DOCSLIB.ORG

Microtubule-Associated Proteins and Glycerol

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Microtubule-Associated Proteins and Glycerol Proc. Nati. Acad. Sci. USA Vol. 74, No. 5, pp. 1860-1864, May 1977 Biochemistry In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol (brain/magnesium ions/cell structure/colchicine) WILHELM HERZOG AND KLAUS WEBER Max Planck Institute for Biophysical Chemistry, D-3400 Goettingen, Federal Republic of Germany Communicated by Manfred Eigen, February 7, 1977 ABSTRACT Microtubule protein from porcine cerebrum (15) used 4 M glycerol in a normal piperazine-N,N'-bis(2-eth- was fractionated into pure tubulin and microtubule-associated anesulfonic acid) (Pipes) assembly buffer, 1 mM in Mg2+ (16). proteins by chromatography on phosphocellulose. In agreement with previous studies, pure tubulin does not form microtubules Because such buffer conditions have been criticized as un- to a significant extent at 370 in normal assembly buffers, which physiological (6), we wondered if self-assembly of homogeneous are characterized by a low concentration of Mg2+ ions. If, tubulin can be achieved reproducibly in the absence of glycerol. however, the Mg2+ concentration is raised to approximately 10 Here we show that phosphocellulose-purified tubulin, free of mM, rapid and extensive self-assembly of pure tubulin into the high-molecular-weight MAPs and of the r factor, can microtubules is observed, provided the tubulin concentration readily assemble into microtubules in the absence of glycerol. is above 2.5 mg/ml. At a protein concentration of 3 mg/ml, the lag period is 1.5 min and the assembly process is virtually Our assembly system requires a tubulin concentration above complete after 6 min at 370. These microtubules are like normal 2.5 mg/ml and a Mg2+ concentration of approximately 10 microtubules-sensitive to calcium ions, colchicine, and low mM. temperature. MATERIALS AND METHODS The controlled dynamic process of assembly and disassembly of microtubules in vivo is an intriguing but still poorly under- Microtubule Protein, Tubulin, and MAPs. Microtubule stood feature of eukaryotic cells. Therefore, much attention has protein from porcine cerebrum was purified by three cycles been focussed on the in vitro assembly of microtubules (for of assembly and disassembly (16) in P-buffer [0.1 M Pipes/2 recent reviews see refs. 1 and 2). It has been generally assumed mM ethylene glycol bis(O-aminoethyl ether)-N,N,N',N'- that in vitro assembly of tubulin into microtubules requires the tetraacetic acid (EGTA)/1 mM MgSO4, pH 6.35 at 23°1 and presence of at least one microtubule-associated protein (3-9). stored at -70°. The first polymerization step was carried out The protein(s) necessary for initiation of microtubule formation in the presence of 4 M glycerol and the two other assembly re- has either specifically been called the r factor (3, 5, 6) or is as- actions were performed in glycerol-free buffer. sumed without further specification to be a microtubule-asso- Tubulin was separated from MAPs by phosphocellulose ciated protein (4-6) or one or both of the high-molecular-weight (Whatman P11) chromatography in the cold (3). Samples (2.5 proteins that are especially predominant in the microtubule- ml) of microtubule protein (20 mg/ml) in P-buffer were slowly associated protein (MAP) fraction (4, 7, 9). Some reports assume loaded on a 20 ml column which had been previously exten- that in vitro assembly of tubulin has an absolute requirement sively equilibrated with P-buffer. The pure tubulin was eluted for a MAP (3, 5, 6, 9), while others indicate that an extremely from the column with P-buffer at a flow rate of 6 ml/hr. The poor but still observable assembly could be noticed in the ab- protein was either used directly or stored in aliquots at a protein sence of MAPs (4, 7-9). concentration of 8-12 mg/ml at -700. All