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Molecular motors and a spectrin matrix associate with Golgi membranes in vitro.

Fath KR, Trimbur GM, Burgess DR - J. Cell Biol. (1997)

Bottom Line: In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules.Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin.Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
Cytoplasmic dynein is a microtubule minus-end-directed motor that is thought to power the transport of vesicles from the TGN to the apical cortex in polarized epithelial cells. Trans-Golgi enriched membranes, which were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isolated Golgi stacks lack dynein but contain myosin-I (Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. J. Cell Biol. 126:661-675). We show now that Golgi stacks in vitro bind dynein supplied from cytosol in the absence of ATP, and bud small membranes when incubated with cytosol and ATP. Cytosolic dynein binds to regions of stacks that are destined to bud because dynein is present in budded membranes, but absent from stacks after budding. Budded membranes move exclusively towards microtubule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resists extraction by ice-cold Triton X-100. In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.

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Cytosolic dynein binding to TX-100-extracted Golgi  stacks is physiologically relevant. (A) Cytosolic dynein that  bound to detergent-extracted Golgi stacks was functional in in  vitro motility assays. Golgi stacks were extracted with 1% TX-100 on ice, pelleted and resuspended in buffer. In a motility  chamber, the detergent ghosts were incubated with cytosol and  with microtubules that were polymerized from the ends of axonemes. This series of video frames shows a membrane ghost (arrow) moving along a microtubule. (B) Dynein binding protein(s)  are extracted from Golgi stack detergent ghosts at alkaline pH.  Golgi stacks were incubated on ice with PEMS (Control); 1%  TX-100 (TX); or 1% TX-100 followed by 0.1 M Na2CO3 (TX →   pH 11.5). The membranes were collected by centrifugation, resuspended in PEMS, incubated with cytosol to allow dynein binding, and repelleted through 0.5 M sucrose. This immunoblot  shows that cytosolic dynein could bind to both unextracted and  TX-100–extracted Golgi membranes, but not to stacks that were  sequentially extracted with TX-100 and Na2CO3.
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Figure 5: Cytosolic dynein binding to TX-100-extracted Golgi stacks is physiologically relevant. (A) Cytosolic dynein that bound to detergent-extracted Golgi stacks was functional in in vitro motility assays. Golgi stacks were extracted with 1% TX-100 on ice, pelleted and resuspended in buffer. In a motility chamber, the detergent ghosts were incubated with cytosol and with microtubules that were polymerized from the ends of axonemes. This series of video frames shows a membrane ghost (arrow) moving along a microtubule. (B) Dynein binding protein(s) are extracted from Golgi stack detergent ghosts at alkaline pH. Golgi stacks were incubated on ice with PEMS (Control); 1% TX-100 (TX); or 1% TX-100 followed by 0.1 M Na2CO3 (TX → pH 11.5). The membranes were collected by centrifugation, resuspended in PEMS, incubated with cytosol to allow dynein binding, and repelleted through 0.5 M sucrose. This immunoblot shows that cytosolic dynein could bind to both unextracted and TX-100–extracted Golgi membranes, but not to stacks that were sequentially extracted with TX-100 and Na2CO3.

Mentions: Golgi stacks were also extracted with 1% TX-100 on ice (releasing 75% of total protein, see below) and analyzed for the presence of dynactin complex and for cytosolic dynein binding (Fig. 4 A). The detergent released most of the Arp1 from the membrane, while little, if any, of the p150Glued was extracted. Immunoblots showed that cytosolic dynein could bind to stacks that were solubilized with cold 1% TX-100 (Fig. 4 B). Cytosolic dynein binding to detergent-extracted stacks was physiologically relevant as shown by two methods. First, the cytosolic dynein that bound to the stack ghosts was functional and could move these membranes on microtubules in in vitro motility assays (Fig. 5 A) at rates of 1.1 ± 0.2 μm/s (mean ± SD; n = 12; at 25°C); rates that were indistinguishable from the rates of unextracted membranes at 1.2 ± 0.5 μm/s (mean ± SD; n = 8; at 25°C) from the same preparation. In the absence of added cytosol, these membrane ghosts neither bound to nor moved on microtubules in these assays. Second, as with the native Golgi stacks, the dynein binding protein(s) on Triton membrane ghosts was extracted at alkaline pH. Cytosolic dynein did not bind to Golgi stacks that had been sequentially extracted with TX-100 and pH 11.5 (Fig. 5 B). These two independent assays suggest that cytosolic dynein is binding to the same peripheral membrane protein(s) in intact and detergent-extracted Golgi stacks.


Molecular motors and a spectrin matrix associate with Golgi membranes in vitro.

