![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 18, Issue 7, 2503-2510, July 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138; Department of Molecular and Cell Biology, and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720; and Department of Biological Chemistry and Molecular Pharmacology, and Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115
Submitted February 16, 2007;
Revised April 10, 2007;
Accepted April 17, 2007
Monitoring Editor: Kerry Bloom
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
125-base pair centromere to bind a single microtubule (MT) of the mitotic spindle (McAinsh et al., 2003
). Structural studies of these subcomplexes coupled with functional experiments have begun to add molecular detail to our picture of kinetochores. For example, the yeast Ndc80 complex forms a 570-Å coiled coil rod with two globular domains at each end (Wei et al., 2005), suggesting a role as a molecular adaptor. The globular domain of the human Ndc80 subunit, Hec1, folds into a MT-binding calponin homology domain (Wei et al., 2007), and the worm Ndc80 homolog directly binds MTs and facilitates association of additional kinetochore complexes (Cheeseman et al., 2006). The yeast DASH complex (also called the Dam1 complex) forms closed rings around MTs (Miranda et al., 2005). DASH can be induced to move along a MT and to translate with the depolymerizing end (Westermann et al., 2005, 2006). The interaction resists several picoNewtons of force (Asbury et al., 2006). DASH may therefore serve to help tether kinetochores to MTs during chromosome segregation.
The DASH complex is necessary for faithful segregation of chromosomes in mitosis. The Saccharomyces cerevisiae complex consists of 10 essential subunits: Dam1p, Duo1p, Dad1p, Dad2p, Spc19, Spc34p, Ask1p, Dad3p, Dad4p, and Hsk3p (Figure 1A) (Cheeseman et al., 2001, 2002; Janke et al., 2002; Li et al., 2002, 2005). Loss of functional DASH results in sister chromatids attached to the same spindle pole body, an arrangement that leads to unequal segregation. The homologous complex in Schizosaccharomyces pombe contains similar subunits and localizes to kinetochores as well as to MT plus ends (Liu et al., 2005; Sanchez-Perez et al., 2005). Although not essential in fission yeast, loss of functional DASH also results in segregation defects. The ring structure observed in vitro may contribute to proper segregation by acting as a processivity factor for kinetochores, allowing chromosomes to remain attached to depolymerizing MT plus ends during anaphase. Rings are commonly found in assemblies that must remain attached to a linear substrate, for example, in the assemblies that carry out DNA replication (Hingorani and O'Donnell, 2000). The mechanism by which the DASH ring binds to and translates along MTs is therefore not only an integral part of how kinetochores work, but also an instance of a molecular solution to a ubiquitous structural challenge.
Negative stain electron microscopy (EM) of MTs decorated with DASH reveals a gap between the inner diameter of DASH and the outer diameter of the MT (Miranda et al., 2005; Westermann et al., 2005, 2006). No discrete mass of DASH can be seen abutting the MT, but polypeptide extensions not visibly contrasted by negative stain may position DASH rings around microtubules. On the MT side, the acidic C-termini of tubulin may project from the surface, but the 10–20 amino acids of these unstructured extensions (Lowe et al., 2001) are unlikely to bridge the 50–100-Å gap of the DASH-MT interface. Because rotary shadowed EM preparations show that DASH is relatively compact, but hydrodynamic measurements yield a Stokes' radius much larger than expected for a globular particle (Miranda et al., 2005), we believe that extended projections are probably present. No obvious candidates for those bridging elements can be deduced from examination of amino acid sequences of DASH components. We have therefore carried out a series of experiments to determine the location and MT-binding properties of flexible, protease-sensitive polypeptides on DASH. This mapping adds detail to models of how DASH binds to and translates along MTs.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The plasmid containing Ask1p with a C-terminal Strep-tag was generated in an analogous manner. Constructs without HSK3 were obtained by omitting the subcloning step incorporating HSK3 from pET21aTr into the expression vector. This procedure was repeated to generate similar plasmids encoding Ask1p, Dad1p, Dad2p, Dad3p, Dad4p, Spc19p, and Spc34p with C-terminal hexahistidine tags. A construct without DAM1 was obtained by omitting the subcloning step incorporating DAM1 from pET3aTr into the expression vector. This procedure generated a plasmid encoding Spc34p with a C-terminal hexahistidine tag and Ask1p with a C-terminal Strep-tag. Vectors with only DAD1 and DAD3 were generated by sequentially subcloning DAD1 and DAD3 from pET3aTr into the compatible restriction sites of the first and third cassettes in pST39 as NheI/BamHI and SacI/KpnI fragments, respectively. This procedure was repeated to generate similar plasmids encoding Dad1p and Dad3p with C-terminal hexahistidine tags.