the systematic studies Recently Lee and Timasheff reported (10) that tubulin free shown in this report were obtained with such tubulin prepa- of higher-molecular-weight polypeptides can assemble into rations, which had been kept frozen for at least 3 weeks. After microtubules provided an assembly buffer containing high the column was washed with three column volumes of P-buffer, concentrations of glycerol (3.4 M) and Mg2+ ions (16 mM) is the MAPs were eluted with P-buffer, 0.8 M in NaCl. The pro- used. Their studies used tubulin purified by the ammonium teins were freed of salt by gel-filtration on Sephadex G-25 sulfate, DEAE-Sephadex method rather than by the poly- equilibrated with P-buffer and used directly or stored in ali- merization-depolymerization procedure. The critical tubulin quots at -700. Polyacrylamide gel electrophoresis in the concentration in this system was found to be higher (1.2 mg/ml) presence of sodium dodecyl sulfate was performed as described than in the assembly system involving MAPs (0.2 mg/ml) previously (17). (11-13). This finding challenged the previous assumption that Miscellaneous Procedures. Polymerization in the presence tubulin assembly can only occur when preexisting nucleation of 1 mM GTP and different Mg2+ concentrations was induced centers or protein factors required to assemble such nucleation by temperature shift from 40 to 370 and measured by light centers are present. The finding of Lee and Timasheff (10) has scattering as described previously (17). In experiments involving been confirmed in two different buffer systems with homo- glycerol a solution of tubulin in P-buffer was mixed in the cold geneous tubulin separated from MAPs by phosphocellulose with an equal aliquot of P-buffer, 8 M in glycerol. Protein was used the determined by the Lowry reagents with bovine serum albumin chromatography. Erickson and Voter (14) high glyc- as a standard. Normal negative staining of microtubules was erol, high Mg2+ buffer previously described (10), whereas we performed as described (17). For thin sectioning, microtubules Abbreviations: MAP, microtubule-associated protein; Pipes, pipera- assembled in vitro were harvested by centrifugation at 32' for zine-N,N'-bis(2-ethanesulfonic acid); EGTA, ethylene glycol bis(fl- 30 min at 100,000 X g. The large pellets obtained were fixed aminoethyl ether)-N,N,N',N'-tetraacetic acid; EDTA, ethylenedi- for 2 hr at 370 in P-buffer containing 1 mM GTP and 2% aminetetraacetic acid. (wt/vol) glutaraldehyde, washed, postfixed with osmium te- 1860 Downloaded by guest on September 28, 2021 Biochemistry: Herzog and Weber Proc. Natl. Acad. Sci. USA 74 (1977) 1861 a)0 _0.c0 60- 0 A l u S (A cm 'C . B o =200- 0z InA. C t-0 20 S B FIG. 1. Sodium dodecyl sulfate gel electrophoresis on a slab gel 0 2 4 6 8 10 12 14 with a 4-15% gradient of acrylamide: (A) 6 ,g of unfractionated mi- time (min) crotubule protein; (B) 5Sag of MAPs; (C) 90 ,4g of purified tubulin. The left-hand arrow indicates the start of the running gel and the right- FIC. 3. Induction of polymerization of pure tubulin as a function hand arrow the dye front. Note the separation of a and (# polypeptides of Mg2+ concentration. Tubulin concentration was 4 mg/ml in P- in (A) and (C). buffer, 1 mM in GTP. Total concentration of MgSO4 in mM: A, 1 in the presence of 1.5mM EDTA; B, 1; C, 2; D, 3; E, 5; F, 8; G, 10; H, 18. troxide, washed, dehydrated, and embedded in Spurr's resin The values for the Mg2+ concentrations are the total final values in the samples. using standard procedures (18). Sections were stained in methanolic uranyl acetate and lead citrate and examined in a Zeiss EM9 electron microscope. merization process of pure tubulin (protein concentration 4 mg/ml). Fig. 3 shows that Mg2+ concentrations above 2 mM (total concentration in the assembly buffer) induce a rapid RESULTS polymerization process, which seems