Acute inactivation of retromer and ESCPE-1 leads to time-resolved defects in endosomal cargo sorting

SNX5 knocksideways in SNX5/SNX6 double-knockout HeLa cells led to the time-resolved appearance of a CI-MPR missorting phenotype, defined by reduced enrichment of the CI-MPR at the Golgin97 or TGN46-labelled TGN, with maximum penetrance at 6 h.

“Consistent with the role of SNX5 in the ESCPE-1-mediated endosome-to-TGN transport of the CI-MPR (Kvainickas et al., 2017; Simonetti et al., 2017, 2019), SNX5 knocksideways in SNX5/SNX6 double-knockout HeLa cells led to the time-resolved appearance of a CI-MPR missorting phenotype as defined by a reduced enrichment of the CI-MPR at the Golgin97 or TGN46-labelled TGN with a maximum penetrance at 6”

• cites Kvainickas et al., 2017 sourceCheck pending
• cites Simonetti et al., 2017 sourceCheck pending
• cites Simonetti et al., 2019 sourceSupported

In H4 neuroglioma cells there is a clear distinction between acute retromer knocksideways and long-term retromer knockout: only the latter increased the steady-state expression of CI-MPR, sortilin and cathepsin D.

“Interestingly, in examining CI-MPR phenotypes in H4 neuroglioma cells, we observed a clear distinction between acute retromer knocksideways and the long-term effects of retromer knockout. Only in the latter did we observe an increase in the steady-state expression of CI-MPR, sortilin and cathepsin D (Fig. S5C).”

Retromer and ESCPE-1 can be specifically and rapidly inactivated by knocksideways, leading to the time-resolved development of selective cargo sorting defects through the endosomal pathway.

“In applying knocksideways, we have established that retromer and ESCPE-1 can be specifically and rapidly inactivated, leading to the time-resolved development of selective cargo sorting defects through the endosomal pathway.”

Retromer knocksideways performed in wild-type HeLa cells, which retain expression of endogenous VPS35 not subject to knocksideways, did not elicit development of a GLUT1 missorting phenotype.

“Moreover, the development of the GLUT1 missorting did not stem from the recruiting of ‘foreign’ proteins to peroxisomes as retromer knocksideways performed in wild-type HeLa cells, which retain expression of endogenous VPS35 that is not subject to knocksideways, did not elicit the development of a GLUT1 missorting phenotype (Fig. S3C,D).”

In time-resolved retromer knocksideways experiments in H4 cells, no significant redistribution of CI-MPR away from the TGN markers Golgin97 and TGN46 was detected.

“In a parallel time-resolved retromer knocksideways experiments, we failed to detect a significant redistribution of CI-MPR away from TGN markers Golgin97 and TGN46 (Fig. 8C–F).”

The endosomal dragging was not complete, as there was still a loss of colocalisation between GFP-FRB-SNX5 and EEA1.

“However, this endosomal ‘dragging’ was not complete, as there was still a loss of colocalisation between GFP–FRB–SNX5 and EEA1 (Fig. 5B,D).”

Despite the increase in CI-MPR signal intensity, retromer-knockout H4 neuroglioma cells did not display a significant change in the quantified Pearson's correlation coefficient between CI-MPR and Golgin97.

“Despite the increase in the CI-MPR signal intensity, retromer-knockout cells did not display a significant change in the quantified Pearson's correlation coefficient between CI-MPR and Golgin97 (Fig. 7A,C).”

Retromer knockout in H4 cells resulted in a pronounced upregulation of CI-MPR protein levels, as shown in three independent clonal lines.

“Biochemical analysis of three independent clonal lines revealed that retromer knockout resulted in a pronounced upregulation of CI-MPR protein levels (Fig. S5B).”

The VPS35-GFP-FRB chimera localises to cytosolic puncta identified as endosomes by colocalisation with EEA1 in VPS35-knockout HeLa cells.

“The VPS35–GFP–FRB chimera localised to cytosolic puncta that were identified as endosomes by means of colocalisation with the endosome marker EEA1 (Fig. S1A).”

The endosomal retrieval and recycling of the retriever-dependent cargo α5β1-integrin was not affected upon retromer knocksideways.

