KDEL receptor regulates secretion by lysosome relocation- and autophagy-dependent modulation of lipid-droplet turnover

Abstract

Inter-organelle signalling has essential roles in cell physiology encompassing cell metabolism, aging and temporal adaptation to external and internal perturbations. How such signalling coordinates different organelle functions within adaptive responses remains unknown. Membrane traffic is a fundamental process in which membrane fluxes need to be sensed for the adjustment of cellular requirements and homeostasis. Studying endoplasmic reticulum-to-Golgi trafficking, we found that Golgi-based, KDEL receptor-dependent signalling promotes lysosome repositioning to the perinuclear area, involving a complex process intertwined to autophagy, lipid-droplet turnover and Golgi-mediated secretion that engages the microtubule motor protein dynein-LRB1 and the autophagy cargo receptor p62/SQSTM1. This process, here named 'traffic-induced degradation response for secretion' (TIDeRS) discloses a cellular mechanism by which nutrient and membrane sensing machineries cooperate to sustain Golgi-dependent protein secretion.

Fig. 1

Endoplasmic reticulum (ER)-to-Golgi cargo transport induces transient lysosome repositioning. a HeLa cells were microinjected in the nucleus to deliver a DNA construct encoding LAMP1-GFP (green fluorescent protein) for its synchronized expression and transport from the ER. Representative images show LAMP1-GFP localization in the ER, Golgi or post-Golgi at the indicated time after microinjection. Cells were stained with DeepRed-Lysotracker, and its radial integrated fluorescence intensity was used to quantify the distribution of lysosomes as described in Methods. The graph shows the percentage of cells depicting cytoplasmically spread or perinuclear lysosome distribution (n = 30 cells). Scale bar, 10 µm. b HeLa cells expressing the human growth hormone fused in tandem to GFP and the polymerization/depolymerization FM domain (hGH-GFP-FM) were subjected to the ER-to-Golgi cargo transport assay. Lysosomes were also stained with DeepRed-Lysotracker. Images were acquired by dual time-lapse to track cargo transport (inset) and lysosome localization before and at the indicated time after addition of D/D solubilizer. The perinuclear distribution of lysosomes was quantified as in a and the values are indicated at the bottom of each image (n = 10 cells). Scale bar, 10 µm. c HeLa cells were examined by electron microscopy when hGH-GFP-FM was localized either at the ER or the Golgi complex. The ratio of lysosomes (red arrowheads) with respect to Golgi stacks (green Gs) was calculated as described in Methods (n = 3 independent experiments), and the values are indicated at the bottom of each image (ER = 1.91 ± 0.61; Golgi = 3.58 ± 0.52***). Scale bar, 500 nm. Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t tests). All t tests were conducted comparing to control cells

Fig. 2

Lysosome repositioning is dependent on the KDELR1 isoform. a To activate KDELR, HeLa cells were incubated for 30 min with 1 µM KDEL-BODIPY peptide. Cells were fixed with glutaraldehyde and processed for electron microscopy analysis. The ratio of lysosomes (red arrowheads) with respect to Golgi stacks (green Gs) was calculated as described in Methods, and the values are indicated at the bottom of each image (three independent experiments). Scale bar, 500 nm. b HeLa cells treated as in a were stained with DeepRed-Lysotracker, and its radial integrated fluorescence intensity was used to quantify the distribution of lysosomes as described in Methods. The graph shows the percentage of cells depicting cytoplasmically spread or perinuclear lysosome distribution. The perinuclear distribution of lysosomes was quantified, and the values are indicated at the bottom of each image (n = 30 cells). Scale bar, 10 µm. c HeLa cells were transfected (insets) to overexpress either of the indicated green fluorescent protein (GFP)-tagged KDELR isoform or GFP-tagged signalling-defective KDELR1-D193N in the presence of KDEL-BODIPY, or with a short hairpin RNA (shRNA) to silence KDELR1 expression in the absence or presence of KDEL-BODIPY. Lysosomes were stained and their distribution quantified as indicated in b. The graph shows the percentage of cells depicting cytoplasmically spread or perinuclear lysosome distribution. The perinuclear distribution of lysosomes was quantified, and the values are indicated at the bottom of each image (n = 30 cells). Scale bar, 10 µm. Data are means ± SEM. ***p < 0.001 (Student’s t tests). All t tests were conducted comparing to control cells