Protein Expression and Purification
DASH complexes were purified with combinations of affinity, ion exchange, and size-exclusion chromatography (Miranda et al., 2005). DASH subcomplexes were purified in the same manner as heterodecameric DASH containing a protein with a C-terminal hexahistidine tag except that the ion exchange column and peptide treatments were omitted. DASH containing Ask1p with a C-terminal Strep-tag was purified in a similar manner with modifications. Cells were lysed in 50 mM phosphate, 500 mM NaCl, 1 mM mercaptoethanol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and Complete Protease Inhibitors (Roche, Indianapolis, IN), pH 7.5. The supernatant was bound to a Strep-Tactin Sepharose (IBA, St. Louis, MO) column and washed in the same buffer without protease inhibitors but including 1 mM ATP and 0.1 mg/ml of the synthetic peptide NRLLLTG. The complex was eluted in the wash buffer supplemented with 2.5 mM desthiobiotin. The eluant was concentrated, fresh ATP added to 1 mM, and additional NRLLLTG peptide added to 250-fold molar excess relative to the DASH. The complex was then purified on a Superose 6 10/300 column (Amersham, Piscataway, NJ) equilibrated in 25 mM HEPES, 500 mM NaCl, 1 mM mercaptoethanol, and 1 mM EDTA, pH 7.4.
Microtubule Decoration
Bovine tubulin was polymerized in the presence of paclitaxel (Miranda et al., 2005). MT cosedimentation assays were performed by mixing 10 µl of 1 mg/ml DASH or subcomplex in 25 mM HEPES, 500 mM NaCl, 1 mM mercaptoethanol, 1 mM EDTA, pH 7.4, with 90 µl of 0.055 mg/ml MTs in 25 mM HEPES, 100 mM NaCl, 1 mM GTP, 10 mM paclitaxel, and 1% dimethyl sulfoxide (DMSO), pH 7.4. Control samples were treated with buffer instead of DASH or subcomplex. All samples were incubated for 20 min at room temperature and then spun at 16,000 x g for 10 min at room temperature.
Limited Proteolysis
DASH at 1 mg/ml and elastase (Sigma, St. Louis, MO) at 0.1 mg/ml, both in 25 mM HEPES, 500 mM NaCl, 1 mM mercaptoethanol, and 1 mM EDTA, pH 7.4, were mixed at a ratio of 9:1. Reactions were incubated at 4°C and quenched by the addition of SDS-PAGE buffer and boiling. The initial time-point sample was treated with buffer instead of elastase. For further purification, 500 µl of DASH proteolyzed for 30 min was fractionated on a Superose 6 10/300 column (Amersham) equilibrated in 25 mM HEPES, 500 mM NaCl, 1 mM mercaptoethanol, and 1 mM EDTA, pH 7.4. For experiments testing the ability of MTs to protect DASH against elastase digestion, 0.05 mg/ml MTs decorated with 0.1 mg/ml DASH was mixed with 0.01 mg/ml elastase in 25 mM HEPES, 150 mM NaCl, 1 mM GTP, 10 µM paclitaxel, 1% DMSO, pH 7.4, at room temperature. The initial time-point sample was treated with buffer instead of elastase. Control samples were treated with buffer instead of MTs. For subtilisin treatment of MTs, 9 vol of polymerized MTs at 5 mg/ml were mixed with 1 vol of 2 mg/ml subtilisin (Sigma) in 80 mM PIPES, 1 mM MgCl2, and 1 mM EGTA, pH 6.9. The reaction was incubated at 30°C for 60 min (Skiniotis et al., 2004), quenched by the addition of 10 mM PMSF from a 100 mM stock in ethanol, spun at 16,000 x g for 10 min, and resuspended in 25 mM HEPES, 100 mM NaCl, 1 mM GTP, 10 µM paclitaxel, 1% DMSO, and 1 mM PMSF, pH 7.4. Control samples were treated with buffer instead of subtilisin. All subsequently used buffers contained 1 mM PMSF. Western blots verifying cleavage of the C-terminus of 
-tubulin were probed with the mAb JDR.3B8 (Sigma), which is specific for that epitope (Banerjee et al., 1988). Similarly, cleavage of the C-terminus of 
-tubulin was verified by probing with the mAb YL1/2 (AbD Serotec, Raleigh, NC), which is specific for the C-terminal tyrosine residue (Wehland et al., 1984).
