Dam1 Complex Research Articles

Deducing Kinetochore Protein Distributions along and Around a Microtubule Plus-End from FRET Measurements

The eukaryotic kinetochore is a sophisticated motor that moves and segregates chromosomes during cell division. As a microtubule-based motor, the kinetochore is distinct from all other motor proteins and microtubule-associated proteins: it attaches end-on to ∼ 40 nm zone behind the plus-end of one or more microtubules, and it acts as a force coupler rather than an active force generator. These characteristics ensure that chromosome movement is tightly coupled with polymer growth and shortening at the plus-end. They also strongly suggest that kinetochore protein architecture, the distributions of the microtubule-binding kinetochore proteins along and around the microtubule plus-end and their dynamics, dictate the molecular mechanisms that generate movement. We have developed a FRET-based approach to determine the nanometer-scale distributions of multiple copies of the three principle microtubule-binding kinetochore complexes: Dam1, Ndc80, and Spc105. Sensitized emission measurements are carried out in metaphase and anaphase budding yeast (Saccharomyces cerevisiae) cells expressing kinetochore proteins labeled with Cerulean (donor) and Venus (acceptor) at desired locations. Preliminary measurements probing the distribution of the Ndc80 complex molecules show that the microtubule-binding head domains of adjacent Ndc80 molecules are ∼ five nm apart, but this distance increases significantly at the other end of the molecule. The Dam1 complex is located more than 10 nm away from these head domains of Ndc80. We are developing Monte Carlo approaches to simulate the energy transfer processes among a known number of donor and acceptor molecules using known protein structures of some of the kinetochore proteins and the cylindrical microtubule lattice. These simulations will identify molecular distributions consistent with the FRET data, and illuminate the structural basis for microtubule end-coupled movement of the kinetochore.

The Ndc80 Loop Region Facilitates Formation of Kinetochore Attachment to the Dynamic Microtubule Plus End

SummaryProper chromosome segregation in mitosis relies on correct kinetochore-microtubule (KT-MT) interactions. The KT initially interacts with the lateral surface of a single MT (lateral attachment) extending from a spindle pole and is subsequently anchored at the plus end of the MT (end-on attachment) [1]. The conversion from lateral to end-on attachment is crucial because end-on attachment is more robust [2–4] and thought to be necessary to sustain KT-MT attachment when tension is applied across sister KTs upon their biorientation [1]. The mechanism for this conversion is still elusive. The Ndc80 complex is an essential component of the KT-MT interface [1, 5], and here we studied a role of the Ndc80 loop region, a distinct motif looping out from the coiled-coil shaft of the complex [6], in Saccharomyces cerevisiae. With deletions or mutations of the loop region, the lateral KT-MT attachment occurred normally; however, subsequent conversion to end-on attachment was defective, leading to failure in sister KT biorientation. The Ndc80 loop region was required for Ndc80-Dam1 interaction and KT loading of the Dam1 complex, which in turn supported KT tethering to the dynamic MT plus end [3, 7]. The Ndc80 loop region, therefore, has an important role in the conversion from lateral to end-on attachment, a crucial maturation step of KT-MT interaction.

The Dam1 ring binds to the E-hook of tubulin and diffuses along the microtubule

There has been much effort in recent years aimed at understanding the molecular mechanism by which the Dam1 kinetochore complex is able to couple microtubule depolymerization to poleward movement. Both a biased diffusion and a forced walk model have been proposed, and several key functional aspects of Dam1-microtubule binding are disputed. Here, we investigate the elements involved in tubulin-Dam1 complex interactions and directly visualize Dam1 rings on microtubules in order to infer their dynamic behavior on the microtubule lattice and its likely relevance at the kinetochore. We find that the Dam1 complex has a preference for native tubulin over tubulin that is lacking its acidic C-terminal tail. Statistical mechanical analysis of images of Dam1 rings on microtubules, applied to both the distance between rings and the tilt angle of the rings with respect to the microtubule axis, supports a diffusive ring model. We also present a cryo-EM reconstruction of the Dam1 ring, likely the relevant assembly form of the complex for energy coupling during microtubule depolymerization in budding yeast. The present studies constitute a significant step forward by linking structural and biochemical observations toward a comprehensive understanding of the Dam1 complex.

An assay to measure the affinity of proteins for microtubules by quantitative fluorescent microscopy

We report a fluorescence-based assay for measuring the affinity of microtubule binding proteins for microtubules. The affinity of any fluorescently tagged protein for taxol-stabilized microtubules can be measured with this assay. We describe the assay and provide a detailed protocol. Using this assay, we found that the affinity of the Dam1 complex for microtubules is decreased by the presence of free unpolymerized tubulin and is sensitive to the salt concentration in the binding buffer. These effects may account for the previous differences in binding affinities reported.