“the endosomal retrieval and recycling of the retriever-dependent cargo α5β1-integrin (McNally et al., 2017) was not affected upon retromer knocksideways (Fig. S3A,B)”

• cites McNally et al., 2017 sourceNot checked yet

ESCPE-1 knockout H4 neuroglioma cells displayed a clear redistribution of CI-MPR to peripheral dispersed puncta.

“In contrast, the ESCPE-1 knockout H4 neuroglioma cells displayed a clear redistribution of CI-MPR to peripheral dispersed puncta (Fig. 7A,D; Fig. S5A).”

Endogenous SNX1, a component of the ESCPE-1 complex, is not rerouted onto peroxisomes after 24 h of rapalog treatment.

“Indeed, we failed to observe the rerouting of endogenous SNX1, a component of the ESCPE-1 complex, onto peroxisomes after 24 h of rapalog treatment (Fig. 2F–H).”

Endogenous VPS26 is rerouted to peroxisomes with a similar kinetic profile to VPS35-GFP-FRB upon retromer knocksideways.

“analysis of the endogenous localisation of VPS26 revealed that it too was rerouted to peroxisomes with a similar kinetic profile to that observed for VPS35–GFP–FRB (the lack of a suitable antibody precluded the equivalent analysis of VPS29) (Fig. 2A,B).”

The FAM21-containing WASH complex is rerouted to peroxisomes upon retromer knocksideways.

“In addition, the major retromer accessory complex, the FAM21-containing WASH complex (Derivery et al., 2009; Gomez and Billadeau, 2009; Harbour et al., 2010; Jia et al., 2012) was also rerouted to peroxisomes upon retromer knocksideways (Fig. 2C,D; Fig. S2B,C).”

Confocal imaging of retromer-knockout H4 neuroglioma cells revealed an enhanced intensity in the staining of endogenous CI-MPR that was not observed in ESCPE-1-knockout cells.

“Interestingly, confocal imaging of the retromer-knockout cells revealed an enhanced intensity in the staining of endogenous CI-MPR that was not observed in the ESCPE-1-knockout cells (Fig. 7A,B).”

When expressed in HeLa cells, GFP-FRB-SNX5 localised to endosomes as defined by colocalisation with EEA1.

“When expressed in HeLa cells, GFP–FRB–SNX5 localised to endosomes as defined by colocalisation with EEA1 (Fig. 5A).”

Retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1.

“While retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1, we failed to observe a quantifiable defect in CI-MPR sorting.”

Following addition of rapalog for 24 h, fixed cell confocal imaging revealed a GLUT1 missorting phenotype, defined by steady-state loss of GLUT1 at the cell surface and enrichment of GLUT1 with LAMP1-positive late endosomes/lysosomes.

“Following the addition of rapalog for 24 h, fixed cell confocal imaging revealed a GLUT1 missorting phenotype, defined by the steady-state loss of GLUT1 at the cell surface and the enrichment of GLUT1 with LAMP1-positive late endosomes/lysosomes (Fig. 3A,B).”

In VPS35-knockout HeLa cells rescued through expression of VPS35-GFP-FRB, the CI-MPR is chiefly localised to the perinuclear TGN, as defined through colocalisation with TGN markers Golgin97 and TGN46.

“In VPS35-knockout HeLa cells rescued through expression of VPS35–GFP–FRB, the CI-MPR is chiefly localised to the perinuclear TGN, as defined through colocalisation with TGN markers Golgin97 and TGN46 (also known as GOLGA1 and TGOLN2, respectively) (Fig. 4A,B).”

Expression of VPS35-GFP-FRB rescued the GLUT1 missorting phenotype in retromer-knockout cells.

“Expression of VPS35–GFP–FRB rescued the GLUT1 missorting phenotype in retromer-knockout cells.”

After rapalog addition, a slight recruitment of EEA1 to the peroxisomal hook was observed, indicating movement of the endosomal compartment to the peroxisomal compartment.

“Interestingly, after rapalog addition, we observed a slight recruitment of EEA1 to the peroxisomal hook, indicating a movement of the endosomal compartment to the peroxisomal compartment (Fig. 5B,C).”

