MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C

  1. Celia Segui-Perez
  2. Daphne A. C. Stapels
  3. Ziliang Ma
  4. Jinyi Su
  5. Elsemieke Passchier
  6. Bart Westendorp
  7. Richard W. Wubbolts
  8. Wei Wu
  9. Jos P. M. van Putten
  10. Karin Strijbis

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Abstract

Glycosylated mucin proteins contribute to the essential barrier function of the intestinal epithelium. The transmembrane mucin MUC13 is an abundant intestinal glycoprotein with important functions for mucosal maintenance that are not yet completely understood. We demonstrate that in human intestinal epithelial monolayers, MUC13 localized to both the apical surface and the tight junction (TJ) region on the lateral membrane. MUC13 deletion resulted in increased transepithelial resistance (TEER) and reduced translocation of small solutes. TEER buildup in ΔMUC13 cells could be prevented by addition of MLCK, ROCK or protein kinase C (PKC) inhibitors. The levels of TJ proteins including claudins and occludin were highly increased in membrane fractions of MUC13 knockout cells. Removal of the MUC13 cytoplasmic tail (CT) also altered TJ composition but did not affect TEER. The increased buildup of TJ complexes in ΔMUC13 and MUC13-ΔCT cells was dependent on PKC. The responsible PKC member might be PKCδ (or PRKCD) based on elevated protein levels in the absence of full-length MUC13. Our results demonstrate for the first time that a mucin protein can negatively regulate TJ function and stimulate intestinal barrier permeability.

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  1. Version published to 10.1242/jcs.261468
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    Reply to the reviewers

    Reviewer 1

    Although this is an interesting, and generally well-performed study, it is primarily observational and there are few mechanistic insights provided into how MUC13 modulates barrier function. The authors propose a presumably direct interaction between MUC13 and PKC, which apparently sequesters PKC, preventing this kinase from triggering PKC-dependent increases in TJ barrier function; however, there is no evidence that a MUC13-PKC interaction occurs, that MUC13 is phosphorylated by PKC, or that phosphorylation of MUC13 has any impact on its function or overall barrier function. Thus, the hypothesis is not directly tested and all observations in this manuscript are generally correlative in nature.

    While the MUC13 cytoplasmic tail contains a putative PKC-binding motif, we indeed do not show a direct interaction between MUC13 and a member of the PKC family in this manuscript. Unfortunately, we have so far not been able to successfully perform (co-)immunoprecipitation of MUC13 with our current anti-MUC13 antibodies.

    To provide more insights into the possible MUC13-PKC interaction, we plan to perform several experiments.

    • First, we will determine the expression levels of the different PKC isotypes (PKC alpha, beta, gamma, delta, epsilon, and zeta) in the HRT18 cell lines by western blot.
    • Next, we will determine the localization of the relevant PKC isoforms and MUC13 by immunofluorescence microscopy. We are curious to see if we can find a colocalization between MUC13 and a PKC member on the lateral or apical membrane. If we can demonstrate a colocalization, we could follow up with a proximity ligation assay, but this would require the MUC13 antibody directed against the cytoplasmic tail (which only detects the lateral population) and might therefore be challenging.
    • Furthermore, since PKC delta protein levels were upregulated in the total lysate of ∆MUC13 cells, we will test a PKC delta-specific inhibitor in the TEER assay.

    Consider quantifying all blots (Fig. 5C, Fig. 6B).

    As suggested, we will quantify both blots.

    Consider using dot-plots for all quantified data.

    The graphs will be altered to include individual measurement points.

    Reviewer 2

    Fig2E showed two bands with different size in the two MUC13 WT control cell lines. They hypothesized that this could be the consequences of glycosylation different patterns. A sample with untransfected HRT18 might be included in the western blot panel. Additionally, what is the 100kDa band?

    Mucin blots are notoriously difficult and these MUC13 blots are the result of a lot of trial and error. We repeated the Western Blot with original HRT18 cells, HRT18 original cell line, as well as the two CRISPR control cells used in the study (WT 1 and WT 2) and one of the full-length MUC13 knockout cells. The higher band was absent from the MUC13 knockout cells, but a small shift in the MUC13 band size can be noted in the WT 1 cells compared to the original and the WT 2 cell lines, possibly indicating a change in the glycosylation pattern. The 100 kDa band remains detectable in all cell lines including the ∆MUC13 cell line, therefore we consider this to be an aspecific background band of the MUC13 antibody. We will add a more extensive Western Blot analysis to the manuscript.

    Did the transfection of the inducible GFP-MUC13 plasmid induce any decrease of Claudin1/3/4 in HRT18 or Caco2 cells? Same question regarding PKCdelta.

    These are indeed interesting questions. We will perform these experiments with our MUC13-overexpression HRT18 cells.

    Reviewer 3

    Moreover, the authors should determine if MUC13∆CT localize to TJs, as suggested by the working model in Figure 7C. The subcellular localization of MUC3∆CT could give critical clues for its function, but Figure 2G fails to provide any information and the authors do not present any additional data concerning the localization of MUC13∆CT. Detection of MUC13 in membrane fractions of WT, MUC13∆CT and cells lacking the mucin domain could be a feasible strategy forward.

    We will perform additional immunofluorescence experiments to determine the subcellular localization of MUC13-∆CT more accurately. However, detection of the extracellular domain by western blot, as suggested, is not possible due to the incompatibility of the extracellular MUC13-directed hybridoma antibody with the western blot technique. We currently do not have a suitable antibody that recognizes the ED and can be used for western blot.

