A LCMT1-PME-1 methylation equilibrium controls mitotic spindle size

Abstract

Leucine carboxyl methyltransferase-1 (LCMT1) and protein phosphatase methylesterase-1 (PME-1) are essential enzymes that regulate the methylation of the protein phosphatase 2A catalytic subunit (PP2AC). LCMT1 and PME-1 have been linked to the regulation of cell growth and proliferation, but the underlying mechanisms have remained elusive. We show here an important role for an LCMT1-PME-1 methylation equilibrium in controlling mitotic spindle size. Depletion of LCMT1 or overexpression of PME-1 led to long spindles. In contrast, depletion of PME-1, pharmacological inhibition of PME-1 or overexpression of LCMT1 led to short spindles. Furthermore, perturbation of the LCMT1-PME-1 methylation equilibrium led to mitotic arrest, spindle assembly checkpoint activation, defective cell divisions, induction of apoptosis and reduced cell viability. Thus, we propose that the LCMT1-PME-1 methylation equilibrium is critical for regulating mitotic spindle size and thereby proper cell division.

Keywords: LCMT1; PME-1; cell division; methylation; mitotic spindle.

Figure 1.

For figure legend, see page 1951. Figure 1 (See previous page). RNAi-mediated perturbation of the LCMT1-PME-1 methylation equilibrium leads to misregulation of spindle size and cell division. (A) siRNA knockdown of LCMT1 or PME-1 expression levels compared to control non-targeting siRNA. Note that the levels of demethylated PP2AC decreases in PME-1 depleted cells and increases in LCMT1 depleted cells. (B) Immunofluorescence microcopy showing that PME-1 depletion leads to abnormally short spindles and LCMT1 depletion leads to abnormally long spindles. Bar = 5 μm. (C) Quantification of the percentage of cells in mitosis showing that depletion of LCMT1 or PME-1 leads to a major increase in cells arrested in mitosis. (D) Quantification of the percentage of mitotic cells with abnormal spindles (short, long, multipolar) showing that depletion of LCMT1 or PME-1 leads to a major increase in abnormal spindles. (E) Quantification of the percentage of mitotic cells with unaligned chromosomes showing that depletion of LCMT1 or PME-1 leads to a major increase in unaligned chromosomes. (F) Outline of metaphase spindle pole-to-pole (PTP) distance and width measurements. (G) Quantification of metaphase spindle PTP distance (in μm) showing that depletion of PME-1 leads to abnormally short spindles and depletion of LCMT1 depletion leads to abnormally long spindles. (H) Quantification of metaphase spindle width (in μm) showing that depletion of PME-1 leads to abnormally short spindle widths, while depletion of LCMT1 had no significant effect on spindle width. (I) Quantification of metaphase spindle volume (in μm³) showing that depletion of PME-1 leads to a decrease in volume and depletion of LCMT1 leads to an increase in volume. (J) Quantification of the total fluorescence intensity of mitotic spindle microtubules for PME-1 or LCMT1-depleted cells, in arbitrary units (A.U.), showing that depletion of PME-1 leads to a decrease in total microtubule polymer, whereas depletion of LCMT1 leads to an increase in total microtubule polymer. (K) Model of the LCMT1-PME-1 methylation equilibrium that regulates spindle size and normal cell division. (C, D, F–J) Data represents average ± SDs of 3 independent experiments. * = P < 0.05, ** = P < 0.005, *** = P < 0.0005, ns = not statistically significant.

Figure 2.

Pharmacological-mediated perturbation of the LCMT1-PME-1 methylation equilibrium leads to misregulation of spindle size and cell division. (A) Pharmacological inhibition of PME-1 with AMZ30 compared to DMSO vehicle control. Note that the levels of demethylated PP2AC decreased in cells treated with AMZ30. (B) Immunofluorescence microcopy showing that AMZ30-treated cells have abnormally short spindles. Bar = 5 μm. (C) Quantification of the percentage of cells in mitosis showing that inhibition of PME-1 with AMZ30 leads to a major increase in cells arrested in mitosis. (D) Quantification showing that treatment of cells with AMZ30 leads to a major increase in the percentage of mitotic cells with abnormal spindles (short, long, multipolar). (E) Quantification showing that treatment of cells with AMZ30 leads to a major increase in the percentage of unaligned chromosomes. (F) Quantification of metaphase spindle PTP distance (in μm) showing that AMZ30-treated cells have abnormally short spindles. (G) Quantification of metaphase spindle width (in μm) showing that AMZ30-treated cells have abnormally short spindle widths. (H) Quantification of metaphase spindle volume (in μm³) showing that treatment with AMZ30 leads to a decrease in volume. (I) Quantification of the total fluorescence intensity of mitotic spindle microtubules for AMZ30-treated cells, in arbitrary units (A.U.), showing that AMZ30 treatment leads to a decrease in total microtubule polymer. (C–I) Data represents average ± SDs of 3 independent experiments. ** = P < 0.005, *** = P < 0.0005.

