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The LKB1 tumor suppressor controls spindle orientation and localization of activated AMPK in mitotic epithelial cells.

Wei C, Bhattaram VK, Igwe JC, Fleming E, Tirnauer JS - PLoS ONE (2012)

Bottom Line: Orientation of mitotic spindles plays an integral role in determining the relative positions of daughter cells in a tissue.RNAi of LKB1 causes spindle misorientation in three-dimensional MDCK cell cysts.Maintaining proper spindle orientation, possibly mediated by effects on the downstream kinase AMPK, could be an important tumor suppressor function of LKB1.

View Article: PubMed Central - PubMed

Affiliation: Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Orientation of mitotic spindles plays an integral role in determining the relative positions of daughter cells in a tissue. LKB1 is a tumor suppressor that controls cell polarity, metabolism, and microtubule stability. Here, we show that germline LKB1 mutation in mice impairs spindle orientation in cells of the upper gastrointestinal tract and causes dramatic mislocalization of the LKB1 substrate AMPK in mitotic cells. RNAi of LKB1 causes spindle misorientation in three-dimensional MDCK cell cysts. Maintaining proper spindle orientation, possibly mediated by effects on the downstream kinase AMPK, could be an important tumor suppressor function of LKB1.

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AMPK inhibition by Compound C causes spindle misorientation and other mitotic defects in MDCK cell cysts.A) Representative spindles from vehicle treated control cysts. Images were rotated in three dimensions to show the cyst lumen, place the apical cell surface at the top of the cell, and place both spindle poles in a single plane. Spindle angle was measured relative to the apical surface. Microtubules are green, actin is red, and DNA is blue. B–D) Representative spindles from cysts treated with 20 µM Compound C for 4 hours prior to fixation. Images in B are displayed as in A. Images in C and D are taken from single lumen cysts, although the lumen is not visible in every image. Insets in D show magnified cells with misattached chromosomes marked by white arrows. Scale bar, 10 µm. E) Quantification of spindle angles. Each dot represents a single spindle angle measurement. Blue bars represent means and standard error of the mean. See text for numbers. P<0.0001 for the difference. F) Data from (E) presented as the % of angles that range from 0° to 30°, 30° to 60°, and 60° to 90°.
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pone-0041118-g005: AMPK inhibition by Compound C causes spindle misorientation and other mitotic defects in MDCK cell cysts.A) Representative spindles from vehicle treated control cysts. Images were rotated in three dimensions to show the cyst lumen, place the apical cell surface at the top of the cell, and place both spindle poles in a single plane. Spindle angle was measured relative to the apical surface. Microtubules are green, actin is red, and DNA is blue. B–D) Representative spindles from cysts treated with 20 µM Compound C for 4 hours prior to fixation. Images in B are displayed as in A. Images in C and D are taken from single lumen cysts, although the lumen is not visible in every image. Insets in D show magnified cells with misattached chromosomes marked by white arrows. Scale bar, 10 µm. E) Quantification of spindle angles. Each dot represents a single spindle angle measurement. Blue bars represent means and standard error of the mean. See text for numbers. P<0.0001 for the difference. F) Data from (E) presented as the % of angles that range from 0° to 30°, 30° to 60°, and 60° to 90°.

Mentions: Compound C treatment for 3.5–4 hours resulted in significant spindle misorientation, with a mean spindle angle in controls of 8±8° (n = 62 spindles from 3 experiments) and a mean angle of 22±19° in Compound C treated cyst cells (n = 70 spindles from 3 experiments; Figure 5). This demonstrates a similar degree of spindle misorientation upon AMPK inhibition as with LKB1 RNAi. Of note, Compound C treatment also caused an increased incidence of monopolar spindles (27% of spindles (22 of 83); Figure 5C) and misattached chromosomes (28% of spindles (17 of 61); Figure 5D), which did not occur with LKB1 RNAi. These findings suggest that AMPK could be a mediator of the role of LKB1 in spindle orientation and could also have additional mitotic roles.


The LKB1 tumor suppressor controls spindle orientation and localization of activated AMPK in mitotic epithelial cells.

