User:MMA12/sandbox

Overview: One of the most important steps in the cell cycle is the separation of the chromosomes during anaphase. This separation occurs when the kinetochores are pulled apart from the centromere of the chromatin. When microtubules depolymerize, the kinetochores move towards the poles of the cell. During microtubule polymerization, the kinetochores move towards the center of the cell. During anaphase, it is paramount that the kinetochores are able to move along depolymerizing microtubule and towards the ends of the cell. Dam1, a protein complex found in the outer kinetochore is vital to this process, because it helps properly attach and orient the microtubules and kinetochore by Dam1 oligomerizing to form a ring around the microtubule. Dam1 is regulated by an Aurora B Kinase, and associates with Ndc80.

Structure of Dam1 Complex: The Dam 1 complex (also called DASH) is a 201 kDa heterodimer, which means that it is made out of is made out of 10 individual subunits. Although all of the subunits have been identified, the functions of each individual subunit have not. Six of the subunits (Dam1p, Dou1p, Spc34p, Spc19p, dad1p and Dad3) play a role in binding to microtubules, and do not oligomerize, while three subunits (Ask1p, Dad2p and Dad4p) do not bind MTs. The remaining subunit, Hsk3p plays a role in organizing subunit oligomerization. In its unbound form, Dam1 is a globular protein. However, in the presence of a microtubule, Dam1 can oligomerize into either a ring formation or a helix, depending on the ratio of Dam1 components to microtubules. However, in vivo, the Dam1 complex will only form a ring containing 12-15 heterodecimers, due to a lower ratio of Dam1components to microtubules.

Ring Structure: When Dam1 oligomerizes around a microtubule, it forms a 50nm ring, whose outer diameter is estimated to be 540Å, and its inner diameter is around 320Å. The inner ring is larger than the microtubule - thus, there is a gap of around 4nm between the two. Electrostatic attraction between the positive charges on the inner surface of the Dam1 complex and the negative charges along the microtubule are responsible for their association. It is thought that these negative charges are found along the GTP lattice of the microtubule. In fact, Dam1 preferentially binds to the GTP/GDP-P1 microtubule state, allowing Dam1 to be sensitive to changes in the microtubule, possibly allowing the kinetochore to preferentially recognize and bind to the microtubule plus end.

Ring Formation: During metaphase, the Ndc80 complex recruits the Dam1 complex to form a ring around the kinetochore. Dam1 ring formation is cooperative; thus, it is much more favorable for a Dam1 ring complex to form than it is for a partial ring complex to form. Oligomerization of the Dam1 complex can be controlled by changing the concentration of Hsk3 levels. When Hsk3 is expressed, oligomerization occurs however, inexpression causes suppression of oligomerization.

When Dam1 switches from its globular form to its ring formation, it must undergo a series of conformational changes. While the exact organization of the Dam1 complex is unknown, we do have some knowledge about how the ring is organized. In the absence of microtubules, Dam1 and Duo1 interact with themselves; that is, Dam1C and Duo1C interact with Dam1N and Duo1N. However, in the event of microtubule binding, the c-terminals of both monomers move to the surface of the Dam1 complex in order to bind to the microtubules. This requires a rearrangement of the Dam1 complex, suggesting that there is movement of other Dam1 complex subunits.

When in ring formation, Dam1p and Duo1p are the only subunits that are directly cross linked to the microtubule, although significant cross linkage is found within the microtubule subunits themselves. Phosphorylation of the Dam1 amino acid S20 (which is found at the interface of two subunits) prevents Dam1 oligermization and destabilizes the ring, although it does not directly affect kinetochore-microtubule binding. Lastly, phosphorylation of the Dam1 terminal sites (S257, S265 and S292) decreases the affinity of Dam1 for the microtubules it must attach to.

Attachment of Kinetochore to microtubule: The Ndc 80 complex, as noted above, is responsible for recruiting Dam1 to the kinetochore. However, it is Kap121, a nuclear transport factor, that is responsible for exporting the Dam1 complex out of the nucleus and bringing it to the spindle microtubules. Additionally, Kap121 is able to protect the Dam1 complex from degradation as it enters the cytoplasm. Yet, Kap121 is not necessary for cytoplasmic stabilization. Thus, there are probably other importins that also help facilitate this process. Furthermore, Kap121 binds specifically to Duo1 and Dam1and stabilizes the entire Dam1 complex even while on the microtubule, although the molecular basis for Kap121 stabilization is not certain.