Kinetochore–microtubule interactions: steps towards bi-orientation

Eukaryotic cells segregate their chromosomes accurately to opposite poles during mitosis, which is necessary for maintenance of their genetic integrity. This process mainly relies on the forces generated by kinetochore-microtubule (KT-MT) attachment. During prometaphase, the KT initially interacts with a single MT extending from a spindle pole and then moves towards a spindle pole. Subsequently, MTs from the other spindle pole also interact with the KT. Eventually, one sister KT becomes attached to MTs from one pole while the other sister to those from the other pole (sister KT bi-orientation). If sister KTs interact with MTs with aberrant orientation, this must be corrected to attain proper bi-orientation (error correction) before the anaphase is initiated. Here, I discuss how KTs initially interact with MTs and how this interaction develops into bi-orientation; both processes are fundamentally crucial for proper chromosome segregation in the subsequent anaphase.

The Dad1 subunit of the yeast kinetochore Dam1 complex is an intrinsically disordered protein

The Dam1 complex is an important part of the yeast kinetochore. It mediates attachment of the chromosome to the mitotic spindle and is involved in chromatid separation initiated at anaphase. It is comprised of 10 individual subunits and has been observed to oligomerize in various ways as it interacts with microtubules, including forming a ring. This work explores the biochemical and biophysical properties of Dad1, one of the Dam1 complex subunits from the human fungal pathogen Candida albicans. Unlike its Saccharomyces cerevisiae counterpart, C. albicans Dad1 can be expressed as a soluble protein in Escherichia coli. Analysis of this protein’s hydrodynamic properties, thermostability and primary sequence have been conducted. As a result, we conclude that isolated Dad1 is an intrinsically disordered protein.

A non-ring-like form of the Dam1 complex modulates microtubule dynamics in fission yeast

The Dam1 complex is a kinetochore component that couples chromosomes to the dynamic ends of kinetochore microtubules (kMTs). Work in the budding yeast Saccharomyces cerevisiae has shown that the Dam1 complex forms a 16-unit ring encircling and tracking the tip of a MT in vitro, consistent with its cellular function as a coupler. Dam1 also forms smaller, nonring patches in vitro that track the dynamic ends of MTs. However, the identity of Dam1's functional form in vivo remains unknown. Here we report a comprehensive in vivo characterization of Dam1 in the fission yeast Schizosaccharomyces pombe. In addition to their dense localizations on kinetochores and spindle MTs during mitosis, we identify that Dam1 is also localized onto cytoplasmic MTs as discrete spots in interphase, providing the unique opportunity to analyze Dam1 oligomers at the single-particle resolution in live cells. Such analysis shows that each oligomer contains one to five copies of Dam1, and is able to "switch-rail" while moving along MTs, precluding the possibility of a 16-unit encircling structure. Dam1 patches track the plus ends of the shortening, but not the elongating, MTs and retard MT depolymerization. Together with Mal3, the EB1-like MT-interacting protein, cytoplasmic Dam1 plays an important role in maintaining proper cell shape. In mitosis, kinetochore-associated Dam1 appears to facilitate kMT depolymerization. Together, our findings suggest that patches, instead of rings, are the physiologically functional forms of Dam1 in pombe. Our findings help establish the benchmark parameters of the Dam1 coupler and elucidate the mechanism of its functions.

Native Capillary Isoelectric Focusing for the Separation of Protein Complex Isoforms and Subcomplexes

Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.

A method for reverse interactome analysis: High‐resolution mapping of interdomain interaction network in Dam1 complex and its specific disorganization based on the interaction domain expression

An experimental methodology that facilitates functional analysis of numerous protein-protein interactions, which have been found in genome-wide interactome researches, has long been awaited. We propose herein an antagonistic inhibition-based approach. The antagonizing polypeptide is generated in the course of interaction domain mapping based on yeast 2-hybrid (Y2H) screening coupled with in vitro convergence of the Y2H-selected fragments, which is performed in a formatted procedure. Using the coupled methodology, we first performed a high-resolution mapping of an interdomain interaction network within budding yeast's Dam1 complex. Dam1 complex is a kinetochore protein complex composed of 10 essential subunits including Spc34p and Spc19p. The high-resolution mapping revealed the overall network structure within the complex for the first time: Dam1 components form into two separated subnetworks on N-terminal scaffolding domains of Spc34p and Spc19p, and the coiled-coil interaction in their C-terminal domains connects the subnetworks. Secondly, we show that the domain fragments converged in the high-resolution mapping acted as potent inhibitors for the endogenous interactions when episomally overexpressed. The in vivo Dam1 interaction targeting with the fragments conferred a similar phenotype on the host cells; a critical and irreversible damage, which was accompanied with disturbed budding and chromosome mis-segregation as a result of disorganized spindle. These phenotypes were strongly related to the cellular function of the Dam1 complex. The results and approach we demonstrated herein not only shed light on the Dam1 molecular architecture but also pave the road to reverse-interactome analysis and discoveries of novel drugs that target disease-related protein-protein interactions.

Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B.

The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore-microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore-microtubule attachment is regulated by conserved signals for error correction.

The Dam1 complex confers microtubule plus end–tracking activity to the Ndc80 kinetochore complex

Kinetochores must remain associated with microtubule ends, as they undergo rapid transitions between growth and shrinkage. The molecular basis for this essential activity that ensures correct chromosome segregation is unclear. In this study, we have used reconstitution of dynamic microtubules and total internal reflection fluorescence microscopy to define the functional relationship between two important budding yeast kinetochore complexes. We find that the Dam1 complex is an autonomous plus end-tracking complex. The Ndc80 complex, despite being structurally related to the general tip tracker EB1, fails to recognize growing ends efficiently. Dam1 oligomers are necessary and sufficient to recruit Ndc80 to dynamic microtubule ends, where both complexes remain continuously associated. The interaction occurs specifically in the presence of microtubules and is subject to regulation by Ipl1 phosphorylation. These findings can explain how the force harvested by Dam1 is transmitted to the rest of the kinetochore via the Ndc80 complex.

Kinetochores Generate Microtubules with Distal Plus Ends: Their Roles and Limited Lifetime in Mitosis

SummaryIn early mitosis, microtubules can be generated at kinetochores as well as at spindle poles. However, the role and regulation of kinetochore-derived microtubules have been unclear. In general, metaphase spindle microtubules are oriented such that their plus ends bind to kinetochores. However, we now have evidence that, during early mitosis in budding yeast, microtubules are generated at kinetochores with distal plus ends. These kinetochore-derived microtubules interact along their length with microtubules that extend from a spindle pole, facilitating kinetochore loading onto the lateral surface of spindle pole microtubules. Once kinetochores are loaded, microtubules are no longer generated at kinetochores, and those that remain disappear rapidly and do not contribute to the metaphase spindle. Stu2 (the ortholog of vertebrate XMAP215/ch-TOG) localizes to kinetochores and plays a central role in regulating kinetochore-derived microtubules. Our work provides insight into microtubule generation at kinetochores and the mechanisms that facilitate initial kinetochore interaction with spindle pole microtubules.

Structure-function insights into the yeast Dam1 kinetochore complex

Faithful segregation of genetic material during cell division requires the dynamic but robust attachment of chromosomes to spindle microtubules during all stages of mitosis. This regulated attachment occurs at kinetochores, which are complex protein organelles that are essential for cell survival and genome integrity. In budding yeast, in which a single microtubule attaches per kinetochore, a heterodecamer known as the Dam1 complex (or DASH complex) is required for proper chromosome segregation. Recent years have seen a burst of structural and biophysical data concerning this interesting complex, which has caught the attention of the mitosis research field. In vitro, the Dam1 complex interacts directly with tubulin and self-assembles into ring structures around the microtubule surface. The ring is capable of tracking with depolymerizing ends, and a model has been proposed whereby the circular geometry of the oligomeric Dam1 complex allows it to couple the depolymerization of microtubules to processive chromosome movement in the absence of any additional energy source. Although it is attractive and simple, several important aspects of this model remain controversial. Additionally, the generality of the Dam1 mechanism has been questioned owing to the fact that there are no obvious Dam1 homologs beyond fungi. In this Commentary, we discuss recent structure-function studies of this intriguing complex.

A Dam1-based artificial kinetochore is sufficient to promote chromosome segregation in budding yeast

Kinetochores are large multiprotein complexes that mediate chromosome segregation in all eukaryotes by dynamically connecting specialized chromosome regions, termed centromeres, to the plus-ends of spindle microtubules. Even the relatively simple kinetochores of the budding yeast Saccharomyces cerevisiae consist of more than 80 proteins, making analysis of their respective roles a daunting task. Here, we have developed a system that allows us to artificially recruit proteins to DNA sequences and determine whether they can provide any aspect of kinetochore function in vivo. We show that artificial recruitment of the microtubule-binding Dam1 complex to a plasmid lacking any centromere DNA is sufficient to confer mitotic stabilization. The Dam1-based artificial kinetochores are able to attach, bi-orient and segregate mini-chromosomes on the mitotic spindle, and they bypass the requirement for essential DNA-binding components of natural kinetochores. Thus, we have built a simplified chromosome segregation system by directly recruiting a microtubule force-transducing component to DNA.

