Limits...
Organelle tethering by a homotypic PDZ interaction underlies formation of the Golgi membrane network.

Sengupta D, Truschel S, Bachert C, Linstedt AD - J. Cell Biol. (2009)

Bottom Line: Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction.Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans.Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

ABSTRACT
Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

Show MeSH

Related in: MedlinePlus

Mitochondria are tethered and not fused into syncytia. GFP-ActA (A and B) or G65-GFP-ActA (C and D) transfected HeLa cells were BFA treated, processed for electron microscopy, and shown at 2 magnifications. GFP-ActA (E–H) or G65-GFP-ActA (I–L) transfected cells were imaged live using a scanning laser microscope. A region of interest (marked in figure) was selected and bleached and recovery was monitored in subsequent frames at 2-s intervals. Bar, 2 µm. Mouse embryonic fibroblasts lacking mitofusin-1/2 and expressing the matrix marker COX-IV-DsRed were transfected with GFP-ActA (M–O) or G65-GFP-ActA (P–R), BFA-treated, and processed to reveal mitochondria (red), and the expressed proteins (green). Bar = 10 µm. An enlarged view of a single optical section is also shown (S–U). Bar = 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2712994&req=5

fig2: Mitochondria are tethered and not fused into syncytia. GFP-ActA (A and B) or G65-GFP-ActA (C and D) transfected HeLa cells were BFA treated, processed for electron microscopy, and shown at 2 magnifications. GFP-ActA (E–H) or G65-GFP-ActA (I–L) transfected cells were imaged live using a scanning laser microscope. A region of interest (marked in figure) was selected and bleached and recovery was monitored in subsequent frames at 2-s intervals. Bar, 2 µm. Mouse embryonic fibroblasts lacking mitofusin-1/2 and expressing the matrix marker COX-IV-DsRed were transfected with GFP-ActA (M–O) or G65-GFP-ActA (P–R), BFA-treated, and processed to reveal mitochondria (red), and the expressed proteins (green). Bar = 10 µm. An enlarged view of a single optical section is also shown (S–U). Bar = 1 µm.

Mentions: Next, electron microscopy was performed on the BFA-treated transfected cells to assess the ultrastructure of the clustered mitochondria. In untransfected cells, and in cells transfected with the GFP-ActA control plasmid, mitochondria were evident throughout the entire cytoplasm and were frequently well separated from each other (Fig. 2, A and B). As expected, the filamentous aspect apparent using fluorescence microscopy was not evident, presumably due to the low probability of obtaining thin sections with longitudinal profiles of membrane tubules. In contrast, cells transfected with G65-GFP-ActA had prominent clusters of mitochondria in the juxtanuclear region and the remaining cytoplasm was essentially devoid of mitochondria (Fig. 2, C and D). Unlike Golgi stacks, which have extended zones of apposition with uniform gap widths, the mitochondria in the clusters were apposed mostly at discrete sites and at a greater distance. Other membranes may be present within the clusters. Nevertheless, an immunofluorescence assay (not depicted) failed to reveal any accumulation in the clusters of calnexin, an ER marker, or ERGIC53, a marker of the intermediate compartment that accumulates in BFA remnants (Seemann et al., 2000). Interestingly, the outer membranes of individual mitochondria appeared distinct from neighboring outer membranes, suggesting maintenance of mitochondrial integrity within the cluster. Absence of syncytia formation was further supported by FRAP experiments. Cells expressing the control construct, GFP-ActA, exhibited an extended mitochondrial network, and when a small region of the network was bleached, fluorescence was rapidly recovered in the bleached structures (Fig. 2, E–H, movie and quantification in Video 1). This is the expected behavior for a contiguous membrane network established by membrane fusion. In contrast, cells expressing the G65-GFP-ActA construct exhibited a juxtanuclear cluster of mitochondria and there was no recovery of fluorescence observed after photobleaching small portions of the clustered membranes (Fig. 2, I–L, movie and quantification in Video 2).


Organelle tethering by a homotypic PDZ interaction underlies formation of the Golgi membrane network.