    The authors introduce an inducible MUC13-GFP fusion protein into WT and ∆MUC13 cells and show that it reverses the enhanced TEER upon MUC13 deletion. Unfortunately, the "Materials and Methods" section lacks adequate information on how this fusion protein was designed. Critical questions are the position of the GFP tag within MUC13, whether the fusion protein is correctly processed in HRT18 cells, and if it localizes to the apical or apico-lateral membrane domains? Figure 2H is of low magnification and fails to provide information on the subcellular localization of the MUC13-GFP fusion protein.

    The materials and methods section will be adjusted to describe all the design details of the fusion protein. The GFP tag was added to the MUC13 C-terminus with a GGGS linker sequence in between. Processing of the fusion protein seems correct as we observed MUC13-GFP localization to both lateral and apical membranes and no access intracellular build up. As suggested by the reviewer, we will add more detailed immunofluorescence pictures to the manuscript.

    Figures 6B-C suggest that PKCdelta levels increase in ∆MUC13 cells, which correlates with higher enrichment of Claudins in membrane fractions. The authors then inhibited PKCdelta and observed reduced recruitment of Claudins to membrane fractions. Since the family of Claudins are differentially regulated by phosphorylation (PMID: 29186552), the authors should investigate the TEER phenotype of WT, ∆MUC13 and MUC13∆CT upon PKC inhibition.

    We must clarify that figures 6C-D are done using the PKC inhibitor targeting all conventional PKCs (alpha, beta, gamma) as well as delta (https://www.tocris.com/products/gf-109203x_0741). We recently obtained a PKCdelta-specific inhibitor which we will test in the TEER build-up experiments.

    Moreover, the authors predict phosphorylation sites in MUC13CT and suggest a link between PKC and MUC13 (Figure. 6A), however no evidence is presented to support this hypothesis. The authors should either determine if PKC phosphorylates MUC13 and if this modification has implication on MUC13 localization and TJ function, or remove statements regarding MUC13 phosphorylation. The data provided suggest that PKC regulates TJ proteins independent of MUC13.

    We will adjust the manuscript to put less emphasis on the putative PKC motifs in the MUC13 cytoplasmic tail. For further details on how we will proceed regarding the possible MUC13-PKC interaction see question 1 from reviewer #1.

    Figure 5C. Quantification of at least 3 independent experiments is required.

    These data will be added to the manuscript.

    Figure 6B. Quantification of at least 3 independent experiments is required.

    These data will be added to the manuscript.

    Reviewer 4

    OPTIONAL: MUC13 is expressed both, in the basolateral membranes and in the apical membrane of intestinal epithelial cells (IECs). Does the authors check the relevance of MUC13 in the formation of microvilli in IECs? Are microvilli different (microvilli staining, number of positive cells to microvilli, length, width or distribution of microvilli) in ΔMUC13 and in MUC13-ΔCT? How the glycocalyx looks like in these cells genetically modified for MUC13?

    HRT18 cells do not seem to develop microvilli. However, we plan to stain these cells with a microvilli-specific antibody (ACTUB). The HRT18 cells express mostly MUC13 and relatively low levels of the larger TM mucin MUC1. To study changes in the glycocalyx, we will stain using a MAL-II antibody which targets α-2,3 sialic acids, which are abundantly present in mucins. In this way, we will determine any big changes in the total glycocalyx that may occur in response to the removal of MUC13.

    In the figure 1D would be nice to represent the co-localization of MUC13 together with occluding in a graph in each Z-stack so you can visualize in which part of the cell is maximum colocalization of these both components.

    These data will be provided.

    In the figure 1E, would be great to compare between the two different MUC13 antibodies the apical fraction stained in HRT18 and Caco-2. Specially in the HRT18 cell line since the first antibody did not label apical MUC13 expression meanwhile the second antibody detects the apical expression in these cells. How much lateral lateral stain the C terminal antibody compare with the extracellular antibody for MUC13 and how much stain apically the C terminal antibody compare with the extracellular antibody? Would be nice to see some comparative results using the intensity by Z-stack and plotting in a graph.

    This is a good suggestion as it is quite intriguing that both MUC13 antibodies seem to target (partially) different MUC13 populations. We will perform co-staining with both MUC13 antibodies to provide information on which MUC13 populations are detected by each antibody (apical vs lateral membrane).

    Manuscript would be improved if in the figure 2H to compare within the same cell line the number of MUC13 positive cells in the WT, number of MUC13 positive cells in WT+pMUC13 and the number of MUC13 positive cells in the ΔMUC13+pMUC13

    We will quantify the percentage of MUC13-GFP positive cells in both the WT and ΔMUC13 backgrounds by either microscopy or flow cytometry.

    In figure 5C would be helpful to plot in a graph the normalized expression of each TJ protein and compare between the different cells used (WT, ΔMUC13 and MUC13-ΔCT) as you did in figure 5A

    We will provide the quantification data of three independent experiments.

    Description of the revisions that have already been incorporated in the transferred manuscript

    Reviewer 1

    In addition, this model does not explain why all kinase inhibitors tested reverse the increase in TER observed in deltaMUC13 cell lines. Does this reflect the lack of inhibitor specificity or the likelihood that many kinases are involved?

    As stated in the manuscript, we think that MLCK, ROCK, and PKC are all essential for TER buildup in the ∆MUC13 cells. Because the roles of MLCK and ROCK are well established, we choose to follow up on the PKC results. We adjusted the text to clarify this point.

    The authors do observe that there is an increase in expression of several tight junction-associated proteins, including the claudins, in deltaMUC13 cells. Affected CLDNs include 1, 2, 3, 4, 7, 12. (1) While it appears the authors are arguing that this increased claudin expression results in increased barrier function, they do not sufficiently highlight the well-known role that CLDN2 has in cation transport, and both CLDN-4 and -7 have also been implicated in paracellular ion flux (although this is apparently cell-type specific). These observations would seem to argue against a simple correlation between claudin expression and tight junction barrier function.