optimally expressed at Microtubule protein was separated into tubulin and MAPs by 10 mM Mg2+ (curve G in Fig. 3). At this concentration the lag phosphocellulose chromatography. Sodium dodecyl sulfate gel period before the onset of detectable polymerization is reduced electrophoresis (Fig. 1) showed that the tubulin fraction was to 1.5 min and assembly is virtually complete after 5 min. free of any polypeptides with molecular weights higher than Higher Mg2+ concentrations (18 mM; curve H of Fig. 3) seem that of tubulin, although a few bands of lower-molecular-weight to inhibit both the initiation process and the extent of poly- proteins could be barely detected on very heavily overloaded merization. The polymerization product behaves like normal slab gels. Our MAP preparation is composed of the prominent microtubules in that it is sensitive to colchicine, to calcium ions, high-molecular-weight MAPs and many proteins in lesser and to exposure to low temperature (Fig. 4). Pure tubulin can amounts migrating faster than MAPs 1 and 2 (4, 9, 15). In again be reversibly polymerized upon temperature shift to 37°. agreement with previous studies (3-5, 9), neither tubulin nor Colchicine or calcium when added prior to the temperature MAPs showed a significant self-assembly in the presence of 1 shift to 37° prevents the self-assembly process. tubulin concentration on the mM Mg2 , although the two together gave a fast and extensive The influence of the assembly polymerization process (Fig. 2) yielding microtubules as judged process in standard buffer (final Mg2+ concentration 10 mM) by electron microscopy. The pure tubulin fraction did, how- is shown in Fig. 5. Increasing the tubulin concentration shortens ever, form microtubules in the presence of 4 M glycerol in the the lag period, increases the slope of the polymerization curves, assembly buffer (curve C in Fig. 2), in agreement with three and results in a higher final value of the extent of the poly- previous reports (10, 14, 15).
Load more

Recommended publications
  • Weber K., Schneider A., Müller N. and Plessmann U
    FEBS 17455 FEBS Letters 393 (1996) 27-30 Polyglycylation of tubulin in the diplomonad Giardia lamblia, one of the oldest eukaryotes Klaus Weber ~,*, Andr6 Schneider b, Norbert Mtiller c, Uwe Plessmann a ~Max-Planck-lnstitute for Biophysical Chemistry, Department of Biochemistry, PO Box 2841, D-37018 Goettingen, Germany u University of Fribourg, Institute for Zoology, Pbrolles, CH 1700 Fribourg, Switzerland c University of Berne, Institute of Parasitology, PO Box 8466, CH 3001 Berne, Switzerland Received 16 July 1996 to search for their evolutionary origin because they are unique Abstract We have searched for post-translational modifications in tubulin of the diplomonad Giardia lamblia, which is a to tubulin, a typical eukaryotic protein. representative of the earliest branches in eukaryotic evolution. Based on ultrastructural characteristics and several molecu- The carboxyterminal peptide of a-tubulin was isolated and lar phylogenies there is general agreement that the diplomo- characterized by automated sequencing and mass spectrometry. nads were among the first branches which emerged from the Some 60% of the peptide is unmodified, while the remainder eukaryotic tree. Diplomonads, like other Archezoa, are shows various degrees of polyglycylation. The number of glycyl thought to have arisen before the acquisition of mitochondria residues in the lateral side chain ranges from 2 to 23. All peptide and to have retained many primitive features of the first nu- species encountered end with alanine-tyrosine, indicating the cleated cells [17-21]. Giardia lamblia is a particularly well- absence of a detyrosination/tyrosination cycle. We conclude that characterized diplomonad. Its cytoskeleton is dominated by tubulin-specific polyglycylation could be as old as tubulin and microtubules.
