Limits...
Human Vam6p promotes lysosome clustering and fusion in vivo.

Caplan S, Hartnell LM, Aguilar RC, Naslavsky N, Bonifacino JS - J. Cell Biol. (2001)

Bottom Line: This effect is reminiscent of that caused by expression of a constitutively activated Rab7.However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7.Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch at the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH(2) terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2-3 microm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

Show MeSH

Related in: MedlinePlus

Dynamics of hVam6p-induced lysosome clustering and fusion. COS-7 (A and C) or HeLa (B) cells were transfected with plasmids encoding GFP–lamp-1 (GFP–lgp120) and HA–hVam6p. 10 h after transfection, live cells were incubated at 37°C and scanned for GFP-expressing cells by confocal microscopy. Live images were acquired at 30-s time intervals and are displayed as inverted images with the time in minutes relative to the start of imaging indicated in the lower left corner (A) or upper left corner (B and C). The dotted oval region of interest (C) outlines juxtanuclear regions that accumulate lysosome clusters and giant lysosomes. Arrows (A and B) point at moving vesicles. Arrowheads (A) point at a lysosome clustering event. n, nucleus. (D) Microtubule depolymerization impairs formation of a unified, giant juxtanuclear lysosomal structure in hVam6p-transfected cells. HeLa cells were transfected with Myc-hVam6p and treated with 0.5 μM nocodazole for 16 h. Cells were then fixed-permeabilized, incubated with a rabbit polyclonal antibody to Myc and a mouse monoclonal antibody to lamp-1, followed by Alexa-488–conjugated donkey antibody to mouse IgG (left, green channel) and Cy3-conjugated donkey anti–rabbit IgG (middle, red channel). Bars, (A and C) 5 μm; (B) 10 μm. Quicktime movie sequence versions of this figure are available at http://www.jcb.org/cgi/content/full/200102142/DC1.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196876&req=5

fig6: Dynamics of hVam6p-induced lysosome clustering and fusion. COS-7 (A and C) or HeLa (B) cells were transfected with plasmids encoding GFP–lamp-1 (GFP–lgp120) and HA–hVam6p. 10 h after transfection, live cells were incubated at 37°C and scanned for GFP-expressing cells by confocal microscopy. Live images were acquired at 30-s time intervals and are displayed as inverted images with the time in minutes relative to the start of imaging indicated in the lower left corner (A) or upper left corner (B and C). The dotted oval region of interest (C) outlines juxtanuclear regions that accumulate lysosome clusters and giant lysosomes. Arrows (A and B) point at moving vesicles. Arrowheads (A) point at a lysosome clustering event. n, nucleus. (D) Microtubule depolymerization impairs formation of a unified, giant juxtanuclear lysosomal structure in hVam6p-transfected cells. HeLa cells were transfected with Myc-hVam6p and treated with 0.5 μM nocodazole for 16 h. Cells were then fixed-permeabilized, incubated with a rabbit polyclonal antibody to Myc and a mouse monoclonal antibody to lamp-1, followed by Alexa-488–conjugated donkey antibody to mouse IgG (left, green channel) and Cy3-conjugated donkey anti–rabbit IgG (middle, red channel). Bars, (A and C) 5 μm; (B) 10 μm. Quicktime movie sequence versions of this figure are available at http://www.jcb.org/cgi/content/full/200102142/DC1.

Mentions: To investigate the processes that led to the formation of lysosomal clusters and large vacuoles over time, we monitored the movement of lysosomes in live cells by time-lapse fluorescence microscopy. To this aim, COS-7 and HeLa cells were transfected with plasmids encoding green fluorescent protein (GFP)–lamp-1 alone or GFP–lamp-1 and hVam6p. In cells transfected with GFP–lamp-1 alone, lysosomes and/or late endosomes exhibited bidirectional movement between the juxtanuclear area and sites in the cell periphery (data not shown). In the doubly transfected cells, the effects of hVam6p on lysosomes were concurrent with the expression of GFP–lamp-1, which made it necessary to image cells already displaying incipient lysosome clustering. At early time points, we could observe peripheral lysosomes moving centripetally toward the center of the cell and attaching to or merging with other lysosomes (Fig. 6 A, arrow, Video 1). More centrally located lysosomes were also observed to cluster and/or fuse with one another (Fig. 6 A, arrowhead, and B; Videos 1 and 2). Over time, these clustering and fusion events lead to the accumulation of large lysosomal structures in the juxtanuclear area (Fig. 6 C; Video 3). These observations suggested that hVam6p causes lysosomes and late endosomes to stick together in tight clusters and fuse, thus preventing them from migrating back to the cell periphery. (Videos 1, 2, and 3 are available at http://www.jcb.org/cgi/content/full/200102142/DC1)


Human Vam6p promotes lysosome clustering and fusion in vivo.