    The reviewer is right about the different functions of claudins. Claudin-2, -4 and -7 have (potentially) pore-forming properties, while the other claudins restrict paracellular passage. It has been previously demonstrated that the magnitude of paracellular ion and water flux is reflected by the specific repertoire of claudin family members (Shashikanth et al., 2022). In this paper, overexpression of claudin-4 was shown to mobilize and affect polymeric strands of claudin-2, thus blocking its channel activity. Our mass spectrometry data demonstrated a striking increase in claudin-1, -2, -3, -4, -7, and -12 in the MUC13 knockout membranes compared to WT. We hypothesize that the claudin repertoire in the MUC13 knockout cells leads to a more restricted paracellular route (as observed in the TEER and tracer experiments). The pore-forming claudins may be subject to “interclaudin interference” therefore leading to restriction of the total paracellular ion and water flux. We have adjusted the text of the manuscript to clarify this point.

    We attempted to investigate claudin-2 expression levels in isolated membranes by Western Blot but were unsuccessful as the antibody did not detect any protein while claudins-1 and -4 could be detected with the same method.

    Furthermore, the authors should note the disconnect between paracellular ion flux mediated by claudins and the flux of markers such as dextrans and lucifer yellow, which can be dissociated from claudin function.

    We acknowledge that the flux of larger particles (the leak pathway) is not regulated by claudins (which regulates the pore pathway). We aimed to assess both the pore and the leak paracellular pathways, by using different techniques including TEER, small solutes (Lucifer Yellow CH), and larger molecules (4 and 70 kDa FITC-Dextrans). HRT18 wild type cells are already very restrictive to the pass of larger molecules (FITC-Dextrans) but are more permeable to smaller solutes such as Lucifer Yellow (400 Da). We observed that removal of the MUC13 cytoplasmic tail did not affect the TEER, but reduced the paracellular passage of Lucifer Yellow, demonstrating that manipulation of MUC13 can affect both the pore and leak pathways. We adjusted to text to include this point.

    The increased expression of claudins in the nominally tail-minus MUC13 without a corresponding change in TER would again seem to argue against a simple correlation;

    MUC13-dCT cells showed consistently increased levels of claudins-1 and -2, but not the other claudins. This claudin repertoire (with high claudins-1 and -2, but lower claudin-3, -4, -7, and -12) is apparently not enough to increase TEER. We think that this again reflects the importance of the total claudin composition for the control of the paracellular pathway.

    Watch the use of decimal points instead of commas (lines 253 and 256).

    Corrected.

    Line 543: MilliQ is not a washing agent (or is it?). (Line 535) We use MilliQ as a final step before mounting the glass slides to remove any possible salt deposition that would affect the visualization by microscopy.

    We have specified this in the text.

    Line 553: TER is the product of total resistance times the area. The units are ohms times area.

    Indeed, we have changed this mistake (line 545).

    Line 630: Please provide the transfer conditions (voltage, amp, watts?) and transfer buffer when describing the Western blot protocol.

    For immunoblotting of MUC13, protein lysates were transferred to 0.2 µm PVDF membranes using the Trans-Blot Turbo Transfer system (Biorad). The transfer was run using the protocol (High MW) which consisted in running for 10 min at 25 volts (V) and 1,3 amperes (A). These experimental data were added to the manuscript.

    Reviewer 2

    My main concern about this manuscript is that the authors analyzed MUC13 role in intestinal homeostasis and function using colorectal cancer cells. As helpful as cancer cells are, we should always be cautious about extrapolating roles in normal intestinal epithelium or IBD pathology. Obviously, these finding are also interesting in a cancer context. Using GEPIA (http://gepia.cancer-pku.cn/), I observed that MUC13 is overexpressed in colorectal cancer COAD-TCGA dataset (compared to normal colon from GTEX). Similar results were obtained previously by Gupta et al. (ref #10). I am aware that this would be difficult to confirm the main findings in a non-cancerous intestinal cell line but this limit (normal intestine using cancer cells) should be at least discussed in the manuscript.

    We appreciate the reviewers’ comments and are aware of the downsides of using cancer-derived cell lines. We have performed the GEPIA analysis ourselves and have an ongoing project about the possible role of MUC13 in colorectal cancer progression. In a separate project, we are collaborating with the Gaultier Laboratory at the University of Virginia which has generated a MUC13 knockout mouse. This model will allow us to study the role of MUC13 in non-cancerous tissue. We recently received intestinal biopsies from these mice which will be stained with MUC13 and claudin antibodies to determine localization in healthy tissue. These experiments will reveal if MUC13 colocalizes with claudin on the lateral membrane in the healthy mouse intestinal tract. In future experiments, we will also address MUC13 localization and function in human intestinal organoids. We have adjusted the discussion to refer to the limitations of using cancer cell lines.

    Massey et al (Micro 2021, PMC7014956) previously showed that MUC13 overexpression increased rigidity in PDAC cells and discussed involvement MUC13 link with EMT. MUC13-Her2 interaction was also associated with decrease of E-cadherin suggesting an EMT phenotype. This should be included in the discussion section.

    The discussion has been adjusted to include the link with EMT.

    The authors performed mass spectrometry analysis. Results are deposited on ProteomeXchange but are not yet publicly released. Among the 1189 membrane protein identified. Did the authors observed alteration of EMT proteins? (decrease of vimentin for example). In the discussion section (lane 347), the authors mentioned the relationship between other membrane bound mucins such as MUC1, MUC4, MUC16 or MUC17 and AJ/TJproteins. Did the authors observed any alteration of these mucin in the mass spectrometry data?

