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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, but not kinesin, binds to isolated  Golgi stacks. (A) Isolated Golgi stacks, which lack dynein and kinesin, were incubated for 15 min at 37°C in motility buffers (in  the absence of ATP) or with buffer including cytosol. A third tube  contained cytosol, but no membranes. The samples were then  chilled and pelleted through 0.5 M sucrose to separate stacks and  bound material from cytosol. All of each pellet and one-half of  the supernatant were immunoblotted with a mixture of dynein IC  and kinesin heavy chain mAbs. Pelleted Golgi stacks incubated in  buffer (Stacks) contained no cytosolic dynein (DIC, dynein IC) or  kinesin (KHC, kinesin heavy chain). Soluble cytosolic kinesin and  dynein remained in the supernatant in cytosol-alone controls (Cytosol). Pelleted stacks that were incubated with cytosol (Stacks +  Cytosol) bound dynein, but not kinesin. (B) Cytosolic dynein  binding to Golgi stacks was also demonstrated on flotation gradients. Golgi stacks and cytosol were incubated as described in A,  then made 25% nycodenz. The samples were overlaid with 20%  nycodenz/PEMS and PEMS. After centrifugation, immunoblots  show that both Golgi cisternae, as indicated by the presence of  α-mannosidase II, and cytosolic dynein were present at the 20%  nycodenz/PEMS interface in samples containing both Golgi  stacks and cytosol (Stacks + Cytosol). Dynein was not detected at  this interface in samples containing Golgi stacks incubated in  buffer (Stacks) or in cytosol alone controls (Cytosol).
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Figure 1: Cytosolic dynein, but not kinesin, binds to isolated Golgi stacks. (A) Isolated Golgi stacks, which lack dynein and kinesin, were incubated for 15 min at 37°C in motility buffers (in the absence of ATP) or with buffer including cytosol. A third tube contained cytosol, but no membranes. The samples were then chilled and pelleted through 0.5 M sucrose to separate stacks and bound material from cytosol. All of each pellet and one-half of the supernatant were immunoblotted with a mixture of dynein IC and kinesin heavy chain mAbs. Pelleted Golgi stacks incubated in buffer (Stacks) contained no cytosolic dynein (DIC, dynein IC) or kinesin (KHC, kinesin heavy chain). Soluble cytosolic kinesin and dynein remained in the supernatant in cytosol-alone controls (Cytosol). Pelleted stacks that were incubated with cytosol (Stacks + Cytosol) bound dynein, but not kinesin. (B) Cytosolic dynein binding to Golgi stacks was also demonstrated on flotation gradients. Golgi stacks and cytosol were incubated as described in A, then made 25% nycodenz. The samples were overlaid with 20% nycodenz/PEMS and PEMS. After centrifugation, immunoblots show that both Golgi cisternae, as indicated by the presence of α-mannosidase II, and cytosolic dynein were present at the 20% nycodenz/PEMS interface in samples containing both Golgi stacks and cytosol (Stacks + Cytosol). Dynein was not detected at this interface in samples containing Golgi stacks incubated in buffer (Stacks) or in cytosol alone controls (Cytosol).

Mentions: Dynein binding to Golgi stacks was performed as described for the in vitro budding assay, except that the ATP and the ATP regenerating system were omitted. In some experiments (see Fig. 1), dynein binding was assayed in conditions that were identical to those used in the in vitro motility assays described below with the omission of ATP. After incubation at 37°C, membranes were pelleted at 259,000 gmax (TLS-55; Beckman) for 30 min through a 75-μl cushion containing 0.5 M sucrose PKM at 4°C. For centrifugation, 11 × 34 mm polycarbonate tubes were used as rotor inserts to hold the 7 × 20 mm polycarbonate tubes, which contained the samples. The smaller tubes were surrounded by a cushion of 50 μl of water to prevent their distortion. The pellet was resuspended and analyzed by SDS-PAGE and immunoblotting.


