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Immunolocalization and Molecular Properties of a High Molecular Weight Microtubule-Bundling Protein (Syncolin) from Chicken Erythrocytes E Feick, R

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Immunolocalization and Molecular Properties of a High Molecular Weight Microtubule-Bundling Protein (Syncolin) from Chicken Erythrocytes E Feick, R Immunolocalization and Molecular Properties of a High Molecular Weight Microtubule-bundling Protein (Syncolin) from Chicken Erythrocytes E Feick, R. Foisner, and G. Wiche Institute of Biochemistry, University of Vienna, 1090 Vienna, Austria Abstract. A protein of apparent molecular weight was displaced from the polymer by salt. Brain as well 280,000 (syncolin), which is immunoreactive with an- as erythrocyte microtubules, reconstituted with taxol tibodies to hog brain microtubule-associated protein from MAP-free tubulin and purified syncolin, were ag- (MAP) 2, was purified from chicken erythrocytes. Im- gregated into dense bundles containing up to 15 Downloaded from http://rupress.org/jcb/article-pdf/112/4/689/1060909/689.pdf by guest on 29 September 2021 munofluorescence microscopy of bone marrow cells microtubules, as determinedby'etectron microscopy. revealed the presence of syncolin in cells at all stages On the ultrastructural level, syncolin molecules were of erythrocyte differentiation. In early erythroblasts visualized as globular or ringlike structures, in con- syncolin was diffusely distributed throughout the trast to the thin, threadlike appearance of filamentous cytoplasm. At later stages it was found along microtu- MAPs, such as brain MAP 2. According to ultrastruc- bules of the marginal band, as confirmed by immuno- tural measurements and gel permeation chromatogra- electron microscopy. The association of syncolin with phy, syncolin's molecular weight was ,',,1 x 106. It is the marginal band was dependent on the integrity of suggested that syncolin's specific function is the cross- microtubules, as demonstrated by temperature- linking of microtubules in the marginal band and, by dependent de- and repolymerization or marginal band implication, the stabilization of this structure typical microtubules. Syncolin cosedimented in a saturable for nucleated (chicken) erythrocytes. manner with microtubules assembled in vitro, and it "~ ArICROTUBULES,one of three prominent filamentous Nucleated erythrocytes are an attractive source for non- l~kif[ elements of the cytoskeleton, consist of tubulin, neuronal microtubule proteins. Subjacent to the plasma • • 1 their main subunit component, and a number of membrane of these cells there is a circumferential ring of associated proteins, called microtubule-associated proteins compactly bundled microtubules, called the marginal band, (MAPs)) In brain, where microtubules are most abun- which is responsible, at least in part, for the generation of dam, two major classes of MAPs have been identified: an asymmetric cell shape (Barrett and Dawson, 1974). fibrous MAPs, comprising high molecular weight MAPs Lately the biochemistry and the ultrastructure of marginal (apparent Mr >~ 280,000), 200-kD MAPs, and tan proteins band microtubules have been studied extensively. Cohen and (apparent Mr = 55,000-68,000), and energy-transducing co-workers (1982) isolated intact marginal bands from MAPs, including kinesin and cytoplasmic dynein (for a re- dogfish erythrocytes by detergent and elastase treatment and cent review see Wiche et al., 1991). Energy-transducing found four tubulin-like proteins, but no MAPs. On the other MAPs are considered to mediate various microtubule-de- hand, an anti-MAP 2-immunoreactive, high molecular pendent motile phenomena, while the proposed major func- weight protein, which was suggested to play a role in mi- tions of fibrous MAPs involve microtubule crosslinking and crotubule crosslinking, was identified in the marginal band stabilization. Contrary to energy-transducing MAPs, which of amphibian and avian erythrocytes (Sloboda and Dicker- are under study using a variety of different tissues and cell sin, 1980; Centonze et al., 1985; Centonze and Sloboda~ types, relatively little is known about the structure and func- 1986). Murphy and co-workers identified a B-tubulin variant tion of fibrous MAPs from sources other than brain. Such from chicken, specifically contained in erythrocytes and studies would seem important, however, considering that thrombocytes, that exhibited 17% overall divergence in its structural diversities of MAP species are probably responsi- amino acid sequence compared with other chicken/3-tubulins ble for differential functions of these proteins, in particular (Murphy and Wallis, 1983a; Murphy et al., 1987). Further- their ability to bind to various tubulin isoforms or other cellu- more, the association of a 67-kD tau protein with the mar- lar interaction partners. ginal band of chicken erythrocytes and the immunofluores- cent staining of the marginal band using a rabbit antibody to hog brain MAP 2 were recently demonstrated (Murphy 1. Abbreviation used in this paper: MAP, microtubule-associated protein. and Wallis, 1983b, 1985). However, only trace amounts of © The Rockefeller University Press, 0021-9525/91/02/689/11 $2.00 The Journal