    [Show full text]
  • BIBLIOGRAPHY Klaus Weber 1. Sund, H., and Weber, K. Größe Und
    BIBLIOGRAPHY Klaus Weber 1. Sund, H., and Weber, K. Größe und Gestalt der β-Galaktosidase aus E. coli. Biochem. Z. 337:24-34 (1963). 2. Wallenfels, K., Sund, H., and Weber, K. Die Untereinheiten der β-Galaktosidase aus E. coli. Biochem. Z. 338:714-727 (1963); Angew. Chemie 75:642 (1963). 2.a Sund, H., Arens, A., Weber, K., and Wallenfels, K. Zur Struktur und Wirkungsweise der Alkoholdehydrogenase aus Hefe. Angew. Chemie 2:144-145 (1963) 3. Weber, K., Sund, H., and Wallenfels, K. Über die Art der Bindung zwischen den Untereinheiten im Molekül der β-Galaktosidase aus E. coli. Biochem. Z. 339:498-500 (1964). 4. Weber, K., and Sund, H. Quaternary structure of catalase from beef liver. Angew. Chemie Intern. Edition 4:597-598 (1965). 5. Gussin, G.N., Capecchi, M.R., Adams, J.M., Argetsinger, J.E., Tooze, J., Weber, K., and Watson, J.D. Protein synthesis directed by RNA phage messengers. Cold Spring Harbor Symp. Quant. Biol. 31:257-271 (1966). 6. Konigsberg, W., Weber, K., Notani, G., and Zinder, N. The isolation and characterization of the tryptic peptides from the f2 bacteriophage coat protein. J. Biol. Chem. 241:2579-2588 (1966). 7.a Sund, H., and Weber, K. The quaternary structure of proteins. Angew. Chemie Intern. Edition 5:231-245 (1966). 7.b Sund, H., and Weber, K. Die Quartärstruktur der Proteine. Angew. Chemie 4:217-232 (1966). 8. Weber, K., Notani, G., Wikler, M., and Konigsberg, W. Amino acid sequence of the f2 coat protein. J. Mol. Biol. 20:423-425 (1966). 9. Sund, H., Weber, K., and Moelbert, E.
    [Show full text]
  • Antibody Against Tubulin: the Specific Visualization of Cytoplasmic
    Proc. Nat. Acad. Sci. USA Vol. 72, No. 2, pp. 459-463, February 1975 Antibody Against Tubulin: The Specific Visualization of Cytoplasmic Microtubules in Tissue Culture Cells (microfilaments/immunofluorescence/colchicine/celi structure) KLAUS WEBER*J, ROBERT POLLACK*, AND THOMAS BIBRINGt * Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724; and t Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee Communicated by Barbara McClintock, November 12, 1974 ABSTRACT Cytoplasmic microtubules in tissue cul- The fibers that can be decorated with antibodies against ture cells can be directly visualized by immunofluores- tubulin disappear when cells are exposed to colchicine or to cence microscopy. Antibody against tubulin from the outer doublets of sea urchin sperm flagella decorates a low temperature, whereas the microfilament fibers do not. -network of fine cytoplasmic fibers in a variety of cell lines of human, monkey, rat, mouse, and chicken origin. These MATERIALS AND METHODS fibers are separate and of uniform thickness and are seen throughout the cytoplasm. The fibers disappear either in Cells. 3T3, an established cell line of mouse embryonic a medium containing colchicine or after subjection of the origin (11), was grown in Dulbecco's modified Eagle's me- cells to low temperature. The same treatments do not dium with 10% calf serum. Secondary chick embryo fibro- destroy the microfilamentous structures that are visual- modified medium ized by means of antibody against actin. When trypsin- blasts were grown in Dulbecco's Eagle's treated enucleated cells are replated and then stained with with 10% fetal calf serum. Growth of monkey BSC-1 cells, antibody against tubulin, the fibers can be seen to tra- enucleation of these cells after cytochalasin B treatment, and verse the entire enucleated cytoplasm.
