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. 2020 Jul 9;15(7):e0235864.
doi: 10.1371/journal.pone.0235864. eCollection 2020.

Cell-type-specific differences in KDEL receptor clustering in mammalian cells

Achim Bauer  1 , Ludger Santen  2 , Manfred J Schmitt  1 , M Reza Shaebani  2 , Björn Becker  1
Affiliations

Cell-type-specific differences in KDEL receptor clustering in mammalian cells

Achim Bauer et al. PLoS One. .

Abstract

In eukaryotic cells, KDEL receptors (KDELRs) facilitate the retrieval of endoplasmic reticulum (ER) luminal proteins from the Golgi compartment back to the ER. Apart from the well-documented retention function, recent findings reveal that the cellular KDELRs have more complex roles, e.g. in cell signalling, protein secretion, cell adhesion and tumorigenesis. Furthermore, several studies suggest that a sub-population of KDELRs is located at the cell surface, where they could form and internalize KDELR/cargo clusters after K/HDEL-ligand binding. However, so far it has been unclear whether there are species- or cell-type-specific differences in KDELR clustering. By comparing ligand-induced KDELR clustering in different mouse and human cell lines via live cell imaging, we show that macrophage cell lines from both species do not develop any clusters. Using RT-qPCR experiments and numerical analysis, we address the role of KDELR expression as well as endocytosis and exocytosis rates on the receptor clustering at the plasma membrane and discuss how the efficiency of directed transport to preferred docking sites on the membrane influences the exponent of the power-law distribution of the cluster size.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
(a) Schematic of the fluorescent model cargo without (top-left, eGFP-RTAE177D) or with (top-right, eGFP-RTAE177D-HDEL) the ER-retention motif HDEL, which ensures the physical interaction with KDELRs. eGFP-RTAE177D lacking the HDEL motif serves as negative control and is unable to bind KDELRs. An additional mammalian enhanced GFP tag was introduced at the N-terminus to monitor the binding/uptake and interacellular transport of the model cargo in living cells. Furthermore, a less toxic variant of the cytotoxic A subunit of ricin (RTAE177D) serves as a second marker to determine the cargo uptake via cell viability. The integrated His-tag ((His)6-Tag) is used for affinity purification of the fluorescent model cargos from E. coli lysates. (bottom row) Schematics of cargo-KDELR interaction at the mammalian cell surface in the presence/absence of the HDEL motif. (b) Confocal laser scanning microscopy of human cell lines treated with eGFP-RTAE177D-HDEL or eGFP-RTAE177D (negative control). In the latter case, the images represent the steady-state regime of receptor cluster development (t ≥ 150 min). Scale bars, 20 μm. (c) Similar to panel (b) but for mouse cell lines.
Fig 2
Fig 2. Evolution of the receptor clusters at the surface of various cell types.
A randomly chosen region of the plasma membrane is shown at different time points. Scale bars, 4 μm.
Fig 3
Fig 3
(a) Time evolution of the density of KDELR cargo signals at the surface of human cell lines after treatment with eGFP-RTAE177D-HDEL. The optimal signal-to-noise ratio, scaled by the cell periphery size, is shown as a function of time. The dashed lines show the fluctuation range of the signal intensity at the steady state. (b) Similar to panel (a) but for mouse cell lines. (c) Duration of transient and growth regimes and the mean relative saturation level of signal density (compared to the largest analyzed cells) in the steady state versus the cell size.
Fig 4
Fig 4. Cluster-size distribution of KDEL receptors at the surface of various cell types.
The power-law exponent in the indicated cell lines varies from α≃2 for HeLa and SH-SY5Y cells to α≃5 for HEK-293T cells.
Fig 5
Fig 5. mRNA levels of three KDELR homologues in various (a) human and (b) mouse cell lines.
Values in the indicated cell lines are scaled to the relative mRNA level of (a) HeLa and (b) L929 cells. Statistical significance is assessed by one-way ANOVA based on biological replicates and at sample sizes of n = 3 (***p ≤ 0.001; **p < 0.01; *p < 0.05; ns, not significant).
Fig 6
Fig 6. Schematic illustration of the receptor cycling model.
Black, green, and red full symbols represent, respectively, the receptors which survive or will be added or eliminated in the next time step. Dashed lines indicate the affected zones by endo/exocytosis events.
Fig 7
Fig 7
(a) Characteristic time ts and (b) relative saturation level fs in the (κendo, κexo) phase space. Single-hashed regions in panels (a) and (b) correspond to the values of κendo and κexo which result in ts or fs values experimentally observed for cluster-forming cells. (c) Double-hashed region shows the approximate extent of κendo and κexo rates in cell lines showing receptor clustering. The full circle represents the reference point (see text). (d) Similar to panel (c) but with different plot range. The solid line corresponds to the threshold line κendo=(-1+1/fsc)κexo, representing the unset of undetectable saturation level in experiments.
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Grants and funding

This work was funded by the Deutsche Forschungsgemeinschaft (DFG) through Collaborative Research Center SFB 1027. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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