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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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CLASPs at the Golgi determine ribbon morphology(a) Control cells have a ribbon-like Golgi (GM130, green) when CLASPs (red, a') are present. (b–c) Golgi (green) morphology is circular when CLASPs (red, b', c') are depleted from cells. (d) Ribbon-like Golgi morphology (white arrow) is restored in CLASP-depleted cells expressing a nonsilenceable GFP-CLASP2C construct (d', false-colored red). The Golgi (GM-130, false-colored green) remains circular in non-expressing cells (hollow arrow). (e–f) Immunostainings of Golgi (GM130, green) and MTs (red). (e) Control cell showing ribbon-like Golgi in the presence of Golgi-associated MTs (white arrow). (f) CLASP-depleted cell (siRNA combination #2) showing circular Golgi morphology and radial centrosomal MT array (white arrow). (g) Ribbon-like Golgi (GM130, false-colored green, hollow yellow arrow) turns circular (yellow arrow) in cells over-expressing GFP-CLASP2C (false-colored red). (h) Golgi circularity depends on CLASPs intensity at the Golgi. Average CLASP intensity at the Golgi in mixed-culture cell plotted against circularity index (n=50, 4 independent experiments). (i) Removal of full-length CLASP results in circular morphology. Average GFP-CLASP2C intensity (pink, lower x-axis) and GFP vector only intensity (blue, top x-axis) in the Golgi area plotted against circularity index (n=50, 3 independent experiments for each condition). Cells with similar overall expression levels are compared, in which GFP-CLASP2c intensity in the Golgi region is 4 to 5 times higher than GFP due to specific accumulation of GFP-CLASP2C in the Golgi area (~1/5 of the cell area). GFP-CLASP2C but not GFP expression leads to circular Golgi morphology.
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Figure 5: CLASPs at the Golgi determine ribbon morphology(a) Control cells have a ribbon-like Golgi (GM130, green) when CLASPs (red, a') are present. (b–c) Golgi (green) morphology is circular when CLASPs (red, b', c') are depleted from cells. (d) Ribbon-like Golgi morphology (white arrow) is restored in CLASP-depleted cells expressing a nonsilenceable GFP-CLASP2C construct (d', false-colored red). The Golgi (GM-130, false-colored green) remains circular in non-expressing cells (hollow arrow). (e–f) Immunostainings of Golgi (GM130, green) and MTs (red). (e) Control cell showing ribbon-like Golgi in the presence of Golgi-associated MTs (white arrow). (f) CLASP-depleted cell (siRNA combination #2) showing circular Golgi morphology and radial centrosomal MT array (white arrow). (g) Ribbon-like Golgi (GM130, false-colored green, hollow yellow arrow) turns circular (yellow arrow) in cells over-expressing GFP-CLASP2C (false-colored red). (h) Golgi circularity depends on CLASPs intensity at the Golgi. Average CLASP intensity at the Golgi in mixed-culture cell plotted against circularity index (n=50, 4 independent experiments). (i) Removal of full-length CLASP results in circular morphology. Average GFP-CLASP2C intensity (pink, lower x-axis) and GFP vector only intensity (blue, top x-axis) in the Golgi area plotted against circularity index (n=50, 3 independent experiments for each condition). Cells with similar overall expression levels are compared, in which GFP-CLASP2c intensity in the Golgi region is 4 to 5 times higher than GFP due to specific accumulation of GFP-CLASP2C in the Golgi area (~1/5 of the cell area). GFP-CLASP2C but not GFP expression leads to circular Golgi morphology.

Mentions: Our data support a model wherein Golgi-derived MTs play a distinct and essential role in Golgi organization (Fig. 4g). Indeed, while in NT-control cells a classic Golgi ribbon associated with Golgi-derived MT array was observed (Fig. 5a,e), in CLASP-depleted cells (Fig. 5b,c,f) MTs formed a radial array and the Golgi showed circular morphology (Fig. 5f). The Golgi shape was reverted back to a ribbon by expression of a non-silenceable GFP-CLASP2α rescue construct in CLASP-depleted cells (Fig. 5d). Dependence of the circular Golgi phenotype on the Golgi-associated CLASP fraction was confirmed by over-expressing GFP-tagged C-terminus of CLASP2 that competes full-length CLASPs off the Golgi (Fig. S3). Similar to CLASP-knockdown, cells over-expressing GFP-CLASP2C had circular Golgi complex (Fig. 5g).


