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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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Microtubules are arranged into intersecting networks on isolated basal membrane patches. (A) GFP-tubulin–labeled microtubules on basal membrane patches prepared from MDCK cells that were polarized on filters. (B) Scanning electron microscopy of MDCK cell basal patches shows cytoplasts (red arrowheads) and open patches (yellow arrowheads). (A and B) Bars, 10 μm. (C) Two examples of GFP-labeled microtubules on MDCK basal patches show the presence of microtubule–microtubule intersections (arrows) and the intersection of multiple microtubules at a focus (yellow arrowhead). (D) Immunofluorescence staining of microtubules on the basal membrane isolated from a polarized Caco-2 cell shows the presence of end-to-side microtubule intersections (arrows). (C and D) Bars, 2.5 μm. (E) An isolated basal membrane from an EpH4 cell shows the presence of a microtubule network with intersections (arrows). Multiple microtubules often intersect at one focus (arrowheads). (F) Scanning electron microscopy of a basal patch shows a few microtubules that remain (boxed and enlarged at right) overlying a dense cytoskeletal network containing actin stress fibers (the microtubules are brighter because they scatter more secondary electrons with their exposed edges). Bar (left), 5 μm. Microtubule ends terminate on the sides of other microtubules and exhibit different conformations. Red arrows point to enlarged ends of microtubules; blue arrows point to ends that curve as they terminate on the sides on other microtubules. Microtubules also show side-to-side interactions (pink arrow). Bar (right), 0.5 μm. (G) Gold labeling of the microtubule network. Microtubules are densely coated with gold beads along their length. Microtubule ends terminate on the sides of other microtubules (red arrows). Microtubules also bundle (pink arrows). Bar, 0.5 μm. (H) Additional examples of the different conformations of microtubule ends contacting the sides of other microtubules. Enlargements present at the end of microtubules are shown by red arrows. Bar (left), 0.5 μm. An end without an enlargement is shown by the blue arrow. Bar (right), 200 nm. A side-to-side interaction is indicated by the pink arrow. (I) Multiple microtubules converging at a focus (yellow arrowhead) and exhibiting end-to-side interactions (red arrows) and side-to-side interactions (pink arrows). Bar, 0.5 μm. (J) GFP-tubulin–labeled microtubules on a nonfixed MDCK basal patch show that the appearance of the intersections is not a result of fixation (arrows). The yellow arrowhead indicates a focus of microtubules; red arrows show end-to-side microtubule intersections; pink arrow shows a side-to-side microtubule interaction. Bar, 1 μm. (K) Tubulin antibody staining (red; left) of GFP-labeled microtubules (far right) shows that GFP fluorescence shows through areas of diminished antibody staining (arrows; merged image in middle). Bar, 2.5 μm.
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fig2: Microtubules are arranged into intersecting networks on isolated basal membrane patches. (A) GFP-tubulin–labeled microtubules on basal membrane patches prepared from MDCK cells that were polarized on filters. (B) Scanning electron microscopy of MDCK cell basal patches shows cytoplasts (red arrowheads) and open patches (yellow arrowheads). (A and B) Bars, 10 μm. (C) Two examples of GFP-labeled microtubules on MDCK basal patches show the presence of microtubule–microtubule intersections (arrows) and the intersection of multiple microtubules at a focus (yellow arrowhead). (D) Immunofluorescence staining of microtubules on the basal membrane isolated from a polarized Caco-2 cell shows the presence of end-to-side microtubule intersections (arrows). (C and D) Bars, 2.5 μm. (E) An isolated basal membrane from an EpH4 cell shows the presence of a microtubule network with intersections (arrows). Multiple microtubules often intersect at one focus (arrowheads). (F) Scanning electron microscopy of a basal patch shows a few microtubules that remain (boxed and enlarged at right) overlying a dense cytoskeletal network containing actin stress fibers (the microtubules are brighter because they scatter more secondary electrons with their exposed edges). Bar (left), 5 μm. Microtubule ends terminate on the sides of other microtubules and exhibit different conformations. Red arrows point to enlarged ends of microtubules; blue arrows point to ends that curve as they terminate on the sides on other microtubules. Microtubules also show side-to-side interactions (pink arrow). Bar (right), 0.5 μm. (G) Gold labeling of the microtubule network. Microtubules are densely coated with gold beads along their length. Microtubule ends terminate on the sides of other microtubules (red arrows). Microtubules