Analysis of mitotic microtubule-associated proteins using mass spectrometry identifies astrin, a spindle-associated protein

Abstract

We purified microtubules from a mammalian mitotic extract and obtained an amino acid sequence from each microtubule-associated protein by using mass spectrometry. Most of these proteins are known spindle-associated components with essential functional roles in spindle organization. We generated antibodies against a protein identified in this collection and refer to it as astrin because of its association with astral microtubule arrays assembled in vitro. Astrin is Ϸ134 kDa, and except for a large predicted coiled-coil domain in its C-terminal region it lacks any known functional motifs. Astrin associates with spindle microtubules as early as prophase where it concentrates at spindle poles. It localizes throughout the spindle in metaphase and anaphase and associates with midzone microtubules in anaphase and telophase. Astrin also localizes to kinetochores but only on those chromosomes that have congressed. Deletion analysis indicates that astrin's primary spindle-targeting domain is at the C terminus, although a secondary domain in the N terminus can target some of the protein to spindle poles. Thus, we have generated a comprehensive list of major mitotic microtubule-associated proteins, among which is astrin, a nonmotor spindle protein.T he spindle is a complex microtubule-based superstructure responsible for chromosome segregation in mitosis and meiosis (1-6). Spindle microtubules are organized in a complex process that requires both motor and nonmotor microtubuleassociated proteins. A great deal of emphasis has been placed on the functional contribution that motor proteins make to spindle organization because of their inherent biological activity. The strong sequence homology of the conserved motor domain in these proteins has facilitated both their rapid identification and characterization during spindle assembly. These investigations have demonstrated that motors of both the kinesin and dynein families play important roles in such varied aspects of spindle organization as spindle bipolarity (6), chromosome movement (7-10), checkpoint regulation (11, 12), spindle pole organization (13-16), and the regulation of microtubule dynamics (17). In contrast, the functional contribution that nonmotor proteins make to spindle organization has not progressed as rapidly. In part, the lag in progress in defining the roles of nonmotor spindle proteins stems from the lack of clearly defined biological activity of nonmotor proteins. A further complication arises from the lack of any clear homology among nonmotor proteins to facilitate their identification. Thus, although some nonmotor spindle proteins have been characterized (18,19), others are less well understood (20,21), and many others may await identification.In this article we use mass spectrometry to identify the proteins associated with microtubules assembled in a cell-free mitotic extract under conditions where Ͼ90% of microtubules are organized into astral arrays. Most proteins identified through this strategy have known functional roles for spindle assembly. A pro...

Protein 4.1R, a Microtubule-associated Protein Involved in Microtubule Aster Assembly in Mammalian Mitotic Extract

“…Cell-free Mitotic Extract-To determine whether 4.1R plays an active role in organizing the mitotic spindle assembly, we first asked whether 4.1R is a component of mitotic asters assembled in a well established in vitro mitotic aster assembly system (25,30,32). In addition to the spectrin-actin-4.1R ternary complex interaction (39, 40), 4.1R exhibits a binary interaction with spectrin (39,41) or actin (42).…”

“…We separated the asters from the soluble components of the extract by centrifugation through a 50% sucrose cushion, which allows for the sedimentation of microtubule asters with sufficient mass (32). Under this condition, a portion of 4.1R, but not actin, was co-sedimented with NuMA, dynactin, and tubulin (Fig.…”

“…The organization of microtubules into spindles is governed largely by the interaction of microtubules and microtubule ends with accessory proteins that regulate microtubule dynamics. Several noncentrosomal proteins such as cytoplasmic dynein (26), dynactin (27), Eg5 (28), HSET (29), ch-TOP (30), cohesin (31), and astrin (32), as well as the structural protein NuMA (33) are involved in focusing microtubules at spindle poles. Researchers are now actively searching for additional proteins that may associate only transiently with spindle poles and for the proteins that are minor components of the spindle poles.…”

Motility proteins and the origin of the nucleus

“…Astrin, too, is a structural protein specific to the spindle, as its name implies. It associates with kinetochores on only those chromosomes that are properly aligned at the metaphase plate (Mack and Compton, 2001).…”

Phosphorylation of Astrin Regulates Its Kinetochore Function

“…Astrin, which contains two coiled coil domains in its C terminus, is associated with spindle MTs as early as prophase and functions in centrosome integrity, spindle formation prior to metaphase chromosome alignment, and chromosome segregation (28,29). Mitotic proteins, including Kinastrin/Skap (27), hNinein (30), cytoplasmic linker-associated protein-1␣ (Clasp1␣) (31), and dynein light chain 8 (32), interact with Astrin and target it to spindle poles or KTs.…”