Phosphorylation In Vitro
One vol of DASH at 1 mg/ml in 25 mM HEPES, 500 mM NaCl, 1 mM mercaptoethanol, 1 mM EDTA, pH 7.4, and 1 vol of human Cdc2-cyclin B (New England Biolabs, Beverly, MA) in 50 mM HEPES, 100 mM NaCl, 1 mM dithiothreitol, 100 µM EDTA, 50% glycerol, and 0.01% Brij 35, pH 7.5, 1 vol of 500 mM Tris, 100 mM MgCl2, 20 mM dithiothreitol, 10 mM EGTA, 50% glycerol, and 0.1% Brij 35, pH 7.5, and 1 vol of 2 mM ATP were diluted into water to a total of 10 vol. Alternatively, 2 vol of human Cdk2-cyclin A (New England Biolabs) were substituted. Reactions were incubated at 4°C overnight. Control samples were treated with water instead of ATP. For gel electrophoresis, samples were quenched by the addition of SDS-PAGE buffer and boiling. EM and mass spectrometry (MS) experiments were performed with reactions including human Cdk2-cyclin A and DASH containing Spc34p with a C-terminal His-tag.
Mass Spectrometry
Peptide maps of proteins in gel bands were obtained by in-gel trypsinolysis and peptide extraction using conventional methods and analysis by matrix-assisted laser desorption ionization time-of-flight MS (Bruker, Billerica, MA). Intact proteins were desalted by polystyrene-divinylbenzene microbore reversed-phase liquid chromatography, and intact masses were measured by matrix-assisted laser desorption ionization time-of-flight MS (Bruker) as well as by electrospray ionization-ion trap MS (Bruker-Agilent, Santa Clara, CA). Phosphorylation was assessed by microwave trypsinolysis (CEM, Matthews, NC) of the proteins of interest for 7 min with 25 W at 50°C followed by desalting on a ZipTip (Millipore, Bedford, MA) and flow-injection analysis of the entire tryptic digest on a 9.4-T electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometer (Bruker).
Electron Microscopy
Negatively stained specimens of decorated MTs were prepared with uranyl formate (Miranda et al., 2005). Samples of MTs decorated by DASH
Hsk3p 6mer contained 0.1 mg/ml MTs and 0.2 mg/ml DASHHsk3p 6mer. Samples of MTs decorated by DASH modified by Cdk were prepared by mixing 5.5 µl of 5 mg/ml MTs in 74 mM PIPES, 1 mM GTP, 1 mM MgCl2, 1 mM EGTA, 100 µM paclitaxel, and 7.5% DMSO, pH 6.9, to 50 µl of a kinase reaction. The sample was spun, resuspended, and then absorbed onto a grid. Scanning transmission electron microscopy (STEM) experiments were performed at Brookhaven National Laboratory. MTs at 0.05 mg/ml were partially decorated with 0.01 mg/ml DASH for observation of single rings (Miranda et al., 2005). The grids were freeze-dried overnight and transferred into the microscope under vacuum. Digital dark-field images were obtained at an operating voltage of 40 kV. Control samples were treated with buffer instead of DASH. Experiments were performed with DASH containing Spc34p with a C-terminal His-tag. Data were analyzed with PCMASS29 (Wall and Simon, 2001), which performs background calculations to subtract from the summed intensity measurements. For analysis, each particle was chosen manually from a 5120 x 5120-Å scan with 10-Å spacing and centered in a 400 x 800-Å box. Two data sets were analyzed, one including all reasonably unencumbered particles based on visual inspection and a smaller collection including only particles that met more stringent visual criteria for background and proximity to other particles. Molecular masses were calibrated using a mass/length value of 13.1 kDa/Å for tobacco mosaic virus, which is included as a control on all grids.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
; Westermann et al., 2005), but eDASH does not (Figure 1C). Nonetheless, MTs do not protect DASH from elastase digestion (Figure 1D). Moreover, the resulting eDASH, which is indistinguishable from eDASH prepared in the absence of MTs, no longer cosediments with MTs in the preparation (data not shown). We would expect contacts between the MT-bound heterodecamers within a ring to be shielded from proteolysis, but not dynamic, flexible extensions across the gap between the ring and MT. We believe that proteolysis eliminates direct DASH-MT interactions of this kind, thereby destabilizing the ring. An alternate possibility, that proteolysis destroys contacts between neighboring heterodecamers around the ring, is less consistent with the apparent lack of any protection from proteolysis in a MT-bound ring of DASH complexes relative to free DASH heterodecamer. We also note that eDASH tends to oligomerize at high concentrations during size-exclusion chromatography (data not shown), a property observed with DASH (Miranda et al., 2005). This oligomerization suggests that the interface between eDASH heterodecamers within a ring may still be intact. Lack of protection of MT-bound DASH from proteolysis also suggests that either the contacting peptides are still exposed while bound to MTs or that the on and off rates of binding to MTs are high enough to free a reasonable fraction of the contacting peptides at any one moment. The width of the gap between the major mass of a DASH ring and the MT surface is 50–100 Å, so a polypeptide extension from a ring subunit might be easily accessible to elastase, an enzyme 40–50 Å in diameter (Shotton and Watson, 1970), even when the tip of the extension is docked against the MT wall. Thus, the cleavage data suggest that exposed, extended parts of DASH bridge the ring and the MT lattice.