    The mass spec analysis was performed on membrane fractions, therefore our dataset will not contain true cytosolic proteins. One of the key EMT proteins, Vimentin, is a cytosolic protein, and indeed it was not found in our dataset. Other EMT-related proteins are shown in the following table. TGF beta 1 was slightly decreased, while E-cadherin and Integrin beta 6 were slightly increased in the ∆MUC13 cells compared to WT cells.

    Gene Name

    Mean WT

    Mean ∆MUC13

    Mean MUC13-∆CT

    TGFBI (TGB beta 1)

    20,54

    16,48

    18,83

    CDH1 (E-cadherin)

    22,69

    24,57

    24,24

    ITGB6 (Integrin beta 6)

    18,86

    21,74

    19,19

    Vimentin - Cytosolic

    CDH2 (Cadherin-2, N-cadherin)

    Mucins are large proteins comprised of densely O-glycosylated mucin domains, which makes them extremely challenging to study by mass spectrometry (MS) (Rangel-Angarita et al., 2021). We did not specifically employ mucin-directed technologies in this dataset, thus making the detection of mucins hard. No mucins other than MUC13 were detected. For MUC13, two peptides corresponding to the EGF-like domains in the extracellular domain, a region that is less densely glycosylated. We added a sentence to the description of the mass spec results to include the EMT proteins and other mucins.

    Minor points:

    Lane 126: HRT18 and Caco2 colon cancer cells instead of intestinal epithelial cells

    Corrected.

    Lane 181 and lane 514: add "full length" MUC13 DNA sequence

    Corrected.

    Lane 234: TEER was measured every 12h. How the authors did observed the largest increase at 42h? Was it 48h? Please clarify.

    We aimed at measuring every 12 h, however the exact measurements were done at 18h, 24h, and 42 h post-infection. We have corrected this in the manuscript.

    Reviewer 3

    Line 43 and 46. "Enterocytes" should be replaced with "intestinal epithelial cells", since enterocytes are themselves a distinct subpopulation of IECs.

    We have changed it in the manuscript.

    Lines 58-60. References in support of the statements should be added.

    We added a reference to this sentence.

    Lines 188-190. Authors comment on "roundness" of different cell lines. If the parameter is critical for the manuscript, the authors should quantify this phenotype.

    The parameter is not critical for the manuscript. We removed the sentence.

    Figure 3A. Staining of cell lines should include panels showing localization of MUC13.

    Co-staining of MUC13 with occludin in HRT18 cell lines can be found in figure 1D, and MUC13 with E-cadherin in supplementary figure 1.

    Lines 323-327 and 390-392. Sentences on these lines contradict each other. The sentences should describe/discuss quantified data presented in Figure 6D.

    The reviewer is right that we should be discussing the quantified data in 6D. We adjusted the sentence in line 323-327.

    Proteomic data sets should be made publicly available on data depositories.

    All proteomics raw data were deposited to the ProteomeXchange Consortium with the dataset identifier PXD029606.

    Reviewer 4

    OPTIONAL: In the figure 2E, is the extracellular antibody still detecting the MUC13-ΔCT?

    No, unfortunately the antibody directed against the MUC13 ED is not compatible with western blot.

    In the figure 2G, would be nice to comment possible reasons why the deletion in the first cell line of the MUC13-CT you can still detect with the extracellular antibody some lateral expression of MUC13 meanwhile in the second cell line, the same deletion (MUC13-CT) you cannot see any lateral MUC13 staining with the extracellular antibody.

    Yes, this is indeed a puzzling finding, especially because the CRISPR deletion is the same in both cell lines. We will add a sentence about possible reduced stability of the MUC13 without CT domain that leads to a different outcome in both cell lines.

    It would be nice that the results from Figure 3H are better explained since it is difficult to follow.

    We adjusted the text to explain the experiment in more detail.

    2. Description of analyses that authors prefer not to carry out

    Reviewer 1

    The authors may be overly reliant on TER measurements. Epithelial cells have two parallel resistive pathways: transcellular and paracellular. TER measure the contribution of both. Thus, an increase in TER could result from a decrease in transcellular ion transport. The authors need to measure transcellular ion flow or selectively measure the junctional resistance in a select set of experiments to rule this possibility out.

    The reviewer is right that TEER is a sum of the resistance of the transcellular and paracellular pathways. However, due to the high resistance of cell membranes, the current predominantly travels via the paracellular route (Elbrecht et al., 2016). For this reason, TEER measurements are widely accepted techniques for the assessment of ions passage through the paracellular pathway (Shen et al., 2011).

    Reviewer 3

    Figure 1C. Caco2 and HRT18 cells exhibit distinct MUC13 expression patterns when probed with an antibody against the MUC13 CT; MUC13 localizes almost exclusively to lateral cell junction in HRT18 cells, while a higher portion of MUC13 is present on the apical surface of Caco2 cells. This observation has two possible explanations: 1) the two cell lines express distinct forms of MUC13, or 2) the two cell lines carry distinct machineries for anchoring MUC13 to apical versus apico-lateral membranes. Thus, The authors should take the opportunity to determine the impact of MUC13 deletion on TEER and TJ function in Caco2 cells. Proteomic analysis and functional assays in Caco2 cells may provide more a general mechanism for how MUC13 regulates TJ proteins.

