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Combinational soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins VAMP8 and Vti1b mediate fusion of antimicrobial and canonical autophagosomes with lysosomes.

Furuta N, Fujita N, Noda T, Yoshimori T, Amano A - Mol. Biol. Cell (2010)

Bottom Line: Knockdown of Vti1b and VAMP8 with small interfering RNAs disturbed the colocalization of LC3 with lysosomal membrane protein (LAMP)1.The invasive efficiency of GAS into cells was not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency was significantly diminished, indicating that antimicrobial autophagy was functionally impaired.Knockdown of Vti1b and VAMP8 also disturbed colocalization of LC3 with LAMP1 in canonical autophagy, in which LC3-II proteins were negligibly degraded.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka 565-0871, Japan.

ABSTRACT
Autophagy plays a crucial role in host defense, termed antimicrobial autophagy (xenophagy), as it functions to degrade intracellular foreign microbial invaders such as group A Streptococcus (GAS). Xenophagosomes undergo a stepwise maturation process consisting of a fusion event with lysosomes, after which the cargoes are degraded. However, the molecular mechanism underlying xenophagosome/lysosome fusion remains unclear. We examined the involvement of endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in xenophagosome/lysosome fusion. Confocal microscopic analysis showed that SNAREs, including vesicle-associated membrane protein (VAMP)7, VAMP8, and vesicle transport through interaction with t-SNAREs homologue 1B (Vti1b), colocalized with green fluorescent protein-LC3 in xenophagosomes. Knockdown of Vti1b and VAMP8 with small interfering RNAs disturbed the colocalization of LC3 with lysosomal membrane protein (LAMP)1. The invasive efficiency of GAS into cells was not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency was significantly diminished, indicating that antimicrobial autophagy was functionally impaired. Knockdown of Vti1b and VAMP8 also disturbed colocalization of LC3 with LAMP1 in canonical autophagy, in which LC3-II proteins were negligibly degraded. In contrast, knockdown of Syntaxin 7 and Syntaxin 8 showed little effect on the autophagic fusion event. These findings strongly suggest that the combinational SNARE proteins VAMP8 and Vti1b mediate the fusion of antimicrobial and canonical autophagosomes with lysosomes, an essential event for autophagic degradation.

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Colocalization of mRFP-LC3 with LAMP1 in VAMP8- and Vti1b-depleted cells. (A) HeLa cells were transfected with siRNA for the control, VAMP7, VAMP8, and Vti1b. At 24 h after transfection, the cells were further transfected with plasmids expressing mRFP-LC3. After 24 h of incubation, the cells were subjected to a starved condition for 180 min, followed by fixation and incubation with anti-LAMP1 antibodies and then observed with a confocal microscope. Cellular DNA was stained with DAPI. Bars, 10 μm. (B) HeLa cells were preloaded with Alexa 488 dextran for marking lysosomes, as described in Materials and Methods. Cellular DNA was stained with DAPI. Bars, 10 μm. (C) The colocalization frequencies of mRFP shown as LAMP1 pixels were determined using LSM Image Browser software (Carl Zeiss) and are presented as the percentage of total number of mRFP pixels. Values are shown as the mean ± SD of >30 images. *p < 0.01 by one-way ANOVA and Scheffé's posttest. (D) The colocalization frequency of mRFP-LC3 shown as Alexa 488 dextran pixels was determined using LSM Image Browser (Carl Zeiss) and presented as the percentage of total number of mRFP pixels. The mean value ± SD of >30 cell images is shown. *p < 0.01 by one-way ANOVA and Scheffé's posttest.
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Figure 8: Colocalization of mRFP-LC3 with LAMP1 in VAMP8- and Vti1b-depleted cells. (A) HeLa cells were transfected with siRNA for the control, VAMP7, VAMP8, and Vti1b. At 24 h after transfection, the cells were further transfected with plasmids expressing mRFP-LC3. After 24 h of incubation, the cells were subjected to a starved condition for 180 min, followed by fixation and incubation with anti-LAMP1 antibodies and then observed with a confocal microscope. Cellular DNA was stained with DAPI. Bars, 10 μm. (B) HeLa cells were preloaded with Alexa 488 dextran for marking lysosomes, as described in Materials and Methods. Cellular DNA was stained with DAPI. Bars, 10 μm. (C) The colocalization frequencies of mRFP shown as LAMP1 pixels were determined using LSM Image Browser software (Carl Zeiss) and are presented as the percentage of total number of mRFP pixels. Values are shown as the mean ± SD of >30 images. *p < 0.01 by one-way ANOVA and Scheffé's posttest. (D) The colocalization frequency of mRFP-LC3 shown as Alexa 488 dextran pixels was determined using LSM Image Browser (Carl Zeiss) and presented as the percentage of total number of mRFP pixels. The mean value ± SD of >30 cell images is shown. *p < 0.01 by one-way ANOVA and Scheffé's posttest.