Caplan S, Hartnell LM, Aguilar RC, Naslavsky N, Bonifacino JS - J. Cell Biol. (2001)

Dynamics of hVam6p-induced lysosome clustering and fusion. COS-7 (A and C) or HeLa (B) cells were transfected with plasmids encoding GFP–lamp-1 (GFP–lgp120) and HA–hVam6p. 10 h after transfection, live cells were incubated at 37°C and scanned for GFP-expressing cells by confocal microscopy. Live images were acquired at 30-s time intervals and are displayed as inverted images with the time in minutes relative to the start of imaging indicated in the lower left corner (A) or upper left corner (B and C). The dotted oval region of interest (C) outlines juxtanuclear regions that accumulate lysosome clusters and giant lysosomes. Arrows (A and B) point at moving vesicles. Arrowheads (A) point at a lysosome clustering event. n, nucleus. (D) Microtubule depolymerization impairs formation of a unified, giant juxtanuclear lysosomal structure in hVam6p-transfected cells. HeLa cells were transfected with Myc-hVam6p and treated with 0.5 μM nocodazole for 16 h. Cells were then fixed-permeabilized, incubated with a rabbit polyclonal antibody to Myc and a mouse monoclonal antibody to lamp-1, followed by Alexa-488–conjugated donkey antibody to mouse IgG (left, green channel) and Cy3-conjugated donkey anti–rabbit IgG (middle, red channel). Bars, (A and C) 5 μm; (B) 10 μm. Quicktime movie sequence versions of this figure are available at http://www.jcb.org/cgi/content/full/200102142/DC1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196876&req=5

fig6: Dynamics of hVam6p-induced lysosome clustering and fusion. COS-7 (A and C) or HeLa (B) cells were transfected with plasmids encoding GFP–lamp-1 (GFP–lgp120) and HA–hVam6p. 10 h after transfection, live cells were incubated at 37°C and scanned for GFP-expressing cells by confocal microscopy. Live images were acquired at 30-s time intervals and are displayed as inverted images with the time in minutes relative to the start of imaging indicated in the lower left corner (A) or upper left corner (B and C). The dotted oval region of interest (C) outlines juxtanuclear regions that accumulate lysosome clusters and giant lysosomes. Arrows (A and B) point at moving vesicles. Arrowheads (A) point at a lysosome clustering event. n, nucleus. (D) Microtubule depolymerization impairs formation of a unified, giant juxtanuclear lysosomal structure in hVam6p-transfected cells. HeLa cells were transfected with Myc-hVam6p and treated with 0.5 μM nocodazole for 16 h. Cells were then fixed-permeabilized, incubated with a rabbit polyclonal antibody to Myc and a mouse monoclonal antibody to lamp-1, followed by Alexa-488–conjugated donkey antibody to mouse IgG (left, green channel) and Cy3-conjugated donkey anti–rabbit IgG (middle, red channel). Bars, (A and C) 5 μm; (B) 10 μm. Quicktime movie sequence versions of this figure are available at http://www.jcb.org/cgi/content/full/200102142/DC1.
Mentions: To investigate the processes that led to the formation of lysosomal clusters and large vacuoles over time, we monitored the movement of lysosomes in live cells by time-lapse fluorescence microscopy. To this aim, COS-7 and HeLa cells were transfected with plasmids encoding green fluorescent protein (GFP)–lamp-1 alone or GFP–lamp-1 and hVam6p. In cells transfected with GFP–lamp-1 alone, lysosomes and/or late endosomes exhibited bidirectional movement between the juxtanuclear area and sites in the cell periphery (data not shown). In the doubly transfected cells, the effects of hVam6p on lysosomes were concurrent with the expression of GFP–lamp-1, which made it necessary to image cells already displaying incipient lysosome clustering. At early time points, we could observe peripheral lysosomes moving centripetally toward the center of the cell and attaching to or merging with other lysosomes (Fig. 6 A, arrow, Video 1). More centrally located lysosomes were also observed to cluster and/or fuse with one another (Fig. 6 A, arrowhead, and B; Videos 1 and 2). Over time, these clustering and fusion events lead to the accumulation of large lysosomal structures in the juxtanuclear area (Fig. 6 C; Video 3). These observations suggested that hVam6p causes lysosomes and late endosomes to stick together in tight clusters and fuse, thus preventing them from migrating back to the cell periphery. (Videos 1, 2, and 3 are available at http://www.jcb.org/cgi/content/full/200102142/DC1)

Bottom Line: This effect is reminiscent of that caused by expression of a constitutively activated Rab7.However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7.Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch at the National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Regulated fusion of mammalian lysosomes is critical to their ability to acquire both internalized and biosynthetic materials. Here, we report the identification of a novel human protein, hVam6p, that promotes lysosome clustering and fusion in vivo. Although hVam6p exhibits homology to the Saccharomyces cerevisiae vacuolar protein sorting gene product Vam6p/Vps39p, the presence of a citron homology (CNH) domain at the NH(2) terminus is unique to the human protein. Overexpression of hVam6p results in massive clustering and fusion of lysosomes and late endosomes into large (2-3 microm) juxtanuclear structures. This effect is reminiscent of that caused by expression of a constitutively activated Rab7. However, hVam6p exerts its effect even in the presence of a dominant-negative Rab7, suggesting that it functions either downstream of, or in parallel to, Rab7. Data from gradient fractionation, two-hybrid, and coimmunoprecipitation analyses suggest that hVam6p is a homooligomer, and that its self-assembly is mediated by a clathrin heavy chain repeat domain in the middle of the protein. Both the CNH and clathrin heavy chain repeat domains are required for induction of lysosome clustering and fusion. This study implicates hVam6p as a mammalian tethering/docking factor characterized with intrinsic ability to promote lysosome fusion in vivo.

Show MeSH
Related in: MedlinePlus