Report |
Address correspondence to Linda Wordeman, Dept. of Physiology and Biophysics, University of Washington, 1959 NE Pacific St., Box 357290, Seattle, WA 98195-7290. Tel.: (206) 543-4135. Fax: (206) 685-0619. E-mail: worde{at}u.washington.edu
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Abstract |
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Key Words: Kin I; XKCM1; kinesins; processivity; diffusional motility
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Introduction |
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The direction of transport and the affinity for MTs are molecular refinements that adapt motile kinesins for specific cellular functions within the realm of intracellular transport, cell division, and MT dynamics (Goldstein and Philp, 1999; Woehlke and Schliwa, 2000). The "neck" domain, a region adjacent to the motor domain (head), is responsible for conferring directionality and processivity to the core motor domain (Case et al., 1997; Henningsen and Schliwa, 1997; Endow and Waligora, 1998; Romberg et al., 1998). MCAK's neck domain is highly conserved within the Kin I subfamily, but is very divergent from other kinesin-related proteins (Vale and Fletterick, 1997; Maney et al., 2001). A truncated MCAK protein, consisting of the neck domain (A182-D246) plus the core motor domain (P247-E581), shows MT depolymerization activity similar to that of the full-length (FL) MCAK (Maney et al., 2001). However, the neck itself does not possess depolymerizing activity (Maney et al., 1998), nor does it confer depolymerization activity to a motile kinesin because a chimera consisting of the MCAK neck fused to the motor domain of a conventional kinesin (KIF5B) exhibits no MT depolymerization activity when expressed in cultured cells (Maney et al., 2001). Finally, the neck domain is not involved in dimerization because the A182-E581 fragment of MCAK, which includes the neck, is a monomer (Maney et al., 2001). What, then, is the role of the neck domain in the MT depolymerization activity of MCAK?
The electrostatic interactions between a positively charged neck domain and the negatively charged COOH terminus of tubulin have been shown to be important for the processivity of conventional kinesins. The addition of positive charges to the neck of a conventional kinesin increases its processivity by perhaps tethering the kinesin near the MT surface (Thorn et al., 2000). Similarly, the positively charged K-loop in the motor domain of the KIF1 single-headed kinesin provides a flexible attachment site for binding to MTs in the weakly bound state (Okada and Hirokawa, 2000; Rogers et al., 2001). Given the high percentage of positively charged amino acids in the MCAK neck region and their strong conservation among Kin I kinesins (Fig. 1 A), it is reasonable to suggest that the neck might be anchoring MCAK to MTs. Here, we used mutational analysis and chimeras to test whether the MCAK neck functions as an electrostatic tether. We found that although the removal of positive charges from the neck region significantly diminished the MT depolymerization activity of MCAK, the insertion of the positively charged K-loop into "neckless" MCAK completely restored its MT depolymerization activity. We propose that the neck, analogous to the K-loop, may interact electrostatically with the negatively charged MT lattice. This interaction may serve to target MCAK to MT ends by diffusional motility. The weak electrostatic interaction between MCAK's neck and MT ends may also play an important role in processivity of MCAK-induced MT depolymerization.
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Results and discussion |
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The removal of positive charges from the neck domain dramatically reduced MT depolymerization activity of MCAK
As expected from previous studies (Maney et al., 2001), cells transfected with neckless MCAK ( A182-D246) displayed a normal MT network. The mean tubulin fluorescence of the
A182-D246 MCAK mutant was similar to that of the EGFP control and equaled 1,825 gray-scale units (Fig. 3 A). Cells transfected with MCAK mutants containing progressively smaller deletions in the neck domain,
A182-E232 and
A182-D218 (Fig. 2 E, arrowhead), also showed normal levels of MT polymer that were similar to that of the EGFP-transfected cells (Fig. 3 A). Hence, these neckless MCAK mutants exhibited severely impaired MT depolymerization activity. Smaller deletions in the neck region (
A182-E201 and
E201-D218) diminished MT depolymerization activity to a lesser degree. (Fig. 3 A). Next, we performed alanine substitutions of the highly conserved positively charged residues in the neck domain (Fig. 1, arrows) to test whether the loss of positive charges in this region affects the depolymerization activity of MCAK. Alanine substitutions of 34 positively charged amino acids resulted in some reduction of MT depolymerization activity (more than 50% of MTs were depolymerized in the transfected cells). Substitutions of 7 to 10 amino acids resulted in much greater inhibition of the depolymerization activity (<20% of MTs were depolymerized; Fig. 3 B). Therefore, the total sum of removed positive charges was of greater importance than the positions of the substituted residues.
