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An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport

View Article: PubMed Central - PubMed

ABSTRACT

Through a network of progressively maturing vesicles, the endosomal system connects the cell’s interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell’s periphery. By drawing the endosomal system’s architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

No MeSH data available.


Related in: MedlinePlus

RNF26 Depletion Disrupts Spatiotemporal Organization of Endosomes(A) Intracellular distribution of LEs (CD63, green) in various cell lines. Representative maximum z projection (3D) overlays with nuclear DAPI (blue) and their corresponding z cross sections along the demarcated line are shown. Cell boundaries are depicted in dashed lines. For other markers, see Figure S1.(B) Effect of RNF26 depletion on distribution of LEs, represented as fractional distances of CD63 vesicles from center of nucleus (distance of pixels from nucleus = faction of distance from nucleus to the plasma membrane [1.0]; mean shown in red). For 3D view, see Movies S1A and S1B.(C) Cell shape analysis for samples in (B), showing total cell area and eccentricity calculated in an automated fashion as described in the Supplemental Experimental Procedures.(D) mRNA levels of RNF26 targeted by two different siRNAs (siRNF26_1 and siRNF26_2) as assessed by qPCR are expressed relative to siC; n = 3.(E) Quantification of the mobile fraction of acidified Lysotracker (LT)-positive vesicles (LTVs) as a function of RNF26; n = 3. For details, refer to the Supplemental Experimental Procedures.(F) Organization and dynamics of LTVs (white) in control (siC) versus RNF26-depleted (siRNF26_1) MelJuSo cells. Left panels: representative single confocal plane fluorescence images taken at the start of time lapse. Right panels: vesicle displacement rates (blue, immobile; red, max mobility) observed over the 297-s time interval. Nuclei and cell boundaries are depicted in dashed lines, and zoom-ins highlight peripheral (PP) and perinuclear (PN) boxed regions. Quantification appears in (E). For LT time lapses, see Movies S2A–S2C. For CD63 time lapses see Movies S2D and S2E.Scale bars, 10 μm. For all figures: n, # of cells analyzed per condition; n, # independent experiments; error bars, SD.
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fig1: RNF26 Depletion Disrupts Spatiotemporal Organization of Endosomes(A) Intracellular distribution of LEs (CD63, green) in various cell lines. Representative maximum z projection (3D) overlays with nuclear DAPI (blue) and their corresponding z cross sections along the demarcated line are shown. Cell boundaries are depicted in dashed lines. For other markers, see Figure S1.(B) Effect of RNF26 depletion on distribution of LEs, represented as fractional distances of CD63 vesicles from center of nucleus (distance of pixels from nucleus = faction of distance from nucleus to the plasma membrane [1.0]; mean shown in red). For 3D view, see Movies S1A and S1B.(C) Cell shape analysis for samples in (B), showing total cell area and eccentricity calculated in an automated fashion as described in the Supplemental Experimental Procedures.(D) mRNA levels of RNF26 targeted by two different siRNAs (siRNF26_1 and siRNF26_2) as assessed by qPCR are expressed relative to siC; n = 3.(E) Quantification of the mobile fraction of acidified Lysotracker (LT)-positive vesicles (LTVs) as a function of RNF26; n = 3. For details, refer to the Supplemental Experimental Procedures.(F) Organization and dynamics of LTVs (white) in control (siC) versus RNF26-depleted (siRNF26_1) MelJuSo cells. Left panels: representative single confocal plane fluorescence images taken at the start of time lapse. Right panels: vesicle displacement rates (blue, immobile; red, max mobility) observed over the 297-s time interval. Nuclei and cell boundaries are depicted in dashed lines, and zoom-ins highlight peripheral (PP) and perinuclear (PN) boxed regions. Quantification appears in (E). For LT time lapses, see Movies S2A–S2C. For CD63 time lapses see Movies S2D and S2E.Scale bars, 10 μm. For all figures: n, # of cells analyzed per condition; n, # independent experiments; error bars, SD.

Mentions: Across cell types, a wide variety of endosomal maturation stages—late (CD63, Figure 1A), early (EEA1) and recycling (TrfR) endosomes, as well as the vesicular arm of the TGN (TGN46) (Figure S1)—tend to cluster into a “cloud” near the nucleus, with only a fraction of each subset extending into the cell’s periphery. How such organization is established and controlled and what purpose it may serve is largely unknown. Given that late endosomes (LEs) constitute central nodes within the endo- and exocytic vesicular network (Huotari and Helenius, 2011), we mined a genome-wide small interfering RNA (siRNA)-based screen for novel factors controlling LE biology (Paul et al., 2011), where silencing the RING finger ubiquitin ligase RNF26 was shown to severely disrupt the intracellular LE organization, leading to marked dispersion of LEs throughout the cytoplasm and even to the tips of cells, without significantly impacting cell shape (Figures 1B–1D; Movies S1A and S1B). These observations cast RNF26 as a potent candidate for control of the LE compartment architecture, prompting us to investigate the role of RNF26 in the organization and function of the perinuclear (PN) cloud.


