusion protein?

Based on the construction strategy of the CRB3-GFP fusion protein (Figure 6D) and its base sequence, we were able to calculate its molecular weight. Then the molecular weight of CRB3-GFP fusion protein was verified by western blotting (Figure 6F and 7A). Meanwhile, exogenous overexpression allowed for the production of the CRB3-GFP fusion protein in large quantities. Due to these features, we could know that the band indicated by the black arrow is most likely CRB3-GFP fusion proteins. In order to check the molecular weight, we have labeled the key molecular weight markers in the new Supplementary Figure 4B.

1. Lines 251-253: this seems like data overinterpretation.

Thank you for your comments. We have revised this sentence in lines 252-254.

1. Lines 260-261: the data showing perturbed gamma tubulin localization is not convincing as data was not quantified.

According to your suggestions, we performed the quantitative analysis of Figure 4C, which is now presented in the new Figure 4E.

1. Figure 5H and Figure 6C: to show that the GCP6 IP actually worked, these blots should be probed also for GCP6.

Thank you for your good suggestions. We have added these blots probed for GCP6 in new Figure 5H and 6C.

1. Figure 5I: how many cells were analyzed in how many experiments?

Our statistical methods for analyzing cellular experiments using IF were essentially the same. We took 3-4 random IF micrographs of each sample in one experiment, and this experiment was repeated three times. The detailed description has been added to the Figure 5 legend. The revised part is in lines 992-994.

1. Figure S5: it looks like GPC6 and Rab11 are localizing all over the cell, are the antibodies used for the IFMs specific for these proteins?

After checking the specificity of these antibodies used for the IFMs, we have decided to delete the corresponding results in the Supplementary Figure 5 and their description in the original manuscript.

1. Lines 43, 89, and 314-315: the claim that CRB3 directly binds Rab11 is not supported by the data. The data provided only shows that these proteins interact indirectly. To show direct interaction, yeast-2-hybrid analysis or pull-down assays with purified proteins would be required.

Thank you for your positive comments. Since we were unable to complete the relevant experiments to demonstrate direct interaction of two proteins, we have revised our conclusions. Replace " CRB3 directly binds Rab11" with " CRB3 binds Rab11" in the manuscript.

1. Figure 6G and lines 314-315: this result is surprising as it indicates GTP- and GDP-locked versions of Rab11 have the same inhibitory effect on CRB3 binding? Please comment, and also indicate how data in Figure 6G was quantified (and how many independent experiments were used for the quantification).

We were also puzzled by the results shown in Figure 6G. Based on the western blotting bands, we suspected that there may have been some issues with the experiment. Specifically, we believed that the inefficient transfection of Flag-Rab11aWT, Flag-Rab11a[Q70L], Flag-Rab11a[S20V], and Flag-Rab11a[S25N] plasmids, as well as the insufficient amount of GFP antibody used in the co-IP experiment, led to the corresponding bands being too weak and masking the true differences.

To address this, we optimized the experimental conditions, strictly increased the experimental control, and repeated the experiment in triplicate. The new results are shown in the revised Figure 6G. The statistics from the three independent experiments revealed that CRB3b had a stronger interaction with Rab11a[Q70L] and Rab11a[S20V], while showing a weaker interaction with Rab11a[S25N], compared to Rab11aWT. As this result, we revised the original manuscript in lines 308-310 and added a detailed description to the Figure 6 legend in lines 1012-1013.

1. Figure 8G: data needs to be quantified.

Thank you for your comments. We replaced the unattractive bands in the western blotting of Figure 8G with better quality ones. The statistical analysis of the Figure 8G data is shown in Supplementary Figure 6.

Further minor comments

1. Abstract should indicate that this study describes conditional knockout of Crb3 in mouse mammary gland epithelial cells.

This is good writing advice. We have added the relevant description in lines 40-42.

1. Line 87: specify which gland (mammary?).

We have modified to " mammary gland" in line 87.

1. Line 140: sentence states that knockout of Crb3 is essential for branching morphogenesis in mammary gland development, I do not think this is correct.

We have removed the inappropriate finding.

1. Line 152: "formed more number" should be "formed more" or "formed higher number of".

We modified "formed more number" to "formed more" in line 154.

1. Lines 157-163: text and logic are difficult to follow for a non-expert.

We have modified the logic of this paragraph, as detailed in lines 158-165.

1. Figure 4A, C: figure resolution could be improved. It is difficult to see what the authors claim these figures are showing.

The clarity of the original images in Figure 4A and C is acceptable, while the images on the right are electronically enlarged. Although there is a decrease in pixels, it can still display our findings.

1. Figure 7D, E: images look pixelated.

The clarity of the original images in Figure 7D and E is acceptable using a laser confocal microscope, while the images on the right are electronically enlarged.

1. Line 222: unclear what authors mean by "detected a series".

We modified "detected a series" to "some important" in line 226.

1. Lines 221-225: which cells were used for the analysis in Fig. S4?

We used MCF10A cells for the analysis in Supplementary Figure 4, and modified its legend in line 1098.

1. Line 245: what is "cytomembrane"?

We modified "cytomembrane" to "cell membrane" in lines 246-247.