In summary, we identified A182-D218 as the smallest neck deletion that would reduce MT depolymerizing activity to the level of the EGFP control (Fig. 3 A). This region contains 13 (36%) positively charged residues and 7 (19%) negatively charged residues (Fig. 1). The net charge of A182-D218 region is positive and equals +6. Alanine substitutions of only 7 positive residues rendered the net charge of A182-D218 segment negative and resulted in greatly diminished MT depolymerization activity (Fig. 3 B). The A182-D218 region contains a highly charged hydrophilic helix (residues R183-Q215) as predicted by SSpro2 server (http://promoter.ics.uci.edu/BRNN-PRED [Baldi et al., 1999]). The helical wheel representation of this helix shows that the positively charged residues lie mostly on one side of the helix, whereas the opposite side contains most of the negatively charged residues found in this neck region (Fig. 1 B).
KIF1A K-loop insertion into neckless MCAK rescues MT depolymerization activity
We have shown that elimination of the positive charges in the MCAK neck region, A182-D218, either by deletions or alanine substitutions inhibits MT depolymerization activity of MCAK. Conversely, Niederstrasser et al. (2002) showed that the interaction between the COOH terminus of tubulin (C-hook) and MCAK is required for MT depolymerization because subtilisin cleavage of the C-hook abolishes depolymerization of MTs by the MCAK homologue, XKCM1. Furthermore, Okada and Hirokawa (2000) have shown that the positively charged K-loop of KIF1A interacts with the negatively charged C-hook and serves as an electrostatic tether to support the diffusion of KIF1A along MT protofilament. The positively charged neck of MCAK might interact with the negatively charged C-hook of tubulin analogously to the KIF1A K-loop. We tested this hypothesis by inserting the K-loop of KIF1A into the MT depolymerization deficient neckless MCAK. We found that MCAK with the A182-D218 neck segment replaced by the KIF1A K-loop, NKNKKKKK, (abbreviated as A182-K-loop-D218 MCAK) depolymerized MTs with the same efficiency as FL MCAK (Figs. 2 F and 4). Moreover, mitotic cells transfected with the A182-NKNKKKKK-D218 MCAK mutant displayed the same mitotic defects (unpublished data) described for overexpression of FL MCAK, namely bundling and depolymerization of spindle MTs, which in turn resulted in prometaphase arrest and an elevated mitotic index in transfected cells (Maney et al., 1998).
None of the MCAK mutations described in this report interfered with centromere or centrosome binding. The nuclear/cytoplasmic localization of the mutants was identical to that of wild-type FL MCAK (unpublished data). All MCAK mutants showed the ability to bind MTs in vivo when cells were extracted before fixation in the absence of nucleotide (unpublished data) in a manner identical to FL MCAK (Maney et al., 1998). This implies that all mutated MCAKs were functional proteins with motor domains able to bind MT lattice in the nucleotide-free state, similar to conventional kinesins.