Fig. 3

The KDELR-dependent cAMP/PKA signalling pathway regulates lysosome repositioning. a HeLa cells were left untreated (Control) or incubated for 15 min at 37 °C with 1 µM KDEL-BODIPY peptide, to activate KDELR, in the absence (KDEL-BODIPY) or presence of either 10 µM R8-Gq peptide (KDEL-BODIPY + R8-Gq peptide) or 10 µM R8-Gs peptide (R8-Gs peptide), or in conjunction to transfection for the expression of a Gq minigene (KDEL-BODIPY + Gq minigene) or a Gs minigene (KDEL-BODIPY + Gs minigene). Cells were stained with DeepRed-Lysotracker, and its radial integrated fluorescence intensity was used to quantify the distribution of lysosomes as described in Methods. The graph shows the percentage of cells depicting cytoplasmically spread or perinuclear lysosome distribution. The perinuclear distribution of lysosomes was quantified, and the values are indicated at the bottom of each image (n = 30 cells). b HeLa cells stably transfected with sh-ADCY9 (insets; red fluorescent protein (RFP)-positive cells; red asterisks) were incubated with Lysotracker green and then either left untreated (Control) or treated with KDEL-BODIPY peptide as in a. Lysosomes distribution was quantified as indicated in a. The graph shows the percentage of cells depicting cytoplasmically spread or perinuclear lysosome distribution. The perinuclear distribution of lysosomes was quantified, and the values are indicated at the bottom of each image (n = 30 cells). c ADCY9-silenced HeLa cells (insets; RFP-positive cells) were incubated with KDEL-BODIPY alone as in a or co-incubated with only the non-hydrolysable cAMP analogue 8-Br-cAMP (100 µM) or with 8-Br-cAMP in conjunction to either of the PKA inhibitors PKI (50 µM) or H89 (30 µM). Lysosome distribution was quantified as indicated in a. The graph shows the percentage of cells depicting cytoplasmically spread or perinuclear lysosome distribution. The perinuclear distribution of lysosomes was quantified, and the values are indicated at the bottom of each image (n = 30 cells). Scale bar, 10 µm. Data are means ± SEM. ***p < 0.001 (Student’s t tests). All t tests were conducted comparing to control cells

Fig. 4

LRB1 dynein light chain drives the lysosome repositioning that is necessary to sustain secretion. a HeLa cells subjected either to control silencing or DynLRB1 silencing (insets; green fluorescent protein (GFP)) were left untreated or incubated for 15 min with 1 µM KDEL-BODIPY peptide to induce lysosome repositioning. Silencing was controlled by determining the messenger RNA (mRNA) level of DynLRB1 (first graph, n = 3 independent experiments). Alternatively, cells were stained with DeepRed-Lysotracker, and its radial integrated fluorescence intensity was used to quantify the distribution of lysosomes as described in Methods. The percentage of cells depicting cytoplasmically spread or perinuclear lysosome distribution was calculated (n = 30 cells). The perinuclear distribution of lysosomes was quantified, and the values are indicated at the bottom of each image (n = 30 cells). Scale bar, 10 µm. b HeLa cells not transfected (Control) or expressing either of the indicated HA-tagged variants of DynLRB1 were either left untreated or incubated with KDEL-BODIPY. Lysosomes were stained, and their distribution quantified as indicated in a, (n = 30 cells). c HeLa cells were transfected to express either of the indicated hemagglutinin (HA)-tagged variants of DynLRB1 and processed for immunogold labelling (10 nm) against the HA-tag and subsequent electron microscopy analysis. DynLRB1 expression was assessed by western blotting using anti-HA or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies, and the corresponding quantification is shown in a bar graph (data are means ± SEM). The ratio of lysosomes (red arrowheads) with respect to Golgi stacks (green Gs) was calculated as described in Methods, and the values are indicated in the table depicted in the fourth image (n = 3 independent experiments). The second and fourth images correspond to a two-fold magnification of the indicated regions of the first and third images, respectively. d Cells expressing LAMP1-mcherry and either KDELR1-GFP or KDELR-D193N-GFP were left without further treatment (Control) or transfected to co-express either of the indicated HA-tagged variants of DynLRB1. Lysosomes were stained, and their distribution quantified as indicated in a, (n = 30 cells). Scale bar, 10 µm. e HeLa cells expressing human growth hormone fused to the polymerization/depolymerization FM domain (hGH-GFP-FM) and co-expressing either DynLRB1-wt-HA (Control) or DynLRB1-S73A-HA were subjected to the ER-to-Golgi transport assay, and images were acquired for 50 min at 5 min interval. The level of cargo on the Golgi complex was quantified (n = 15 cells). Data are means ± SEM. **p < 0.01; ***p < 0.001 (Student’s t tests). All t tests were conducted comparing to control cells. wt Wild type

