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A size filter at the Golgi regulates apical membrane protein sorting

Nature Cell Biology (2024)Cite this article

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Abstract

Despite decades of research, apical sorting of epithelial membrane proteins remains incompletely understood. We noted that apical cytoplasmic domains are smaller than those of basolateral proteins; however, the reason for this discrepancy is unknown. Here we used a synthetic biology approach to investigate whether a size barrier at the Golgi apparatus might hinder apical sorting of proteins with large cytoplasmic tails. We focused on Crb3, Ace2 and Muc1 as representative apical proteins with short cytoplasmic tails. By incorporating a streptavidin-binding peptide, these proteins can be trapped in the endoplasmic reticulum until addition of biotin, which triggers synchronous release to the Golgi and subsequent transport to the apical cortex. Strikingly, increasing the size of their cytoplasmic domains caused partial mislocalization to the basolateral cortex and significantly delayed Golgi departure. Moreover, N-glycosylation of ‘large’ Crb3 was delayed, and ‘small’ Crb3 segregated into spatially distinct Golgi regions. Biologically, Crb3 forms a complex through its cytoplasmic tail with the Pals1 protein, which could also delay departure, but although associated at the endoplasmic reticulum and Golgi, Pals1 disassociated before Crb3 departure. Notably, a non-dissociable mutant Pals1 hampered the exit of Crb3. We conclude that, unexpectedly, a size filter at the Golgi facilitates apical sorting of proteins with small cytoplasmic domains and that timely release of Pals1, to reduce cytoplasmic domain size, is essential for normal Crb3 sorting.

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Fig. 1: A survey of apical versus basolateral membrane proteins reveals a significant cytoplasmic size domain difference.
Fig. 2: Synchronizing anterograde traffic of the apical protein Crb3.
Fig. 3: Cytoplasmic domain size regulates delivery and dynamics of apical trafficking.
Fig. 4: Apical proteins with small cytoplasmic domains are preferentially sorted into a distinct region of the Golgi.
Fig. 5: Size filter generalizability to other apical cargo and cell types.
Fig. 6: Pals1 dissociates from Crb3 at the Golgi just before Crb3 departure.
Fig. 7: Dissociation of Pals1 is essential for timely exit of Crb3 from the Golgi.

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Data availability

All data are available in the main text or the supplementary materials. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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Acknowledgements

We thank F. Perez, G. Church, F. Martín-Belmonte, F. Zhang, L.-E. Jao, D. Trono, P. Tsoulfas and N. Hacohen for the kind gift of vectors, and L. Lavis for the kind gift of JF-Halo and JF-SNAP dyes. We also thank Macara lab members for valuable comments and advice. This work was funded by National Institutes of Health grant GM070902. Superresolution Nikon SoRa imaging was performed through the Vanderbilt Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, DK59637, EY08126 and 1S10MH130456-01A1). Some figure diagrams (Figs. 2e, 3b,g,l, 4a,d,f and 6b) were created in part with Biorender.com.

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  1. Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, USA

    Christian de Caestecker & Ian G. Macara

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  1. Christian de Caestecker
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Contributions

C.d.C. proposed the model, designed and built the experimental system, and performed the experiments. I.G.M. supervised the project.

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Correspondence to Ian G. Macara.

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Extended data

Extended Data Fig. 1 Expression of RUSH constructs and creation/validation of CRISPR knock-in RUSH construct.

(a) Confocal imaging of exogenous SBP-HaloTag-Crb3 RUSH in cells also expressing trans-Golgi reporter GalT-EGFP at time points indicated. Maximum intensity projections. Scalebar 10 µm. Representative micrographs from N = 2 independent experiments. (b) Left: Schematic of CRISPR/Cas9 gene editing strategy to insert the SBP-HaloTag sequence into Crb3 exon 2, which encodes amino acids immediately following the signal peptide of Crb3. In red: guide RNA targeting and approximate cut site. In magenta: SBP-HaloTag insert. In blue: homology arms flanking the insert to facilitate homology directed repair (HDR). Right: FACS gating to isolate JF549-Halo+ clones. Full gating strategy available in Supplementary Information 1 (c) Genotyping for SBP-Halo HDR alleles in putative knock-in clones. PCR product lengths: wild-type allele: 385 bp; HDR allele: 1408 bp; nonhomologous end-joining allele: ~1700 & 2100 bp (none shown). Genotyping performed once. (d) Confocal microscopy of EpH4 Crb3SBP-Halo/SBP-Halo cells at steady state labeled with JF549-Halo. Maximum intensity projection or XZ orthogonal view. Scalebar 10 µm. Representative micrograph from N = 3 independent experiments. (e) Near-TIRF of EpH4 Crb3SBP-Halo/SBP-Halo cells at steady state labeled with JF549-Halo and imaged live, at either steady state (junctional) or with the StrKDEL hook added (ER localized). Representative images are maximum intensity projections in time over a 30 second span, from N = 3 independent experiments. Top row: raw data. Bottom row: temporal color coded using Fiji. Scalebar 10 µm. Unprocessed gels are available in Source data.

