The microtubule-destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells

Abstract

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.

Figures

Figure 1

GFP-XKCM1 overexpression destabilizes microtubules in PtK2 cells. Cells were transfected with GFP or GFP-XKCM1 then fixed and processed for immunofluorescence at 48 h posttransfection. Digital images were taken using identical microscope settings and exposure times. (A) Control cells expressing GFP and corresponding interphase MT arrays. (B) Cell expressing a low level of GFP-XKCM1 and corresponding MT array. Note the nonexpressing cells with normal MT arrays to the left. (C) Cell expressing a high level of GFP-XKCM1 and corresponding MT array. Bar, 20 μm.

Figure 2

Increased overexpression of GFP-XKCM1 decreases the level of microtubule polymer. The average pixel intensities in both the fluorescein channel (GFP or GFP-XKCM1) and the Texas Red channel (MTs) were measured for each cell and then plotted as a single point on the graphs. (A) Plot of cells expressing different levels of GFP (n = 107 cells). (B) Plot of cells expressing different levels of GFP-XKCM1 (n = 164 cells). The arrow indicates the level of GFP-XKCM1 intensity at which “maximal destabilization” of MTs occurred.

Figure 3

Inhibitory anti-XKCM1 antibody rescues the effect of GFP-XKCM1 overexpression. PtK2 cells were transfected with either GFP or GFP-XKCM1 and coinjected with antibody plus X-rhodamine tubulin at 48 h posttransfection. Cells were then fixed at 1–3 h postinjection. (A) Cells expressing GFP coinjected with labeled tubulin and random IgG antibody. (B) Cell expressing GFP-XKCM1 and coinjected with labeled tubulin and a random IgG antibody. Note soluble tubulin pool in cell expressing GFP-XKCM1 vs. normal MT arrays in two nonexpressing cells. (C) Cells expressing GFP and coinjected with inhibitory anti-XKCM1 antibody and labeled tubulin. (D) Cells expressing GFP-XKCM1 and coinjected with labeled tubulin and inhibitory anti-XKCM1 antibody. Note cytoplasmic aggregations of GFP-XKCM1 and corresponding normal interphase arrays. Bar, 20 μm.

Figure 4

Overexpression of GFP-XKCM1 increases dynamic instability of microtubules. PtK2 cells were transfected with GFP or GFP-XKCM1 and microinjected with labeled tubulin at 24 h posttransfection. Individual MTs were visualized in live cells at 1–3 h posttransfection. Rhodamine MTs in representative cells expressing GFP (A) or GFP-XKCM1 (B). Asterisks lie next to the MTs represented in C–F. Individual MTs in live cells expressing either GFP (C) or GFP-XKCM1 (D) were imaged for MT dynamics. Actual image times are in the top right corner. White arrowheads denote the end of the MT. (E) Life history plot of the MT shown in C in a cell expressing GFP. Black arrowheads correspond to the three image time points. (F) Life history plot of the MT shown in D in a cell expressing GFP-XKCM1. Black arrowheads correspond to the three image time points. Bars, 5 μm.

Figure 5

Injection of inhibitory anti-XKCM1 antibody increases microtubule density. PtK2 cells were microinjected with antibody and fixed and processed for immunofluorescence at 1–3 h postinjection. (A) Cell injected with nonimmune IgG antibody and corresponding MT array. Note similar MT arrays of uninjected cells. (B) Cell injected with inhibitory anti-XKCM1 antibody and corresponding MT array. Note slight increase in density of perinuclear MTs in injected cells compared with uninjected cells to the right. Bar, 20 μm.

Figure 6

Inhibition of KCM1 suppresses dynamic instability of microtubules. PtK2 cells were coinjected with antibody and rhodamine-labeled tubulin. Individual MTs were visualized in live cells at 1–3 h posttransfection. Rhodamine MTs in representative cells injected with nonimmune IgG antibody (A) or inhibitory anti-XKCM1 antibody (B). The asterisks lie next to the MTs represented in C–F. Individual MTs in live cells injected with either IgG antibody (C) or inhibitory anti-XKCM1 antibody (D) were imaged for MT dynamics. Actual image times are in the bottom right corner. White arrowheads denote the end of the MT. (E) Life history plot of the MT shown in C in a cell injected with control IgG. Black arrowheads correspond to the three image time points. (F) Life history plot of the MT shown in D in a cell injected with anti-XKCM1 antibody. Black arrowheads correspond to the three image time points. Bars, 5 μm.

Figure 7

Overexpression of GFP-XKCM1 perturbs spindle morphology in PtK2 cells. Cells were transfected with GFP or GFP-XKCM1 and processed for immunofluorescence microscopy at 72 h posttransfection. MTs are in red and DNA is in blue. Optical Z-series images were collected using a Z-axis motor and three-dimensionally reconstructed in MetaMorph. (A) Prometaphase spindle in a cell expressing GFP. (B) Monoastral spindle in a cell overexpressing GFP-XKCM1. (C) Monopolar spindle in a cell overexpressing GFP-XKCM1. (D) Prometaphase-like spindle in a cell overexpressing GFP-XKCM1. Arrowheads point between unseparated centrosomes. Arrows denote centromere localization of GFP-XKCM1. Bar, 10 μm.

Figure 8

Inhibition of KCM1 in mitosis leads to a mitotic delay in PtK2 cells. Prophase cells were microinjected with either nonimmune IgG or inhibitory anti-XKCM1 antibody. At 30 min postinjection, cells were fixed and processed for fluorescence of injected antibodies, MTs, and DNA. The percentage of cells in each stage of mitosis was determined and plotted for IgG-injected cells (dotted line) vs. cells injected with anti-XKCM1 antibody (solid line).

Figure 9

Inhibition of KCM1 in mitosis perturbs spindle morphology in PtK2 cells. Prophase cells were microinjected with either nonimmune IgG or inhibitory anti-XKCM1 antibody. At 30 min postinjection, cells were fixed and processed for fluorescence of injected antibodies, MTs (red), and DNA (blue). Optical Z-series images were collected using a Z-axis motor and three-dimensionally reconstructed in MetaMorph. (A) Prometaphase spindle in a cell injected with IgG. (B) Hairy prometaphase spindle in a cell injected with anti-XKCM1. (C) Monoastral spindle in a cell injected with anti-XKCM1. Note lack of XKCM1 staining at centromeres and spindle poles in B and C. Bar, 10 μm.