Microtubule Binding of DASH Subcomplexes
Characterization of the MT-binding properties of DASH subcomplexes suggests which proteins are likely to contribute extended arms to the MT-DASH interface. We have purified a variety of distinct subcomplexes by deleting different subunits from our coexpression vector (Miranda, 2006). DASHHsk3p 3mer, which contains Ask1p, Dad2p, and Dad4p (Figure 2A), does not cosediment with MTs (Figure 3A), but DASHHsk3p 6mer, which contains Dam1p, Duo1p, Spc34p, Spc19p, Dad1p, and Dad3p (Figure 2A), does (Figure 3B). The limited proteolysis experiments suggest Ask1p, Dam1p, and Duo1p as candidates for providing MT bridges. Because DASHHsk3p 3mer contains Ask1p and does not cosediment with MTs, we conclude that this subunit is not sufficient for establishing a functional DASH-MT interaction. DASHHsk3p 6mer contains both Dam1p and Duo1p, suggesting that the extensions cleaved by elastase on these two proteins are sufficient for association with MTs. Comparing EM images of undecorated (Figure 4A) and decorated MTs (Figure 4B) reveals an unorganized clustering of DASHHsk3p 6mer on the surface of the MT rather than organized rings. Extended elements of Dam1p, the C-terminus of Duo1p, or both are sufficient to form some interaction with MTs, but ring assembly and MT binding remain separable functions.
|
|
|
Dam1p contains Ask1p, Spc34p, Spc19, Dad2p, Dad4p, and Hsk3p (Figure 2B). MS analysis of the species between Spc34p and Spc19p identified 41% of Spc34p; mapped tryptic peptides spanned residues 99–274 out of 295. Edman degradation yielded the sequence MKRNRR, indicating that the observed band corresponds to an N-terminal truncation beginning at residue 93. The limited proteolysis experiments and MT cosedimentation assays of DASHHsk3p subcomplexes predict that DASHDam1p 6mer will not bind MTs, and we have confirmed this prediction (Figure 3C). During coexpression experiments, we also observed frequent overexpression of a Dad1p/Dad3p heterodimer relative to DASH (data not shown). The Dad1p/Dad3p heterodimer can be expressed and purified independently (Figure 2C). This subcomplex does not interact with MTs (Figure 3D), again consistent with previous conclusions. The absence of Dam1p, Duo1p, Dad1p, and Dad3p from DASHDam1p 6mer is noteworthy. Because Dad1p and Dad3p form a separable structural unit, Dam1p and Duo1p may form another distinct structural unit, a suggestion supported by their joint role in providing flexible extensions from MT interaction. The sum of the cosedimentation experiments is consistent with the interpretation that Dam1p and Duo1p cooperate to form the principal connection between DASH and MTs.
