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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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Localization of γ-tubulin on basal patches. Basal patches prepared from GFP-tubulin–expressing MDCK cells polarized on filters were stained for γ-tubulin (red). γ-Tubulin is localized along microtubules, at branch points (arrows), and in association with the cortex independently of microtubules. Bar, 5 μm.
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fig6: Localization of γ-tubulin on basal patches. Basal patches prepared from GFP-tubulin–expressing MDCK cells polarized on filters were stained for γ-tubulin (red). γ-Tubulin is localized along microtubules, at branch points (arrows), and in association with the cortex independently of microtubules. Bar, 5 μm.

Mentions: Generally, new microtubules grew without shrinking and did not exhibit dynamic instability until they reached the margin of the cortical patch (Video 4); a similar microtubule behavior was reported in fibroblasts (Komarova et al., 2002). Microtubules often grew over points where other microtubule ends were located (Fig. 4 E and Video 5), indicating that there are points on the cortex or microtubules for microtubule binding and stabilization. We have identified many proteins that decorate the length of microtubules and that are present at microtubule–microtubule and microtubule–cortex intersections, including EB1, p150glued, Kap3, and APC; of these proteins, we showed that antibodies to APC can block exogenous microtubule assembly on the patch (Reilein and Nelson, 2005). At present, the identity of proteins that regulate microtubule nucleation on the sides of other microtubules or on the cortex is poorly understood. γ-Tubulin has been shown recently to nucleate microtubule growth on the sides of other microtubules in fungi and plants (Janson et al., 2005; Murata et al., 2005). Microtubule branches arise on the plant cell cortex when γ-tubulin is recruited from the cytoplasm to the side of a microtubule (Murata et al., 2005). Examination of γ-tubulin staining on basal membrane patches showed that γ-tubulin is localized along the length of microtubules, at branch points, and on the cortex independently of microtubules (Fig. 6). Typical centrosomal staining by the γ-tubulin antibody as well as noncentrosomal staining was observed in intact MDCK cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200505071/DC1); note that a large pool of noncentrosomal γ-tubulin is found both in soluble and insoluble fractions in cells (Moudjou et al., 1996).


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)

Localization of γ-tubulin on basal patches. Basal patches prepared from GFP-tubulin–expressing MDCK cells polarized on filters were stained for γ-tubulin (red). γ-Tubulin is localized along microtubules, at branch points (arrows), and in association with the cortex independently of microtubules. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Localization of γ-tubulin on basal patches. Basal patches prepared from GFP-tubulin–expressing MDCK cells polarized on filters were stained for γ-tubulin (red). γ-Tubulin is localized along microtubules, at branch points (arrows), and in association with the cortex independently of microtubules. Bar, 5 μm.
Mentions: Generally, new microtubules grew without shrinking and did not exhibit dynamic instability until they reached the margin of the cortical patch (Video 4); a similar microtubule behavior was reported in fibroblasts (Komarova et al., 2002). Microtubules often grew over points where other microtubule ends were located (Fig. 4 E and Video 5), indicating that there are points on the cortex or microtubules for microtubule binding and stabilization. We have identified many proteins that decorate the length of microtubules and that are present at microtubule–microtubule and microtubule–cortex intersections, including EB1, p150glued, Kap3, and APC; of these proteins, we showed that antibodies to APC can block exogenous microtubule assembly on the patch (Reilein and Nelson, 2005). At present, the identity of proteins that regulate microtubule nucleation on the sides of other microtubules or on the cortex is poorly understood. γ-Tubulin has been shown recently to nucleate microtubule growth on the sides of other microtubules in fungi and plants (Janson et al., 2005; Murata et al., 2005). Microtubule branches arise on the plant cell cortex when γ-tubulin is recruited from the cytoplasm to the side of a microtubule (Murata et al., 2005). Examination of γ-tubulin staining on basal membrane patches showed that γ-tubulin is localized along the length of microtubules, at branch points, and on the cortex independently of microtubules (Fig. 6). Typical centrosomal staining by the γ-tubulin antibody as well as noncentrosomal staining was observed in intact MDCK cells (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200505071/DC1); note that a large pool of noncentrosomal γ-tubulin is found both in soluble and insoluble fractions in cells (Moudjou et al., 1996).

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