Source data

Extended Data Fig. 2 Crb3 RUSH experiments ± enlarged cytoplasmic domain.

(a) Golgi trafficking dynamics of SBP-EGFP-Crb3 versus SBP-Halo-Crb3. n = 182 total cells. N = 3 independent experiments. Two-tailed Student’s t-test on means. (b) SBP-Halo-Crb3-FKBP localizes apically in the absence of SBP-EGFP-Crb3. Representative image from N = 2 independent experiments. Scalebar = 10 µm. (c) Lateralization index quantification strategy, related to Fig. 3e, f. Scalebar = 10 µm. (d) Related to Fig. 3g–k, using the same methodology but with SNAPtagx2-FRB as recruitable cargo in the presence of CID. n = 89 total cells. N = 3 independent experiments. Two-tailed Student’s t-test on means. (e) Related to Fig. 3m, n: Still images from FRAP experiments of SBP-EGFP-Crb3 vs SBP-HaloTag-Crb3-FKBP in the presence of SNAPx1-FRB and CID. FRAP performed at the endoplasmic reticulum (left) or Golgi apparatus 15 min into RUSH (right). FRAP at time 0 s as indicated in the left corner. Related to Fig. 3l, m. Scalebars 2 µm. (f) Extended data related to Fig. 3d–g, using the same methods but with mCherry-FRB as recruitable cargo, and either vehicle or CID. Vehicle: n = 95 total cells. N = 2 experiments. Two-tailed Welch’s t-test. CID: n = 75 total cells. N = 3 experiments. Two-tailed Student’s t-test or one-way ANOVA. (g) Golgi trafficking dynamics of mutant SBP-EGFP-Crb3-E117STOP, lacking the PDZ-binding motif and adaptor protein binding site, versus full length SBP-Halo-Crb3. n = 110 total cells. N = 3 experiments. Student’s t-test on experimental means. Source numerical data are available in source data.

Source data

Extended Data Fig. 3 Super-resolution imaging of Crb3 RUSH constructs at the Golgi.

(a) SoRA super-resolution images of Golgi at 30 min post-initiation of RUSH, for EGFP-Crb3 and Halo-Crb3-FKBP with co-expressed SNAPtagx1-FRB but in the absence of dimerizer (conditions in which there is no delay in Golgi transit). Images show Crb3 distributions versus Golgi sub-compartment markers GALT-mRuby3 (trans) or MGAT2-mRuby3 (cis/medial). Maximum intensity projections. Scalebar 2 µm. (b) Same as A but in the presence of CID. (c) Related to Fig. 4e. Left: Quantification of N-glycosylated “mature” SBP-Halo-Crb3-FKBP in the presence of SNAPx1-FRB ± dimerizer. N = 5 independent experiments. One-way anova + Šídák multiple comparison test between matched time points. Matched timepoint n.s. p = 0.9928; 0.0845; 0.0795; 0.3134; 0.3507; 0.3518. Right: PNGase F treatment demonstrating collapse of higher molecular weight band to the lower molecular weight band. Source numerical data and unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 Ace2 and Muc1 RUSH data; Crb3 RUSH in MDCK cells.

(a) Related to Fig. 5: Aggregated Golgi depletion time data for Ace2 and Ace2-FKBP in polarized EpH4 monolayers, in the presence of SNAP-FRB ± CID. Each point is a cell, and each line joins paired measurements within the same cell. Datapoints are colored by experiment, with diamonds representing the mean of each experiment. n = 209; 208; 231 total cells. N = 5; 4; 5 independent experiments. (b) Same as A but for Muc1 and Muc1-FKBP. n = 96; 95; 135 total cells. N = 4; 3; 3 independent experiments. (c) Same as A but for Crb3 and Crb3-FKBP in polarized MDCKII monolayers. n = 71; 101; 83 total cells. N = 3; 3; 3 independent experiments In all datasets, points are colored by experiment, and each circular point represents a single cell. Colored diamonds represent experimental means. Crossbar indicates mean of experimental means, with error bars representing SD. Two-sided Student’s t-test performed on experimental means. Source numerical data are available in source data.

Source data

Extended Data Fig. 5 Pals1 colocalization with Crb3; creation of CRISPR SNAPtag Pals1 knock-in.