of Cell Biology, Volume 112, Number 4, February 1991 689-699 689 MAP 2-related proteins were found in extracts of chicken (bed volume: 0.5 x 5 cm) equilibrated with buffer C plus 0.1 mM PMSE erythrocytes by immunoblotting, and no MAP 2 was ob- After the column was washed with buffer C, adsorbed proteins were eluted with buffer C containing 0.1 mM PMSF and 0.5 M NaC1. FOr all experi- served in preparations of in vitro assembled microtubules ments, preparations of syncolin were generally dialyzed against 25 mM fractionated by SDS-PAGE (Murphy and WaUis, 1983b). MES, pH 6.7, 1 mM EGTA, 1 mM MgC12, and 0.1 mM PMSF at 4°C Here we report the isolation and partial characterization for3 h. of a chicken erythrocyte high molecular weight (280,000) MAP that is associated with marginal band microtubules. Immunoreagents Because of its specific microtubule bundling activity, as Antibodies to hog brain MAP 2 or to syncolin from chicken erythrocytes demonstrated in vitro, it is suggested that this protein be were raised in rabbits using crushed gel pieces containing the immunogens called syncolin, in reference to the Greek word for sticking after their purification by preparative SDS-PAGE. The specificity and titer together. of the antisera were routinely assessed by immunoblotting; there was no crossreactivity of the anti-MAP 2 antiserum with any of the MAP 1 sub- components from hog brain. Affinity purification of anti-MAP 2 antibodies Materials and Methods by adsorption to purified hog brain MAP 2 coupled to Affi-Gel 15 (Bio-Rad Laboratories, Inc., Richmond, CA) (method a) was done according to the instructions provided by the manufacturer. Bound antibodies were eluted Preparation of Microtubule Proteinsfrom with 200 mM glycine/HCl, pH 2.6, and 150 mM NaC1, followed by im- Brain Tissues mediate neutralization with 1 M Tris. Affinity purification of anti-syneolin or anti-MAP 2 antibodies by adsorption to and elution from electrophoreti- Microtubule proteins were prepared from hog or chicken brain by two cy- cally purified syncolin and hog brain MAP 2, respectively, immobilized on cles of temperature-dependent polymerization and depolymerization ac- nitrocellulose sheets (method b) was per'formed following published proce- Downloaded from http://rupress.org/jcb/article-pdf/112/4/689/1060909/689.pdf by guest on 29 September 2021 cording to the method of Karr et al. (1979). Purified tubulin and total MAP dures (Olmsted, 1981; Wiche et al., 1984). fractions were prepared by chromatography of microtubule proteins on pbosphocellulose columns (Whatman, Maidstone, UK) (Weingarten et al., 1975). Hog brain MAP 2 was separated from MAP 1 by chromatography Electrophoresis and Immunoblotting on hydroxyapatite (Calbiochem Corp., La Jolla, CA) (Kuznetsov et al., Proteins were electrophoretically separated on SDS 7.5 % polyacrylamide 1981) and further puKfied by DEAE-Sepbarose CL-6B chromatography gels (Laemmli, 1970). Electrotransfer to nitrocellulose sheets (15 h at 300 (Pharmacia, Uppsala, Sweden) (Foisner and Wicbe, 1985). Purified pro- mA, followed by 3 h at 800 mA) was done in 25 mM Tris, pH 8,3, 192 mM teins were used fresh for experiments or frozen in liquid nitrogen and kept glycine, and 10% methanol (Towbin et al., 1979) at room temperature. The at -70°C until use. nitrocellulose sheets were blocked with I% milk powder in 10 mM Tris/HC1, pH 8, and 150 mM NaC1 (TBS) for 2 h, before 60-rain incuba- Purification of Tubulin and Syncolinfrom tions with various antibodies dissolved in TBS plus 0.2% Tween 20. Bound Chicken Erythrocytes antibodies were detected using secondary antibodies conjugated to alkaline phospbatase and color development (Proto-Blot Immunoscreenilltg System) Blood from 4-5-wk-old chickens was obtained by decapitation of animals according to the instructions provided by the manufacturer (Promega Bio- and collection of blood in beakers containing sodium citrate, pH 7.4, and tec, Madison, WI). sodium chloride at final concentrations of I and 0.9%, respectively. Red cells were collected by centrifugation at 1,000 g for 10 rain at room tempera- ture. The supernatant and the buffy coat, consisting mainly of leukocytes, Immunofluorescence Microscopy were removed by aspiration. After washing in 0.9 % sodium chloride the red Erythroid cells from chicken were prepared and processed for im- cells were centrifuged and the pellets were resuspended in an equal volume munofluorescence microscopy essentially as described by Murphy et al. of ice-cold 100 mM MES, pH 7, 0.5 mM MgC12, 1 mM EGTA, 0.52 M (1986), with the following modifications: cells obtained from whole blood sucrose, 1 mM PMSF, 5 mM DTT, and 1 mM GTP (buffer A). The cells or bone marrow were extracted with 0.2% Triton X-100 in microtubule were then disrupted by sonication (five times for 30 s, at 60-s intervals) stabilizing buffer (Osborn and Weber, 1982) for 15 s and treated first with using a microtip sonicator (Branson Sonifier, Danbury, CT) at 6/7 maxi- 0.5 % formaldehyde for 20 rain and then with methanol at -20°C for 6 rain. mum output. Soluble cell extracts were obtained from these homogenates After rehydration with 137 mM NaCI, 2.7 mM KC1, 1.5 mM KH2PO4, and by centrifugetion at 70,000 g for 45 rain at 4oc.
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