    [Show full text]
  • Curriculum Vitae ANTHONY PAUL BRETSCHER
    Curriculum Vitae ANTHONY PAUL BRETSCHER Personal: Address: Department of Molecular Biology and Genetics Weill Institute for Cell and Molecular Biology Weill Hall Room 257 Cornell University Ithaca, NY 14853-7202 Telephone: 607-255-5713 Fax: 607-255-5961 e-mail: [email protected] Web site: http://www.mbg.cornell.edu/cals/mbg/faculty- staff/faculty/bretscher.cfm Education: 1971 BA University of Cambridge, UK. Experimental Physics 1974 MA University of Cambridge, UK 1974 PhD University of Leeds. Bacterial Genetics. Advisor: Dr. Simon Baumberg 1974-1977 EMBO Postdoctoral Fellow, Stanford University, CA Advisor: Dr. A. Dale Kaiser 1977-1980 Max Planck Society Fellow, Max Planck Institute for Biophysical Chemistry. Goettingen, Germany. Advisor: Dr. Klaus Weber. Academic Appointments: 1980-1981 Assistant Professor, Department of Cell Biology, Southwestern Medical School, Dallas, TX 1981-1999 Assistant (1981-1987), Associate (1987-1993), Professor (1993-1999) Section of Biochemistry, Molecular and Cell Biology, Cornell University 1999-present Professor of Cell Biology, Department of Molecular Biology and Genetics, Cornell University, NY 2007-present Member, Weill Institute for Cell and Molecular Biology Administrative Appointments: 2007-2011 Associate Director, Weill Institute for Cell and Molecular Biology Society Membership and Honors: 1980- present American Society for Cell Biology 1982-present American Association for the Advancement of Science (AAAS) 2009 Elected Fellow, AAAS 2010 Elected Fellow, American Academy of Microbiology National
    [Show full text]
  • Curriculum Vitae ANTHONY PAUL
    Curriculum Vitae ANTHONY PAUL BRETSCHER Personal: Address: Department of Molecular Biology and Genetics Weill Institute for Cell and Molecular Biology Weill Hall Room 257 Cornell University Ithaca, NY 14853-7202 Telephone: 607-255-5713 Fax: 607-255-5961 e-mail: [email protected] Web site: http://www.mbg.cornell.edu/cals/mbg/faculty- staff/faculty/bretscher.cfm Date of Birth: September 8, 1950 Place of Birth: Harwell, Berkshire, England Citizenship: USA, United Kingdom and Switzerland Marital Status: Married Janice Sperbeck, 5.21.1983 Children: Heidi (b. Nov. 1, 1986), Erika (b. April 24, 1991) Home Address: 293 Ellis Hollow Creek Road, Ithaca, NY 14850 Education: 1971 BA University of Cambridge, UK. Experimental Physics 1974 MA University of Cambridge, UK 1974 PhD University of Leeds. Bacterial Genetics. Advisor: Dr. Simon Baumberg 1974-1977 EMBO Postdoctoral Fellow, Stanford University, CA Advisor: Dr. A. Dale Kaiser 1977-1980 Max Planck Society Fellow, Max Planck Institute for Biophysical Chemistry. Goettingen, Germany. Advisor: Dr. Klaus Weber. Academic Appointments: 1980-1981 Assistant Professor, Department of Cell Biology, Southwestern Medical School, Dallas, TX 1981-1999 Assistant (1981-1987), Associate (1987-1993), Professor (1993-1999) Section of Biochemistry, Molecular and Cell Biology, Cornell University 1999-present Professor of Cell Biology, Department of Molecular Biology and Genetics, Cornell University, NY 2007-present Member, Weill Institute for Cell and Molecular Biology Administrative Appointments: 2007-2011 Associate
    [Show full text]
  • Electrophoretic Analysis for the Separation of Muscle Protein of Fiddler Crabs of Pakistan
    Int. J. Biol. Res., 5(2): 77-81, 2017. ELECTROPHORETIC ANALYSIS FOR THE SEPARATION OF MUSCLE PROTEIN OF FIDDLER CRABS OF PAKISTAN Noor-Us-Saher1*, Sahir Odhano1 and Mustafa Kamal2 1Centre of Excellence in Marine Biology, University of Karachi, Karachi 2Department of Biotechnology, University of Karachi, Karachi *Corresponding author’s email: [email protected] ABSTRACT Fiddler crabs muscle tissue was used to estimate molecular weight of protein through SDS-PAGE electrophoresis. A detailed study was carried out from Sandspit, Karachi area to observe the relative mobility and molecular weight of proteins by using standard marker BSA. Results showed that all four species of fiddler crabs vary apart from each other by their molecular weight and their relative mobility. Austruca analyses revealed a maximum number of bands (total seven). While, A. annulipes showed 4. Out of 7 bands found heavier in size (>66 kDa) while three bands from each species shown as smaller in size (<66 kDa) from standard marker BSA. During the current study unveiled that the BSA can effectively be used for calculating the molecular weight of protein in family Ocypodidae. KEYWORDS: Fiddler crabs, SDS-PAGE, BSA, Sandspit, Karachi. INTRODUCTION Polyacrylamide gel electrophoresis (PAGE) is widely used technique in biochemistry, molecular biology and biotechnology to separate biological macromolecules such as proteins or nucleic acids according to their electrophoretic mobility. To separate the protein sodium dodecyl sulfate (SDS) is applied to sample for protein transformation into a linear form and convey negative charge to linearize the protein, therefore, this procedure is well known as SDS-PAGE (Roy and Kumar, 2014).