An ER-Associated Pathway Defines Endosomal Architecture for Controlled Cargo Transport
RNF26 Depletion Disrupts Spatiotemporal Organization of Endosomes(A) Intracellular distribution of LEs (CD63, green) in various cell lines. Representative maximum z projection (3D) overlays with nuclear DAPI (blue) and their corresponding z cross sections along the demarcated line are shown. Cell boundaries are depicted in dashed lines. For other markers, see Figure S1.(B) Effect of RNF26 depletion on distribution of LEs, represented as fractional distances of CD63 vesicles from center of nucleus (distance of pixels from nucleus = faction of distance from nucleus to the plasma membrane [1.0]; mean shown in red). For 3D view, see Movies S1A and S1B.(C) Cell shape analysis for samples in (B), showing total cell area and eccentricity calculated in an automated fashion as described in the Supplemental Experimental Procedures.(D) mRNA levels of RNF26 targeted by two different siRNAs (siRNF26_1 and siRNF26_2) as assessed by qPCR are expressed relative to siC; n = 3.(E) Quantification of the mobile fraction of acidified Lysotracker (LT)-positive vesicles (LTVs) as a function of RNF26; n = 3. For details, refer to the Supplemental Experimental Procedures.(F) Organization and dynamics of LTVs (white) in control (siC) versus RNF26-depleted (siRNF26_1) MelJuSo cells. Left panels: representative single confocal plane fluorescence images taken at the start of time lapse. Right panels: vesicle displacement rates (blue, immobile; red, max mobility) observed over the 297-s time interval. Nuclei and cell boundaries are depicted in dashed lines, and zoom-ins highlight peripheral (PP) and perinuclear (PN) boxed regions. Quantification appears in (E). For LT time lapses, see Movies S2A–S2C. For CD63 time lapses see Movies S2D and S2E.Scale bars, 10 μm. For all figures: n, # of cells analyzed per condition; n, # independent experiments; error bars, SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4930482&req=5

fig1: RNF26 Depletion Disrupts Spatiotemporal Organization of Endosomes(A) Intracellular distribution of LEs (CD63, green) in various cell lines. Representative maximum z projection (3D) overlays with nuclear DAPI (blue) and their corresponding z cross sections along the demarcated line are shown. Cell boundaries are depicted in dashed lines. For other markers, see Figure S1.(B) Effect of RNF26 depletion on distribution of LEs, represented as fractional distances of CD63 vesicles from center of nucleus (distance of pixels from nucleus = faction of distance from nucleus to the plasma membrane [1.0]; mean shown in red). For 3D view, see Movies S1A and S1B.(C) Cell shape analysis for samples in (B), showing total cell area and eccentricity calculated in an automated fashion as described in the Supplemental Experimental Procedures.(D) mRNA levels of RNF26 targeted by two different siRNAs (siRNF26_1 and siRNF26_2) as assessed by qPCR are expressed relative to siC; n = 3.(E) Quantification of the mobile fraction of acidified Lysotracker (LT)-positive vesicles (LTVs) as a function of RNF26; n = 3. For details, refer to the Supplemental Experimental Procedures.(F) Organization and dynamics of LTVs (white) in control (siC) versus RNF26-depleted (siRNF26_1) MelJuSo cells. Left panels: representative single confocal plane fluorescence images taken at the start of time lapse. Right panels: vesicle displacement rates (blue, immobile; red, max mobility) observed over the 297-s time interval. Nuclei and cell boundaries are depicted in dashed lines, and zoom-ins highlight peripheral (PP) and perinuclear (PN) boxed regions. Quantification appears in (E). For LT time lapses, see Movies S2A–S2C. For CD63 time lapses see Movies S2D and S2E.Scale bars, 10 μm. For all figures: n, # of cells analyzed per condition; n, # independent experiments; error bars, SD.
Mentions: Across cell types, a wide variety of endosomal maturation stages—late (CD63, Figure 1A), early (EEA1) and recycling (TrfR) endosomes, as well as the vesicular arm of the TGN (TGN46) (Figure S1)—tend to cluster into a “cloud” near the nucleus, with only a fraction of each subset extending into the cell’s periphery. How such organization is established and controlled and what purpose it may serve is largely unknown. Given that late endosomes (LEs) constitute central nodes within the endo- and exocytic vesicular network (Huotari and Helenius, 2011), we mined a genome-wide small interfering RNA (siRNA)-based screen for novel factors controlling LE biology (Paul et al., 2011), where silencing the RING finger ubiquitin ligase RNF26 was shown to severely disrupt the intracellular LE organization, leading to marked dispersion of LEs throughout the cytoplasm and even to the tips of cells, without significantly impacting cell shape (Figures 1B–1D; Movies S1A and S1B). These observations cast RNF26 as a potent candidate for control of the LE compartment architecture, prompting us to investigate the role of RNF26 in the organization and function of the perinuclear (PN) cloud.

View Article: PubMed Central - PubMed

ABSTRACT

Through a network of progressively maturing vesicles, the endosomal system connects the cell’s interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell’s periphery. By drawing the endosomal system’s architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

No MeSH data available.


Related in: MedlinePlus