Autoantibodies from a patient with scleroderma CREST recognized kinetochores of the higher plant Haemanthus.

Abstract

Human autoantibodies from a patient with scleroderma CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, scierodactyly, telangiectasia) were used to immunostain kinetochores on chromosomes in endosperm of the seed of the monocot Haemanthus katherinae Bak. Kinetochores of mitotic chromosomes and prekinetochores of interphase cells were specifically stained using conventional indirect immunofluorescence procedures as well as a nonfading immunogold-silver-enhanced technique and analyzed by fluorescence and video microscopy. In interphase, prekinetochores were either single or double structures depending on the stage of the cell cycle but became quadruple (two distinct stained dots on each chromatid) in mid-to-late prophase. In favorable preparations ofprometaphase chromosomes, multiple subunits could be resolved within each sister kinetochore suggesting a compound organization. Western blot analysis demonstrated common epitopes in centromeric peptides of HeLa and Haemanthus cell extracts. Although the molecular mass of individual polypeptides differed in the two species, the presence of shared epitopes indicates striking conservation of centromere/ kinetochore components throughout evolution.The centromere is a specialized domain located at the primary constriction of most eukaryotic chromosomes and is intimately involved in chromosome segregation (for review, see refs. 1 and 2). The spindle fibers (microtubule bundle), which are instrumental for chromosome segregation, associate with a localized segment of the centromere called the kinetochore. The centromere and kinetochore in some plant and animal cells may not be localized at a single focus, instead being diffuse or polycentric in its distribution along the chromosome. Chromosomes lacking functional kinetochores fail to undergo normal movement and are excluded from the telophase nucleus, often forming micronuclei. Structural rearrangements leading to the formation of two centromeres on the same chromosome (dicentrics) are unstable, resulting in an anaphase bridge, breakage, and fusion cycle (3).Studies of mammalian centromeres/kinetochores have provided the most detailed information on kinetochore structure, composition, and function. The typical mammalian centromere remains unseparated until the onset of anaphase and the kinetochore has a characteristic trilaminar structure with microtubules generally attached to outer-most layer (for review, see refs. 1 and 2).The discovery that human autoantibodies from patients with the CREST (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, telangiectasia) variant of scleroderma react with the kinetochore (4-7) and recognize a small family of centromeric proteins (6,8,9) has facilitated the biochemical dissection of this segment of the mammalian chromosome. Moreover, CREST autoantibodies are useful immunostaining reagents because they recognize inactive kinetochores in interphase nuclei (prekinetochores) as well as functional kinetochores on mitotic chromosomes (5). Although anti-ki...

Titin as a Chromosomal Protein

“…Identification of titin as a chromosomal component Human autoimmune sera have been used successfully to identify a wide range of nuclear antigens, including both single-and double-stranded DNA, RNA, histones, small nuclear RNA-binding proteins, transcription factors, nuclear larnins, topo I and II, heterochromatin-associated proteins and centromere proteins (Tan, 1982;Earnshaw and Rothfield, 1985;Tan et al, 1987;Tan, 1989;Mole-Bajer et al, 1990;Earnshaw and Rattner, 1991;Tan, 1991;Fritzler, 1997;Saunders et al, 1993;Shibata et al, 1993;Bejarano and Valdivia, 1996;Spain et al, 1997;Sugiura et al, 1997). We recently identified titin as a chromosomal component using a human autoimmune scleroderma serum (Machado, 1998;Machado et al, 1998).…”

Immunocytochemical visualization of the centromeres during male and female meiosis in Lilium longiflorum

“…However, it was recently demonstrated that some proteins required for sister chromatid cohesion are localized specifically in the centromere region during metaphase I and anaphase I (Kerrebrock et al 1995;Moens and Spyropoulos 1995). Unfortunately, the sera from patients with CREST rarely recognize plant centromeres (Mole-Bajer et al 1990;Palevitz 1990;Houben et al 1995). Therefore, no biochemical comparisons have been reported between the mitotic and meiotic centromere proteins of higher plants.…”

Structure and Evolution of Plant Centromeres

“…Results indicated that a few of the sera recognized centromeric regions of plants, implying that at least a portion of the centromeric proteins is shared among plants and animals (Mole-Bajer et al 1990 ;Houben et al 1995) . In plants, the first centromeric protein that was confirmed to be localized to the centromere region was CENP-C homologs in maize .…”