1. Lines 246-250: wording is unclear/difficult to understand.

We have modified this paragraph, as detailed in lines 248-251.

1. Line 273: should "regimented" be "sedimented"?

We modified "regimented" to "sedimented" in line 274.

1. Line 287-288: sentence does not make sense.

We have removed this sentence.

1. Figure 5A: it would be desirable to show the original dataset (Excel file) used for generating this figure.

To maintain data integrity, we should provide the original dataset (Excel file). However, there are some unpublished data in this file that we must withhold for the time being. If needed, the corresponding author can be requested to provide the file.

1. Lines 298-299: wording is unclear.

We have modified this sentence, as detailed in lines 296-298.

1. Lines 285-287: replace "instead of" with "but not".

We modified "instead of" to "but not" in line 286.

1. For all IFMs showing merged images of the green and red channel, please also show the red and green channel separately.

Most of our fluorescence images are presented separately for each channel in this manuscript, with only a few merged images due to space limitations. This type of presentation is commonly used in published papers.

1. Lines 326 and 327: replace "bonded" with "bound".

We have modified in lines 322-323.

1. Lines 327-328 and 361-364: wording is unclear/grammatically incorrect.

We have modified these paragraphs, as detailed in line 323 and lines 357-360.

1. Line 342: what is meant by "the combination of"?

We modified "the combination of" to "the binding of" in line 338.

1. Line 365: localization of what?

This means "subcellular localization" in lines 360-361.

Reviewer # 2

Major points

1. CRB3 is present in mammals as 2 isoforms, A and B, originating from alternative splicing. In this study, the authors never mention this fact and when using approaches to KO or KD CRB3A/B they are likely to deplete both isoforms which have been shown to have different C-terminal domains and functions (Fan et al., 2007). This is also important for the CRB3 antibodies used in the study since according to the material and methods section they are either against the extracellular domain common to both isoforms or the intracellular domain which is only similar in the domain close to transmembrane between the 2 isoforms. Since the antibodies used in each figure are not detailed it is impossible to know if the authors are detecting CRB3A or B or both. Please provide the information and correct for the actual isoform detected in the data and conclusions.

Thanks for your positive comments. In mammals, CRB3 has two isoforms, CRB3a and CRB3b, distinguished by alternative splicing within the fourth exon of the CRB3 gene, which in turn produces a protein with 23 amino acid differences at the C terminus. Both CRB3a and CRB3b have mostly identical amino acid sequences, and have indistinguishable molecular weight sizes. As a result, the knockout mouse construction strategy and the design principles of RNAi sequences target both CRB3a and CRB3b. This is described in lines 100-104 and lines 149-150. Additionally, commercially available antibodies detect both CRB3a and CRB3b, as mentioned in line 123 and lines 636-637 in revised manuscript.

However, it should be noted that our CRB3 overexpression, as shown in the CRB3 structural domain in Figure 6D, refers specifically to the sequence of CRB3b. As a result, we have updated the original manuscript as well as the legends of Figures 3C, 3E, 4A, 5A, 5B, 6D-G, 7A, 7B and Supplementary Figure 2F-H, 3A, 4B, 6B to reflect this change. All instances of overexpressed CRB3 have been changed to CRB3b.

1. CRB3A and B have been localized in the cilium itself (Fan et al., 2004; 2007) but in the study CRB3A/B does not enter the cilium but is localized in the basal body (figure 4). How the authors reconcile these different localizations?

Indeed, we found that CRB3 is mainly localized at the basal body of the primary cilium, which differs from previous reports in the literature (Curr Biol. 2004;14(16):1451-61 and J Cell Biol. 2007;178(3):387-98). However, upon closer examination of one of these reports (Curr Biol. 2004;14(16):1451-61), it appears that CRB3 was actually scattered on the primary cilia, with a strong focus at the basal body. Additionally, in rat kidney collecting ducts, the localization of CRB3 on primary cilia was significantly reduced, with obvious localization at the basal body. Another study (J Cell Biol. 2007;178(3):387-98) also reported the co-localization of CRB3b and γ-tubulin in MDCK cells, which is consistent with our conclusion. We further verified the co-localization of CRB3 with the centrosome by overexpressing CRB3b in mammary epithelial cells, indicating that CRB3 mainly localizes to the basal body of the primary cilium. This information is discussed in the Discussion section of the manuscript (lines 400-410).

1. The authors use GFP-CRB3A/B, it is not stated which isoform, over-expression to localize CRB3A/B in MCF10A cells (figure 4A). The levels of expression appear to be very high in the GFP panel and it is likely that the secretory pathway of the cells is clogged with GFP-CRB3A/B in transit from the ER to the plasma membrane. Thus, the colocalization with pericentrin might be due to the accumulation of ER and Golgi around the centrosome. This colocalization should be done with the endogenous CRB3A/B and with a better resolution.

