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A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton.

Gao Y, Sztul E - J. Cell Biol. (2001)

Bottom Line: We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro.Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells.The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.

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FTCD targets to the Golgi complex and organelles into fibers. COS-7 cells were transfected with GFP-tagged (A) or -untagged (B and C) rFTCD and grown for 18–48 h. (A) Cells were processed for immunofluorescence with polyclonal anti-GM130 antibodies. (B and C) Cells were processed for immunofluorescence with monoclonal anti-FTCD and polyclonal anti-GM130 antibodies. In cells expressing low levels of FTCD (A and B), the recombinant FTCD is concentrated in the Golgi region. In cells expressing high levels of FTCD (C), the recombinant FTCD also localizes to thick fibers originating from the Golgi region. (D) COS-7 cells were transfected with GFP–FTCD for 48 h and fractionated by equilibrium density centrifugation. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC, and immunoblotted with anti-FTCD antibodies to detect the 58-kD endogenous FTCD and the 86-kD recombinant GFP–FTCD. The NC was also probed with anti-GM130 antibodies. The relative recovery of endogenous FTCD, GFP–FTCD, and GM130 in each lane were quantitatively evaluated by densitometry. Endogenous FTCD and GFP–FTCD were predominantly recovered in the high density sucrose load (fractions 9–15) containing soluble cytosolic proteins. A proportion of FTCD and GFP–FTCD were recovered in low density sucrose (fractions 5–7) enriched in the Golgi marker GM130.
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Figure 3: FTCD targets to the Golgi complex and organelles into fibers. COS-7 cells were transfected with GFP-tagged (A) or -untagged (B and C) rFTCD and grown for 18–48 h. (A) Cells were processed for immunofluorescence with polyclonal anti-GM130 antibodies. (B and C) Cells were processed for immunofluorescence with monoclonal anti-FTCD and polyclonal anti-GM130 antibodies. In cells expressing low levels of FTCD (A and B), the recombinant FTCD is concentrated in the Golgi region. In cells expressing high levels of FTCD (C), the recombinant FTCD also localizes to thick fibers originating from the Golgi region. (D) COS-7 cells were transfected with GFP–FTCD for 48 h and fractionated by equilibrium density centrifugation. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC, and immunoblotted with anti-FTCD antibodies to detect the 58-kD endogenous FTCD and the 86-kD recombinant GFP–FTCD. The NC was also probed with anti-GM130 antibodies. The relative recovery of endogenous FTCD, GFP–FTCD, and GM130 in each lane were quantitatively evaluated by densitometry. Endogenous FTCD and GFP–FTCD were predominantly recovered in the high density sucrose load (fractions 9–15) containing soluble cytosolic proteins. A proportion of FTCD and GFP–FTCD were recovered in low density sucrose (fractions 5–7) enriched in the Golgi marker GM130.

Mentions: To initiate the inquiry into the intracellular function of FTCD, we transiently expressed GFP-tagged and -untagged full-length FTCD in COS-7 cells. As shown in Fig. 3 A, in cells expressing low levels of GFP–FTCD, the recombinant protein was predominantly localized to the Golgi area, where it colocalized with GM130. In addition, faint filamentous staining extending from the Golgi region in a pattern similar to that of endogenous FTCD was detected. The faithfulness of GFP–FTCD distribution was also analyzed by fractionation after cell disruption. As shown in Fig. 3 D, the distribution of GFP–FTCD paralleled that of endogenous FTCD. Both were predominantly recovered at the bottom of the gradient (fractions 9–15), representing the high density sucrose load and containing soluble cytosolic proteins. Only a small amount of GFP–FTCD and endogenous FTCD was recovered in the low density sucrose (fractions 5–7) enriched in the Golgi membrane marker GM130. These results are analogous to those seen following homogenization of rat liver, where FTCD was predominantly found in the cytosolic fraction, with only a small proportion (<10%) cofractionating with Golgi membranes (Bloom and Brashear 1989; Gao et al. 1998).


A novel interaction of the Golgi complex with the vimentin intermediate filament cytoskeleton.

Gao Y, Sztul E - J. Cell Biol. (2001)

FTCD targets to the Golgi complex and organelles into fibers. COS-7 cells were transfected with GFP-tagged (A) or -untagged (B and C) rFTCD and grown for 18–48 h. (A) Cells were processed for immunofluorescence with polyclonal anti-GM130 antibodies. (B and C) Cells were processed for immunofluorescence with monoclonal anti-FTCD and polyclonal anti-GM130 antibodies. In cells expressing low levels of FTCD (A and B), the recombinant FTCD is concentrated in the Golgi region. In cells expressing high levels of FTCD (C), the recombinant FTCD also localizes to thick fibers originating from the Golgi region. (D) COS-7 cells were transfected with GFP–FTCD for 48 h and fractionated by equilibrium density centrifugation. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC, and immunoblotted with anti-FTCD antibodies to detect the 58-kD endogenous FTCD and the 86-kD recombinant GFP–FTCD. The NC was also probed with anti-GM130 antibodies. The relative recovery of endogenous FTCD, GFP–FTCD, and GM130 in each lane were quantitatively evaluated by densitometry. Endogenous FTCD and GFP–FTCD were predominantly recovered in the high density sucrose load (fractions 9–15) containing soluble cytosolic proteins. A proportion of FTCD and GFP–FTCD were recovered in low density sucrose (fractions 5–7) enriched in the Golgi marker GM130.
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Related In: Results  -  Collection