Figure 3.

Overexpression-mediated perturbation of the LCMT1-PME-1 methylation equilibrium leads to misregulation of spindle size and cell division. (A) Overexpression of LAP-LCMT1 or LAP-PME-1 compared to non-induced control cells. Note that the levels of demethylated PP2AC decreased in cells overexpressing LAP-LCMT1 and increased in cells overexpressing LAP-PME-1. (B) Immunofluorescence microcopy showing that LAP-PME-1 overexpression leads to abnormally long spindles and LAP-LCMT1 overexpression leads to abnormally short spindles. Bar = 5 μm. (C) Quantification of the percentage of cells in mitosis showing that overexpression of LAP-PME-1 or LAP-LCMT1 leads to a major increase in cells arrested in mitosis. (D) Quantification showing that overexpression of LAP-LCMT1 or LAP-PME-1 leads to a major increase in the percentage of abnormal spindles (short, long, multipolar). (E) Quantification showing that overexpression of LAP-LCMT1 or LAP-PME-1 leads to a major increase in the percentage of unaligned chromosomes. (F) Quantification of metaphase spindle PTP distance (in μm) showing that LAP-PME-1 overexpression leads to abnormally long spindles and LAP-LCMT1 overexpression leads to abnormally short spindles. (G) Quantification of metaphase spindle width (in μm) showing that overexpression of LAP-LCMT1 leads to a decrease in spindle width, while overexpression of LAP-PME-1 had no significant effect on spindle width. (H) Quantification of metaphase spindle volume (in μm³) showing that overexpression of LAP-PME-1 leads to an increase in volume and LAP-LCMT1 overexpression leads to a decrease in volume. (I) Quantification of the total fluorescence intensity of mitotic spindle microtubules for LAP-PME-1 or LAP-LCMT1 overexpressing cells, in arbitrary units (A.U.), showing that overexpression of LAP-LCMT1 leads to a decrease in total microtubule polymer, whereas overexpression of LAP-PME-1 leads to an increase in total microtubule polymer. (C–I) Data represents average ± SDs of 3 independent experiments. * = P < 0.05, ** = P < 0.005, *** = P < 0.0005, ns = not statistically significant.

Figure 4.

Analyzing the consequences of perturbing the LCMT1-PME-1 methylation equilibrium. (A–C) Fixed-cell immunofluorescence microcopy showing that the spindle assembly checkpoint is activated (Bub1 remains localized to kinetochores) in LCMT1 or PME-1 depleted cells (A), PME-1 inhibited cells (B) and LCMT1 or PME-1 overexpressing cells (C). Bar = 5 μm. (D–F) Quantification of normalized percent cell viability for LCMT1 or PME-1-depleted (D), AMZ30-treated (E), or LCMT1 or PME-1-overexpressing cells (F). Data represents average ± SDs of 3 independent experiments. * = P < 0.05, ** = P < 0.005.

Figure 5.

Analyzing the consequences of perturbing the LCMT1-PME-1 equilibrium by live-cell time-lapse microscopy. (A) Live-cell imaging of siControl, siLCMT1 or siPME-1 treated cells. (B) Live-cell imaging of control DMSO or AMZ30 treated cells. (C) Live-cell imaging of control, LAP-LCMT1 overexpressing or LAP-PME-1 overexpressing cells. (A–C) Bar = 20 μm. Time is in minutes. See also Supplemental Movies S1-S8. (D–F) Quantification of the live-cell imaging data from A-C for the percentage of cells undergoing mitotic arrest (MA), defective cell divisions (DD) and cell death in mitosis (DM). Data represents average ± SDs of 3 independent experiments. (G–I) Quantification of live-cell imaging data from A-C for the length of time cells spent in mitosis, cell rounding through cell abscission. Data represents average ± SDs of 3 independent experiments. ** = P < 0.005, *** = P < 0.0005.