    Yes, this would be a great line of investigation. However, we aimed to knockout MUC13 in Caco-2 cell lines (with the same CRISPR/Cas9 protocol as the HRT18 cells) but were unable to obtain Caco-2 knockout clones. We think this might be a consequence of the poor capability of Caco-2 cells to grow as single colonies (a required step in the protocol). Another option is Caco-2 MUC13 knockout cells have reduced viability.

    The authors generate cell lines that either lack MUC13 or express MUC13 lacking the cytoplasmic domain. Loss of MUC13 cells resulted in enhanced TEER and increased recruitment of TJ proteins to membrane fractions. MUC13∆CT cells show moderate recruitment of TJ proteins to membranes and no increase in TEER but inhibit paracellular diffusion of Luciferase Yellow across monolayers. Figure 3A suggests that Occludin redistributes to tricellular junctions in ∆MUC13 cells, whereas it is found more laterally in WT and MUC13∆CT cells. These finding suggest that full-length MUC13 interferes with TJ protein complexes. However the impact of the extracellular and intracellular (CT) domains is not fully elucidated. Does the O-glycosylated mucin domain interfere with the extracellular domains Occludin and Claudins? The authors should clarify the contribution of the mucin domain to the observed phenotype, for example by performing the described experiments in a cell line expressing MUC13 lacking the mucin domain.

    Mucins are type I membrane proteins with the N-terminal part of the protein on the extracellular site. Therefore, a CRISPR method to specifically remove the glycosylated domain but leave the remainder of the protein in frame is challenging. An additional difficulty is that the ED contains a lot of repeats, complicating the design of specific guide RNAs. To specifically address the contribution of the glycosylated domain, we could complement the MUC13 knockout cell with a construct lacking the ED. However, this would not be comparable to the endogenous MUC13∆CT cell line presented in this manuscript. In future studies, we will strive to address the functions of the different MUC13 domains in more detail.

    Figure 5A. Turnover of TJ proteins in membrane fractions occurs faster than over a period of 1-3 days (PMID: 18474622). The authors should determine TJ protein turnover over a period of minutes and hours.

    We acknowledge the findings in this interesting paper concerning the continuous remodeling of tight junctions. However, the readout of our biotinylation assay is degradation and the timeframe of degradation turns out to be days and not hours. Within this timeframe remodeling is taking place but it cannot be captured in the total lysate.

    Reviewer 4

    OPTIONAL: The authors show that the probiotic Lactobacillus plantarum increase epithelial barrier independently of MUC13. Have the authors considered to use other probiotics as Lactobacillus paracasei (10.3389/fcimb.2015.00026), Akkermansia muciniphila (10.1038/emm.2017.282) or some metabolic products from intestinal microbiota as short-chain fatty acids (SCFAs) (10.3389/fphys.2021.650313) to check what is the role of MUC13 and if it is related with other microbe or microbiota metabolite?

    Thank you for the suggestion. We have an ongoing project in which we investigate the impact of different probiotic bacteria and plan to investigate whether they have an impact on the epithelial barrier function in a MUC13-dependent manner. This study will lead to a separate publication.

    OPTIONAL: The authors successfully delete MUC13 in IECs, both, full length and the cytosolic tail. Have the authors considered targeting the deletion of the PTS domain in MUC13? Could affect that something different from paracellular trafficking as the extracellular detection of microbes and microbial products?

    Removal of a domain in the extracellular domain of MUC13 with CRISPR is challenging because mucins are type I membrane proteins, the repeats and possible frameshift, as described above.

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    Referee #4

    Evidence, reproducibility and clarity

    This work highlight the importance of the transmembrane mucin MUC13 in the control of the intestinal epithelial integrity by the negative regulation of tight junction (TJ) proteins mediated by Protein Kinase C (PKC). To achieve this conclusion, the authors designed CRISPR/Cas9 strategie to generate two types of HRT18 and Caco-2 MUC13 knockout cell lines: one to delete the full MUC13 length (ΔMUC13) and other to target the deletion of the MUC13 cytoplasmic tail (MUC13-ΔCT). More, they design MUC13-GFP plasmid to overexpress MUC13 in WT cells and to rescue the ΔMUC13 cells.

    The key conclusions of this project are:

    • Transepithelial electrical resistance (TEER) was upregulated in ΔMUC13 with no changes in MUC13-ΔCT compared with the control. The rescue of ΔMUC13 by MUC13-GFP plasmid reduce the TEER to values similar to the WT cells. More, ΔMUC13 and MUC13-ΔCT were more restrictive in the paracellular translocation of small tracers than WT cells suggesting the negative correlation of MUC13 and paracellular conductance of small ions together with higher TEER.
    • Absence of MUC13 leads to an upregulation of TJ proteins, which explain the decrease of the paracelullar ion traffic
    • Upregulation of TEER in cells lacking MUC13 is dependent on MLCK, ROCK and PKC kinases meanwhile the upregulation of TJ proteins is mediated by PKC proteins. Finally, the authors intend to address in the future the molecular link between MUC13 and PKC during TJ regulation.

    The conclusions and the claims from the authors of this work are supported by the data where they extensively test the close relation between the transmembrane mucin MUC13 and the intestinal epithelial integrity.