    [Show full text]
  • The Major Microfilament-Associated Protein of the Intestinal Microvillus
    Proc. Nati. Acad. Sci. USA Vol. 76, No. 5, pp. 2321-2325, May 1979 Cell Biology Villin: The major microfilament-associated protein of the intestinal microvillus (brush border/membrane attachment/a-actinin/immunofluorescence microscopy/immunoferritin label) ANTHONY BRETSCHER AND KLAUS WEBER Max Planck Institute for Biophysical Chemistry, D-3400 Gottingen, West Germany Communicated by Hugh E. Huxley, February 14, 1979 ABSTRACT The major protein associated with actin in the from the finding that the crossfilaments have molecular di- microfilament core of intestinal microvilli has been purified. mensions similar to purified muscle a-actinin (8), together with This protein, for which we propose the name villin, has a poly- the of a-actinin in microvilli as peptide molecular weight of approximately 95,000. Two argu- a brief report on presence ments suggest that villin may be the microvillus crossfilament judged by immunofluorescence microscopy (9), as well as the protein that links the microfilament core laterally down its presence in the brush border of a polypeptide with a molecular length to the cytoplasmic side of the plasma membrane. First, weight similar to that of a-actinin-i.e., 100,000 (4). In addi- electron microscopy shows that crossfilaments stay attached tion, indirect evidence from other systems has suggested that to isolated membrane-free microvillus cores. Calculation of the a-actinin may be involved in microfilament-membrane an- expected abundance of the crossfilament protein shows that only villin is present in sufficient quantity to account for these chorage (9-11). However, because a-actinin has never been structures. Second, decoration of microvillus cores by antibodies purified from nonmuscle tissues and its presence has only been to either actin or villin, followed by ferritin-labeled second inferred by immunological techniques, there exists no direct antibody in a sandwich procedure, results in specific labeling evidence for its role in microfilament-membrane attach- of the cores in both cases.
    [Show full text]
  • Visualization of a System of Filaments 7-10 Nm Thick in Cultured Cells of An
    Proc. Natl. Acad. Sci. USA Vol. 74, No. 6, pp. 2490-2494, June 1977 Cell Biology Visualization of a system of filaments 7-10 nm thick in cultured cells of an epithelioid line (Pt K2) by immunofluorescence microscopy (intermediate filaments/tonofilaments/keratin/autoantibodies/mitotic drugs) MARY OSBORN*, WERNER W. FRANKEt, AND KLAUS WEBER* * Max Planck Instittit fiir Biophysikalische Chemie, G6ttingen; and t Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany Communicated by J. Brachet, April 5, 1977 ABSTRACT During our studies with antibodies against MATERIALS AND METHODS structural proteins of the cytoskeleton of eukaryotic cells we have observed that sera from many normal rabbits decorate a Pt K2 cells, originally derived from rat kangaroo kidney epi- fiber system in cells of the established rat kangaroo cell line Pt thelium and Nettro 2a cells, a neuroblastoma line from mouse, K2. The display and organization of these fibers are different were from the American Type Culture Collection. Pt K2 cells from those of microfilament bundles (decorated by antibody were maintained in Eagle's minimal essential medium sup- to actin) and microtubules (decorated by antibody to tubulin). plemented with nonessential amino acids, glutamine, sodium This new fiber system can be further distinguished by its resis- tance to reorganization when cells are treated with Colcemid pyruvate, and 10% (vol/vol) fetal calf serum. Neuro 2a cells or cytochalasin B. The decoration of this fiber system is not were grown in Dulbecco's modified Eagle's medium containing detected if Pt K2 cells are fixed with formaldehyde. Such sera 10% fetal calf serum. Growth conditions for mouse 3T3 cells also appear to decorate swirls of perinuclear fibers in mouse have been described (6).