In vitro MT depolymerization activity of MCAK neck mutants
The rescue of the MT depolymerizationdeficient neckless MCAK by K-loop insertion suggests that the MCAK neck is affecting the interactions between motor and MTs rather than regulating the depolymerization activity. To test this hypothesis further, we replicated our depolymerization assays in vitro. We expressed and purified the following proteins: core motor plus neck (A182-S583); core motor plus alanine-substituted neck (A182-Ala-S583 with substituted residues indicated by arrows in Fig. 1 A); core motor plus truncated neck (D218-S583); core motor plus K-loop in substitution for the neck (A182-K-loop-D218-S583); core motor plus poly-lysine (5K) in substitution for the neck (A1825K-D218-S583); and core motor domain of MCAK (I253-S583; Fig. 5
A). The purified proteins were added to the taxol-stabilized MTs and were incubated at 24 ± 1°C for 10 min in the presence of 1 mM ATP. The depolymerization of tubulin polymer was measured as the percentage of tubulin released in the supernatant after subtraction of tubulin in supernatant of the no-motor control reaction and normalizing the supernatant and pellet fractions. Fig. 5 B shows the supernatants and pellets of the in vitro depolymerization reactions containing 100 nM active motor proteins and 1,500 nM taxol MTs. Although the core motor plus neck (A182-S583) depolymerizes 92 ± 4% of tubulin polymer, the core motor plus alanine-substituted neck, A182-Ala-S583 depolymerized only 10 ± 4% of tubulin polymer (Fig. 5 B). The truncated neck plus core motor (D218-S583) lacks the positively charged helix found in the FL neck (Fig. 1 B). This protein depolymerized only 13 ± 5% of tubulin polymer. Therefore, removal of positive charges from the neck either by alanine substitutions or deletions dramatically suppresses the MT depolymerization activity of this kinesin, but does not eliminate it completely in vitro. Although analogous neckless MCAK mutants ( A182-D246,
A182-E232, and
A182-D218) expressed to high levels in vivo displayed no detectable depolymerization activity (Fig. 2 E, 3A), in corroboration with our in vitro data, extremely high levels of expression showed a slightly disassembled MT network (unpublished data).
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The rescue of the MT depolymerizationdeficient neckless MCAK by K-loop insertion suggests that the MCAK neck might function analogously to the K-loop. The K-loop of KIF1A mediates diffusional motility (Okada and Hirokawa, 2000; Rogers et al., 2001). Similarly, we propose that the neck of MCAK may support diffusional motility to transport MCAK along the MT protofilament toward MT ends where Kin Imediated MT depolymerization occurs (Desai et al., 1999). The neck of MCAK may also use diffusional motility to confer accelerated kinetics or processivity to the core motor domain (unpublished data).
Moores et al. (2002) have clearly shown that a conserved core motor domain of a Plasmodium falciparum Kin I kinesin (pKinI) can disassemble MTs. However, the assay conditions (extremely low ionic strength) were chosen to maximize electrostatic interactions. Maximizing the electrostatic interactions and raising the stoichiometry of Kin I/tubulin (1:10) obviates the need for processivity or diffusional motility despite the fact that it is essential for depolymerization under physiological conditions. We have duplicated the low ionic strength conditions used by Moores et al. (2002). The decrease in salt concentration from 60 to 7 mM KCl in BRB20 buffer produced a twofold increase of tubulin polymer depolymerized by the core motor (Fig. 5 C). This shows that the core motor of MCAK (I253-S583) is also a definite but enfeebled depolymerizer. Direct comparison of the neck plus motor (A182-S583) with the conserved core motor (I253-S583) shows that the neck plus motor is a tremendously more effective depolymerizer than the core motor (Fig. 5, B and C).
In summary, we demonstrated that deletions or alanine substitutions of positively charged residues of A182-D218 neck region drastically reduced the MT depolymerization activity. Insertion of the positively charged KIF1A K-loop or poly-lysine rescued the neckless MCAK mutants. We propose that the neck acts as an electrostatic tether. This electrostatic tether may increase the rate of MT depolymerization by either increasing the on-rate of MCAK to MT ends (perhaps by mediating diffusional motility) or decreasing the off-rate of MCAK from MTs (perhaps by conferring processivity to the core motor domain; unpublished data). The data presented here do not allow us to distinguish between diffusional targeting and processivity. Mediation of diffusional targeting and processivity are two distinct and not mutually exclusive hypotheses for the contribution of the neck to the MT depolymerization activity of MCAK.
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Materials and methods |
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Cell transfection and immunofluorescence
CHO AA8 Tet-OffTM cells (CLONTECH Laboratories, Inc.) were grown in 90% MEM, 10% FBS, and 100 µg/ml G418 at 37°C, 5% CO2. 1 d before transfection, cells were plated onto 12-mm coverslips at low density (2 x 104 cells/cm2). Transfections were done with ExGen 500 transfection reagent (MBI Fermentas). Cells were cultured for 24 h after transfection and fixed with 1% PFA in precooled methanol for 10 min. The cells were then incubated with mouse antitubulin DM1
(Sigma-Aldrich) at 1:50 dilution for 1 h and Texas red antimouse antibodies (Jackson ImmunoResearch Laboratories) at 1:100 dilution in PBS plus 0.1% Triton X-100 and 1% BSA for 1 h. Cells were washed with PBS, stained with DAPI, and mounted in Vectashield® mounting medium (Vector Laboratories). Analysis was done with a microscope (model FX-A; Nikon) equipped with 60x/1.4 NA Plan Apo oil objective. The digital images were acquired with a cooled CCD camera (SenSys; Photometrics) controlled by QED camera software (QED Imaging, Inc.).