Wei C, Bhattaram VK, Igwe JC, Fleming E, Tirnauer JS - PLoS ONE (2012)

AMPK inhibition by Compound C causes spindle misorientation and other mitotic defects in MDCK cell cysts.A) Representative spindles from vehicle treated control cysts. Images were rotated in three dimensions to show the cyst lumen, place the apical cell surface at the top of the cell, and place both spindle poles in a single plane. Spindle angle was measured relative to the apical surface. Microtubules are green, actin is red, and DNA is blue. B–D) Representative spindles from cysts treated with 20 µM Compound C for 4 hours prior to fixation. Images in B are displayed as in A. Images in C and D are taken from single lumen cysts, although the lumen is not visible in every image. Insets in D show magnified cells with misattached chromosomes marked by white arrows. Scale bar, 10 µm. E) Quantification of spindle angles. Each dot represents a single spindle angle measurement. Blue bars represent means and standard error of the mean. See text for numbers. P<0.0001 for the difference. F) Data from (E) presented as the % of angles that range from 0° to 30°, 30° to 60°, and 60° to 90°.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3399794&req=5

pone-0041118-g005: AMPK inhibition by Compound C causes spindle misorientation and other mitotic defects in MDCK cell cysts.A) Representative spindles from vehicle treated control cysts. Images were rotated in three dimensions to show the cyst lumen, place the apical cell surface at the top of the cell, and place both spindle poles in a single plane. Spindle angle was measured relative to the apical surface. Microtubules are green, actin is red, and DNA is blue. B–D) Representative spindles from cysts treated with 20 µM Compound C for 4 hours prior to fixation. Images in B are displayed as in A. Images in C and D are taken from single lumen cysts, although the lumen is not visible in every image. Insets in D show magnified cells with misattached chromosomes marked by white arrows. Scale bar, 10 µm. E) Quantification of spindle angles. Each dot represents a single spindle angle measurement. Blue bars represent means and standard error of the mean. See text for numbers. P<0.0001 for the difference. F) Data from (E) presented as the % of angles that range from 0° to 30°, 30° to 60°, and 60° to 90°.
Mentions: Compound C treatment for 3.5–4 hours resulted in significant spindle misorientation, with a mean spindle angle in controls of 8±8° (n = 62 spindles from 3 experiments) and a mean angle of 22±19° in Compound C treated cyst cells (n = 70 spindles from 3 experiments; Figure 5). This demonstrates a similar degree of spindle misorientation upon AMPK inhibition as with LKB1 RNAi. Of note, Compound C treatment also caused an increased incidence of monopolar spindles (27% of spindles (22 of 83); Figure 5C) and misattached chromosomes (28% of spindles (17 of 61); Figure 5D), which did not occur with LKB1 RNAi. These findings suggest that AMPK could be a mediator of the role of LKB1 in spindle orientation and could also have additional mitotic roles.

Bottom Line: Orientation of mitotic spindles plays an integral role in determining the relative positions of daughter cells in a tissue.RNAi of LKB1 causes spindle misorientation in three-dimensional MDCK cell cysts.Maintaining proper spindle orientation, possibly mediated by effects on the downstream kinase AMPK, could be an important tumor suppressor function of LKB1.

View Article: PubMed Central - PubMed

Affiliation: Department of Epidemiology, The University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America.

ABSTRACT
Orientation of mitotic spindles plays an integral role in determining the relative positions of daughter cells in a tissue. LKB1 is a tumor suppressor that controls cell polarity, metabolism, and microtubule stability. Here, we show that germline LKB1 mutation in mice impairs spindle orientation in cells of the upper gastrointestinal tract and causes dramatic mislocalization of the LKB1 substrate AMPK in mitotic cells. RNAi of LKB1 causes spindle misorientation in three-dimensional MDCK cell cysts. Maintaining proper spindle orientation, possibly mediated by effects on the downstream kinase AMPK, could be an important tumor suppressor function of LKB1.

Show MeSH
Related in: MedlinePlus