VPS35-knockout H4 neuroglioma cells display an upregulation of lysosomal hydrolases and lysosomal hydrolase receptors.

“VPS35-knockout H4 neuroglioma cells display an upregulation of lysosomal hydrolases and lysosomal hydrolase receptors.”

In the continued presence of rapalog, peroxisome-rerouted VPS35-GFP-FRB is retained on peroxisomes for up to 24 h.

“In VPS35-knockout HeLa cells transiently transfected to express PEX–Myc–3×FKBP and VPS35–GFP–FRB, the addition of 100 nM of rapalog established that rerouting of VPS35–GFP–FRB from EEA1-positive endosomes to peroxisomes was achieved within 10 min and was complete by 30 min (Fig. 1C–E) – in the continued presence of rapalog the peroxisome rerouted VPS35–GFP–FRB was retained on this organelle (maxim”

A significant amount of the WASH complex is retained on endosomes even after retromer knocksideways.

“Supporting evidence that a sub-population of the WASH complex is associated with endosomes independently of retromer (McNally et al., 2017; MacDonald et al., 2018), a significant amount of the WASH complex was retained on endosomes even after retromer knocksideways (Fig. 2C,E; Fig. S2B,D,E).”

• cites McNally et al., 2017 sourceNot checked yet
• cites MacDonald et al., 2018 sourceCheck pending

After addition of rapalog and initiation of retromer knocksideways, no quantifiable alteration in the steady-state distribution of the CI-MPR was observed over a time frame where endosomal missorting of internalised GLUT1 was readily observed.

“After the addition of rapalog and initiation of retromer knocksideways, we failed to observe a quantifiable alteration in the steady-state distribution of the CI-MPR (Fig. 4A–D) over a time frame where the endosomal missorting of internalised GLUT1 was readily observed (Fig. 3C,D).”

The increases in CI-MPR, sortilin and cathepsin D protein levels were not observed across three independent ESCPE-1-knockout H4 cell lines.

“These increases in protein levels were not observed across three independent ESCPE-1-knockout H4 cell lines (Fig. 7E–I).”

The observed upregulation of CI-MPR, sortilin and cathepsin D expression in H4 neuroglioma cells likely reflects retromer's regulatory role, through a complex compensation in lysosomal function induced by long-term retromer knockout.

“The observed upregulation of CI-MPR, sortilin and cathepsin D expression in H4 neuroglioma cells therefore likely reflects this regulatory role, through a complex compensation in lysosomal function induced by long-term retromer knockout.”

The PX domains of SNX5 and SNX6 directly bind to the ΦxΩxΦ(x)nΦ sorting motif in CI-MPR to mediate endosome-to-TGN transport.

“Of these proteins, it is the PX domains of SNX5 and SNX6 that directly bind to the ΦxΩxΦ(x)nΦ sorting motif (where Φ represents hydrophobic amino acids) in CI-MPR to mediate endosome-to-TGN transport (Simonetti et al., 2019).”

• cites Simonetti et al., 2019 sourceNot checked yet

The rapalog AP21967 has a lower affinity to endogenous mTOR than rapamycin.

“we utilised a modified version of FRB (T2098L) that enables the induction of heterodimerisation by rapalog (AP21967), a compound that has a lower affinity to endogenous mTOR than rapamycin (Clackson et al., 1998).”

• cites Clackson et al., 1998 sourceNot checked yet

These data establish a method for acute knocksideways of a functional VPS35-GFP-FRB construct.

“Together, these data establish a method for the acute knocksideways of a functional VPS35–GFP–FRB construct.”

These data support that it is the specific removal and inactivation of retromer that causes the time-resolved development of the observed GLUT1 missorting phenotype.

“Together, these data support that it is the specific removal and inactivation of retromer that causes the time-resolved development of the observed GLUT1 missorting phenotype.”

• AssumptionRests on an assumption: Peroxisomes are not implicated in retromer biology.

The study establishes a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting.

“Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting.”

Given that endosome-to-TGN transport of the CI-MPR is considered to occur over about 20 to 30 min, acute perturbation of retromer function does not appear to lead to a detectable defect in the endosomal sorting of the CI-MPR.