Fath KR, Trimbur GM, Burgess DR - J. Cell Biol. (1997)

Cytosolic dynein binding to TX-100-extracted Golgi  stacks is physiologically relevant. (A) Cytosolic dynein that  bound to detergent-extracted Golgi stacks was functional in in  vitro motility assays. Golgi stacks were extracted with 1% TX-100 on ice, pelleted and resuspended in buffer. In a motility  chamber, the detergent ghosts were incubated with cytosol and  with microtubules that were polymerized from the ends of axonemes. This series of video frames shows a membrane ghost (arrow) moving along a microtubule. (B) Dynein binding protein(s)  are extracted from Golgi stack detergent ghosts at alkaline pH.  Golgi stacks were incubated on ice with PEMS (Control); 1%  TX-100 (TX); or 1% TX-100 followed by 0.1 M Na2CO3 (TX →   pH 11.5). The membranes were collected by centrifugation, resuspended in PEMS, incubated with cytosol to allow dynein binding, and repelleted through 0.5 M sucrose. This immunoblot  shows that cytosolic dynein could bind to both unextracted and  TX-100–extracted Golgi membranes, but not to stacks that were  sequentially extracted with TX-100 and Na2CO3.
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Figure 5: Cytosolic dynein binding to TX-100-extracted Golgi stacks is physiologically relevant. (A) Cytosolic dynein that bound to detergent-extracted Golgi stacks was functional in in vitro motility assays. Golgi stacks were extracted with 1% TX-100 on ice, pelleted and resuspended in buffer. In a motility chamber, the detergent ghosts were incubated with cytosol and with microtubules that were polymerized from the ends of axonemes. This series of video frames shows a membrane ghost (arrow) moving along a microtubule. (B) Dynein binding protein(s) are extracted from Golgi stack detergent ghosts at alkaline pH. Golgi stacks were incubated on ice with PEMS (Control); 1% TX-100 (TX); or 1% TX-100 followed by 0.1 M Na2CO3 (TX → pH 11.5). The membranes were collected by centrifugation, resuspended in PEMS, incubated with cytosol to allow dynein binding, and repelleted through 0.5 M sucrose. This immunoblot shows that cytosolic dynein could bind to both unextracted and TX-100–extracted Golgi membranes, but not to stacks that were sequentially extracted with TX-100 and Na2CO3.
Mentions: Golgi stacks were also extracted with 1% TX-100 on ice (releasing 75% of total protein, see below) and analyzed for the presence of dynactin complex and for cytosolic dynein binding (Fig. 4 A). The detergent released most of the Arp1 from the membrane, while little, if any, of the p150Glued was extracted. Immunoblots showed that cytosolic dynein could bind to stacks that were solubilized with cold 1% TX-100 (Fig. 4 B). Cytosolic dynein binding to detergent-extracted stacks was physiologically relevant as shown by two methods. First, the cytosolic dynein that bound to the stack ghosts was functional and could move these membranes on microtubules in in vitro motility assays (Fig. 5 A) at rates of 1.1 ± 0.2 μm/s (mean ± SD; n = 12; at 25°C); rates that were indistinguishable from the rates of unextracted membranes at 1.2 ± 0.5 μm/s (mean ± SD; n = 8; at 25°C) from the same preparation. In the absence of added cytosol, these membrane ghosts neither bound to nor moved on microtubules in these assays. Second, as with the native Golgi stacks, the dynein binding protein(s) on Triton membrane ghosts was extracted at alkaline pH. Cytosolic dynein did not bind to Golgi stacks that had been sequentially extracted with TX-100 and pH 11.5 (Fig. 5 B). These two independent assays suggest that cytosolic dynein is binding to the same peripheral membrane protein(s) in intact and detergent-extracted Golgi stacks.

Bottom Line: In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules.Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin.Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
Cytoplasmic dynein is a microtubule minus-end-directed motor that is thought to power the transport of vesicles from the TGN to the apical cortex in polarized epithelial cells. Trans-Golgi enriched membranes, which were isolated from primary polarized intestinal epithelial cells, contain both the actin-based motor myosin-I and dynein, whereas isolated Golgi stacks lack dynein but contain myosin-I (Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1994. J. Cell Biol. 126:661-675). We show now that Golgi stacks in vitro bind dynein supplied from cytosol in the absence of ATP, and bud small membranes when incubated with cytosol and ATP. Cytosolic dynein binds to regions of stacks that are destined to bud because dynein is present in budded membranes, but absent from stacks after budding. Budded membranes move exclusively towards microtubule minus-ends in in vitro motility assays. Extraction studies suggest that dynein binds to a Golgi peripheral membrane protein(s) that resists extraction by ice-cold Triton X-100. In the presence of cytosol, these membrane ghosts can move towards the minus-ends of microtubules. Detergent-extracted Golgi stacks and TGN-containing membranes are closely associated with an amorphous matrix composed in part of spectrin and ankyrin. Although spectrin has been proposed to help link dynein to organellar membranes, we found that functional dynein may bind to extracted membranes independently of spectrin and ankyrin.

Show MeSH
Related in: MedlinePlus