Limited Proteolysis of Microtubules
We have previously reported preliminary EM experiments suggesting that removal of the acidic C-termini of both and tubulin by subtilisin does not abolish DASH binding (Miranda et al., 2005). The opposite conclusion has been drawn by others on the basis of EM and fluorescence binding assays (Westermann et al., 2005). Our treatment of MTs with subtilisin alters electrophoretic mobility of both tubulin subunits on a gel (Figure 5A). Although this mobility shift is often a sufficient indicator of removal of both the and tubulin termini, we have also verified cleavage with epitope-specific monoclonal antibodies. The JDR.3B8 antibody detects the C-termini of tubulin on mock-treated MTs, but not on subtilisin-treated MTs (Figure 5B). Similarly, the YL1/2 antibody detects the intact subunit C-terminus, but almost all of the epitope is removed by subtilisin treatment (Figure 5C). DASH binds to subtilisin-digested MTs with similar affinity as it does to mock-digested MTs (Figure 5D). Moreover, we observe DASH rings with the same frequency on mock-treated (Figure 5E) and subtilisin-treated MTs (Figure 5F). The acidic C-termini of and tubulin are therefore not essential for proper formation of the DASH-MT interface, and we suggest that the flexible extensions of DASH dock against the cylindrical wall of the microtubule.
|
), and mutation of both putative phosphorylation sites, S216 and S250, yields defects in anaphase spindle MT dynamics and chromosome segregation (Higuchi and Uhlmann, 2005). Incubation of DASH with Cdks in vitro results in a detectable electrophoretic mobility shift for only one component, Ask1p (Supplementary Figure 3, A and B). Time-of-flight MS of unmodified and modified Ask1p yields a molecular weight of 32058 (expected 32071) and 32132 (expected 32151) Da, respectively. The shift of 74 (expected 82) Da suggests that one and only one site is modified; no masses corresponding to unphosphorylated or diphosphorylated species are observed. Peptide mass mapping of modified Ask1p found the phosphorylated peptide QLAHEEHINNDGDNDDENSNNIESSPLK, which contains S250. The unphosphorylated form of the peptide was not found. A peptide containing S216, ISLLLQQQYGSSSSMVPSPIVPNK, is observed in the unphosphorylated state with no detection of a mass corresponding to the phosphorylated form. Phosphorylated DASH cosediments with MTs as efficiently as unphosphorylated DASH (data not shown). EM experiments also show that DASH phosphorylated by Cdk still forms rings around MTs with a similar frequency as does unphosphorylated DASH (data not shown). Cdks therefore modify DASH in vitro only on S250 of Ask1p, and this phosphorylation event does not affect ring assembly. We conclude that the anaphase defects seen when S250 is mutated must involve regulation of some other aspect of DASH activity.
Scanning Transmission Electron Microscopy
We have determined the molecular masses of MTs decorated with DASH rings by direct mass measurement with STEM. Substoichiometric amounts of DASH relative to tubulin were bound to MTs in order to favor the formation of single rings spaced sufficiently apart from each other to allow mass measurement of each particle. Unstained, lyophilized preparations were examined in the STEM. Images of particles from dark field micrographs were boxed into 400 x 800-Å arrays for analysis; high background as a result of binding conditions and specimen damage from mechanical manipulations resulted in only a small number of particles suitable for analysis. MTs appear as cylinders without the characteristic tubulin heterodimer repeat pattern probably because of radiation damage (Figure 6A). Our measurements of a 400-Å-long MT segment yielded a molecular mass of 8 MDa (Table 1), close to the 7.8 MDa expected for a 14 protofilament MT. We performed similar measurements for MT segments decorated with DASH. MTs with single (Figure 6B) and double rings (Figure 6C) yielded molecular masses of 13 and 18 MDa, respectively. The SD of our measurements ranged from 1 to 2 MDa, 4–13% depending on the data set. The precision and accuracy of our measurements are similar to those obtained with kinesin-MT complexes (Hoenger et al., 2000). We calculate the molecular mass of a ring as 5 MDa, with propagated errors ranging from 0.5 to 2.5 MDa, 10–60%, depending on the data set (Table 1). The molecular mass of one DASH heterodecamer is 0.2 MDa; each DASH ring therefore contains 25 ± 5 heterodecamers.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), and the rings can indeed translate quite freely (Asbury et al., 2006). The processivity factors that keep a DNA replication complex from dissociating slide loosely along the double helix (Hingorani and O'Donnell, 2000). A similar electrostatic sliding has been suggested for DASH (Westermann et al., 2006), but the observations reported here lead us to seek an alternative mechanism. In particular, two results favor direct contacts. First, we find that the negatively charged tubulin C-termini on the MT surface do not appear to be essential for ring formation. Second, the DASHHsk3p 6mer subcomplex, which contains Dam1p and Duo1p, binds without forming rings. Were encirclement the only property holding DASH components onto a MT, we would expect ring formation to be essential for binding. We also note that sliding clamps in DNA replication need an energy-dependent clamp loader, whereas DASH rings assemble spontaneously around MTs.
