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Golgi-derived CLASP-dependent microtubules control Golgi organization and polarized trafficking in motile cells.

Miller PM, Folkmann AW, Maia AR, Efimova N, Efimov A, Kaverina I - Nat. Cell Biol. (2009)

Bottom Line: These Golgi-derived microtubules draw Golgi ministacks together in tangential fashion and are crucial for establishing continuity and proper morphology of the Golgi complex.We propose that specialized functions of these two microtubule arrays arise from their specific geometries.Further, we demonstrate that directional post-Golgi trafficking and cell migration depend on Golgi-associated CLASPs, suggesting that correct organization of the Golgi complex by microtubules is essential for cell polarization and motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

ABSTRACT
Microtubules are indispensable for Golgi complex assembly and maintenance, which are integral parts of cytoplasm organization during interphase in mammalian cells. Here, we show that two discrete microtubule subsets drive two distinct, yet simultaneous, stages of Golgi assembly. In addition to the radial centrosomal microtubule array, which positions the Golgi in the centre of the cell, we have identified a role for microtubules that form at the Golgi membranes in a manner dependent on the microtubule regulators CLASPs. These Golgi-derived microtubules draw Golgi ministacks together in tangential fashion and are crucial for establishing continuity and proper morphology of the Golgi complex. We propose that specialized functions of these two microtubule arrays arise from their specific geometries. Further, we demonstrate that directional post-Golgi trafficking and cell migration depend on Golgi-associated CLASPs, suggesting that correct organization of the Golgi complex by microtubules is essential for cell polarization and motility.

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Golgi assembly depends on directionality of two MT subsets and on dynein activity(a–b) Overlaid GFP-EB3 (green) and mCherry-GT (red) video frames within 2.5 min. (a) EB3 tracks in a control cell illustrate radial centrosomal (yellow arrow) and tangential Golgi-associated (blue arrowhead) MT arrays. Box is enlarged in (a') for mCherry-GT and in (a”) for GFP-EB3. (b) Radial centrosomal (yellow arrow) EB3 tracks in CLASP-depleted cell (siRNA combination #1). Box is enlarged in (b') for mCherry-GT and in (b”) for GFP-EB3. (c) Video frames illustrating minus-end directed mini-stack movement (yellow arrow) along Golgi-nucleated MTs upon nocodazole washout in mCherry-GT (red) and GFP-EB3 (green) expressing NT control cells. MT plus end, asterisk. Time after nocodazole removal is shown. (d) Video frames illustrating nocodazole washout in cell over-expressing GFP-P50 (not shown) and mCherry-GT (black). Mini-stacks move toward the cell periphery along forming MTs due to kinesin activity (asterisks). Time after nocodazole removal is shown. (e) Fold increase of Golgi particle size upon nocodazole washout based on live cell imaging of control (n=7, 6 independent experiments) and GFP-P50 over-expressing (n=8, 7 independent experiments) cells. Error bars, standard error. *P<0.001, unpaired Student's t-test. (f) Average Golgi particle area (μm2) in nocodazole (time 0) and upon 60 min washout in fixed samples of control, GFP-P50 over-expressing, and RFP-CC1 over-expressing cells. n=50 for each condition, 4 independent experiments. Error bars, standard error. *P<0.001, **P<0.01 unpaired Student's t-test. (g) Role of Golgi-associated MTs in Golgi ribbon assembly (model).
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Figure 4: Golgi assembly depends on directionality of two MT subsets and on dynein activity(a–b) Overlaid GFP-EB3 (green) and mCherry-GT (red) video frames within 2.5 min. (a) EB3 tracks in a control cell illustrate radial centrosomal (yellow arrow) and tangential Golgi-associated (blue arrowhead) MT arrays. Box is enlarged in (a') for mCherry-GT and in (a”) for GFP-EB3. (b) Radial centrosomal (yellow arrow) EB3 tracks in CLASP-depleted cell (siRNA combination #1). Box is enlarged in (b') for mCherry-GT and in (b”) for GFP-EB3. (c) Video frames illustrating minus-end directed mini-stack movement (yellow arrow) along Golgi-nucleated MTs upon nocodazole washout in mCherry-GT (red) and GFP-EB3 (green) expressing NT control cells. MT plus end, asterisk. Time after nocodazole removal is shown. (d) Video frames illustrating nocodazole washout in cell over-expressing GFP-P50 (not shown) and mCherry-GT (black). Mini-stacks move toward the cell periphery along forming MTs due to kinesin activity (asterisks). Time after nocodazole removal is shown. (e) Fold increase of Golgi particle size upon nocodazole washout based on live cell imaging of control (n=7, 6 independent experiments) and GFP-P50 over-expressing (n=8, 7 independent experiments) cells. Error bars, standard error. *P<0.001, unpaired Student's t-test. (f) Average Golgi particle area (μm2) in nocodazole (time 0) and upon 60 min washout in fixed samples of control, GFP-P50 over-expressing, and RFP-CC1 over-expressing cells. n=50 for each condition, 4 independent experiments. Error bars, standard error. *P<0.001, **P<0.01 unpaired Student's t-test. (g) Role of Golgi-associated MTs in Golgi ribbon assembly (model).