Mentions: To determine whether lysosomal enzyme failure in VAMP8- and Vti1b-depleted cells is the cause of this phenotype, the distribution of mRFP-LC3 in lytic compartments was further examined. RFP signals were clearly merged with LAMP1 in the control and VAMP7-depleted cells after 180 min of starvation (Figure 8, A and C), whereas their coexistence was merely observed in VAMP8- and Vti1b-depleted cells. Next, we used another lysosome marker, dextran (Bright et al., 2005), and preloaded HeLa cells with Alexa 488 dextran for marking lysosomes via endocytosis and then starved them for 180 min, after which the colocalization of dextran with mRFP-LC3 was examined as described previously (Kimura et al., 2007). This assay also indicated that colocalization of the markers for autophagosomes and lysosomes was significantly inhibited by siRNA for VAMP8- and Vti1b, respectively (Figure 8, B and D).


Combinational soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins VAMP8 and Vti1b mediate fusion of antimicrobial and canonical autophagosomes with lysosomes.

Furuta N, Fujita N, Noda T, Yoshimori T, Amano A - Mol. Biol. Cell (2010)

Colocalization of mRFP-LC3 with LAMP1 in VAMP8- and Vti1b-depleted cells. (A) HeLa cells were transfected with siRNA for the control, VAMP7, VAMP8, and Vti1b. At 24 h after transfection, the cells were further transfected with plasmids expressing mRFP-LC3. After 24 h of incubation, the cells were subjected to a starved condition for 180 min, followed by fixation and incubation with anti-LAMP1 antibodies and then observed with a confocal microscope. Cellular DNA was stained with DAPI. Bars, 10 μm. (B) HeLa cells were preloaded with Alexa 488 dextran for marking lysosomes, as described in Materials and Methods. Cellular DNA was stained with DAPI. Bars, 10 μm. (C) The colocalization frequencies of mRFP shown as LAMP1 pixels were determined using LSM Image Browser software (Carl Zeiss) and are presented as the percentage of total number of mRFP pixels. Values are shown as the mean ± SD of >30 images. *p < 0.01 by one-way ANOVA and Scheffé's posttest. (D) The colocalization frequency of mRFP-LC3 shown as Alexa 488 dextran pixels was determined using LSM Image Browser (Carl Zeiss) and presented as the percentage of total number of mRFP pixels. The mean value ± SD of >30 cell images is shown. *p < 0.01 by one-way ANOVA and Scheffé's posttest.
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Figure 8: Colocalization of mRFP-LC3 with LAMP1 in VAMP8- and Vti1b-depleted cells. (A) HeLa cells were transfected with siRNA for the control, VAMP7, VAMP8, and Vti1b. At 24 h after transfection, the cells were further transfected with plasmids expressing mRFP-LC3. After 24 h of incubation, the cells were subjected to a starved condition for 180 min, followed by fixation and incubation with anti-LAMP1 antibodies and then observed with a confocal microscope. Cellular DNA was stained with DAPI. Bars, 10 μm. (B) HeLa cells were preloaded with Alexa 488 dextran for marking lysosomes, as described in Materials and Methods. Cellular DNA was stained with DAPI. Bars, 10 μm. (C) The colocalization frequencies of mRFP shown as LAMP1 pixels were determined using LSM Image Browser software (Carl Zeiss) and are presented as the percentage of total number of mRFP pixels. Values are shown as the mean ± SD of >30 images. *p < 0.01 by one-way ANOVA and Scheffé's posttest. (D) The colocalization frequency of mRFP-LC3 shown as Alexa 488 dextran pixels was determined using LSM Image Browser (Carl Zeiss) and presented as the percentage of total number of mRFP pixels. The mean value ± SD of >30 cell images is shown. *p < 0.01 by one-way ANOVA and Scheffé's posttest.
Mentions: To determine whether lysosomal enzyme failure in VAMP8- and Vti1b-depleted cells is the cause of this phenotype, the distribution of mRFP-LC3 in lytic compartments was further examined. RFP signals were clearly merged with LAMP1 in the control and VAMP7-depleted cells after 180 min of starvation (Figure 8, A and C), whereas their coexistence was merely observed in VAMP8- and Vti1b-depleted cells. Next, we used another lysosome marker, dextran (Bright et al., 2005), and preloaded HeLa cells with Alexa 488 dextran for marking lysosomes via endocytosis and then starved them for 180 min, after which the colocalization of dextran with mRFP-LC3 was examined as described previously (Kimura et al., 2007). This assay also indicated that colocalization of the markers for autophagosomes and lysosomes was significantly inhibited by siRNA for VAMP8- and Vti1b, respectively (Figure 8, B and D).