Systematic analysis of the MT-binding properties of limited proteolysis products and various subcomplexes defines the nature of the DASH-MT interface. Polypeptide arms from both DASH and MTs could contribute to a molecular bridge across the gap observed between a DASH ring and the MT in EM images, but we determined that DASH alone makes functionally significant contacts across this space. Specifically, Dam1p, Duo1p, or both are probably responsible for MT binding (Figure 7A). Cleavage of these proteins with elastase in limited proteolysis experiments abrogates the DASH-MT interaction; presence of these proteins in a subcomplex allows MT-binding. No other subunits meet both criteria. Our conclusion is consistent with previously reported observations from cosedimentation assays. In vitro–translated Dam1p binds MTs (Hofmann et al., 1998), and a 138-amino acid truncation of the Dam1p C-terminus slightly lowers the affinity of recombinant DASH for MTs (Westermann et al., 2005). Our experiments also show that upon removal of the acidic C-termini of both and tubulin, DASH still binds to and forms rings around MTs. A contrary conclusion was reached with different MT proteolysis protocols, cleavage verification methods, and binding assays (Westermann et al., 2005); we cannot rationalize the difference in results. Our data suggest a DASH-MT interface in which extensions from DASH rings reach across a gap between the ring and MT and dock on the MT wall (Figure 7, B and C).
|
20–30 heterodecamers, thereby defining the multiplicity of potential MT-binding contacts. This number is somewhat larger than other estimates. An apparent 16-fold symmetry of rare EM images of DASH bound to MTs led to the conclusion that rings contain 16 heterodecamers (Westermann et al., 2006); the possibility of 32 copies was not considered. Fluorescence measurements of Ask1p copy number in vivo suggest 16–20 heterodecamers (Joglekar et al., 2006). STEM is a direct measurement of the molecular mass of an isolated particle and depends on no assumptions other than appropriate calibration of the instrument. In any case, all the various measurements lead to the conclusion that the polyvalent DASH ring contains more sets of MT-binding extensions than the number of MT protofilaments. We note that polyvalent attachment through extended arms or loops is fully compatible with diffusive motion, provided that the individual interactions are weak and that an individual arm may reach multiple tubulin docking sites. A range of potential attachment directions is consistent with the variable geometry with which DASH can decorate MTs in vitro, as rings and helices of various pitch. The lack of protection from proteolysis of Dam1p and Duo1p suggests that the cleavable extensions on these subunits are in dynamic equilibrium between the on and off positions. Indeed, all of the arms are unlikely to be engaged at once as the resulting avidity would probably yield too strong an attachment for translation along a MT. Thus, we imagine that at any moment only a fraction of the arms contact the MT. As the population of attached arms shifts, the ring will undergo diffusional translation along the MT, moving in one direction or the other at each step depending on whether a preponderance of the newly attaching arms lie toward one end or the other.
Molecular motors have been thought to move the kinetochore actively toward the poles during anaphase (Hyman et al., 1992), but various lines of evidence suggest that motors are not essential in mitosis. Chromosome movement in S. cerevisiae, as measured by transient sister separation during metaphase, is unaffected by individual deletion of any of the nuclear kinesin-like motors (Tytell and Sorger, 2006). Poleward kinetochore movement in S. pombe during anaphase is unaffected by deletion all three known minus end–directed motors (Grishchuk and McIntosh, 2006). Experiments with chromosomes and MTs in vitro demonstrate that MT depolymerization alone is sufficient to drive chromosome segregation toward the MT minus end (Koshland et al., 1988).
At least two models have been proposed to explain how MT depolymerization drives chromosome motion in anaphase. The conformational wave model (Koshland et al., 1988) suggests that the curling of protofilaments at the depolymerizing end of a MT exerts force directly on the kinetochore, a process that propagates poleward. The diameter of the DASH ring would prevent it from falling off during depolymerization, making DASH a suitable force transducer (Miranda et al., 2005; Westermann et al., 2005). The biased-diffusion model (Hill, 1985) presumes that multiple attachment sites are present and that the energy required to move from one site on the MT lattice to another is sufficiently small to allow rapid diffusion of the kinetochore. At the depolymerizing plus end, new attachment sites are selectively available on one side of the ring and not the other, biasing diffusion toward the minus end. Curling could also contribute, especially if the docking sites were at the protofilament interface. Our picture of associating and dissociating bridges between the body of the DASH ring and the MT surface is compatible with either model.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
Present address: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158.