also bundle (pink arrows). Bar, 0.5 μm. (H) Additional examples of the different conformations of microtubule ends contacting the sides of other microtubules. Enlargements present at the end of microtubules are shown by red arrows. Bar (left), 0.5 μm. An end without an enlargement is shown by the blue arrow. Bar (right), 200 nm. A side-to-side interaction is indicated by the pink arrow. (I) Multiple microtubules converging at a focus (yellow arrowhead) and exhibiting end-to-side interactions (red arrows) and side-to-side interactions (pink arrows). Bar, 0.5 μm. (J) GFP-tubulin–labeled microtubules on a nonfixed MDCK basal patch show that the appearance of the intersections is not a result of fixation (arrows). The yellow arrowhead indicates a focus of microtubules; red arrows show end-to-side microtubule intersections; pink arrow shows a side-to-side microtubule interaction. Bar, 1 μm. (K) Tubulin antibody staining (red; left) of GFP-labeled microtubules (far right) shows that GFP fluorescence shows through areas of diminished antibody staining (arrows; merged image in middle). Bar, 2.5 μm.

Mentions: Microtubules in columnar epithelial cells are organized in the apico–basal axis of the cell into parallel bundles with their plus ends oriented toward the basal membrane and weblike networks on the apical and basal cortex (Fig. 1, A–C; Bacallao et al., 1989; Gilbert et al., 1991; Meads and Schroer, 1995; Eaton et al., 1996). High resolution imaging of microtubules on the basal cortex reveals a network of microtubules formed by intersections that arise from the termination of one microtubule on the side of another or from two microtubules crossing over one another (Fig. 1, D and E). A similar microtubule network is found on the basal cortex of different types of polarized epithelial cells, including Caco-2 cells derived from colonic epithelium (Figs. 1 E and 2 D) and EpH4 cells derived from mammary epithelium (Fig. 2 E).


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)

Microtubules are arranged into intersecting networks on isolated basal membrane patches. (A) GFP-tubulin–labeled microtubules on basal membrane patches prepared from MDCK cells that were polarized on filters. (B) Scanning electron microscopy of MDCK cell basal patches shows cytoplasts (red arrowheads) and open patches (yellow arrowheads). (A and B) Bars, 10 μm. (C) Two examples of GFP-labeled microtubules on MDCK basal patches show the presence of microtubule–microtubule intersections (arrows) and the intersection of multiple microtubules at a focus (yellow arrowhead). (D) Immunofluorescence staining of microtubules on the basal membrane isolated from a polarized Caco-2 cell shows the presence of end-to-side microtubule intersections (arrows). (C and D) Bars, 2.5 μm. (E) An isolated basal membrane from an EpH4 cell shows the presence of a microtubule network with intersections (arrows). Multiple microtubules often intersect at one focus (arrowheads). (F) Scanning electron microscopy of a basal patch shows a few microtubules that remain (boxed and enlarged at right) overlying a dense cytoskeletal network containing actin stress fibers (the microtubules are brighter because they scatter more secondary electrons with their exposed edges). Bar (left), 5 μm. Microtubule ends terminate on the sides of other microtubules and exhibit different conformations. Red arrows point to enlarged ends of microtubules; blue arrows point to ends that curve as they terminate on the sides on other microtubules. Microtubules also show side-to-side interactions (pink arrow). Bar (right), 0.5 μm. (G) Gold labeling of the microtubule network. Microtubules are densely coated with gold beads along their length. Microtubule ends terminate on the sides of other microtubules (red arrows). Microtubules also bundle (pink arrows). Bar, 0.5 μm. (H) Additional examples of the different conformations of microtubule ends contacting the sides of other microtubules. Enlargements present at the end of microtubules are shown by red arrows. Bar (left), 0.5 μm. An end without an enlargement is shown by the blue arrow. Bar (right), 200 nm. A side-to-side interaction is indicated by the pink arrow. (I) Multiple microtubules converging at a focus (yellow arrowhead) and exhibiting end-to-side interactions (red arrows) and side-to-side interactions (pink arrows). Bar, 0.5 μm. (J) GFP-tubulin–labeled microtubules on a nonfixed MDCK basal patch show that the appearance of the intersections is not a result of fixation (arrows). The yellow arrowhead indicates a focus of microtubules; red arrows show end-to-side microtubule intersections; pink arrow shows a side-to-side microtubule interaction. Bar, 1 μm. (K) Tubulin antibody staining (red; left) of GFP-labeled microtubules (far right) shows that GFP fluorescence shows through areas of diminished antibody staining (arrows; merged image in middle). Bar, 2.5 μm.