Bottom Line: Knockdown of Vti1b and VAMP8 with small interfering RNAs disturbed the colocalization of LC3 with lysosomal membrane protein (LAMP)1.The invasive efficiency of GAS into cells was not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency was significantly diminished, indicating that antimicrobial autophagy was functionally impaired.Knockdown of Vti1b and VAMP8 also disturbed colocalization of LC3 with LAMP1 in canonical autophagy, in which LC3-II proteins were negligibly degraded.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Frontier Biology, Osaka University Graduate School of Dentistry, Suita-Osaka 565-0871, Japan.

ABSTRACT
Autophagy plays a crucial role in host defense, termed antimicrobial autophagy (xenophagy), as it functions to degrade intracellular foreign microbial invaders such as group A Streptococcus (GAS). Xenophagosomes undergo a stepwise maturation process consisting of a fusion event with lysosomes, after which the cargoes are degraded. However, the molecular mechanism underlying xenophagosome/lysosome fusion remains unclear. We examined the involvement of endocytic soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) in xenophagosome/lysosome fusion. Confocal microscopic analysis showed that SNAREs, including vesicle-associated membrane protein (VAMP)7, VAMP8, and vesicle transport through interaction with t-SNAREs homologue 1B (Vti1b), colocalized with green fluorescent protein-LC3 in xenophagosomes. Knockdown of Vti1b and VAMP8 with small interfering RNAs disturbed the colocalization of LC3 with lysosomal membrane protein (LAMP)1. The invasive efficiency of GAS into cells was not altered by knockdown of VAMP8 or Vti1b, whereas cellular bactericidal efficiency was significantly diminished, indicating that antimicrobial autophagy was functionally impaired. Knockdown of Vti1b and VAMP8 also disturbed colocalization of LC3 with LAMP1 in canonical autophagy, in which LC3-II proteins were negligibly degraded. In contrast, knockdown of Syntaxin 7 and Syntaxin 8 showed little effect on the autophagic fusion event. These findings strongly suggest that the combinational SNARE proteins VAMP8 and Vti1b mediate the fusion of antimicrobial and canonical autophagosomes with lysosomes, an essential event for autophagic degradation.

Show MeSH
Related in: MedlinePlus