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)

CLASPs at the Golgi determine ribbon morphology(a) Control cells have a ribbon-like Golgi (GM130, green) when CLASPs (red, a') are present. (b–c) Golgi (green) morphology is circular when CLASPs (red, b', c') are depleted from cells. (d) Ribbon-like Golgi morphology (white arrow) is restored in CLASP-depleted cells expressing a nonsilenceable GFP-CLASP2C construct (d', false-colored red). The Golgi (GM-130, false-colored green) remains circular in non-expressing cells (hollow arrow). (e–f) Immunostainings of Golgi (GM130, green) and MTs (red). (e) Control cell showing ribbon-like Golgi in the presence of Golgi-associated MTs (white arrow). (f) CLASP-depleted cell (siRNA combination #2) showing circular Golgi morphology and radial centrosomal MT array (white arrow). (g) Ribbon-like Golgi (GM130, false-colored green, hollow yellow arrow) turns circular (yellow arrow) in cells over-expressing GFP-CLASP2C (false-colored red). (h) Golgi circularity depends on CLASPs intensity at the Golgi. Average CLASP intensity at the Golgi in mixed-culture cell plotted against circularity index (n=50, 4 independent experiments). (i) Removal of full-length CLASP results in circular morphology. Average GFP-CLASP2C intensity (pink, lower x-axis) and GFP vector only intensity (blue, top x-axis) in the Golgi area plotted against circularity index (n=50, 3 independent experiments for each condition). Cells with similar overall expression levels are compared, in which GFP-CLASP2c intensity in the Golgi region is 4 to 5 times higher than GFP due to specific accumulation of GFP-CLASP2C in the Golgi area (~1/5 of the cell area). GFP-CLASP2C but not GFP expression leads to circular Golgi morphology.
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Figure 5: CLASPs at the Golgi determine ribbon morphology(a) Control cells have a ribbon-like Golgi (GM130, green) when CLASPs (red, a') are present. (b–c) Golgi (green) morphology is circular when CLASPs (red, b', c') are depleted from cells. (d) Ribbon-like Golgi morphology (white arrow) is restored in CLASP-depleted cells expressing a nonsilenceable GFP-CLASP2C construct (d', false-colored red). The Golgi (GM-130, false-colored green) remains circular in non-expressing cells (hollow arrow). (e–f) Immunostainings of Golgi (GM130, green) and MTs (red). (e) Control cell showing ribbon-like Golgi in the presence of Golgi-associated MTs (white arrow). (f) CLASP-depleted cell (siRNA combination #2) showing circular Golgi morphology and radial centrosomal MT array (white arrow). (g) Ribbon-like Golgi (GM130, false-colored green, hollow yellow arrow) turns circular (yellow arrow) in cells over-expressing GFP-CLASP2C (false-colored red). (h) Golgi circularity depends on CLASPs intensity at the Golgi. Average CLASP intensity at the Golgi in mixed-culture cell plotted against circularity index (n=50, 4 independent experiments). (i) Removal of full-length CLASP results in circular morphology. Average GFP-CLASP2C intensity (pink, lower x-axis) and GFP vector only intensity (blue, top x-axis) in the Golgi area plotted against circularity index (n=50, 3 independent experiments for each condition). Cells with similar overall expression levels are compared, in which GFP-CLASP2c intensity in the Golgi region is 4 to 5 times higher than GFP due to specific accumulation of GFP-CLASP2C in the Golgi area (~1/5 of the cell area). GFP-CLASP2C but not GFP expression leads to circular Golgi morphology.
Mentions: Our data support a model wherein Golgi-derived MTs play a distinct and essential role in Golgi organization (Fig. 4g). Indeed, while in NT-control cells a classic Golgi ribbon associated with Golgi-derived MT array was observed (Fig. 5a,e), in CLASP-depleted cells (Fig. 5b,c,f) MTs formed a radial array and the Golgi showed circular morphology (Fig. 5f). The Golgi shape was reverted back to a ribbon by expression of a non-silenceable GFP-CLASP2α rescue construct in CLASP-depleted cells (Fig. 5d). Dependence of the circular Golgi phenotype on the Golgi-associated CLASP fraction was confirmed by over-expressing GFP-tagged C-terminus of CLASP2 that competes full-length CLASPs off the Golgi (Fig. S3). Similar to CLASP-knockdown, cells over-expressing GFP-CLASP2C had circular Golgi complex (Fig. 5g).

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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