Sorting nexin 17 (SNX17) links endosomal sorting to Eps15 homology domain protein 1 (EHD1)–mediated fission machinery

SNX17 interacts with a multitude of receptors in a sequence-specific manner to regulate their recycling.

“Notably, SNX17 interacts with a multitude of receptors in a sequence-specific manner to regulate their recycling.”

• cites [9] Donoso, 2009, Polarized traffic of LRP1 involves AP1B and SNX17 operating on Y-dependent sorting motifs in different pathways, Mol. Biol. Cell sourcePartly supported

  The Introduction reports that SNX17 binds several LDLR-family members, and the Results (p. 490) link a specific LRP1 motif to SNX17-dependent recycling. The paper does not establish that SNX17 regulates the recycling of a multitude of different receptors. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [10] Osborne, 2015, SNX17 affects T cell activation by regulating TCR and integrin recycling, J. Immunol sourceCheck pending
• cites [11] Steinberg, 2012, SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways, J. Cell Biol sourceSupported
• cites [12] Stockinger, 2002, The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor, EMBO J sourcePartly supported

  The Results (page unavailable) show that SNX17 binds the intracellular tails of several LDL-receptor-family members but not megalin, supporting selective interaction. The Introduction and Discussion (pages unavailable) report enhanced LDLR endocytosis and suggest faster movement through endocytic or recycling compartments. They do not establish a specific binding sequence or show that SNX17 regulates recycling of all the receptors it binds. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

The SNX17 PX domain does not interact with EHD1.

“no interaction with EHD1 was observed with this lipid-binding domain”

EHD1 and SNX17 reside in a complex in cells.

“These data demonstrate that EHD1 and SNX17 reside in a complex in cells.”

• Also stated in: § Results · plain

  “In addition, IP with anti-EHD1 antibodies also led to detection of SNX17 (IP EHD1).”

EHD1 interaction with SNX17 depends on the presence of the SNX17 FERM B and FERM C domains.

“Recruitment of EHD1 to the SE leads to its direct interaction with SNX17 and depends on the presence of the SNX17 FERM B and FERM C domains.”

SNX17-containing endosomes are larger in EHD1-depleted cells than in WT cells.

“SNX17-containing endosomes were larger in EHD1-depleted cells than in WT cells”

EHD1 depletion leads to enlarged sorting endosome structures.

“Indeed, EHD1 depletion leads to enlarged SE structures, supporting a role of EHD1 in SE fission.”

SNX17 and EHD1 partially colocalize on endosomes.

“surface rendering and quantification of overlap volumes indicated that SNX17 and EHD1 partially colocalize on endosomes”

EHD1 and SNX17 directly interact.

“Coimmunoprecipitation experiments and in vitro assays provided evidence that EHD1 and SNX17 directly interact.”

• Also stated in: § Results · hedged

  “These data support the notion that EHD1 and SNX17 reside within a complex in cells and are capable of direct interaction.”

EHD1 undergoes recruitment to endosomal membranes upon induction of receptor-mediated endocytosis.

“Here we demonstrate that EHD1 undergoes recruitment to endosomal membranes upon induction of receptor-mediated endocytosis.”

Inducing internalization of LRP1 leads to recruitment of cytoplasmic EHD1 to endosomal membranes.

“We also found that inducing internalization of a SNX17 cargo receptor, low-density lipoprotein receptor–related protein 1 (LRP1), led to recruitment of cytoplasmic EHD1 to endosomal membranes.”

The overlap between SNX17 and EHD1 on endosomes increases upon LRP1 internalization.

“and that this overlap further increases upon LRP1 internalization.”

Recruitment of EHD1 to the sorting endosome leads to its direct interaction with SNX17.

“Recruitment of EHD1 to the SE leads to its direct interaction with SNX17 and depends on the presence of the SNX17 FERM B and FERM C domains.”

IP with anti-EHD1 antibodies led to detection of SNX17.

“In addition, IP with anti-EHD1 antibodies also led to detection of SNX17 (IP EHD1).”

Mutation of SNX17 W321 to W321A has no effect on its ability to bind EHD1.

“mutation of SNX17 to W321A had no effect on its ability to bind EHD1”

Induced recruitment of EHD1 to SNX17-containing endosomes led to increased colocalization.

“As demonstrated in Fig. S5, A–F, insets), induced recruitment of EHD1 to SNX17-containing endosomes led to increased colocalization.”

SNX17 showed only a modest mean ~20% recruitment upon induction of LRP1 internalization.