    Major comments:

    • OPTIONAL: The authors show that the probiotic Lactobacillus plantarum increase epithelial barrier independently of MUC13. Have the authors considered to use other probiotics as Lactobacillus paracasei (10.3389/fcimb.2015.00026), Akkermansia muciniphila (10.1038/emm.2017.282) or some metabolic products from intestinal microbiota as short-chain fatty acids (SCFAs) (10.3389/fphys.2021.650313) to check what is the role of MUC13 and if it is related with other microbe or microbiota metabolite?
    • OPTIONAL: MUC13 is expressed both, in the basolateral membranes and in the apical membrane of intestinal epithelial cells (IECs). Does the authors check the relevance of MUC13 in the formation of microvilli in IECs? Are microvilli different (microvilli staining, number of positive cells to microvilli, length, width or distribution of microvilli) in ΔMUC13 and in MUC13-ΔCT? How the glycocalyx looks like in these cells genetically modified for MUC13?
    • OPTIONAL: The authors successfully delete MUC13 in IECs, both, full length and the cytosolic tail. Have the authors considered targeting the deletion of the PTS domain in MUC13? Could affect that something different from paracellular trafficking as the extracellular detection of microbes and microbial products?
    • OPTIONAL: In the figure 2E, is the extracellular antibody still detecting the MUC13- ΔCT?

    Minor comments:

    • In the figure 1D would be nice to represent the co-localization of MUC13 together with occluding in a graph in each Z-stack so you can visualize in which part of the cell is maximum colocalization of these both components.
    • In the figure 1E, would be great to compare between the two different MUC13 antibodies the apical fraction stained in HRT18 and Caco-2. Specially in the HRT18 cell line since the first antibody did not label apical MUC13 expression meanwhile the second antibody detects the apical expression in these cells. How much lateral lateral stain the C terminal antibody compare with the extracellular antibody for MUC13 and how much stain apically the C terminal antibody compare with the extracellular antibody? Would be nice to see some comparative results using the intensity by Z-stack and plotting in a graph.
    • In the figure 2G, would be nice to comment possible reasons why the deletion in the first cell line of the MUC13-CT you can still detect with the extracellular antibody some lateral expression of MUC13 meanwhile in the second cell line, the same deletion (MUC13-CT) you cannot see any lateral MUC13 staining with the extracellular antibody.
    • Manuscript would be improved if in the figure 2H to compare within the same cell line the number of MUC13 positive cells in the WT, number of MUC13 positive cells in WT+pMUC13 and the number of MUC13 positive cells in the ΔMUC13+pMUC13
    • It would be nice that the results from Figure 3H are better explained since it is difficult to follow.
    • In figure 5C would be helpful to plot in a graph the normalized expression of each TJ protein and compare between the different cells used (WT, ΔMUC13 and MUC13-ΔCT) as you did in figure 5A

    Significance

    This is a novel study where the authors directly correlate the lack of MUC13 expression with paracellular transport and tight junction proteins. This study describe the high correlation between the transmembrane mucin MUC13 and the integrity of the intestinal epithelium. Therefore, this project is highly valuable not only for the scientific research nowadays, but for future investigations of the intestinal epithelial physiology and biochemistry.

    The strengths parts of this study are the different cell constructs including the full deletion of MUC13 and the targeting deletion of MUC13 cytosolic tail. Then, they have been able to directly correlate lack of MUC13 with paracellular traffic where PKC intracellular signal is involved. A limitation of the study is the lack of a cell line lacking the extracellular domain of MUC13, which could give some clues about the direct relation of this membrane mucin with the outer world of the cell (i.e. bacteria).

    My field of expertise is intestinal epithelial defense including, but not limited, to mucins.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The authors describe a novel function for transmembrane mucin MUC13 in regulation of tight junctions (TJs) that create an impermeable cell monolayer that allows. paracellular diffusion of very small molecules. The authors use cultured intestinal epithelial cell monolayers. to demonstrate that MUC13 localizes to the apical aspects of IECs as well laterally to tight junctions. CRSIPR/Cas-mediated deletion of MUC13 increased transepithelial resistance (TEER) and reduced the extent of paracellular diffusion of <0.5 kDa molecules across the monolayer. Proteomic analysis revealed that specific TJ proteins are enriched in cell membrane fractions upon deletion of MUC13, while pharmacological inhibition of PKC involved in actomyosin contractility, resulted in loss of TJ proteins from cell membranes and TEER reduction. See major comments for a detailed discussion concerning the findings.

    Major comments:

