Mapping the kinetochore MAP functions required for stabilizing microtubule attachments to chromosomes during metaphase

Abstract

In mitosis, faithful chromosome segregation is orchestrated by the dynamic interactions between the spindle microtubules (MTs) emanating from the opposite poles and the kinetochores of the chromosomes. However, the precise mechanism that coordinates the coupling of the kinetochore components to dynamic MTs has been a long-standing question. Microtubule-associated proteins (MAPs) regulate MT nucleation and dynamics, MT-mediated transport and MT cross-linking in cells. During mitosis, MAPs play an essential role not only in determining spindle length, position, and orientation but also in facilitating robust kinetochore-microtubule (kMT) attachments by linking the kinetochores to spindle MTs efficiently. The stability of MTs imparted by the MAPs is critical to ensure accurate chromosome segregation. This review primarily focuses on the specific function of nonmotor kinetochore MAPs, their recruitment to kinetochores and their MT-binding properties. We also attempt to synthesize and strengthen our understanding of how these MAPs work in coordination with the kinetochore-bound Ndc80 complex (the key component at the MT-binding interface in metaphase and anaphase) to establish stable kMT attachments and control accurate chromosome segregation during mitosis.

Keywords: MAPs; Ndc80; chromosomes; kinetochores; microtubules; mitosis.

Figure 1:. A detailed view of various kinetochore MAPs across phyla, dissociating their kinetochore and microtubule localizations and functions.

A schematic representation of several microtubule-associated proteins (MAPs) ranging from those in yeast to those in humans based on their function or classification within a family. Multiple interactions are shown to depict the localization of each MAP either directly or indirectly at both the kinetochores and MTs. Kinetochore localization specifically refers to the ability of the indicated MAPs to bind to the Ndc80 complex of the KMN network unless otherwise specified. The binding to the human Ska complex to kinetochores is thought to require the loop domain of Ndc80 (A1). Ska binds to MTs directly, while it has been reported to bind to the plus-ends with the help of the +TIP, EB1 (A2). Human Cdt1 also localizes to kinetochores with the help of the loop domain of the Ndc80 complex (B1), while it binds to MTs directly (B2). MT-binding of Cdt1 is regulated by Aurora B kinase phosphorylation (B2). Human Astrin/SKAP localizes to kinetochores but whether it binds to the Ndc80 complex directly or indirectly is not clear (C1). Human Astrin binds to MTs through its binding partner, SKAP (C2). For chTOG, MT-binding has been shown to be dependent on another MAP, TACC3, and is also thought to require Cyclin B/Cdc2 function (D2). However, it is not yet clear whether Cyclin B binds directly to the Ndc80 complex to recruit chTOG to kinetochores (D1). In the context of the XMAP215 family members, fission yeast Dis1 has been shown to be directly dependent on the loop domain for localizing to kinetochores (E1), while Alp14 localization to the loop is dependent on its binding partner, Alp7/TACC (H1). However, for budding yeast Stu2, the region of the Ndc80 complex that is required for kinetochore localization is not clear (E1). As far as MT-binding is concerned, it has been well established that all XMAP215 members, including chTOG in humans, bind directly to MTs (E2, G2 & H2). For Stu2, binding to plus-ends also depends on the yeast EB1 homolog (E2). The Dam1 ring complex in budding yeast binds to an MT directly (F2) and localizes to the kinetochore by binding to multiple Ndc80 regions, including the loop domain (F1). The proteins and protein complexes, which are color-coded identically to those presented in Fig. 2, are drawn in arbitrary shapes for the purpose of depiction only and are not to the scale. The Ska complex constitutes three subunits, Ska1, 2 and 3, but for brevity, clarity and ease of reading, the complex is represented as a singular unit (yellow ovals). Additionally, the region within Ndc80, where the MAPs bind, is not precisely marked because the exact details of the interaction interface have not been resolved in many cases.

Figure 2:. A summary of the MAP functions at the kMT interface that are required for the stabilization of kMT attachment in yeast and humans.

The formation of robust kMT attachments is enabled by the MAPs recruited to kinetochores through the interaction with the Ndc80 complex. A. In humans, Cdt1 binds to Ndc80 through its loop domain. Ska also directly binds to Ndc80 through both the loop and CH domains. Both Cdt1 and Ska enhance the formation of Ndc80-mediated stable kMT attachments at the kMT interface. The C-terminus of Astrin in complex with SKAP binds to a yet unidentified region of kinetochore Ndc80, while the Astin-SKAP complex binds to a MT through the SKAP MT-binding domain. The XMAP125 homolog chTOG binds to MTs either directly or by the mediation of another MAP, TACC3. The interaction between chTOG and the Ndc80 complex is likely indirect and mediated through Cyclin B1. Cyclin B1 is recruited to kinetochores by Ndc80, but it is also not yet known whether it exhibits a direct interaction. chTOG, TACC3 and clathrin have been found to be important for recruiting each other to spindle MTs. B. In budding yeast, Dam1/DASH complexes form oligomers and/or ring structures around a MT after being recruited to kinetochores by binding to the loop and CH domains of Ndc80. The XMAP125 homolog Stu2 directly binds to kinetochore Ndc80 and the MT-tracking protein EB1, but the details of how Stu2 interacts with Ndc80 remain unclear. C. In fission yeast, the XMAP125 homolog Dis1 is loaded onto kinetochores through direct interaction with the loop domain where it interacts with EB1 located at the MT plus-end. EB1 interacts with Ndc80 in vitro, but the functional relevance of this interaction in vivo is still unclear. The other XMAP125 homolog, Alp14, forms a complex with Alp7/TACC and is recruited to kinetochores by interacting with the Ndc80 loop domain. Black arrows indicate the interaction between proteins, but the site on Ndc80 has not yet established. Red arrows indicate that the interaction between two proteins that has not yet been characterized.