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fig2: Microtubules are arranged into intersecting networks on isolated basal membrane patches. (A) GFP-tubulin–labeled microtubules on basal membrane patches prepared from MDCK cells that were polarized on filters. (B) Scanning electron microscopy of MDCK cell basal patches shows cytoplasts (red arrowheads) and open patches (yellow arrowheads). (A and B) Bars, 10 μm. (C) Two examples of GFP-labeled microtubules on MDCK basal patches show the presence of microtubule–microtubule intersections (arrows) and the intersection of multiple microtubules at a focus (yellow arrowhead). (D) Immunofluorescence staining of microtubules on the basal membrane isolated from a polarized Caco-2 cell shows the presence of end-to-side microtubule intersections (arrows). (C and D) Bars, 2.5 μm. (E) An isolated basal membrane from an EpH4 cell shows the presence of a microtubule network with intersections (arrows). Multiple microtubules often intersect at one focus (arrowheads). (F) Scanning electron microscopy of a basal patch shows a few microtubules that remain (boxed and enlarged at right) overlying a dense cytoskeletal network containing actin stress fibers (the microtubules are brighter because they scatter more secondary electrons with their exposed edges). Bar (left), 5 μm. Microtubule ends terminate on the sides of other microtubules and exhibit different conformations. Red arrows point to enlarged ends of microtubules; blue arrows point to ends that curve as they terminate on the sides on other microtubules. Microtubules also show side-to-side interactions (pink arrow). Bar (right), 0.5 μm. (G) Gold labeling of the microtubule network. Microtubules are densely coated with gold beads along their length. Microtubule ends terminate on the sides of other microtubules (red arrows). Microtubules also bundle (pink arrows). Bar, 0.5 μm. (H) Additional examples of the different conformations of microtubule ends contacting the sides of other microtubules. Enlargements present at the end of microtubules are shown by red arrows. Bar (left), 0.5 μm. An end without an enlargement is shown by the blue arrow. Bar (right), 200 nm. A side-to-side interaction is indicated by the pink arrow. (I) Multiple microtubules converging at a focus (yellow arrowhead) and exhibiting end-to-side interactions (red arrows) and side-to-side interactions (pink arrows). Bar, 0.5 μm. (J) GFP-tubulin–labeled microtubules on a nonfixed MDCK basal patch show that the appearance of the intersections is not a result of fixation (arrows). The yellow arrowhead indicates a focus of microtubules; red arrows show end-to-side microtubule intersections; pink arrow shows a side-to-side microtubule interaction. Bar, 1 μm. (K) Tubulin antibody staining (red; left) of GFP-labeled microtubules (far right) shows that GFP fluorescence shows through areas of diminished antibody staining (arrows; merged image in middle). Bar, 2.5 μm.
Mentions: Microtubules in columnar epithelial cells are organized in the apico–basal axis of the cell into parallel bundles with their plus ends oriented toward the basal membrane and weblike networks on the apical and basal cortex (Fig. 1, A–C; Bacallao et al., 1989; Gilbert et al., 1991; Meads and Schroer, 1995; Eaton et al., 1996). High resolution imaging of microtubules on the basal cortex reveals a network of microtubules formed by intersections that arise from the termination of one microtubule on the side of another or from two microtubules crossing over one another (Fig. 1, D and E). A similar microtubule network is found on the basal cortex of different types of polarized epithelial cells, including Caco-2 cells derived from colonic epithelium (Figs. 1 E and 2 D) and EpH4 cells derived from mammary epithelium (Fig. 2 E).

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