Dynamics of Centromere and Kinetochore ProteinsImplications for Checkpoint Signaling and Silencing

Abstract

A missing component of this model, however, is a set of proteins responsible for the recruitment, production, Introduction and release of a wait signal, i.e., the factors that facilitate the proposed catalysis. One important characteristic of Genomic integrity is maintained by a number of checkcomponents of such a catalytic kinetochore scaffold points that act during each cell cycle. The prevention would be a long residence time at unattached kinetoof gross chromosome missegregation and the resulting chores and therefore a long recovery time after photoaneuploidy is carried out by the mitotic checkpoint (also bleaching. In addition, this catalytic machinery would referred to as the spindle assembly checkpoint; for rebe expected to be deactivated by microtubule attachview, see [1, 2] Figures 2B and 2F). The raised against human Mad1 yielded maximal estimates small cycling pool showed rapid kinetics (t 1/2 ϭ 6.6 s Ϯ ( Figure 1A) of the levels of the HsMad1 fusions to be 1.1 SEM) that were not significantly different from recovapproximately 1.5-fold and 5-fold (for C-terminal and ery times measured at kinetochores in normal mitoses N-terminal fusions, respectively) that of the endogenous or in nocodazole-blocked mitotic cells ( Figure 2F). After Mad1 level. However, because the antibody affinity for nuclear reassembly in early G1 and throughout inthe PTK2 Mad1 (PtMad1) homolog is very likely to be terphase, Mad1 was stably bound at the nuclear envesignificantly lower than for hsMad1, the actual levels lope (Figures 2A and 2D). Neither N-nor C-terminal YFP were almost certainly significantly lower than these estifusions recovered significantly within 15 min after photomates, which is consistent with the absence of an effect bleaching (R% Ͻ increase (approximately 15%) in the length of mitosis ochores, both C-and N-terminal fusions of Mad2 partially recovered at kinetochores during normal mitoses (see Figure S1). Immunoblotting with an antibody to hsMad2 ( Figure 3A) permitted maximal estimates of ap-( Figure 3C; R% ϭ 60.0% Ϯ 2.7 SEM; t 1/2 ϭ 10.8 s Ϯ 1.2 SEM, n ϭ 60) or in nocodazole-treated, mitotic checkproximately 20-fold and 5-fold the level of endogenous PtMad2 for the C-terminal and N-terminal fusions, repoint-arrested cells ( Figure 3F; R% ϭ 53.8% Ϯ 3.1 SEM; t 1/2 ϭ 6.4 s Ϯ 0.7 SEM, n ϭ 59). Mad2 that accumulated spectively. Because this antibody almost certainly has lower affinity for PtMad2, it is highly likely that the actual near spindle poles after microtubule attachment continued to cycle as rapidly and to a similar recovery percentlevels were much lower than these estimates, consistent with absence of the kinetochore-independent mitotic age as Mad2 Figure 5C, lanes 2 and 7). Mad2/YFP fusions constitutive centromeric protein, CENP-C, and the kinetochore kinase Bub1, known to be essential for the mialso coprecipitated endogenous Mad2 both in randomly cycling and in mitotic cells ( Figure 5C, lanes 3 and 8) Bub1: R% Ͻ 10%, t 1/2 Ͼ 15 min, n ϭ 21) or when the kinetochores; Mad1 is stably bound, whereas a...

In Vivo Dynamics ofDrosophilaNuclear Envelope Components

“…The release of GFP-Mad2 at an early stage of Drosophila NPC disassembly in prophase and the late recruitment of Mad1 and Mad2 at the NE corroborate previous observations in Xenopus and human cells (Chen et al, 1996;Shah et al, 2004). However, we show in addition that relocalization of Drosophila Mad1 and Mad2 to the NE occurs in two steps, with their nuclear import preceding their NE association.…”

SAC‐ing mitotic errors: How the spindle assembly checkpoint (SAC) plays defense against chromosome mis‐segregation

“…2) [reviewed in . For example, Bub1p binds to Bub3p throughout the cell cycle [reviewed in [Howell et al, 2000[Howell et al, , 2004Shah et al, 2004]. The association of SAC proteins with kinetochores is important for SAC function [Kadura et al, 2005;Taylor et al, 1998;Vanoosthuyse et al, 2004] perhaps because it alters the binding interactions between SAC proteins and influences the formation of the mitotic checkpoint complex (MCC) [Sudakin et al, 2001], which imposes a cell cycle arrest by binding to the Anaphase Promoting Complex (APC).…”

Structure of an intermediate conformer of the spindle checkpoint protein Mad2

“…Fluorescence recovery after photobleaching (FRAP) revealed that there were two pools of Mad2 at kinetochores in mammalian cells: a more resident pool that corresponded to the Mad1-C-Mad2 core complex and a fast-exchanging pool that corresponded to a second Mad2 molecule bound to the Mad1-C-Mad2 core through asymmetric dimerization (32). This second Mad2 molecule is likely in the I-Mad2 conformation because I-Mad2-CMad2 is the most stable asymmetric Mad2 dimer in vitro (24).…”