Differential expression of a phosphoepitope at the kinetochores of moving chromosomes

Abstract

Abstract. A phosphorylated epitope is differentially expressed at the kinetochores of chromosomes in mitotic cells and may be involved in regulating chromosome movement and cell cycle progression. During prophase and early prometaphase, the phosphoepitope is expressed equally among all the kinetochores. In mid-prometaphase, some chromosomes show strong labeling on both kinetochores; others exhibit weak or no labeling; while in other chromosomes, one kinetochore is intensely labeled while its sister kinetochore is unlabeled. Chromosomes moving toward the metaphase plate express the phosphoepitope strongly on the leading kinetochore but weakly on the trailing kinetochore. This is the first demonstration of a biochemical difference between the two kinetochores of a single chromosome. During metaphase and anaphase, the kinetochores are unlabeled. At metaphase, a single misaligned chromosome can inhibit further progression into anaphase. Misaligned chromosomes express the phosphoepitope strongly on both kinetochores, even when all the other chromosomes of a cell are assembled at the metaphase plate and lack expression. This phosphoepitope may be involved in regulating chromosome movement to the metaphase plate during prometaphase and may be part of a cell cycle checkpoint by which the onset of anaphase is inhibited until complete metaphase alignment is achieved.

Microtubule-dependent Changes in Assembly of Microtubule Motor Proteins and Mitotic Spindle Checkpoint Proteins at PtK1 Kinetochores

“…Important for kinetochore function is the modification of kinetochore assembly produced by kinetochore and nonkinetochore spindle microtubules. There is qualitative evidence that unattached prometaphase kinetochores are larger in width (Rieder, 1982;Salmon, 1989;Cassimeris et al, 1990) and have greater amounts of microtubule motor proteins and mitotic spindle checkpoint proteins (Gorbsky and Ricketts, 1993;Chen et al, 1996;Echeverri et al, 1996;Li and Benezra, 1996;Taylor and McKeon, 1997;Yao et al, 1997Yao et al, , 2000Chan et al, 1998;Chen et al, 1998;Dujardin et al, 1998;Jablonksi et al, 1998;Waters et al, 1998;Martinez-Exposito et al, 1999; than metaphase kinetochores with their full complement of kinetochore microtubules. The enhanced assembly may be important for the recruiting kinetochore microtubules, increasing force production, and generating a strong "wait-anaphase" signal (Rieder and Salmon, 1998;Howell et al, 2000).…”

“…Another factor related to the spindle checkpoint is the 3F3/2 antigen that has been detected by electron microscopy in the interzone between inner and outer plates (Campbell and Gorbsky, 1995). The 3F3/2 antibody recognizes a phosphorylated epitope at unattached kinetochores not under tension (Gorbsky and Ricketts, 1993;Campbell and Gorbsky, 1995;Nicklas et al, 1995). By comparing the localization of Mad2 to BubR1, CENP-E, cytoplasmic dynein, 3F3/2 antigen, and CREST antigens at kinetochores in nocodazole-treated cells, we initially show that Mad2 is also localized to the outer kinetochore domain.…”

Mad2 and BubR1 Function in a Single Checkpoint Pathway that Responds to a Loss of Tension

“…A tension-sensitive phosphorylation at the kinetochore detected by the 3F3/2 antibody has been shown to become dephosphorylated in response to tension (Gorbsky and Ricketts, 1993;Nicklas et al, 1995). Kinetochores exhibit a "memory" of their 3F3/2 phosphorylation state after lysis (Campbell et al, 2000), reflecting a chemical change that takes place at kinetochores in response to tension.…”

Lysed cell models and isolated chromosomes for the study of kinetochore/centromere biochemistry in vitro

“…Initially this antibody was characterized as binding preferentially to proteins that had been thiophosphorylated using ATP-γS in kinase reactions (7). We later discovered that this antibody labeled a small subset of endogenous phosphoproteins, some of which were concentrated at kinetochores (8). Intriguingly the binding of this antibody to different kinetochores within a single cell differed depending on the extent of congression of the chromosome to the metaphase plate.…”