Mentions: Because MT orientation appeared important for Golgi stack clustering in nocodazole washout assays, we further addressed the role of MT orientation in Golgi organization. MT directionality was detected as described in Efimov et al [7]. In brief, RPE1 cells were transfected with GFP-EB3 to mark MT plus-tips and mCherry-GT to label the Golgi, and time-lapse video sequences were recorded. Distinct centrosomal MT and Golgi-derived MT arrays were detected in NT-control cells (Fig. 4a,a”). Interestingly, while centrosomal MTs showed clear radial geometry, MTs formed at the Golgi were predominantly tangential. Golgi ribbons (Fig. 4a') were aligned along Golgi-derived MT tracks suggesting that they were primarily formed via tangential mini-stack linking and fusion (Fig. 4a).


Golgi-derived CLASP-dependent microtubules control Golgi organization and polarized trafficking in motile cells.

Miller PM, Folkmann AW, Maia AR, Efimova N, Efimov A, Kaverina I - Nat. Cell Biol. (2009)

Golgi assembly depends on directionality of two MT subsets and on dynein activity(a–b) Overlaid GFP-EB3 (green) and mCherry-GT (red) video frames within 2.5 min. (a) EB3 tracks in a control cell illustrate radial centrosomal (yellow arrow) and tangential Golgi-associated (blue arrowhead) MT arrays. Box is enlarged in (a') for mCherry-GT and in (a”) for GFP-EB3. (b) Radial centrosomal (yellow arrow) EB3 tracks in CLASP-depleted cell (siRNA combination #1). Box is enlarged in (b') for mCherry-GT and in (b”) for GFP-EB3. (c) Video frames illustrating minus-end directed mini-stack movement (yellow arrow) along Golgi-nucleated MTs upon nocodazole washout in mCherry-GT (red) and GFP-EB3 (green) expressing NT control cells. MT plus end, asterisk. Time after nocodazole removal is shown. (d) Video frames illustrating nocodazole washout in cell over-expressing GFP-P50 (not shown) and mCherry-GT (black). Mini-stacks move toward the cell periphery along forming MTs due to kinesin activity (asterisks). Time after nocodazole removal is shown. (e) Fold increase of Golgi particle size upon nocodazole washout based on live cell imaging of control (n=7, 6 independent experiments) and GFP-P50 over-expressing (n=8, 7 independent experiments) cells. Error bars, standard error. *P<0.001, unpaired Student's t-test. (f) Average Golgi particle area (μm2) in nocodazole (time 0) and upon 60 min washout in fixed samples of control, GFP-P50 over-expressing, and RFP-CC1 over-expressing cells. n=50 for each condition, 4 independent experiments. Error bars, standard error. *P<0.001, **P<0.01 unpaired Student's t-test. (g) Role of Golgi-associated MTs in Golgi ribbon assembly (model).
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Related In: Results  -  Collection