Fig. 5

The fusion of autophagosomes and lysosomes is regulated by KDELR and DynLRB1. a H4 cells transfected to express the pH-sensitive autophagy flux marker LC3 (mcherry-GFP-LC3) were left untreated (Control) or incubated for 15 or 30 min at 37 °C with 1 µM KDEL-BODIPY peptide alone, or in combination with 10 µM R8-Gs peptide. The graph depicts the quantification of cells with autophagosomes that were regarded as puncta showing mcherry and green fluorescent protein (GFP) fluorescence (n = 50 cells). b H4 cells transfected as in a were subjected to nutrient starvation for 1 h to induce autophagosome formation, and then incubated with cell-permeable peptides as in a. The graph depicts quantification of cells (n = 50 cells) with autophagosomes as indicated in a. c Quantification of the ratio between autolysosomes and autophagosomes in cells treated as indicated in b. Autolysosome/autophagosome ratio was calculated by automatic counting of red (mcherry, pH-insensitive) and green (GFP, pH-sensitive) positive puncta (n = 50 cells). d–h HeLa cells were co-transfected to express mcherry-GFP-LC3 and either of the indicated DynLRB1-HA (hemagglutinin) variants, and either left untreated (d, g and h) or incubated for 30 min with 1 µM KDEL-BODIPY (e and f). Insets show linescans (25-length × 4-width pixels) of representative regions depicting the presence of red and green fluorescent emission profiles of autophagic puncta. Red and green emission identifies puncta as autophagosomes, and lack of green emission identifies puncta as autolysosomes (n = 3 independent experiments). Scale bars, 10 µm. i Quantification of the ratio between autolysosomes and autophagosomes as indicated in c in cells (n = 50 cells) treated as indicated in d–h. Data are means ± SEM. *p < 0.05; ***p < 0.001 (Student’s t tests). All t tests were conducted comparing to control cells

Fig. 6

ATG5-dependent autophagy is required to sustain protein secretion. a HeLa cells expressing human growth hormone fused to the polymerization/depolymerization FM domain (hGH-GFP-FM) were subjected to control silencing (Control) or ATG5 silencing and further subjected to the endoplasmic reticulum (ER)-to-Golgi cargo transport assay. Cargo trafficking was tracked by time-lapse fluorescence microscopy. Representative images show hGH-GFP-FM localization in the ER, Golgi or post-Golgi at the indicated times after addition of DD-solubilizer (n = 15 cells). Scale bar, 10 µm. The graph depicts the quantification of the level of cargo at the Golgi complex during the time of cargo trafficking from images as those shown in a, highlighting in yellow the timeframe of typical cargo localization in the Golgi complex or post-Golgi b. HeLa cells expressing hGH-GFP-FM were subjected to the same treatments shown in a, and the medium (Secreted) and cellular extracts were collected at the indicated times after addition of DD-solubilizer. Proteins from the medium and cellular extracts were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting using antibodies to the proteins indicated on the right. The graph depicts the quantification of the ratio between secreted and total hGH-GFP-FM detected by western blotting (n = 3 independent experiments). Data are means ± SEM. GFP green fluorescent protein

Fig. 7

The autophagy cargo receptor p62/SQSTM1 is required to sustain protein secretion during lysosome repositioning. a HeLa cells expressing human growth hormone fused to the polymerization/depolymerization FM domain (hGH-GFP-FM) were subjected to the endoplasmic reticulum (ER)-to-Golgi cargo transport assay and then subjected to immuno-staining to detect endogenous p62/SQSTM1. Representative images show p62/SQSTM1 puncta distribution at the indicated times of hGH-GFP-FM localization in the ER, Golgi or post-Golgi (insets) after addition of DD-solubilizer. The graph depicts the quantification of the number of p62/SQSTM1 puncta at the times indicated in the images (n = 50 cells). b HeLa cells expressing hGH-GFP-FM were transfected to co-express p62/SQSTM1-wt-mcherry or the p62/SQSTM1-S182A-mcherry mutant. After 16 h, cells were subjected to the ER-to-Golgi cargo transport assay. Representative images show the localization of hGH-GFP-FM and the distribution of puncta containing either of the p62/SQSTM1-mcherry variant, in conditions as those shown in a. The graphs depicts either the quantification of hGH-GFP-FM in the Golgi complex or the quantification of the number of puncta containing either of the p62/SQSTM1-mcherry variant at the times indicated in the images (n = 15 cells). c HeLa cells transfected to express either of the indicated p62/SQSTM1-mcherry variants were incubated with Lysotracker. Cells were nutrient-starved by incubation for 1 h in Hank’s balanced salt solution. At the indicated times after, images were acquired to calculate the level of co-localization between lysosomes and puncta containing either of the indicated p62/SQSTM1-mcherry variant (n = 15 cells). d HeLa cells were transfected and incubated with Lysotracker as in c, but maintained in regular culture medium. Images were acquired to calculate the level of co-localization in steady-state conditions between lysosomes and puncta containing either of the indicated p62/SQSTM1-mcherry variant. The quantification is shown in the graph at the right of the panel (n = 50 cells). e Mouse LMR7.5 T-lymphocytes were subjected to control silencing (shControl) or silencing of KDELR1 using either of the indicated short hairpin RNA (shRNA). Cells were subjected to an antigen-peptide presentation assay using the indicated concentrations of antigen for the indicated periods of time. To determine T-lymphocyte activation, the level of secreted interleukin-2 (IL-2) was estimated using an enzyme-linked immunosorbent assay (ELISA) assay (n = 3 independent expriments). f Mouse LMR7.5 T-lymphocytes were transfected to express p62/SQSTM1-mcherry-wt or p62/SQSTM1-mcherry-S182A. After transfection, cells were subjected to an antigen-peptide presentation assay and to estimation of T-lymphocyte activation as shown in e (n = 3 independent experiments). Data are means ± SEM. Scale bar, 10 µm. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t tests). wt Wild type