Thank you for your comments. We were also interested in the co-localization of endogenous CRB3 and centrosome proteins. However, the only commercial CRB3 antibody available is the rabbit species, and the pericentrin antibody (Abcam, ab4448) that is very useful is also the rabbit species. We had difficulty finding commercial centrosome-associated antibodies for other species. Therefore, we examined the co-localization of endogenous CRB3 with γ-tubulin in Figure 4C and combined the results with those of exogenous CRB3 to illustrate the co-localization of CRB3 with centrosomes.

1. The staining for CRB3A/B in figure 4C (red) is striking with a very strong accumulation in an undefined intracellular structure and the authors do not provide any explanation for such a difference with the GFP-CRB3A/B just above.

Thank you for your good suggestions. The immunofluorescence images of GFP-CRB3 in Figure 4a were obtained using a fluorescence microscope, while the images of endogenous CRB3 were obtained using a laser confocal microscope. The fluorescence microscope excites a fluorescent dye to emit a signal, which is amplified into a visible light signal and presents a full fluorescent signal. In Figure 4a, we can clearly see the full distribution of exogenous CRB3 in MCF10A cells, including its tight junctional localization consistent with previous reports in the literature and its co-localization with centrosomal proteins. On the other hand, laser confocal microscopy uses a laser as the light source to excite the fluorescence within the sample point by point. It employs a precision pinhole filtering technique with strong laminar imaging capabilities. In the specific analysis of endogenous CRB3 co-localization studies with centrosomes and primary cilium, signals at tight junctions must be excluded. Therefore, Figure 4c represents the fluorescence signal at the level of intracellular CRB3 co-localization with γ-tubulin. The two methods use different detection means and techniques, and are not directly comparable.

1. The staining in figure 4E is also different from those shown in figure 4F in which the CRB3A/B staining is right at the base of the axoneme while it is not the case in figure 4E where we can see a red dot close to but not right at the base of the axoneme.

Thank you for your comments. The new Figure 4F displays the localization relationship between CRB3 and primary cilium, analyzed using laser confocal microscopy. With the unique single-level detection function of this microscope, the problem of level selection may cause the red dots to appear close to, rather than right at the basal body of the primary cilium. However, the new Figure 4G, based on the use of 3D reconstruction scanning technique, clearly demonstrates the localization of CRB3 at the basal body of the primary cilium under the same cells and conditions.

1. The authors claim that CRB3A/B interacts directly with Rab11 but they only show co-immunoprecipitation experiments from cell lysates which do not support direct interactions. The only way to show a direct interaction is to produce both proteins in vitro. Thus, the term direct interaction should be removed.

Thank you for your positive comments. Since we were unable to complete the relevant experiments to demonstrate direct interaction of two proteins, we have revised our conclusions. Replace " CRB3 directly binds Rab11" with " CRB3 binds Rab11" in the manuscript.

1. In addition, the authors claim (Line 251/252) that Rab11 is necessary for the transport of CRB3A/B but they should KD Rab11 to show this.

Thank you for your good suggestions. It is essential to observe CRB3 trafficking after knockdown Rab11. However, in Figure 5C, we used the endocytosis inhibitor dynasore, which also inhibits Rab11-positive endosomes. This result shows that dynasore can significantly inhibit CRB3 trafficking in MCF10A cells. We believe that this experiment partially demonstrates that inhibiting Rab11 function can affect CRB3 trafficking.

1. The domain of CRB3A/B that is necessary for the interaction with Rab11 is the N-terminal part of the extracellular domain. This domain is thus inside the transport vesicles and not accessible from the cytoplasm. Given that Rab11 is a cytoplasmic protein, how the 2 proteins could interact across the membrane? The authors do not even discuss this essential point for their hypothesis.

Thank you for your positive comments. As shown in the schematic model in Figure 9, we believe that when cells form tight junctions, CRB3 is primarily located on the cell membrane. Subsequently, endosomes are involved in the intracellular degradation process of CRB3 on the cell membrane. Intracellular CRB3 can bind to Rab11 through the extracellular domain, which in turn participates in primary cilia assembly. We have made detailed modifications to lines 418-421.

1. Figures are not numbered.

Thank you for your comments. We have updated the numbers in the original manuscript as well as the legends of Figures 1D, 1E, 2B, 2D, 2F, 2G, 3B, 3D, 3F-H, 4B, 4E, 5I, 6, 8G and Supplementary Figure 1E, 2, 3C, 4A, 5B, 6.

Minor points

1. The authors cite several studies showing that a down regulation of CRB3A/B in human cells promotes cancer but other studies show the contrary: Lin et al., 2015 for example. Please discuss these discrepancies.

Thanks for your good suggestion. We have included additional studies with contrasting results in the discussion section, specifically in lines 378-380.

1. Line 98: "exhibit smaller" smaller than what?

We modified "exhibit smaller" to "exhibit smaller size" in line 97.

1. Line 152: "form more number, ..." ???

We modified "formed more number" to "formed more" in line 154.

1. Line 180: "Compared with the control, the number of cells with primary cilium was significantly increased ». To me it is the contrary! This part is not clear at all. Please rewrite.

We have revised the sentence in lines 183-185.

1. Authors should check and review extensively for improvements to the use of English.

Thanks for your good writing advice. We have carefully reviewed and revised the entire manuscript to improve its readability.