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Figure 3: FTCD targets to the Golgi complex and organelles into fibers. COS-7 cells were transfected with GFP-tagged (A) or -untagged (B and C) rFTCD and grown for 18–48 h. (A) Cells were processed for immunofluorescence with polyclonal anti-GM130 antibodies. (B and C) Cells were processed for immunofluorescence with monoclonal anti-FTCD and polyclonal anti-GM130 antibodies. In cells expressing low levels of FTCD (A and B), the recombinant FTCD is concentrated in the Golgi region. In cells expressing high levels of FTCD (C), the recombinant FTCD also localizes to thick fibers originating from the Golgi region. (D) COS-7 cells were transfected with GFP–FTCD for 48 h and fractionated by equilibrium density centrifugation. An equivalent amount of each fraction was processed by SDS-PAGE, transferred to NC, and immunoblotted with anti-FTCD antibodies to detect the 58-kD endogenous FTCD and the 86-kD recombinant GFP–FTCD. The NC was also probed with anti-GM130 antibodies. The relative recovery of endogenous FTCD, GFP–FTCD, and GM130 in each lane were quantitatively evaluated by densitometry. Endogenous FTCD and GFP–FTCD were predominantly recovered in the high density sucrose load (fractions 9–15) containing soluble cytosolic proteins. A proportion of FTCD and GFP–FTCD were recovered in low density sucrose (fractions 5–7) enriched in the Golgi marker GM130.
Mentions: To initiate the inquiry into the intracellular function of FTCD, we transiently expressed GFP-tagged and -untagged full-length FTCD in COS-7 cells. As shown in Fig. 3 A, in cells expressing low levels of GFP–FTCD, the recombinant protein was predominantly localized to the Golgi area, where it colocalized with GM130. In addition, faint filamentous staining extending from the Golgi region in a pattern similar to that of endogenous FTCD was detected. The faithfulness of GFP–FTCD distribution was also analyzed by fractionation after cell disruption. As shown in Fig. 3 D, the distribution of GFP–FTCD paralleled that of endogenous FTCD. Both were predominantly recovered at the bottom of the gradient (fractions 9–15), representing the high density sucrose load and containing soluble cytosolic proteins. Only a small amount of GFP–FTCD and endogenous FTCD was recovered in the low density sucrose (fractions 5–7) enriched in the Golgi membrane marker GM130. These results are analogous to those seen following homogenization of rat liver, where FTCD was predominantly found in the cytosolic fraction, with only a small proportion (<10%) cofractionating with Golgi membranes (Bloom and Brashear 1989; Gao et al. 1998).

Bottom Line: We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro.Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells.The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.

ABSTRACT
The integration of the vimentin intermediate filament (IF) cytoskeleton and cellular organelles in vivo is an incompletely understood process, and the identities of proteins participating in such events are largely unknown. Here, we show that the Golgi complex interacts with the vimentin IF cytoskeleton, and that the Golgi protein formiminotransferase cyclodeaminase (FTCD) participates in this interaction. We show that the peripherally associated Golgi protein FTCD binds directly to vimentin subunits and to polymerized vimentin filaments in vivo and in vitro. Expression of FTCD in cultured cells results in the formation of extensive FTCD-containing fibers originating from the Golgi region, and is paralleled by a dramatic rearrangements of the vimentin IF cytoskeleton in a coordinate process in which vimentin filaments and FTCD integrate into chimeric fibers. Formation of the FTCD fibers is obligatorily coupled to vimentin assembly and does not occur in vim(-/-) cells. The FTCD-mediated regulation of vimentin IF is not a secondary effect of changes in the microtubule or the actin cytoskeletons, since those cytoskeletal systems appear unaffected by FTCD expression. The assembly of the FTCD/vimentin fibers causes a coordinate change in the structure of the Golgi complex and results in Golgi fragmentation into individual elements that are tethered to the FTCD/vimentin fibers. The observed interaction of Golgi elements with vimentin filaments and the ability of FTCD to specifically interacts with both Golgi membrane and vimentin filaments and promote their association suggest that FTCD might be a candidate protein integrating the Golgi compartment with the IF cytoskeleton.

Show MeSH
Related in: MedlinePlus