Address correspondence to: Stephen C. Harrison (harrison{at}crystal.harvard.edu).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988). A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin. J. Biol. Chem 263, 3029–3034.
Cheeseman, I. M., Anderson, S., Jwa, M., Green, E. M., Kang, J., Yates, J. R., 3rd, Chan, C. S., Drubin, D. G., and Barnes, G. (2002). Phospho-regulation of kinetochore-microtubule attachments by the Aurora kinase Ipl1p. Cell 111, 163–172.[CrossRef][Medline]
Cheeseman, I. M., Brew, C., Wolyniak, M., Desai, A., Anderson, S., Muster, N., Yates, J. R., Huffaker, T. C., Drubin, D. G., and Barnes, G. (2001). Implication of a novel multiprotein Dam1p complex in outer kinetochore function. J. Cell Biol 155, 1137–1145.
Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M., and Desai, A. (2006). The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127, 983–997.[CrossRef][Medline]
Grishchuk, E. L., and McIntosh, J. R. (2006). Microtubule depolymerization can drive poleward chromosome motion in fission yeast. EMBO J 25, 4888–4896.[CrossRef][Medline]
Higuchi, T., and Uhlmann, F. (2005). Stabilization of microtubule dynamics at anaphase onset promotes chromosome segregation. Nature 433, 171–176.[CrossRef][Medline]
Hill, T. L. (1985). Theoretical problems related to the attachment of microtubules to kinetochores. Proc. Natl. Acad. Sci. USA 82, 4404–4408.
Hingorani, M. M., and O'Donnell, M. (2000). A tale of toroids in DNA metabolism. Nat. Rev. Mol. Cell Biol 1, 22–30.[Medline]
Hoenger, A., Thormahlen, M., Diaz-Avalos, R., Doerhoefer, M., Goldie, K. N., Muller, J., and Mandelkow, E. (2000). A new look at the microtubule binding patterns of dimeric kinesins. J. Mol. Biol 297, 1087–1103.[CrossRef][Medline]
Hofmann, C., Cheeseman, I. M., Goode, B. L., McDonald, K. L., Barnes, G., and Drubin, D. G. (1998). Saccharomyces cerevisiae Duo1p and Dam1p, novel proteins involved in mitotic spindle function. J. Cell Biol 143, 1029–1040.
Hyman, A. A., Middleton, K., Centola, M., Mitchison, T. J., and Carbon, J. (1992). Microtubule-motor activity of a yeast centromere-binding protein complex. Nature 359, 533–536.[CrossRef][Medline]
Janke, C., Ortiz, J., Tanaka, T. U., Lechner, J., and Schiebel, E. (2002). Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation. EMBO J 21, 181–193.[CrossRef][Medline]
Joglekar, A. P., Bouck, D. C., Molk, J. N., Bloom, K. S., and Salmon, E. D. (2006). Molecular architecture of a kinetochore-microtubule attachment site. Nat. Cell Biol 8, 581–585.[CrossRef][Medline]
Koshland, D. E., Mitchison, T. J., and Kirschner, M. W. (1988). Polewards chromosome movement driven by microtubule depolymerization in vitro. Nature 331, 499–504.[CrossRef][Medline]
Li, J. M., Li, Y., and Elledge, S. J. (2005). Genetic analysis of the kinetochore DASH complex reveals an antagonistic relationship with the ras/protein kinase A pathway and a novel subunit required for Ask1 association. Mol. Cell. Biol 25, 767–778.
Li, Y., Bachant, J., Alcasabas, A. A., Wang, Y., Qin, J., and Elledge, S. J. (2002). The mitotic spindle is required for loading of the DASH complex onto the kinetochore. Genes Dev 16, 183–197.