    [Show full text]
  • Encor Biotechnology Inc
    4949 SW 41st Blvd. Suites 40 & 50 Gainesville, FL 32608 Tel: (352) 372 7022 Fax: (352) 372 7066 [email protected] Catalogue MCA-2D1: Mouse Monoclonal Antibody to Vimentin-VIM The Immunogen: Vimentin is a protein subunit of 10nm or intermediate filaments, which are major components of the cytoskeleton in most cell types. Vimentin is the major intermediate filament subunit found in mesenchymal cells, and was first named and characterized in a collaborative study from the labs of German scientists Werner Franke and Klaus Weber (1). The name derives from the Latin “Vimentum“, meaning arrays of flexible rods such as in lattices, filigrees and wicker-work, which describes the intermediate filament network quite well. Vimentin is also found in many cell types in tissue culture, most notably fibroblasts, and in developing neuronal and astrocytic precursor cells in the central nervous system. Many cell lines such as HEK293, HeLa, 3T3 and Cos cells contain prominent vimentin networks. Vimentin frequently forms copolymers with other intermediate filament proteins, such as GFAP (in astrocytes, ependymal cells and neural stem cells), with desmin (in muscle and endothelial cells) and neurofilament proteins (in developing neurons). A E151K point mutation in the vimentin gene was shown to be causative of an autosomal dominant pulverulent cataract disease, but so far only in a single patient (2). Vimentin is a major protein of eye lens and cornea, and this mutation renders the molecule unable assemble into normal 10nm filaments. Antibodies to vimentin are useful in studies of stem cells and generally to reveal the filamentous cytoskeleton. The immunogen used to generate our antibody was recombinant human vimentin expressed in and purified from E.
    [Show full text]
  • The Organization of Titin Filaments in the Half-Sarcomere
    The Organization of Titin Filaments in the Half-Sarcomere Revealed by Monoclonal Antibodies in Immunoelectron Microscopy: A Map ofTen Nonrepetitive Epitopes Starting at the Z Line Extends Close to the M Line Dieter O. Fiirst, Mary Osborn, Riidiger Nave, and Klaus Weber Max-Pianck-Institute for Biophysical Chemistry, D-34 G6ttingen, Federal Republic of Germany Abstract. mAbs specific for titin or nebulin were antibodies decorating at or just before the Z line (T20, characterized by immunoblotting and fluorescence mi- T21) specifically recognized the insoluble titin TI com- croscopy. Immunoelectron microscopy on relaxed ponent but did not recognize TII, a proteolytic deriva- chicken breast muscle revealed unique transverse strip- tive. All other titin antibodies recognized TI and TII. ing patterns. Each of the 10 distinct titin antibodies Thus titin molecules appear as polar structures lacking provided a pair of delicate decoration lines per sarco- over large regions repetitive epitopes. One physical mere. The position of these pairs was centrally sym- end seems related to Z line anchorage, while the other metric to the M line and was antibody dependent. The may bind close to the M line. Titin epitopes influenced results provided a linear epitope map, which starts at by the contractional state of the sarcomere locate be- the Z line (antibody T20), covers five distinct posi- tween the N1 line and the A-I junction (T4, T1, Tll). tions along the I band (T21, T12, T4, T1, Tll), the A-I We discuss the results in relation to titin molecules junction (T3), and three distinct positions within the A having half-sarcomere lengths.
