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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 mini-stacks clustering by Golgi-derived and centrosomal MTs(a–b) Video frames illustrating MT formation in 3GFP-EMTB (green) and mCherry-Rab6 (red) expressing RPE1 cells upon nocodazole washout. Time after nocodazole removal is shown. (a) Control cell, MTs at Golgi mini-stacks and the centrosome. (b) CLASP-depleted cell (siRNA combination #1), MTs at the centrosome. Areas in boxes are enlarged below. (c) enlarged box from (a) showing MT nucleation (chevron) at Golgi mini-stacks (red), binding of mini-stacks to MT (yellow arrow), and transport along MT (white arrow) resulting in clustering along Golgi-nucleated MT. mCherry-Rab6 (red), GFP-EB3 (green). Note transport of a mini stack toward cell periphery and subsequent tangential linking. (d) Mini-stack clustering from (c), mCherry-Rab6 alone. Blue arrow, mini-stack where MT nucleates. Yellow arrow, transported mini-stack. (e) Enlarged box from (b) showing centrosomal MTs growing (chevron), binding to 2 mini-stacks subsequently (blue and yellow arrows), and transport (white arrow) of the second mini-stack resulting in radial clustering in peri-centrosomal area. mCherry-Rab6 (red), GFP-EB3 (green). (f) Mini-stack clustering from (e), mCherry-Rab6 alone. Blue arrow, mini-stack proximal to the centrosome. Yellow arrow, transported mini-stack.
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Figure 3: Golgi mini-stacks clustering by Golgi-derived and centrosomal MTs(a–b) Video frames illustrating MT formation in 3GFP-EMTB (green) and mCherry-Rab6 (red) expressing RPE1 cells upon nocodazole washout. Time after nocodazole removal is shown. (a) Control cell, MTs at Golgi mini-stacks and the centrosome. (b) CLASP-depleted cell (siRNA combination #1), MTs at the centrosome. Areas in boxes are enlarged below. (c) enlarged box from (a) showing MT nucleation (chevron) at Golgi mini-stacks (red), binding of mini-stacks to MT (yellow arrow), and transport along MT (white arrow) resulting in clustering along Golgi-nucleated MT. mCherry-Rab6 (red), GFP-EB3 (green). Note transport of a mini stack toward cell periphery and subsequent tangential linking. (d) Mini-stack clustering from (c), mCherry-Rab6 alone. Blue arrow, mini-stack where MT nucleates. Yellow arrow, transported mini-stack. (e) Enlarged box from (b) showing centrosomal MTs growing (chevron), binding to 2 mini-stacks subsequently (blue and yellow arrows), and transport (white arrow) of the second mini-stack resulting in radial clustering in peri-centrosomal area. mCherry-Rab6 (red), GFP-EB3 (green). (f) Mini-stack clustering from (e), mCherry-Rab6 alone. Blue arrow, mini-stack proximal to the centrosome. Yellow arrow, transported mini-stack.

Mentions: To determine whether CLASPs support Golgi mini-stack clustering at the cell periphery via Golgi-derived MT formation, we directly followed MT involvement in this process. RPE1 cells were co-transfected with variable Golgi stack markers and 3GFP-EMTB to label MTs [16]. As expected, nocodazole washout assay in NT-control cells revealed MT nucleation at individual Golgi mini-stacks as well as at the centrosome (Fig. 3a, c–d; Movie 3). To avoid overlap between G-stage and C-stage, we specifically followed G-stage clustering events at the cell periphery before centrosomal MTs extended into this area. As MTs formed at Golgi mini-stacks polymerized toward nearby mini-stacks, the latter ones were transported into close proximity of the former that allowed subsequent mini-stack linking. Transport of mini-stacks during the G-stage occurred in random directions, often tangential relative to radial centrosomal array.


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 mini-stacks clustering by Golgi-derived and centrosomal MTs(a–b) Video frames illustrating MT formation in 3GFP-EMTB (green) and mCherry-Rab6 (red) expressing RPE1 cells upon nocodazole washout. Time after nocodazole removal is shown. (a) Control cell, MTs at Golgi mini-stacks and the centrosome. (b) CLASP-depleted cell (siRNA combination #1), MTs at the centrosome. Areas in boxes are enlarged below. (c) enlarged box from (a) showing MT nucleation (chevron) at Golgi mini-stacks (red), binding of mini-stacks to MT (yellow arrow), and transport along MT (white arrow) resulting in clustering along Golgi-nucleated MT. mCherry-Rab6 (red), GFP-EB3 (green). Note transport of a mini stack toward cell periphery and subsequent tangential linking. (d) Mini-stack clustering from (c), mCherry-Rab6 alone. Blue arrow, mini-stack where MT nucleates. Yellow arrow, transported mini-stack. (e) Enlarged box from (b) showing centrosomal MTs growing (chevron), binding to 2 mini-stacks subsequently (blue and yellow arrows), and transport (white arrow) of the second mini-stack resulting in radial clustering in peri-centrosomal area. mCherry-Rab6 (red), GFP-EB3 (green). (f) Mini-stack clustering from (e), mCherry-Rab6 alone. Blue arrow, mini-stack proximal to the centrosome. Yellow arrow, transported mini-stack.
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Figure 3: Golgi mini-stacks clustering by Golgi-derived and centrosomal MTs(a–b) Video frames illustrating MT formation in 3GFP-EMTB (green) and mCherry-Rab6 (red) expressing RPE1 cells upon nocodazole washout. Time after nocodazole removal is shown. (a) Control cell, MTs at Golgi mini-stacks and the centrosome. (b) CLASP-depleted cell (siRNA combination #1), MTs at the centrosome. Areas in boxes are enlarged below. (c) enlarged box from (a) showing MT nucleation (chevron) at Golgi mini-stacks (red), binding of mini-stacks to MT (yellow arrow), and transport along MT (white arrow) resulting in clustering along Golgi-nucleated MT. mCherry-Rab6 (red), GFP-EB3 (green). Note transport of a mini stack toward cell periphery and subsequent tangential linking. (d) Mini-stack clustering from (c), mCherry-Rab6 alone. Blue arrow, mini-stack where MT nucleates. Yellow arrow, transported mini-stack. (e) Enlarged box from (b) showing centrosomal MTs growing (chevron), binding to 2 mini-stacks subsequently (blue and yellow arrows), and transport (white arrow) of the second mini-stack resulting in radial clustering in peri-centrosomal area. mCherry-Rab6 (red), GFP-EB3 (green). (f) Mini-stack clustering from (e), mCherry-Rab6 alone. Blue arrow, mini-stack proximal to the centrosome. Yellow arrow, transported mini-stack.
Mentions: To determine whether CLASPs support Golgi mini-stack clustering at the cell periphery via Golgi-derived MT formation, we directly followed MT involvement in this process. RPE1 cells were co-transfected with variable Golgi stack markers and 3GFP-EMTB to label MTs [16]. As expected, nocodazole washout assay in NT-control cells revealed MT nucleation at individual Golgi mini-stacks as well as at the centrosome (Fig. 3a, c–d; Movie 3). To avoid overlap between G-stage and C-stage, we specifically followed G-stage clustering events at the cell periphery before centrosomal MTs extended into this area. As MTs formed at Golgi mini-stacks polymerized toward nearby mini-stacks, the latter ones were transported into close proximity of the former that allowed subsequent mini-stack linking. Transport of mini-stacks during the G-stage occurred in random directions, often tangential relative to radial centrosomal array.

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