Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis - PubMed (original) (raw)

Differential functional interplay of TOGp/XMAP215 and the KinI kinesin MCAK during interphase and mitosis

Per Holmfeldt et al. EMBO J. 2004.

Abstract

XMAP215/TOGp family members and KinI kinesins are conserved microtubule (MT)-regulatory proteins, and have been viewed as possessing prominent antagonistic stabilizing/destabilizing activities that must be balanced. Here, interdependencies between TOGp and the KinI kinesin MCAK were analyzed in human leukemia cells. A system was established that permits inducible overexpression in homogeneous cell populations that simultaneously synthesize interfering short hairpin RNAs. We present evidence that the functional interplay of TOGp and MCAK proteins is manifested as three distinct phenotypes during the cell cycle. The first involves a role for TOGp in protecting spindle MTs from MCAK activity at the centrosome, which appears essential to prevent the formation of disorganized multipolar spindles. The second phenotype involves TOGp-dependent counteraction of excessive MCAK activity during mitosis, which recapitulates the previously established plus-end specific counteractive activities in vitro. The third involves an unexpected destabilization of the interphase MTs by overexpressed TOGp, a phenotype that requires endogenous MCAK. We hypothesize that TOGp-dependent prevention of MCAK-mediated spindle disorganization, as evidenced by depletion experiments, reflects a primary physiological role for TOGp in human somatic cells.

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Figures

Figure 1

Figure 1

Protein expression and cell proliferation of K562 leukemia cell lines producing interfering shRNA. (A) Cells were transfected with a replicating shuttle vector that directs synthesis of shRNA designed to target CaMKIIγ, MCAK or TOGp. Nontransfected cells were counter-selected by culturing in the presence of hygromycin, which killed off most nontransfected cells within 3 days. Total cellular lysates were analyzed by immunoblots using the indicated antibodies for detection. (B) Viable cells grown in the presence of hygromycin were determined on the days indicated. The data plotted represent the mean of triplicate determinations. All data in this and subsequent figures have been reproduced in at least three independent transfection experiments.

Figure 2

Figure 2

Cell cycle phenotypes of K562 leukemia cell lines producing interfering shRNA. DNA content was determined by flow cytometry 4 days after transfection with either Vector-Co or the indicated shRNA-producing derivative(s). The mitotic index is given as a percentage of total cells in each panel. The middle panels show the distribution of the indicated types of mitotic figure and the data plotted represent the mean of duplicate determinations (_n_=300). The lower panels show representative images of mitotic figures double-stained for DNA and MTs. Bar: 10 μm.

Figure 3

Figure 3

Only two of the poles of type B multipolar spindles of TOGp-depleted cells contain pericentrin, but all spindle pools contain NuMA. Transfected K562 cell lines producing shRNA-TOGp were generated as in Figure 1, and cultured for 4 days. (A) Representative epifluorescence image of a mitotic cell stained with anti-α-tubulin (green) and anti-pericentrin (red). (B) The distribution of the number of spindle poles in individual cells with type B multipolar spindles, as determined by pericentrin-labeled dots positioned at the spindle pole. The data are the average of the means of duplicate determinations (_n_=200 mitotic cells). (C) Representative epifluorescence image of a mitotic cell stained with anti-α-tubulin (green) and anti-NuMA (red). Inspection of individual mitotic TOGp-depleted cells revealed the association of NuMA with the center of each aster in all cells analyzed (_n_=100). Bar: 10 μm.

Figure 4

Figure 4

MCAK expression is required for generation of the type B multipolar spindles in TOGp-depleted cells. Cells were transfected either with pMEP vector alone or with a mix of replicating shuttle vectors that direct constitutive synthesis of shRNA-TOGp and shRNA-MCAK, and inducible expression of MCAK, as described under Materials and methods. After 4 days of culture, Cd2+ was added for 24 h to specifically induce ectopic MCAK from the hMTIIa promoter of the pMEP vector. Upper panels show immunoblots of total cellular lysates using the indicated antibody for detection prior to or after induced MCAK expression. Lower panels show the distribution of the indicated types of mitotic figure after 0 and 24 h of induced MCAK expression. The data plotted represent the means of duplicate determinations (_n_=250).

Figure 5

Figure 5

Overexpressed TOGp counteracts spindle disruption by overexpressed MCAK. K562 cells were transfected with pMEP-MCAK or Vector-Co and either additional Vector-Co (−) or pMEP-TOGp (+). The effect of ectopic TOGp and MCAK, either expressed alone or in combination, during spindle formation was analyzed after 20 h of Cd2+ induction. The upper panels show immunoblots of cell lysates using the indicated antibody for detection. The distribution of DNA content is shown in the absence or presence (inserts) of the mitosis-blocking drug paclitaxel (1 μ

M

). The mitotic index is given as a percentage of total cells in each panel. Cells were categorized with respect to frequencies of bipolar, monoastral, multipolar and Type I spindles by inspection of cells double-stained for DNA and MTs. Examples of type I abnormal spindles spanning the different degrees of severity observed are shown at the bottom (bar: 10 μm). Data represent the means of duplicate determinations (_n_=400 mitotic cells).