    1. Figure 1C. Caco2 and HRT18 cells exhibit distinct MUC13 expression patterns when probed with an antibody against the MUC13 CT; MUC13 localizes almost exclusively to lateral cell junction in HRT18 cells, while a higher portion of MUC13 is present on the apical surface of Caco2 cells. This observation has two possible explanations: 1) the two cell lines express distinct forms of MUC13, or 2) the two cell lines carry distinct machineries for anchoring MUC13 to apical versus apico-lateral membranes. Thus, The authors should take the opportunity to determine the impact of MUC13 deletion on TEER and TJ function in Caco2 cells. Proteomic analysis and functional assays in Caco2 cells may provide more a general mechanism for how MUC13 regulates TJ proteins.
    2. The authors generate cell lines that either lack MUC13 or express MUC13 lacking the cytoplasmic domain. Loss of MUC13 cells resulted in enhanced TEER and increased recruitment of TJ proteins to membrane fractions. MUC13∆CT cells show moderate recruitment of TJ proteins to membranes and no increase in TEER but inhibit paracellular diffusion of Luciferase Yellow across monolayers. Figure 3A suggests that Occludin redistributes to tricellular junctions in ∆MUC13 cells, whereas it is found more laterally in WT and MUC13∆CT cells. These finding suggest that full-length MUC13 interferes with TJ protein complexes. However the impact of the extracellular and intracellular (CT) domains is not fully elucidated. Does the O-glycosylated mucin domain interfere with the extracellular domains Occludin and Claudins? The authors should clarify the contribution of the mucin domain to the observed phenotype, for example by performing the described experiments in a cell line expressing MUC13 lacking the mucin domain. Moreover, the authors should determine if MUC13∆CT localize to TJs, as suggested by the working model in Figure 7C. The subcellular localization of MUC3∆CT could give critical clues for its function, but Figure 2G fails to provide any information and the authors do not present any additional data concerning the localization of MUC13∆CT. Detection of MUC13 in membrane fractions of WT, MUC13∆CT and cells lacking the mucin domain could be a feasible strategy forward.
    3. The authors introduce an inducible MUC13-GFP fusion protein into WT and ∆MUC13 cells and show that it reverses the enhanced TEER upon MUC13 deletion. Unfortunately, the "Materials and Methods" section lacks adequate information on how this fusion protein was designed. Critical questions are the position of the GFP tag within MUC13, whether the fusion protein is correctly processed in HRT18 cells, and if it localizes to the apical or apico-lateral membrane domains? Figure 2H is of low magnification and fails to provide information on the subcellular localization of the MUC13-GFP fusion protein.
    4. Figures 6B-C suggest that PKCdelta levels increase in ∆MUC13 cells, which correlates with higher enrichment of Claudins in membrane fractions. The authors then inhibited PKCdelta and observed reduced recruitment of Claudins to membrane fractions. Since the family of Claudins are differentially regulated by phosphorylation (PMID: 29186552), the authors should investigate the TEER phenotype of WT, ∆MUC13 and MUC13∆CT upon PKC inhibition. Moreover, the authors predict phosphorylation sites in MUC13CT and suggest a link between PKC and MUC13 (Figure. 6A), however no evidence is presented to support this hypothesis. The authors should either determine if PKC phosphorylates MUC13 and if this modification has implication on MUC13 localization and TJ function, or remove statements regarding MUC13 phosphorylation. The data provided suggest that PKC regulates TJ proteins independent of MUC13.

    Minor comments:

    1. Line 43 and 46. "Enterocytes" should be replaced with "intestinal epithelial cells", since enterocytes are themselves a distinct subpopulation of IECs.
    2. Line 59. The authors should note that MUC13 does not have a canonical SEA domain that generates a cleaved heterodimer (PMID: 16369486).
    3. Lines 58-60. References in support of the statements should be added.
    4. Lines 188-190. Authors comment on "roundness" of different cell lines. If the parameter is critical for the manuscript, the authors should quantify this phenotype.
    5. Figure 3A. Staining of cell lines should include panels showing localization of MUC13.
    6. Figure 5A. Turnover of TJ proteins in membrane fractions occurs faster than over a period of 1-3 days (PMID: 18474622). The authors should determine TJ protein turnover over a period of minutes and hours.
    7. Figure 5C. Quantification of at least 3 independent experiments is required.
    8. Figure 6B. Quantification of at least 3 independent experiments is required.
    9. Lines 323-327 and 390-392. Sentences on these lines contradict each other. The sentences should describe/discuss quantified data presented in Figure 6D.
    10. Proteomic data sets should be made publicly available on data depositories.

    Significance

    Mucins participate in critical functions in the human intestine. Gel-forming mucins form the mucus layers that separate the gut microbiota from the underlying intestinal epithelial cells (IECs) (PMID: 18806221). Transmembrane mucins are instead anchored to the plasma membrane of various populations of IECs (PMID: 32169835; PMID: 28052300). Despite its discovery over 20 years ago, the functional role of MUC13 in the intestinal epithelium is still debated. MUC13 is expressed in human small intestine and colon under baseline conditions and is dysregulated during inflammation and tumorigenesis, as described by the authors. Thus, understanding how MUC13 expression and localization impact cell function is of great importance for elucidating its function in health and disease. Studies so far have identified transmembrane mucins as biophysical barriers against bacteria (PMID: 33596425) or facilitators of bacterial invasion (PMID: 33824202). The current manuscript can potentially offer novel conceptual insights into how transmembrane mucins govern the integrity of the epithelial monolayer that serves as a firewall between the multitude of microbes in the gut lumen and the immune system. Such insights have implication for both basic and clinical research on inflammatory bowel disease (IBD) and colorectal cancer (CRC). However, while the authors present convincing data that deletion of MUC13 enhances TEER and recruitment of TJ proteins, the study in its current form fail to provide mechanistic proof of how MUC13 impacts individual TJ proteins. Moreover, it is not clear if findings in a specific cultured cell line (HRT18) can be extrapolated to other frequently used intestinal cell lines (e.g. Caco2) and IECs in an in vivo setting. The latter is particularly important since the authors argue that their findings have important implication in intestinal inflammation and cancer.

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    Referee #2

    Evidence, reproducibility and clarity

    Summar: In this manuscript, Segui-Perez and colleagues investigated the role of MUC13 in tight junctions and trans-epithelial barrier function in colon cancer cells. The authors showed that MUC13 is highly expressed throughout the intestine at the apical and lateral membrane. They established Crispr/Cas9 HRT18 cells in which MUC13 (deltaMUC13) or the cytoplasmic tail of MUC13 were deleted. They also performed rescue experiments using GFP-MUC13 constructs. The authors observed that deletion of MUC13 promoted TEER for bigger particles and strengthen tight junctions. Analysis of membrane composition by mass spectrometry showed an upregulation of TJ proteins (Claudins) that is dependent of PKCdelta.

