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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 occurs in 2 stages upon mitotic exit(a) Video frames illustrating post-mitotic Golgi assembly in mCherry-Rab6 expressing RPE1 cells. Time zero marks approximate onset of telophase. Boxed area is enlarged below. (b) Post-mitotic Golgi particle size based on live imaging experiments in NT-control (n=4, 4 independent experiments) cells. Average fold increase of Golgi particles relative to time zero is shown. Error bars, standard error. *P<0.001, unpaired Student's t-test. (c) Enlarged box from (a) showing Golgi mini-stack (red) clustering (6–9', blue and yellow arrows indicate two separate clusters) prior to re-location toward the centrosome (10').
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Figure 2: Golgi assembly occurs in 2 stages upon mitotic exit(a) Video frames illustrating post-mitotic Golgi assembly in mCherry-Rab6 expressing RPE1 cells. Time zero marks approximate onset of telophase. Boxed area is enlarged below. (b) Post-mitotic Golgi particle size based on live imaging experiments in NT-control (n=4, 4 independent experiments) cells. Average fold increase of Golgi particles relative to time zero is shown. Error bars, standard error. *P<0.001, unpaired Student's t-test. (c) Enlarged box from (a) showing Golgi mini-stack (red) clustering (6–9', blue and yellow arrows indicate two separate clusters) prior to re-location toward the centrosome (10').

Mentions: Next we visualized Golgi assembly in untreated cells after mitotic exit (Fig. 2; Movie 2). In mCherryRab6- or GFP-GM130-expressing cells exiting mitosis, the average size of detected Golgi mini-stacks (0.34±0.01μm) did not differ from the size of mini-stacks in nocodazole-treated cells (0.35±0.02μm), indicating that these mini-stacks were likely assembled in MT-independent manner by fusion and stacking of membranes brought together by simple diffusion [8]. During subsequent Golgi assembly, distinct G- and C- stages were easily distinguishable by analysis of the Golgi particle size increase (Fig. 2b). Mini-stack clustering in G-stage often occurred at the cell periphery away from the centrosome (Fig. 2c), suggestive of the role of Golgi-derived MTs that were detected at the periphery of cells exiting mitosis (Fig. S2). The Golgi assembled faster, and temporal separation between G- and C-stages was less distinct than in nocodazole washout assay. Possibly, the efforts of centrosomal and Golgi-derived MTs were better coordinated in a rounded mitotic cell than in a spread cell. Because CLASPs are indispensable for mitosis, we did not directly test their role in post-mitotic assembly; our nocodazole washout results, however, suggest that G-stage Golgi clustering in this scenario is likely CLASP-dependent.


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 occurs in 2 stages upon mitotic exit(a) Video frames illustrating post-mitotic Golgi assembly in mCherry-Rab6 expressing RPE1 cells. Time zero marks approximate onset of telophase. Boxed area is enlarged below. (b) Post-mitotic Golgi particle size based on live imaging experiments in NT-control (n=4, 4 independent experiments) cells. Average fold increase of Golgi particles relative to time zero is shown. Error bars, standard error. *P<0.001, unpaired Student's t-test. (c) Enlarged box from (a) showing Golgi mini-stack (red) clustering (6–9', blue and yellow arrows indicate two separate clusters) prior to re-location toward the centrosome (10').
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Related In: Results  -  Collection

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Figure 2: Golgi assembly occurs in 2 stages upon mitotic exit(a) Video frames illustrating post-mitotic Golgi assembly in mCherry-Rab6 expressing RPE1 cells. Time zero marks approximate onset of telophase. Boxed area is enlarged below. (b) Post-mitotic Golgi particle size based on live imaging experiments in NT-control (n=4, 4 independent experiments) cells. Average fold increase of Golgi particles relative to time zero is shown. Error bars, standard error. *P<0.001, unpaired Student's t-test. (c) Enlarged box from (a) showing Golgi mini-stack (red) clustering (6–9', blue and yellow arrows indicate two separate clusters) prior to re-location toward the centrosome (10').
Mentions: Next we visualized Golgi assembly in untreated cells after mitotic exit (Fig. 2; Movie 2). In mCherryRab6- or GFP-GM130-expressing cells exiting mitosis, the average size of detected Golgi mini-stacks (0.34±0.01μm) did not differ from the size of mini-stacks in nocodazole-treated cells (0.35±0.02μm), indicating that these mini-stacks were likely assembled in MT-independent manner by fusion and stacking of membranes brought together by simple diffusion [8]. During subsequent Golgi assembly, distinct G- and C- stages were easily distinguishable by analysis of the Golgi particle size increase (Fig. 2b). Mini-stack clustering in G-stage often occurred at the cell periphery away from the centrosome (Fig. 2c), suggestive of the role of Golgi-derived MTs that were detected at the periphery of cells exiting mitosis (Fig. S2). The Golgi assembled faster, and temporal separation between G- and C-stages was less distinct than in nocodazole washout assay. Possibly, the efforts of centrosomal and Golgi-derived MTs were better coordinated in a rounded mitotic cell than in a spread cell. Because CLASPs are indispensable for mitosis, we did not directly test their role in post-mitotic assembly; our nocodazole washout results, however, suggest that G-stage Golgi clustering in this scenario is likely CLASP-dependent.

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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