“showed only a modest mean ˜20% recruitment upon induction of LRP1 internalization (Fig. 3, C and D, and quantified in F).”

EHD1 recruitment to SE is independent of SNX17.

“Overall, these data suggest that EHD1 recruitment to SE is independent of SNX17”

• Also stated in: § Discussion · hedged

  “However, because SNX17 knockdown had little impact on the recruitment of EHD1 to endosomes, this suggests the following scenario (Fig. 7).”

The percentage of SNX17 surface volume overlap with EHD1 increases significantly by 2- to 3-fold upon LRP1 uptake.

“As indicated, the percentage of SNX17 surface volume overlap with EHD1 increases significantly by 2- to 3-fold upon LRP1 uptake.”

• Also stated in: § Discussion · plain

  “Remarkably, however, we observed a 2- to 3-fold increase in the surface overlap volume following 30-min internalization of LRP1.”

At steady state, in the absence of select uptake, EHD1 is visualized in the cytoplasm and on a smattering of puncta and small tubules, consistent with sorting endosomes (SE).

“At steady state, in the absence of select uptake, we visualized EHD1 in the cytoplasm and on a smattering of puncta and small tubules, consistent with SE (Fig. 3A, see inset for more detail).”

Inducing LRP1 internalization and EHD1 recruitment leads to significantly more SNX17 overlap with EHD1.

“and that inducing LRP1 internalization and EHD1 recruitment leads to significantly more SNX17 overlap with EHD1.”

Binding of SNX17 to EHD1 occurs through the SNX17 atypical FERM domain.

“Binding of SNX17 to EHD1 occurs through the SNX17 atypical FERM domain”

EHD1 knockout cells appeared to have larger SNX17-containing endosomes.

“As shown in the representative micrographs (Fig. 6, A and B), EHD1 knockout cells indeed appeared to have larger SNX17-containing endosomes.”

In the absence of EHD1, the ratio of larger to smaller endosomes is higher compared with EHD1-GFP-containing cells.

“further highlights the notion that, in the absence of EHD1, the ratio of larger to smaller endosomes is higher compared with EHD1-GFP–containing cells (Fig. 6E, see shift to the right for the knockout in the inset).”

Addition of a control antibody to the V5 epitope had no significant impact on EHD1 recruitment compared with LRP1 uptake.

“addition of a control antibody to the V5 epitope had no significant impact on EHD1 recruitment compared with LRP1 uptake (Fig. S4, A–C, and quantified in D).”

Quantification of total surface volume of EHD1-containing endosomes in cells that internalized LRP1 compared with untreated cells showed a mean ~300% increase per cell.

“Using 3D surface rendering of z-stacks with IMARIS software, we quantified the total surface volume of EHD1-containing endosomes in cells that internalized LRP1 compared with untreated cells, and we observed a mean ˜300% increase per cell (Fig. 3E).”

In the absence of EHD1, SNX17-containing endosomes increase in size.

“our data demonstrate that, in the absence of EHD1, SNX17-containing endosomes increase in size”

Receptor-mediated endocytosis of the LRP1 receptor induces massive recruitment of EHD1 to endosomal membranes.

“Our study demonstrates that receptor-mediated endocytosis of the LRP1 receptor induces massive recruitment of EHD1 to endosomal membranes.”

Incubating cells with an antibody to the SNX17-sorted cargo receptor LRP1 to induce its internalization dramatically increases the number and size of EHD1-containing puncta and small tubules.

“we observed a dramatic increase in the number and size of EHD1-containing puncta and small tubules (Fig. 3B, see inset for more detail).”

At steady state, ~20% of SNX17 overlaps with EHD1.

“Overall, these data support the notion that ˜20% of SNX17 overlaps with EHD1 at steady state”

• Also stated in: § Discussion · boosted

  “Our data clearly show that ˜20% of SNX17 and EHD1 display overlap in 3D at steady state.”

We identified a novel direct interaction between the sorting nexin SNX17 and the endosomal fission protein EHD1.

“Overall, we identified a novel direct interaction between the sorting nexin SNX17 and the endosomal fission protein EHD1.”

The EHD1 EH domain is not required for interaction with SNX17.

“These results support the notion that the EHD1 EH domain is not required for interaction with SNX17.”

The interaction between SNX17 and EHD1 provides a molecular explanation to couple the endosomal sorting and fission machineries.

“Our data are consistent with a new model of endosomal fission in which the interaction between SNX17 and EHD1 provides a molecular explanation to couple the endosomal sorting and fission machineries.”