Connecting with Ska, a key complex at the kinetochore–microtubule interface

Connecting with Ska, a key complex at the kinetochore–microtubule interface

Ab initio reconstruction of helical samples with heterogeneity, disorder and coexisting symmetries

We describe modifications of the single particle helical reconstruction approach devised for the analysis of a sample that could not be processed with existing methods due to its variable and short range helical order. The added steps of reference-free two-dimensional image classification and alignment, and automated microtubule removal from images, have particular application to proteins or protein complexes that assemble around microtubules. The method was successfully applied to the Dam1 complex, an essential component of the yeast kinetochore that couples replicated chromosomes to spindle microtubules during mitosis. Because of its novel mode of binding, which does not involve a footprint on the microtubule lattice, new steps to deal with the disorder and heterogeneity of the Dam1 complex assembly were required to gain structural information about this complex both routinely and efficiently.

The Human Kinetochore Ska1 Complex Facilitates Microtubule Depolymerization-Coupled Motility

Mitotic chromosome segregation requires that kinetochores attach to microtubule polymers and harness microtubule dynamics to drive chromosome movement. In budding yeast, the Dam1 complex couples kinetochores with microtubule depolymerization. However, a metazoan homolog of the Dam1 complex has not been identified. To identify proteins that play a corresponding role at the vertebrate kinetochore-microtubule interface, we isolated a three subunit human Ska1 complex, including the previously uncharacterized protein Rama1 that localizes to the outer kinetochore and spindle microtubules. Depletion of Ska1 complex subunits severely compromises proper chromosome segregation. Reconstituted Ska1 complex possesses two separable biochemical activities: direct microtubule binding through the Ska1 subunit, and microtubule-stimulated oligomerization imparted by the Rama1 subunit. The full Ska1 complex forms assemblies on microtubules that can facilitate the processive movement of microspheres along depolymerizing microtubules. In total, these results demonstrate a critical role for the Ska1 complex in interacting with dynamic microtubules at the outer kinetochore.

Different assemblies of the DAM1 complex follow shortening microtubules by distinct mechanisms

Mitotic chromosomes segregate at the ends of shortening spindle microtubules (MTs). In budding yeast, the Dam1 multiprotein complex supports this dynamic attachment, thereby contributing to accurate chromosome segregation. Purified Dam1 will track the end of a depolymerizing MT and can couple it to microbead transport in vitro. The processivity of such motions has been thought to depend on rings that the Dam1 complex can form around MTs, but the possibility that alternative coupling geometries contribute to these motilities has not been considered. Here, we demonstrate that both rings and nonencircling Dam1 oligomers can track MT ends and enable processive cargo movement in vitro. The coupling properties of these two assemblies are, however, quite different, so each may make a distinct contribution to chromosome motility.

Dam1 complexes go it alone on disassembling microtubules

Kinetochores maintain a mechanical grip on disassembling microtubule plus ends, possibly through a 16-member Dam1 ring that acts as a sliding clamp. It turns out, however, that a ring is not required for maintaining grip: individual Dam1 complexes in vitro can diffuse on the microtubule lattice and track shortening microtubule tips.

Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation

During mitosis, kinetochores form persistent attachments to microtubule tips and undergo corrective detachment in response to phosphorylation by Ipl1 (Aurora B) kinase. The Dam1 complex is required to establish and maintain bi-oriented attachment to microtubule tips in vivo, and it contains multiple sites phosphorylated by Ipl1 (Refs 2, 3, 4, 5, 6, 7, 8, 9, 10). Moreover, a number of kinetochore-like functions can be reconstituted in vitro with pure Dam1 complex. These functions are believed to derive from the ability of the complex to self-assemble into rings. Here we show that rings are not necessary for dynamic microtubule attachment, Ipl1-dependent modulation of microtubule affinity or the ability of Dam1 to move processively with disassembling microtubule tips. Using two fluorescence-based assays, we found that the complex exhibited a high affinity for microtubules (Kd of approximately 6 nM) that was reduced by phosphorylation at Ser 20, a single Ipl1 target residue in Dam1. Moreover, individual complexes underwent one-dimensional diffusion along microtubules and detached 2.5-fold more frequently after phosphorylation by Ipl1. Particles consisting of one to four Dam1 complexes - too few to surround a microtubule - were captured and carried by disassembling tips. Thus, even a small number of binding elements could provide a dynamic, phosphoregulated microtubule attachment and thereby facilitate accurate chromosome segregation.