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Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells.

Reilein A, Yamada S, Nelson WJ - J. Cell Biol. (2005)

Bottom Line: Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network.However, the addition of increased microtubule stabilization at microtubule-microtubule and microtubule-cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ.These results define minimal parameters for the self-organization of an acentrosomal microtubule network.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Mechanisms underlying the organization of centrosome-derived microtubule arrays are well understood, but less is known about how acentrosomal microtubule networks are formed. The basal cortex of polarized epithelial cells contains a microtubule network of mixed polarity. We examined how this network is organized by imaging microtubule dynamics in acentrosomal basal cytoplasts derived from these cells. We show that the steady-state microtubule network appears to form by a combination of microtubule-microtubule and microtubule-cortex interactions, both of which increase microtubule stability. We used computational modeling to determine whether these microtubule parameters are sufficient to generate a steady-state acentrosomal microtubule network. Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network. However, the addition of increased microtubule stabilization at microtubule-microtubule and microtubule-cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ. These results define minimal parameters for the self-organization of an acentrosomal microtubule network.

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Basal microtubules transport endocytic vesicles. Closed arrows denote initial vesicle positions, and open arrows indicate current locations of vesicles. (A) A basal cytoplast isolated from polarized GFP-tubulin–expressing MDCK cells that had been incubated with LysoTracker red (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200505071/DC1). (B) A basal cytoplast isolated from polarized cells expressing clathrin light chain–DsRed (Video 2). The yellow and white arrows show two examples of vesicles that move on microtubules. Bars, 5 μm.
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fig3: Basal microtubules transport endocytic vesicles. Closed arrows denote initial vesicle positions, and open arrows indicate current locations of vesicles. (A) A basal cytoplast isolated from polarized GFP-tubulin–expressing MDCK cells that had been incubated with LysoTracker red (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200505071/DC1). (B) A basal cytoplast isolated from polarized cells expressing clathrin light chain–DsRed (Video 2). The yellow and white arrows show two examples of vesicles that move on microtubules. Bars, 5 μm.

Mentions: The basolateral membrane of polarized epithelial cells is a site of endocytosis and transcytosis of internalized vesicles (Perret et al., 2005). To determine whether basal microtubules are functional in endocytic vesicle transport, we prepared basal cytoplasts from polarized cells that had been incubated with LysoTracker red to label acidified vesicles or from cells that had been transfected with clathrin-DsRed to label endocytic vesicles. Both LysoTracker red– (Fig. 3 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200505071/DC1) and clathrin-DsRed–labeled vesicles (Fig. 3 B and Video 2) translocated along basal microtubules at rates ranging from 0.2 to 0.5 μm/s, which is consistent with microtubule motor-driven activities. To quantify the percentage of vesicles that moved, we considered only those patches in which some vesicles moved to be sure that they were active and had sealed over to form cytoplasts. We counted vesicles that moved at least 1.3 μm in directed lateral movements over a period of 1 min and found that 21% of LysoTracker red–labeled vesicles (24/114 vesicles in 11 basal cytoplasts) exhibited directed lateral movement. The percentage of clathrin vesicles moving was more difficult to determine because of the lower intensity of some clathrin-DsRed spots. However, an approximate estimate showed that 7–20% of clathrin spots moved per minute. Note that it has been reported that in whole cells imaged by total internal reflection fluorescence microscopy, only 2% of clathrin spots moved laterally per minute at rates of microtubule motors in CHO cells (Rappoport et al., 2003), and 7% moved in CV-1 cells (Keyel et al., 2004). We conclude that the microtubule network in basal cytoplasts from polarized MDCK cells is functional in the transport of acidified and clathrin-containing endocytic vesicles.


Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells.

Reilein A, Yamada S, Nelson WJ - J. Cell Biol. (2005)

Basal microtubules transport endocytic vesicles. Closed arrows denote initial vesicle positions, and open arrows indicate current locations of vesicles. (A) A basal cytoplast isolated from polarized GFP-tubulin–expressing MDCK cells that had been incubated with LysoTracker red (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200505071/DC1). (B) A basal cytoplast isolated from polarized cells expressing clathrin light chain–DsRed (Video 2). The yellow and white arrows show two examples of vesicles that move on microtubules. Bars, 5 μm.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171299&req=5

fig3: Basal microtubules transport endocytic vesicles. Closed arrows denote initial vesicle positions, and open arrows indicate current locations of vesicles. (A) A basal cytoplast isolated from polarized GFP-tubulin–expressing MDCK cells that had been incubated with LysoTracker red (Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200505071/DC1). (B) A basal cytoplast isolated from polarized cells expressing clathrin light chain–DsRed (Video 2). The yellow and white arrows show two examples of vesicles that move on microtubules. Bars, 5 μm.
Mentions: The basolateral membrane of polarized epithelial cells is a site of endocytosis and transcytosis of internalized vesicles (Perret et al., 2005). To determine whether basal microtubules are functional in endocytic vesicle transport, we prepared basal cytoplasts from polarized cells that had been incubated with LysoTracker red to label acidified vesicles or from cells that had been transfected with clathrin-DsRed to label endocytic vesicles. Both LysoTracker red– (Fig. 3 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200505071/DC1) and clathrin-DsRed–labeled vesicles (Fig. 3 B and Video 2) translocated along basal microtubules at rates ranging from 0.2 to 0.5 μm/s, which is consistent with microtubule motor-driven activities. To quantify the percentage of vesicles that moved, we considered only those patches in which some vesicles moved to be sure that they were active and had sealed over to form cytoplasts. We counted vesicles that moved at least 1.3 μm in directed lateral movements over a period of 1 min and found that 21% of LysoTracker red–labeled vesicles (24/114 vesicles in 11 basal cytoplasts) exhibited directed lateral movement. The percentage of clathrin vesicles moving was more difficult to determine because of the lower intensity of some clathrin-DsRed spots. However, an approximate estimate showed that 7–20% of clathrin spots moved per minute. Note that it has been reported that in whole cells imaged by total internal reflection fluorescence microscopy, only 2% of clathrin spots moved laterally per minute at rates of microtubule motors in CHO cells (Rappoport et al., 2003), and 7% moved in CV-1 cells (Keyel et al., 2004). We conclude that the microtubule network in basal cytoplasts from polarized MDCK cells is functional in the transport of acidified and clathrin-containing endocytic vesicles.

Bottom Line: Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network.However, the addition of increased microtubule stabilization at microtubule-microtubule and microtubule-cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ.These results define minimal parameters for the self-organization of an acentrosomal microtubule network.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

ABSTRACT
Mechanisms underlying the organization of centrosome-derived microtubule arrays are well understood, but less is known about how acentrosomal microtubule networks are formed. The basal cortex of polarized epithelial cells contains a microtubule network of mixed polarity. We examined how this network is organized by imaging microtubule dynamics in acentrosomal basal cytoplasts derived from these cells. We show that the steady-state microtubule network appears to form by a combination of microtubule-microtubule and microtubule-cortex interactions, both of which increase microtubule stability. We used computational modeling to determine whether these microtubule parameters are sufficient to generate a steady-state acentrosomal microtubule network. Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network. However, the addition of increased microtubule stabilization at microtubule-microtubule and microtubule-cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ. These results define minimal parameters for the self-organization of an acentrosomal microtubule network.

Show MeSH
Related in: MedlinePlus