Quantitation of MT depolymerization activity in vivo
To quantify the microtubule (MT) depolymerization activity, we collected digital images of the transfected cells at the same exposure for each set of GFP and MT images and below saturation level of the camera (the 4,096 gray-value maximum). All images were saved as 12-bit TIFF images for quantification by NIH Image 1.62 software and Microsoft Excel. Mean EGFP or tubulin fluorescence intensities over the entire cellular area were measured as the average gray value within the area. Mean fluorescence over the cell-free area in each image was subtracted from mean fluorescence over the cellular area to correct for background fluorescence. All analyzed cells were in interphase and were free of aggregates of overexpressed protein. 2090 transfected cells were measured for each construct with >500 cells quantified overall. To quantify the MT polymer left in the transfected cells, we assigned an MT polymer value of 0% to the cells transfected with full-length (FL) MCAK, which displayed no MTs and a mean tubulin fluorescence intensity equal to 734 gray-scale units. Similar to nontransfected cells, EGFP-transfected cells showed normal levels of MT polymer, which was given a 100% value. The mean tubulin fluorescence of EGFP-transfected cells was equal to 1,902 gray-scale units. To quantify the percentage of MT polymer left in the cells transfected with mutant MCAK constructs, we used the following formula: {[(the mean tubulin fluorescence intensity of mutant-transfected cells 734)/1,168] x 100%} where 1168 is the difference between EGFP control and FL MCAK mean gray values.
In vitro MT depolymerization assays
The bacterially expressed COOH-terminally 6his-tagged proteins were purified as described in Maney et al. (2001). The active motor concentration was determined as the concentration of nucleotide-binding sites because there is one ATP-binding site per each monomeric motor protein analyzed in this study in vitro. The concentration of nucleotide-binding sites was measured radiometrically as described by Coy et al. (1999). The depolymerization of taxol-stabilized MTs, polymerized from bovine tubulin (Cytoskeleton, Inc.), was performed as described in Maney et al. (2001). In brief, for the assay shown in Fig. 5 A, 100 nM of the active motor proteins in 20 µl of the elution buffer (250 mM imidazole, pH 7.0, 300 mM KCl, 0.2 mM MgCl2, 0.01 mM Mg-ATP, 1 mM DTT, and 20% glycerol) was mixed with 1,500 nM taxol-stabilized MTs in 80 µl of BRB80 (80 mM Pipes, pH 6.8, 1 mM EGTA, and 1 mM MgCl2), 12.5 µM taxol, 1 mM DTT, and 1.25 mM Mg-ATP incubated at 24 ± 1°C for 10 min, and then centrifuged at 30 psi for 10 min. For the assay shown in Fig. 5 B, 100 nM of the active motor proteins in 2.3 µl of the elution buffer was mixed with 1,500 nM taxol-stabilized MTs in 97.7 µl of BRB20 (20 mM Pipes, pH 6.8, 1 mM EGTA, and 1mM MgCl2), 10.2 µM taxol, 1 mM DTT, 10 µg/ml BSA, and 1.02 mM Mg-ATP with or without addition of KCl up to 60 mM, incubated at 24 ± 1°C for 10 min, and then centrifuged at 30 psi for 10 min. Supernatants and pellets were assayed for the presence of tubulin on Coomassie-stained SDS-polyacrylamide gels (Novex). Gels were calibrated and quantified using NIH Image 1.62 software. The percentage of tubulin in the supernatant of no-motor control reactions was subtracted from the percentage of tubulin in supernatant of the motor-containing reactions. The percentages of tubulin released in supernatant of the motor-containing reactions were then normalized with the percentages of tubulin in the corresponding pellet fractions.
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Footnotes |
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Acknowledgments |
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This work was supported by the National Institutes of Health grant (GM53654A) and the Department of Defense grant (DAMD17-01-1-0450) to L. Wordeman.
Submitted: 17 May 2002
Revised: 16 October 2002
Accepted: 17 October 2002
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References |
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