Sengupta D, Truschel S, Bachert C, Linstedt AD - J. Cell Biol. (2009)

Mitochondria are tethered and not fused into syncytia. GFP-ActA (A and B) or G65-GFP-ActA (C and D) transfected HeLa cells were BFA treated, processed for electron microscopy, and shown at 2 magnifications. GFP-ActA (E–H) or G65-GFP-ActA (I–L) transfected cells were imaged live using a scanning laser microscope. A region of interest (marked in figure) was selected and bleached and recovery was monitored in subsequent frames at 2-s intervals. Bar, 2 µm. Mouse embryonic fibroblasts lacking mitofusin-1/2 and expressing the matrix marker COX-IV-DsRed were transfected with GFP-ActA (M–O) or G65-GFP-ActA (P–R), BFA-treated, and processed to reveal mitochondria (red), and the expressed proteins (green). Bar = 10 µm. An enlarged view of a single optical section is also shown (S–U). Bar = 1 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2712994&req=5

fig2: Mitochondria are tethered and not fused into syncytia. GFP-ActA (A and B) or G65-GFP-ActA (C and D) transfected HeLa cells were BFA treated, processed for electron microscopy, and shown at 2 magnifications. GFP-ActA (E–H) or G65-GFP-ActA (I–L) transfected cells were imaged live using a scanning laser microscope. A region of interest (marked in figure) was selected and bleached and recovery was monitored in subsequent frames at 2-s intervals. Bar, 2 µm. Mouse embryonic fibroblasts lacking mitofusin-1/2 and expressing the matrix marker COX-IV-DsRed were transfected with GFP-ActA (M–O) or G65-GFP-ActA (P–R), BFA-treated, and processed to reveal mitochondria (red), and the expressed proteins (green). Bar = 10 µm. An enlarged view of a single optical section is also shown (S–U). Bar = 1 µm.
Mentions: Next, electron microscopy was performed on the BFA-treated transfected cells to assess the ultrastructure of the clustered mitochondria. In untransfected cells, and in cells transfected with the GFP-ActA control plasmid, mitochondria were evident throughout the entire cytoplasm and were frequently well separated from each other (Fig. 2, A and B). As expected, the filamentous aspect apparent using fluorescence microscopy was not evident, presumably due to the low probability of obtaining thin sections with longitudinal profiles of membrane tubules. In contrast, cells transfected with G65-GFP-ActA had prominent clusters of mitochondria in the juxtanuclear region and the remaining cytoplasm was essentially devoid of mitochondria (Fig. 2, C and D). Unlike Golgi stacks, which have extended zones of apposition with uniform gap widths, the mitochondria in the clusters were apposed mostly at discrete sites and at a greater distance. Other membranes may be present within the clusters. Nevertheless, an immunofluorescence assay (not depicted) failed to reveal any accumulation in the clusters of calnexin, an ER marker, or ERGIC53, a marker of the intermediate compartment that accumulates in BFA remnants (Seemann et al., 2000). Interestingly, the outer membranes of individual mitochondria appeared distinct from neighboring outer membranes, suggesting maintenance of mitochondrial integrity within the cluster. Absence of syncytia formation was further supported by FRAP experiments. Cells expressing the control construct, GFP-ActA, exhibited an extended mitochondrial network, and when a small region of the network was bleached, fluorescence was rapidly recovered in the bleached structures (Fig. 2, E–H, movie and quantification in Video 1). This is the expected behavior for a contiguous membrane network established by membrane fusion. In contrast, cells expressing the G65-GFP-ActA construct exhibited a juxtanuclear cluster of mitochondria and there was no recovery of fluorescence observed after photobleaching small portions of the clustered membranes (Fig. 2, I–L, movie and quantification in Video 2).

Bottom Line: Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction.Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans.Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.

ABSTRACT
Formation of the ribbon-like membrane network of the Golgi apparatus depends on GM130 and GRASP65, but the mechanism is unknown. We developed an in vivo organelle tethering assaying in which GRASP65 was targeted to the mitochondrial outer membrane either directly or via binding to GM130. Mitochondria bearing GRASP65 became tethered to one another, and this depended on a GRASP65 PDZ domain that was also required for GRASP65 self-interaction. Point mutation within the predicted binding groove of the GRASP65 PDZ domain blocked both tethering and, in a gene replacement assay, Golgi ribbon formation. Tethering also required proximate membrane anchoring of the PDZ domain, suggesting a mechanism that orientates the PDZ binding groove to favor interactions in trans. Thus, a homotypic PDZ interaction mediates organelle tethering in living cells.

Show MeSH
Related in: MedlinePlus