“Given that the endosome-to-TGN transport of the CI-MPR is considered to occur over a period of ∼20 to 30 min (Seaman, 2004), the acute perturbation of retromer function does not appear to lead to a detectable defect in the endosomal sorting of the CI-MPR (Kvainickas et al., 2017; Simonetti et al., 2017).”

• cites Seaman, 2004 sourceNot checked yet
• cites Kvainickas et al., 2017 sourceCheck pending
• cites Simonetti et al., 2017 sourceCheck pending

The metazoan equivalent assembly has evolved into two functionally distinct complexes, the retromer (VPS26-VPS35-VPS29) and the ESCPE-1 (SNX1/SNX2 and SNX5/SNX6) complexes.

“An increasing body of biochemical, cellular and in vivo functional data are consistent with the equivalent metazoan assembly having evolved into two functionally distinct complexes, the retromer (VPS26–VPS35–VPS29) and the ESCPE-1 (SNX1/SNX2 and SNX5/SNX6) complexes (Norwood et al., 2011; Swarbrick et al., 2011; Kvainickas et al., 2017; Simonetti et al., 2017, 2019; Strutt et al., 2019).”

• cites Norwood et al., 2011 sourceNot checked — no full text of the source
• cites Swarbrick et al., 2011 sourceCheck pending
• cites Kvainickas et al., 2017 sourceCheck pending

These data support that in metazoans retromer and ESCPE-1 have evolved into functionally distinct complexes.

“These data therefore support the in vivo evidence that in metazoans retromer and ESCPE-1 have evolved into functionally distinct complexes (Kvainickas et al., 2017; Simonetti et al., 2017, 2019; Strutt et al., 2019).”

• cites Kvainickas et al., 2017 sourceCheck pending
• cites Simonetti et al., 2017 sourceCheck pending
• cites Simonetti et al., 2019 sourceNot checked yet

The knockout phenotype may in part reflect retromer's role as a master regulator of the activity state of endosomal RAB7 through binding to the RAB7 GAP TBC1D5.

“In part, this phenotype may reflect the role of retromer as a master regulator (Jimenez-Orgaz et al., 2018) of the activity state of endosomal RAB7 through binding to the RAB7 GAP TBC1D5 (Seaman et al., 2009, 2018; Jimenez-Orgaz et al., 2018; Kvainickas et al., 2019).”

• cites Jimenez-Orgaz et al., 2018 sourceNot checked yet
• cites Seaman et al., 2009, 2018Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Kvainickas et al., 2019 sourceNot checked yet

The time-resolved analysis establishes that the ESCPE-1 complex is the primary mediator of sequence-dependent endosome-to-TGN sorting of CI-MPR.

“While not excluding a role for retromer in the known complexities of CI-MPR sorting (Seaman, 2018), our time-resolved analysis establishes that the ESCPE-1 complex is the primary mediator of sequence-dependent endosome-to-TGN sorting of this receptor.”

• cites Seaman, 2018Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The designed VPS35 knocksideways provides a method for acute and selective rerouting of retromer and its functionally coupled accessory proteins away from endosomes to neighbouring peroxisomes.

“Overall, the designed VPS35 knocksideways provides a method for the acute and selective rerouting of retromer (and its functionally coupled accessory proteins) away from endosomes to neighbouring peroxisomes.”

• AssumptionRests on an assumption: Peroxisomes are not implicated in retromer biology.

The knocksideways approach provides a method to acutely perturb the function of sorting complexes in a time frame that better aligns with the dynamic nature of endosomal membrane trafficking.

“this approach provides a method to acutely perturb the function of sorting complexes in a time frame that better aligns with the dynamic nature of endosomal membrane trafficking.”

These data establish that acute perturbation of the ESCPE-1 complex leads to a missorting of CI-MPR.

“Together, these data establish that acute perturbation of the ESCPE-1 complex leads to a missorting of CI-MPR.”

This technically distinct approach provides further data supporting the diversification of retromer and ESCPE-1 into two functionally distinct sorting complexes.

“This technically distinct approach therefore provides further data to support the diversification of retromer and ESCPE-1 into two functionally distinct sorting complexes.”

The data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting.

“Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting.”