Li, Y., and Elledge, S. J. (2003). The DASH complex component Ask1 is a cell cycle-regulated Cdk substrate in Saccharomyces cerevisiae. Cell Cycle 2, 143–148.[Medline]
Liu, X., McLeod, I., Anderson, S., Yates, J. R., 3rd, and He, X. (2005). Molecular analysis of kinetochore architecture in fission yeast. EMBO J 24, 2919–2930.[CrossRef][Medline]
Lowe, J., Li, H., Downing, K. H., and Nogales, E. (2001). Refined structure of alpha beta-tubulin at 3.5 A resolution. J. Mol. Biol 313, 1045–1057.[CrossRef][Medline]
McAinsh, A. D., Tytell, J. D., and Sorger, P. K. (2003). Structure, function, and regulation of budding yeast kinetochores. Annu. Rev. Cell Dev. Biol 19, 519–539.[CrossRef][Medline]
Miranda, J. J., De Wulf, P., Sorger, P. K., and Harrison, S. C. (2005). The yeast DASH complex forms closed rings on microtubules. Nat. Struct. Mol. Biol 12, 138–143.[CrossRef][Medline]
Miranda, J. L. (2006). Structure of the Yeast DASH Complex, a Kinetochore-Microtubule Interface, Cambridge, MA: Harvard University, Ph.D. Thesis.
Sanchez-Perez, I., Renwick, S. J., Crawley, K., Karig, I., Buck, V., Meadows, J. C., Franco-Sanchez, A., Fleig, U., Toda, T., and Millar, J. B. (2005). The DASH complex and Klp5/Klp6 kinesin coordinate bipolar chromosome attachment in fission yeast. EMBO J 24, 2931–2943.[CrossRef][Medline]
Shotton, D. M., and Watson, H. C. (1970). The three-dimensional structure of crystalline porcine pancreatic elastase. Philos. Trans. R. Soc. Lond. B Biol. Sci 257, 111–118.[CrossRef][Medline]
Skiniotis, G., Cochran, J. C., Muller, J., Mandelkow, E., Gilbert, S. P., and Hoenger, A. (2004). Modulation of kinesin binding by the C-termini of tubulin. EMBO J 23, 989–999.[CrossRef][Medline]
Tytell, J. D., and Sorger, P. K. (2006). Analysis of kinesin motor function at budding yeast kinetochores. J. Cell Biol 172, 861–874.
Wall, J. S., and Simon, M. N. (2001). Scanning transmission electron microscopy of DNA-protein complexes. Methods Mol. Biol 148, 589–601.[Medline]
Wehland, J., Schroder, H. C., and Weber, K. (1984). Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2. EMBO J 3, 1295–1300.[Medline]
Wei, R. R., Al-Bassam, J., and Harrison, S. C. (2007). The Ndc80/HEC1 complex is a contact point for kinetochore-microtubule attachment. Nat. Struct. Mol. Biol 14, 54–59.[CrossRef][Medline]
Wei, R. R., Sorger, P. K., and Harrison, S. C. (2005). Molecular organization of the Ndc80 complex, an essential kinetochore component. Proc. Natl. Acad. Sci. USA 102, 5363–5367.
Westermann, S., Avila-Sakar, A., Wang, H. W., Niederstrasser, H., Wong, J., Drubin, D. G., Nogales, E., and Barnes, G. (2005). Formation of a dynamic kinetochore- microtubule interface through assembly of the Dam1 ring complex. Mol. Cell 17, 277–290.[CrossRef][Medline]
Westermann, S., Wang, H. W., Avila-Sakar, A., Drubin, D. G., Nogales, E., and Barnes, G. (2006). The Dam1 kinetochore ring complex moves processively on depolymerizing microtubule ends. Nature 440, 565–569.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  E. L. Grishchuk, A. K. Efremov, V. A. Volkov, I. S. Spiridonov, N. Gudimchuk, S. Westermann, D. Drubin, G. Barnes, J. R. McIntosh, and F. I. Ataullakhanov The Dam1 ring binds microtubules strongly enough to be a processive as well as energy-efficient coupler for chromosome motion PNAS, October 7, 2008; 105(40): 15423 - 15428. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. L. Grishchuk, I. S. Spiridonov, V. A. Volkov, A. Efremov, S. Westermann, D. Drubin, G. Barnes, F. I. Ataullakhanov, and J. R. McIntosh Different assemblies of the DAM1 complex follow shortening microtubules by distinct mechanisms PNAS, May 13, 2008; 105(19): 6918 - 6923. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Efremov, E. L. Grishchuk, J. R. McIntosh, and F. I. Ataullakhanov In search of an optimal ring to couple microtubule depolymerization to processive chromosome motions PNAS, November 27, 2007; 104(48): 19017 - 19022. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