Once Dam1 has been recruited to and stabilized on the microtubule, one Ndc80 complex binds to two Dam1 rings. Thus, the Ndc80 complex forms a bridge between the two Dam1 rings, which keeps them separated at a distance of about 33.1nm away from each other, ensuring that the Dam1 rings are in the correct biorientation. The daughter cells of cells that have importer ratios of Ndc80:Dam1 complexes, or insufficient distance between the two Dam1 rings display incorrect chromosome distributions.

In order for Ndc80 and Dam1 to properly bind, three regions of Ncd80 must interact at three sites on Dam1 - the Ask1p, Spc34p and Dam1p sites. Once bound to Ndc80, Dam1p and Ask1p are positioned near the microtubule’s surface, while Spc34p is positioned away from the surface; also, it is believed that Ndc80 is positioned under the Dam1 ring closest to the kinetochore and above the Dam1 ring farthest from the kinetochore.

Functions of Dam1 Complex: Stabilization of the microtubule: Kinetochores must remain attached to microtubules despite microtubule dynamic instability. Dam1 plays an important role in forming and maintaining kinetochore-microtubule attachment, despite microtubule polymerization and depolymerization. Furthermore, Dam1 helps to promote microtubule assembly and stability. Dam1 is localized to the end of the microtubule, and as the microtubule shrinks, the Dam1 complex slides away from the shrinking edge of the microtubule, dragging the kinetochore with it.

While in vivo, there is a fixed amount of Dam1 complexes per microtubule, experiments have shown that the higher the concentration of Dam1 rings at the end of the microtubule, the slower depolymerization occurs at the ends of the microtubule, indicating that Dam1 plays a vital role in the regulation of microtubule dissociation. Dam1 stabilizes microtubules through lateral interactions with the microfilaments that make up a microtubule. Since the rings bind perpendicular to the microfilament sections, they can strengthen interprotofilament interactions, and prevent them from peeling; thus stabilizing the microtubule.

Movement Coupling: Dam1functions also couples the movement of the depolymerizing microtubule to the moment of the kinetochore. During microtubule depolymerization, the plus ends of the microtubules curve outwards at variable degrees. Dam1, in conjunction with Ndc80, is able to harness the force of microtubule depolymerization. The current model of how this happens is called the wave model. In this model, microtubule depolymerization generates a “power stroke” on Dam1, which pushes the complex upwards, causing Dam1 to exert a pulling force on the kinetochore. The average force that Dam1 can exert is around ~9pN; however, when coupled with Ndc80, it can transmit six times that load.

Other Functions: In addition to stabilizing the microtubules, and helping to generate the force to move the chromosomes, the Dam1 complex is able to recruit other components of the kinetochore and it allows the kinetochore to bi-orient to the microtubule. In meiosis, repression of HSK3 gene expression promotes the proper segregation of chromosomes during anaphase of Meiosis 1.

Regulation by Aurora Kinase:The Dam1 and Ndc80 complexes are both regulated but he Aurora B Kinase Ipl1. When the Aurora B kinase de-phosphorylates Dam1, it strengthens the attachment between microtubule and kinetochore, but when it phosphorylates Dam1, these connections are broken. Moreover, the Aurora Ipl1 kinase is thought to “facilitate the establishment of kinetochore biorientation by promoting turnover of kinetochore-microtubule attachments” in addition to detaching kinetochores from microtubules in the absence of tension. It is likely that the kinase’s activity acts as a binary switch that causes the microtubule kinetochore attachment to either be fully attached, or fully unattached. Ipl1 phosphorylates Dam1, which causes a decrease in binding between Dam1 and Ndc80, without disturbing the localization of Ndc80 or Dam1’s ability to bind to the microtubule. Dam1 can be phosphorylated at multiple sites: Dam1p (at residues S20, S257, S265, S292), Ask1p (at residue S200) and Spc34p (at residue T199). When it is phosphorylates Dam1p at residue S20, oligomerization of Dam1 decreases, preventing the complex from maintaining its ring form.

Equivalence to Ska in humans

It has been hypothesized that Dam1 is the yeast equivalent of human protein Ska 1. In fact, phylogenetic profiles of these two proteins show that they appear inversely amongst eukaryotes. Additionally, all the subunits of Ska and Dam1 are homologues, giving a basis to the idea that these proteins play a similar role within the cells in which they are found.

Category:Dam1