Fig. 8

KDELR1 modulate lipid droplet turnover during activation of lysosome repositioning. a HeLa cells untreated (Control) or transfected with KDELR1, KDELR1-D193N or short hairpin RNA (shRNA) against KDELR1 were fixed and lipid droplets (LDs) were stained and a quantitative analysis of the number of LDs was calculated (n = 50 cells). b H4 cells untreated (Control) or transfected to express either of the indicated p62/SQSTM1-mcherry variants and LDs were stained and quantified (n = 50 cells). c H4 cells were transfected to transiently express p62/SQSTM1-mcherry-wt (wild type), followed by LD staining. Live-cell images were acquired, and the number of interactions between LDs and p62/SQSTM1-mcherry-wt puncta and of the joint speeds of movement were calculated as indicated in Methods (n = 3 independent experiments). d H4 cells were transfected to transiently express p62/SQSTM1-mcherry-S182E or p62/SQSTM1-mcherry-S182A, followed by LD staining and live-cell imaging acquisition and analysis (n = 3 independent experiments) as indicated in c. e H4 cells were transfected to transiently co-express p62/SQSTM1-mcherry-wt and either of the indicated hemagglutinin (HA)-tagged DynLRB1 variants, followed by LD staining and live-cell imaging acquisition and analysis as indicated in c. The number of interactions and the joint speeds of movement (n = 3 independent experiments) was calculated. f HeLa cells were transfected either to express DynLRB1-HA-wt (DynLRB1-HA) or with an shRNA against DynLRB1 (shDynLRB1). Cells were fixed and LDs were stained and immunofluorescence to detect DynLRB1-HA-wt. The graph shows the quantification of the number and fluorescence intensity of LDs of cells subjected to the indicated treatments (n = 30 cells). g H4 cells were transiently transfected to express p62/SQSTM1-wt or p62/SQSTM1-S182A and were stained for LDs, and images were acquired every 5 min under PKA activation and subsequent PKA inhibition (n = 15 cells). (h) H4 cells were transiently transfected to express p62/SQSTM1-wt or p62/SQSTM1-S182A and were stained for LDs, with images acquired every 5 min under KDELR activation and subsequent PKA inhibition (n = 15 cells). i H4 cells transfected as in b–h were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot against actin (load control) and mCherry (p62/SQSTM1). Normalized mCherry/actin signal is shown on the plot bars (n = 3 independent experiments). Data are means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001 (Student’s t tests). All t tests were conducted comparing to control cells

Fig. 9

Autophagy-dependent lipid droplet turnover affects protein secretion. a HeLa cells expressing human growth hormone fused to the polymerization/depolymerization FM domain (hGH-GFP-FM) were left untreated (Control) or subjected to ATG5 silencing (ATG5 silenced) and kept in normal media (Control and ATG5 silenced) or incubated for 4h in media supplemented with 100 µM palmitic acid (third image), followed by lipid droplet (LD) staining with BODIPY. Scale bar, 10 µm. The graph shows the quantification of the number and fluorescence intensity of LDs (n = 1000 LDs) of cells (n = 30 cells) subjected to the indicated treatments, from images as those shown at the left. b HeLa cells expressing hGH-GFP-FM were subjected to ATG5 silencing and left without further transfection (upper two panels) or transfected to co-express either p62/SQSMT1-mcherry-S182A or DynLRB1-HA-S73A. Cells were kept in normal media (Control, first panel) or incubated for 4 h in media supplemented with 100 µM palmitic acid (lower three panels), followed by the ER-to-Golgi transport assay. Cells were fixed (upper three panels) or fixed and permeabilized, followed by immuno-staining with antibody to detect DynLRB1-HA-S73A. Representative images show hGH-GFP-FM localization in the endoplasmic reticulum (ER), Golgi or post-Golgi at the indicated times after addition of DD-solubilizer. The respective graph in each panel depicts the quantification of hGH-GFP-FM in the Golgi complex at the times indicated in images as those shown at the left (n = 30 cells). Scale bar, 10 µm. Data are means ± SEM. ***p < 0.001 (Student’s t tests). All t tests were conducted comparing to control cells. HA hemagglutinin