(a) Images of SBP-HaloTag-Crb3 RUSH cells fixed after 30 minutes biotin, and immunostained against endogenous protein targets as indicated. Confocal maximum intensity projections. Scalebars 10 µm. N = 3 independent experiments. (b) Left: Immunoblot of SBP-HaloTag-Crb3 RUSH cells transduced with pLentiCRISPRv2 virus with nontargeting (NT), or Pals1 sgRNAs, performed once. Right: Images of nontargeting, or Pals1 deleted SBP-HaloTag-Crb3 RUSH cells fixed after 120 minutes biotin, and immunostained against endogenous Pals1; N = 2 independent experiments. (c) Left: Schematic of CRISPR/Cas9 gene editing strategy to insert the SNAPtag sequence into Mpp5 (Pals1) exon 3, the first coding exon of the gene. In red: guide RNA targeting and approximate cut site. In magenta: SNAPtag insert. In blue: homology arms flanking the insert to facilitate homology directed repair (HDR). Right: FACS gating to isolate JF552-SNAPcp+ clones. Full gating strategy available in Supplementary Information 1 (d) Genotyping for SNAPtag HDR alleles in putative knock-in clones. PCR product lengths: wild-type allele: 1054 bp; HDR allele: 1606 bp; nonhomologous end-joining allele: ~3300 & ~2400 bp. Genotyping was performed once (e) Near-TIRF or Confocal microscopy of EpH4 Crb3SBP-Halo/SBP-Halo Mpp5SNAPtag/SNAPtag double knock-in cells at steady state labeled with JFX646-Halo and JF552-SNAPcp. Representative images from N = 3 independent experiments. Scalebars 5 µm and 10 µm. Source numerical data and unprocessed gels are available in source data.

Extended Data Fig. 6 RUSH quantification strategy.

(a) RUSH normalization and quantification strategy, as detailed in Methods: “RUSH quantification”. (b) RUSH heatmap visualization, as detailed in Methods: “RUSH Heatmap Visualization”.

Supplementary information

Supplementary Information

Supplementary Information 1. FACS gating strategy for EpH4 CRISPR–Cas9 knock-in generation of SBP–Halo–Crb3 and SNAPtag–Pals1, related to Extended Data Figs. 1b and 5c.

Reporting Summary

Peer Review File

Supplementary Video 1

Related to Fig. 2c: RUSH of exogenous SBP–Halo–Crb3 in polarized EpH4 cells. Top: confocal maximum intensity projection. Bottom: denoised XZ orthogonal view. Scale bar, 10 µm. Time formatting: hh:mm:ss.

Supplementary Video 2

Related to Fig. 2d: RUSH of endogenous SBP–Halo–Crb3 in polarized EpH4 cells. Confocal Z slice. Scale bar, 10 µm. Time formatting: hh:mm:ss.

Supplementary Video 3

Related to Fig. 2e: RUSH of exogenous SBP–Halo–Crb3 and SBP–EGFP–Ecadherin in polarized EpH4 cells. Confocal denoised XZ orthogonal view. Scale bar, 10 µm. Time formatting: hh:mm:ss.

Supplementary Video 4

Related to Fig. 3h: RUSH of exogenous SBP–EGFP–Crb3 and SBP–Halo–Crb3–FKBP in polarized EpH4 cells. Top: with SNAPx1–FRB and vehicle. Middle: with SNAPx1–FRB and chemically inducible heterodimerizer (CID). Bottom: with SNAPx3–FRB and CID. Confocal maximum intensity projection. Scale bar, 10 µm. Time formatting: hh:mm:ss.

Supplementary Video 5

Related to Fig. 5e: RUSH of exogenous SBP–mNeonGreen–Ace2 and SBP–Halo–Ace2–FKBP in polarized EpH4 cells. Top: with SNAPx1–FRB and vehicle. Middle: with SNAPx1–FRB and chemically inducible heterodimerizer (CID). Bottom: with SNAPx3–FRB and CID. Confocal maximum intensity projection. Scale bar, 10 µm. Time formatting: hh:mm:ss.

Supplementary Video 6

Related to Fig. 6c: RUSH of exogenous SBP–Halo–Crb3 in polarized EpH4 cells also expressing mApple–Pals1. Confocal maximum intensity projection. Scale bar, 10 µm. Time formatting: hh:mm:ss.

Supplementary Video 7

Related to Fig. 6d: RUSH of exogenous of exogenous SBP–Halo–Crb3 in polarized EpH4 cells also expressing mApple–Pals1. Note evolution of Pals1-positive, Crb3-negative puncta around 17:45. Denoised 3D Alpha projection. Bounding box is 60 µm × 60 µm. Time formatting: hh:mm:ss.

Supplementary Table 1

List of reagents used.

Source data

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Unprocessed western blots and gels.

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de Caestecker, C., Macara, I.G. A size filter at the Golgi regulates apical membrane protein sorting. Nat Cell Biol (2024). https://doi.org/10.1038/s41556-024-01500-0

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