A novel mitotic spindle pole component that originates from the cytoplasm during prophase.

Abstract

Abstract. Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the ll5/ll0-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.

Two major autoantigen—Antibody systems of the mitotic spindle apparatus

“…Some of these sera displayed additional bands. Anti-NuMA-2 sera (lanes [11][12][13][14][15][16] showed no characteristic reactivity by immunoblot. b, HeLa cell whole lysate separated by 5% SDS-PAGE and electrotransferred to nitrocellulose.…”

“…The use of cycling cells, some of which may be in mitosis, as substrate for antinuclear antibody (ANA) IIF screening has made it possible to recognize autoantibodies to the mitotic apparatus. Some of these autoantibodies have been shown to stain discrete and well-defined sites of the mitotic apparatus, such as the centrosomal area (11)(12)(13), the intercellular bridge (14)(15)(16), and the mitotic spindles and poles…”

“…Using rare sera from patients with scleroderma, a few proteins have already been defined as autoantigens in the mitotic apparatus. SPA (spindle pole-related antigen) was identified as a conserved centrosomal 59-kd protein (12,22), POPA (prophaseoriginating polar antigen) was characterized as a human 110-115-kd centrosomal autoantigen (13), and 2 intercellular bridge proteins of 36 kd and 38 kd were also characterized as autoantigens (14,23).…”

Recruitment: The ins and outs of spindle pole formation

“…Recruited components of spindle poles have been identified in tissue culture cells and in developing sea urchin embryos using autoimmune sera from patients with scleroderma [CalarcoGillam et al, 1983;Sager et al, 1986;Leslie and Wilson, 1987;Leslie, 19891. In sea urchin embryos, recruited proteins form a core sphere of approximately 4 pm diameter whose surface is near the inferred minus ends of spindle microtubules.…”

“…One might speculate that it has an important role in the organization of spindle poles. The recruitment of autoimmune antigens is temporally correlated with spindle microtubule assembly [Sager et al, 1986;Leslie, 19891. The location of recruited antigens close to the minus ends of spindle microtubules is also consistent with an important role in microtubule organization.…”