• AssumptionRests on an assumption: The mechanisms by which SNX17-labeled vesicles that contain sorted receptors bud and undergo vesicular fission from sorting endosomes remain elusive.
• AssumptionRests on an assumption: The mechanism by which EHD1 is coupled to various receptors and regulates their recycling remains unknown.
• AssumptionRests on an assumption: Direct interactions between EHD1 and the cytoplasmic tails of receptors have not been identified.
• AssumptionRests on an assumption: The mechanism of EHD1 recruitment and coupling to sorting endosomes has not been elucidated.
• AssumptionRests on an assumption: The mechanisms of coupling SNX17 and its cargo receptors to specific fission machinery at the sorting endosome remain unknown.
• AssumptionRests on an assumption: EHD1 appears to be recruited to sorting endosomes to facilitate fission of cargo in conjunction with SNX17-based cargo sorting.

EHD1 plays a role in sorting endosome fission.

“Indeed, EHD1 depletion leads to enlarged SE structures, supporting a role of EHD1 in SE fission.”

EHD1 depletion impairs SNX17-mediated endosomal fission.

“suggesting that EHD1 depletion impairs SNX17-mediated endosomal fission.”

The SNX17 atypical FERM domain directly binds EHD1.

“These experiments suggest that the SNX17 atypical FERM domain directly binds to EHD1.”

Ultimately, EHD1 might play a significant role in fission of vesicles slated for recycling.

“Ultimately, EHD1 might play a significant role in fission of vesicles slated for recycling.”

The developed method using IMARIS surface rendering and Kiss and Run software converts 3D images of cells with EHD1-GFP and immunostained endogenous SNX17 to "3D surfaces," allowing effective filtering of most cytoplasmic protein haze.

“Accordingly, we developed a method using IMARIS surface rendering and Kiss and Run software in which 3D images of cells with EHD1-GFP and immunostained endogenous SNX17 were converted to “3D surfaces,” allowing us to effectively filter out most of the cytoplasmic protein haze.”

EHD1 recruitment to SE may rely on other interaction partners that are localized to SE.

“and may rely on other interaction partners that are localized to SE.”

Internalization of an SNX17-sorted receptor can lead to EHD1 recruitment to SE, potentially to facilitate fission events and recycling.

“These data suggest that internalization of an SNX17-sorted receptor can lead to EHD1 recruitment to SE, potentially to facilitate fission events and recycling.”

EHD1 binding to SNX17 is atypical because most of the EHD1 interaction partners identified to date interact via NPF motifs.

“EHD1 binding to SNX17 is atypical because most of the EHD1 interaction partners identified to date interact via NPF motifs”

• cites [22] Naslavsky, 2005, C-terminal EH-domain-containing proteins: consensus for a role in endocytic trafficking, EH?, J. Cell Sci sourceNot checked — no full text of the source
• cites [23] Naslavsky, 2011, EHD proteins: key conductors of endocytic transport, Trends Cell Biol sourceNot checked yet

  A check is scheduled.

• cites [47] Kieken, 2010, Mechanism for the selective interaction of C-terminal Eps15 homology domain proteins with specific Asn-Pro-Phe-containing partners, J. Biol. Chem sourceCheck pending

The increase in SNX17-containing endosome size in the absence of EHD1 is likely a result of impaired fission.

“likely as a result of impaired fission.”

Use of multiple z-sections and measurement of voxel overlap in this system provides the most accurate assessment of colocalization within the cell.

“Following extensive calibrations and comparisons with both 2D and 3D measurements of Pearson’s correlation and Manders overlap coefficient, we found that use of multiple z-sections and measurement of voxel overlap in this system provides the most accurate assessment of colocalization within the cell.”

SNX17 can couple internalized receptors with EHD1, a protein implicated in membrane fission.

“Here we demonstrated for the first time that a key sorting nexin, SNX17, can couple internalized receptors with EHD1, a protein implicated in membrane fission.”

This is the first evidence that EHD1 and endosomal fission machinery can be recruited to endosomal membranes by a specific physiologic signal.

“Indeed, to our knowledge, this is the first evidence that EHD1 and endosomal fission machinery can be recruited to endosomal membranes by a specific physiologic signal.”

SNX17 and EHD1 likely localize to common endosomal membrane complexes slated for budding and fission.

“Although it remains unclear whether SNX17 can simultaneously bind both receptors and EHD1, these proteins likely localize to common endosomal membrane complexes slated for budding and fission.”