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Figure 4: Golgi assembly depends on directionality of two MT subsets and on dynein activity(a–b) Overlaid GFP-EB3 (green) and mCherry-GT (red) video frames within 2.5 min. (a) EB3 tracks in a control cell illustrate radial centrosomal (yellow arrow) and tangential Golgi-associated (blue arrowhead) MT arrays. Box is enlarged in (a') for mCherry-GT and in (a”) for GFP-EB3. (b) Radial centrosomal (yellow arrow) EB3 tracks in CLASP-depleted cell (siRNA combination #1). Box is enlarged in (b') for mCherry-GT and in (b”) for GFP-EB3. (c) Video frames illustrating minus-end directed mini-stack movement (yellow arrow) along Golgi-nucleated MTs upon nocodazole washout in mCherry-GT (red) and GFP-EB3 (green) expressing NT control cells. MT plus end, asterisk. Time after nocodazole removal is shown. (d) Video frames illustrating nocodazole washout in cell over-expressing GFP-P50 (not shown) and mCherry-GT (black). Mini-stacks move toward the cell periphery along forming MTs due to kinesin activity (asterisks). Time after nocodazole removal is shown. (e) Fold increase of Golgi particle size upon nocodazole washout based on live cell imaging of control (n=7, 6 independent experiments) and GFP-P50 over-expressing (n=8, 7 independent experiments) cells. Error bars, standard error. *P<0.001, unpaired Student's t-test. (f) Average Golgi particle area (μm2) in nocodazole (time 0) and upon 60 min washout in fixed samples of control, GFP-P50 over-expressing, and RFP-CC1 over-expressing cells. n=50 for each condition, 4 independent experiments. Error bars, standard error. *P<0.001, **P<0.01 unpaired Student's t-test. (g) Role of Golgi-associated MTs in Golgi ribbon assembly (model).
Mentions: Because MT orientation appeared important for Golgi stack clustering in nocodazole washout assays, we further addressed the role of MT orientation in Golgi organization. MT directionality was detected as described in Efimov et al [7]. In brief, RPE1 cells were transfected with GFP-EB3 to mark MT plus-tips and mCherry-GT to label the Golgi, and time-lapse video sequences were recorded. Distinct centrosomal MT and Golgi-derived MT arrays were detected in NT-control cells (Fig. 4a,a”). Interestingly, while centrosomal MTs showed clear radial geometry, MTs formed at the Golgi were predominantly tangential. Golgi ribbons (Fig. 4a') were aligned along Golgi-derived MT tracks suggesting that they were primarily formed via tangential mini-stack linking and fusion (Fig. 4a).

Bottom Line: These Golgi-derived microtubules draw Golgi ministacks together in tangential fashion and are crucial for establishing continuity and proper morphology of the Golgi complex.We propose that specialized functions of these two microtubule arrays arise from their specific geometries.Further, we demonstrate that directional post-Golgi trafficking and cell migration depend on Golgi-associated CLASPs, suggesting that correct organization of the Golgi complex by microtubules is essential for cell polarization and motility.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Vanderbilt University Medical Center, Nashville, TN 37232, USA.

ABSTRACT
Microtubules are indispensable for Golgi complex assembly and maintenance, which are integral parts of cytoplasm organization during interphase in mammalian cells. Here, we show that two discrete microtubule subsets drive two distinct, yet simultaneous, stages of Golgi assembly. In addition to the radial centrosomal microtubule array, which positions the Golgi in the centre of the cell, we have identified a role for microtubules that form at the Golgi membranes in a manner dependent on the microtubule regulators CLASPs. These Golgi-derived microtubules draw Golgi ministacks together in tangential fashion and are crucial for establishing continuity and proper morphology of the Golgi complex. We propose that specialized functions of these two microtubule arrays arise from their specific geometries. Further, we demonstrate that directional post-Golgi trafficking and cell migration depend on Golgi-associated CLASPs, suggesting that correct organization of the Golgi complex by microtubules is essential for cell polarization and motility.

Show MeSH