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

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

Cytosolic dynein, but not kinesin, binds to isolated  Golgi stacks. (A) Isolated Golgi stacks, which lack dynein and kinesin, were incubated for 15 min at 37°C in motility buffers (in  the absence of ATP) or with buffer including cytosol. A third tube  contained cytosol, but no membranes. The samples were then  chilled and pelleted through 0.5 M sucrose to separate stacks and  bound material from cytosol. All of each pellet and one-half of  the supernatant were immunoblotted with a mixture of dynein IC  and kinesin heavy chain mAbs. Pelleted Golgi stacks incubated in  buffer (Stacks) contained no cytosolic dynein (DIC, dynein IC) or  kinesin (KHC, kinesin heavy chain). Soluble cytosolic kinesin and  dynein remained in the supernatant in cytosol-alone controls (Cytosol). Pelleted stacks that were incubated with cytosol (Stacks +  Cytosol) bound dynein, but not kinesin. (B) Cytosolic dynein  binding to Golgi stacks was also demonstrated on flotation gradients. Golgi stacks and cytosol were incubated as described in A,  then made 25% nycodenz. The samples were overlaid with 20%  nycodenz/PEMS and PEMS. After centrifugation, immunoblots  show that both Golgi cisternae, as indicated by the presence of  α-mannosidase II, and cytosolic dynein were present at the 20%  nycodenz/PEMS interface in samples containing both Golgi  stacks and cytosol (Stacks + Cytosol). Dynein was not detected at  this interface in samples containing Golgi stacks incubated in  buffer (Stacks) or in cytosol alone controls (Cytosol).
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Related In: Results  -  Collection

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Figure 1: Cytosolic dynein, but not kinesin, binds to isolated Golgi stacks. (A) Isolated Golgi stacks, which lack dynein and kinesin, were incubated for 15 min at 37°C in motility buffers (in the absence of ATP) or with buffer including cytosol. A third tube contained cytosol, but no membranes. The samples were then chilled and pelleted through 0.5 M sucrose to separate stacks and bound material from cytosol. All of each pellet and one-half of the supernatant were immunoblotted with a mixture of dynein IC and kinesin heavy chain mAbs. Pelleted Golgi stacks incubated in buffer (Stacks) contained no cytosolic dynein (DIC, dynein IC) or kinesin (KHC, kinesin heavy chain). Soluble cytosolic kinesin and dynein remained in the supernatant in cytosol-alone controls (Cytosol). Pelleted stacks that were incubated with cytosol (Stacks + Cytosol) bound dynein, but not kinesin. (B) Cytosolic dynein binding to Golgi stacks was also demonstrated on flotation gradients. Golgi stacks and cytosol were incubated as described in A, then made 25% nycodenz. The samples were overlaid with 20% nycodenz/PEMS and PEMS. After centrifugation, immunoblots show that both Golgi cisternae, as indicated by the presence of α-mannosidase II, and cytosolic dynein were present at the 20% nycodenz/PEMS interface in samples containing both Golgi stacks and cytosol (Stacks + Cytosol). Dynein was not detected at this interface in samples containing Golgi stacks incubated in buffer (Stacks) or in cytosol alone controls (Cytosol).
Mentions: Dynein binding to Golgi stacks was performed as described for the in vitro budding assay, except that the ATP and the ATP regenerating system were omitted. In some experiments (see Fig. 1), dynein binding was assayed in conditions that were identical to those used in the in vitro motility assays described below with the omission of ATP. After incubation at 37°C, membranes were pelleted at 259,000 gmax (TLS-55; Beckman) for 30 min through a 75-μl cushion containing 0.5 M sucrose PKM at 4°C. For centrifugation, 11 × 34 mm polycarbonate tubes were used as rotor inserts to hold the 7 × 20 mm polycarbonate tubes, which contained the samples. The smaller tubes were surrounded by a cushion of 50 μl of water to prevent their distortion. The pellet was resuspended and analyzed by SDS-PAGE and immunoblotting.

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