    Major points:

    1. My main concern about this manuscript is that the authors analyzed MUC13 role in intestinal homeostasis and function using colorectal cancer cells. As helpful as cancer cells are, we should always be cautious about extrapolating roles in normal intestinal epithelium or IBD pathology. Obviously, these finding are also interesting in a cancer context. Using GEPIA (http://gepia.cancer-pku.cn/), I observed that MUC13 is overexpressed in colorectal cancer COAD-TCGA dataset (compared to normal colon from GTEX). Similar results were obtained previously by Gupta et al. (ref #10). I am aware that this would be difficult to confirm the main findings in a non-cancerous intestinal cell line but this limit (normal intestine using cancer cells) should be at least discussed in the manuscript.
    2. Massey et al (Micro 2021, PMC7014956) previously showed that MUC13 overexpression increased rigidity in PDAC cells and discussed involvement MUC13 link with EMT. MUC13-Her2 interaction was also associated with decrease of E-cadherin suggesting an EMT phenotype. This should be included in the discussion section.
    3. Fig2E showed two bands with different size in the two MUC13 WT control cell lines. They hypothesized that this could be the consequences of glycosylation different patterns. A sample with untransfected HRT18 might be included in the western blot panel. Additionally, what is the 100kDa band?
    4. The authors performed mass spectrometry analysis. Results are deposited on ProteomeXchange but are not yet publicly released. Among the 1189 membrane protein identified. Did the authors observed alteration of EMT proteins? (decrease of vimentin for example). In the discussion section (lane 347), the authors mentioned the relationship between other membrane bound mucins such as MUC1, MUC4, MUC16 or MUC17 and AJ/TJproteins. Did the authors observed any alteration of these mucin in the mass spectrometry data?
    5. Did the transfection of the inducible GFP-MUC13 plasmid induce any decrease of Claudin1/3/4 in HRT18 or Caco2 cells? Same question regarding PKCdelta.

    Minor points:

    1. Lane 126: HRT18 and Caco2 colon cancer cells instead of intestinal epithelial cells
    2. Lane 181 and lane 514: add "full length" MUC13 DNA sequence
    3. Lane 234: TEER was measured every 12h. How the authors did observed the largest increase at 42h? Was it 48h? Please clarify.

    Significance

    This manuscript is relevant as basic research for both the mucin field and for the intestinal epithelium field. It brings conceptual hypothesis about the role of MUC13 that is less characterized than MUC1 or MUC4.

    I have been working on mucins for over 20 years. I found this work well done and very interesting.

    I feel that the conclusions are mostly supported by the results. The one semantic limit is that this work is based on cancer cell lines and it is a little bit speculative to extrapolate the finding on normal intestinal epithelium.

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    Referee #1

    Evidence, reproducibility and clarity

    The manuscript by Sequi-Perez explores a somewhat novel role for the transmembrane mucin MUC13 in regulating the tight junction barrier. They report that MUC13 is localized, in part, to the apical junctional complex; that depletion of MUC13 increases TER and expression of claudins and OCLN in a membrane fraction; and that partial removal of MUC13 cytoplasmic domain also results in increased expression of OCLN and claudins, but without a corresponding change in TER. Finally, the authors hypothesize that PKCdelta may act in conjunction with MUC13 to regulate paracellular flux in intestinal epithelial cell lines.

    Major points:

    1. Although this is an interesting, and generally well-performed study, it is primarily observational and there are few mechanistic insights provided into how MUC13 modulates barrier function. The authors propose a presumably direct interaction between MUC13 and PKC, which apparently sequesters PKC, preventing this kinase from triggering PKC-dependent increases in TJ barrier function; however, there is no evidence that a MUC13-PKC interaction occurs, that MUC13 is phosphorylated by PKC, or that phosphorylation of MUC13 has any impact on its function or overall barrier function. Thus, the hypothesis is not directly tested and all observations in this manuscript are generally correlative in nature. In addition, this model does not explain why all kinase inhibitors tested reverse the increase in TER observed in deltaMUC13 cell lines. Does this reflect the lack of inhibitor specificity or the likelihood that many kinases are involved?
    2. The authors do observe that there is an increase in expression of several tight junction-associated proteins, including the claudins, in deltaMUC13 cells. Affected CLDNs include 1, 2, 3, 4, 7, 12. (1) While it appears the authors are arguing that this increased claudin expression results in increased barrier function, they do not sufficiently highlight the well-known role that CLDN2 has in cation transport, and both CLDN-4 and -7 have also been implicated in paracellular ion flux (although this is apparently cell-type specific). These observations would seem to argue against a simple correlation between claudin expression and tight junction barrier function. (2) The increased expression of claudins in the nominally tail-minus MUC13 without a corresponding change in TER would again seem to argue against a simple correlation; (3) Furthermore, the authors should note the disconnect between paracellular ion flux mediated by claudins and the flux of markers such as dextrans and lucifer yellow, which can be dissociated from claudin function.
    3. The authors may be overly reliant on TER measurements. Epithelial cells have two parallel resistive pathways: transcellular and paracellular. TER measure the contribution of both. Thus, an increase in TER could result from a decrease in transcellular ion transport. The authors need to measure transcellular ion flow or selectively measure the junctional resistance in a select set of experiments to rule this possibility out.

    Minor points:

    1. Watch the use of decimal points instead of commas (lines 253 and 256).
    2. Consider quantifying all blots (Fig. 5C, Fig. 6B).
    3. Line 543: MilliQ is not a washing agent (or is it?).
    4. Line 553: TER is the product of total resistance times the area. The units are ohms times area.
    5. Line 630: Please provide the transfer conditions (voltage, amp, watts?) and transfer buffer when describing the Western blot protocol.
    6. Consider using dot-plots for all quantified data.

    Significance

    This study advances the fields of mucin biology and tight junction barrier function in an incremental manner. The study is well done, but there are few mechanistic insights into how MUC13 modulates paracellular flux in cultured gut epithelial cells.

  12. Version published to 10.1101/2022.10.27.513982 on bioRxiv