    [Show full text]
  • Tubulin-Tyrosine Ligase Has a Binding Site on Fi-Tubulin
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Tubulin-Tyrosine Ligase Has a Binding Site on fi-Tubulin: A Two-domain Structure of the Enzyme Juergen Wehland and Klaus Weber Max Planck Institute for Biophysical Chemistry, D-3400 Goettingen, Federal Republic of Germany Abstract. Tubulin-tyrosine ligase and a~-tubulin form accessibility of this site in tubulin and in microtubules a tight complex which is conveniently monitored by seems to explain why ligase acts preferentially on un- glycerol gradient centrifugation. Using two distinct polymerized tubulin. Ligase exposed to VS-protease is ligase monoclonal antibodies, several subunit-specific converted to a nicked derivative. This is devoid of tubulin monoclonal antibodies, and chemical cross- enzymatic activity but still forms the complex with linking, a ligase-binding site was identified on tubulin. Gel electrophoresis documents both 30- and a 13-tubulin. This site is retained when the carboxy- 14-kD domains, each which is immunologically and terminal domains of both tubulin subunits are removed biochemically distinct and seems to cover the entire by subtilisin treatment. The ligase-tubulin complex is molecule. The two domains interact tightly under also formed when ligase is added to al3-tubulin carry- physiological conditions. The 30-kD domain carries ing the monoclonal antibody YL 1/2 which binds only the binding sites for 13-tubulin and ATP. The 14-kD to the carboxyl end of tyrosinated a-tubulin. The domain can possibly form an additional part of the 13-tubulin-binding site described here explains the catalytic site as it harbors the epitope for the extreme substrate specificity of ligase, which does not monoclonal antibody ID3 which inhibits enzymatic act on other cellular proteins or carboxy-terminal pep- activity but not the formation of the ligase-tubulin tides derived from detyrosinated a-tubulin.
    [Show full text]
  • Tissue-Specific Co-Expression and in Vitro Heteropolymer Formation of the Two Small Branchiostoma Intermediate Filament Proteins A3 and B2
    doi:10.1006/jmbi.2001.5298availableonlineathttp://www.idealibrary.comon J. Mol. Biol. (2002) 316, 127±137 Tissue-Specific Co-expression and in vitro Heteropolymer Formation of the Two Small Branchiostoma Intermediate Filament Proteins A3 and B2 AntonKarabinos1,JuÈrgenSchuÈnemann1,DavidA.D.Parry2 andKlausWeber1* 1Max Planck Institute for The two small intermediate ®lament (IF) proteins A3 and B2 of the Biophysical Chemistry cephalochordate Amphioxus were investigated. Blot overlays indicated a Department of Biochemistry heterotypic interaction pattern of the recombinant proteins. While the Am Fassberg 11 individual proteins formed only aggregates, the stoichiometric mixture 37077 Goettingen, Germany formed obligatory heteropolymeric ®laments. Mutant proteins with a single cysteine residue in equivalent positions gave rise to ®laments that 2Institute of Fundamental oxidize to the disul®de-linked heterodimer, which can again form IF. Sciences, Massey University Thus the A3/B2 ®laments, which are expressed in the intestinal Palmerston North, New epithelium, are based on a hetero coiled coil. This keratin-like assembly Zealand process of A3 plus B2 was unexpected, since previous evolutionary tree calculations performed by two laboratories on the various Amphioxus IF proteins identi®ed keratin I and II orthologs but left the A/B group as a separate branch. We discuss obvious evolutionary aspects of the Amphioxus IF multigene family, including the previously made obser- vation that B1, the closest relative of B2, forms homopolymeric IF in vitro and is, like vertebrate type III proteins, expressed in mesodermally derived tissues. # 2002 Elsevier Science Ltd. Keywords: Amphioxus; Branchiostoma; cephalochordate; coiled coil; *Corresponding author developmental expression Introduction IV spans ®ve neuronal IF proteins. Two large mol- ecular mass proteins (synemin and paranemin), and the two IF proteins of the eye lens (phakinin A variety of epidermal fragility syndromes are and ®lensin) fall outside these subfamilies.
    [Show full text]