Figure 6

Figure 6

TOGp and MCAK do not exert antagonizing activities in human interphase cells. (A) Transfected K562 cell lines producing the indicated shRNA derivative were generated as in Figure 1. MT content among G1 interphase cells was analyzed on the indicated days by multi-parameter flow cytometry analysis. (B) The fraction of polymerized tubulin was analyzed as in panel (A) after 2 h, in the presence of graded concentrations of the microtubule-destabilizing drug nocodazole. Open bars represent Vector-Co cells and filled bars represent cells after 5 days of TOGp depletion. (C) Cells were cotransfected as indicated, with either Vector-Co or a replicating shuttle vector directing constitutive synthesis of the TOGp-specific interfering shRNA combined with either Vector-Co (−) or pMEP-MCAK (+) (see Materials and methods). After 5 days of culture, Cd2+ was added for 8 h to specifically induce ectopic TOGp from the hMTIIa promoter of the pMEP vector. Upper panels show immunoblots of total cellular lysates using the indicated antibody for detection. Lower panels show MT content among G1 interphase cells after 0 and 8 h of induced expression. It should be noted that the transfection protocol was designed for higher MCAK expression, as compared to the experiments shown in Figures 3 and 5 (see Materials and methods). All data plotted represent the means of duplicate determinations.

Figure 7

Figure 7

Overexpressed TOGp destabilizes interphase MTs by an MCAK-dependent mechanism. (A) Cell lysates from Vector-Co- or pMEP-TOGp-transfected K562 cells were analyzed by immunoblots, using the indicated antibodies for detection, after the indicated time of Cd2+-induced expression. Arbitrary quantification was obtained from serial dilutions of cell lysates, which revealed four- to six-fold increased expression of TOGp (24 h) and nonsignificant alterations in endogenous tubulin levels (lower panel). (B) The fraction of polymerized tubulin was analyzed at the indicated times after Cd2+ induction of Vector-Co- and pMEP-TOGp-transfected cells. (C) Cells were cotransfected as indicated with either Vector-Co or a replicating shuttle vector directing constitutive synthesis of the indicated interfering shRNAs combined with either Vector-Co (−) or pMEP-TOGp (+) (see Materials and methods). After 4 days of culture, Cd2+ was added for 8 h to specifically induce ectopic TOGp from the hMTIIa promoter of the pMEP vector. The upper panels show immunoblots of total cellular lysates using the indicated antibody for detection. Lower panels show MT content among G1 interphase cells after 0 and 8 h of induced expression. (D) Cells were transfected with the indicated pMEP4 derivative as described in Figure 5, and the upper panels show immunoblots of cell lysates using the indicated antibody for detection. The effect of ectopic TOGp and MCAK, either expressed alone or in combination, on MT-polymer content was analyzed after 8 h of Cd2+ induction. All data plotted represent the means of duplicate determinations.

Figure 8

Figure 8

Model of TOGp and MCAK interdependence during interphase and mitosis. (1) Depiction of how TOGp from its centrosomal (pink oval) location is postulated to be essential for protection of spindle MTs against the destabilizing activity of MCAK. The predominant centrosomal location of TOGp is consistent with the observation that TOGp protects spindle MTs from MCAK activity at the centrosome. Moreover, a postulated loss of plus-end protection by TOGp depletion would predict accumulation of type I spindles, which are indicative of excess catastrophe promotion at the plus-end (Segerman et al, 2003), and not the observed disorganized type B multipolar spindles in TOGp-depleted cells. This protective function of TOGp at the centrosome is only evident during mitosis, which is consistent with an indirect attachment of minus-ends of spindle MTs to centrosomes, while interphase MTs are embedded in the centrosome and thereby protected from MCAK activity. This level of TOGp function is suggested by depletion experiments. (2) Depiction of how TOGp expressed at elevated levels protects free spindle MT plus-ends from excess MCAK-dependent destabilization. This mechanism of TOGp function is consistent with analysis of overexpressed phenotypes during mitosis. (3) Depiction of how TOGp may mediate MCAK-dependent destabilization of interphase MTs. This level of TOGp function is suggested by the fact that destabilization of interphase MTs by overexpressed TOGp is dependent on the endogenous MCAK protein. We hypothesize that mechanism 1, which has support from depletion experiments, probably reflects the most physiologically important role of TOGp in human somatic cells, while the other two mechanisms may only be relevant in human K563 leukemia cells under conditions of overexpression.

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