Louis Morejohn - Academia.edu (original) (raw)
Papers by Louis Morejohn
Planta, Feb 1, 1987
The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's... more The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24 ~ C with an apparent inhibition constant (Ki) of 1.4.10-4 M for rose tubulin and an apparent Ki= 8.8" 10-v M for brain tubulin. The binding of [3H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2-3 h to reach equilibrium at 24 ~ C, and was not affected by vinblastine sulfate. The binding of [3H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7.102 M-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24 ~ C. The values for brain tubulin were K=2.46" 106 M-1 and r=0.45 at 37 ~ C. The binding of [3H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to
The Plant Cell, Sep 1, 1993
An understanding of the regulation of microtubule polymerization and dynamics in plant cells requ... more An understanding of the regulation of microtubule polymerization and dynamics in plant cells requires biochemical information on the structures, functions, and molecular interactions of plant tubulin and microtubule-associated proteins (MAPs) that regulate microtubule function. We have probed the regulatory domain and polymerization domain of purified maize tubulin using MAP2, an extensively characterized mammalian neumnal MAP. MAP2 bound to the surface of preformed, taxol-stabilized maize microtubules, with binding saturation occurring with one MAP2 molecule per five to six tubulin dimers, as it does with mammalian microtubules. MAP2 binding and dissociation analyses revealed two affinity classes of binding sites on maize microtubules: a high-affinity site 12 dimers apart that may be homologous to the cognate mammalian MAP2 binding site and an additional low-affinity site also 12 dimers apart that may be homologous to the mammalian tau binding site. MAPS corrected in vitro folding errors in taxol-stabilized maize microtubules and reduced the critical concentration of maize tubulin polymerization eightfold, from 8.3 to 1.0 pM. However, MAPS dissociated much more readily from maize microtubules than from mammalian microtubules and induced the assembly of maize tubulin into aberrant helical ribbon polymers that remained stable for prolonged periods. Our results indicated that MAPS binds to maize tubulin via a partially specific, low-fidelity interaction that reflects unique structural and functional properties of the polymerization and regulatory domains of plant tubulin and possibly of the tubulin binding domains of undocumented MAPs that regulate microtubule function in plant cells.
The Plant Cell, Dec 1, 1994
Cell and Tissue Research, May 1, 1979
The filamentous cytoskeletons of epidermal cells of the bullfrog (Rana catesbeiana) were investig... more The filamentous cytoskeletons of epidermal cells of the bullfrog (Rana catesbeiana) were investigated by electron microscopy. Following treatment with trypsin, sheets of epithelium were removed from swatches of abdominal skin. Trypsinization produces differential effects on the ultrastructure of the various cell layers. The desmosomes of all layers, except those of the stratum corneum, are split by trypsinization and the resulting desmosomal plaques fastened to tonofilaments are retracted into cells to form deep "inpouchings" of the plasma membranes, while tonofilament bundles become diffuse. Epidermal sheets were gently homogenized to form a suspension of cell remnants with damaged plasma membranes as indicated by vital dye exclusion tests and electron microscopy. Cytoskeletons retain their shapes, yet the lateral distances between individual tonofilaments within bundles appear to increase, thus forming diffuse lacelike structures. These observations support the suggestion that tonofilament bundles, when fastened to desmosomes, have elastic properties. The possible role of the cytoskeletons in the maintenance of cell size and shape in an ion-transporting epithelium is discussed.
Proceedings of the National Academy of Sciences of the United States of America, Mar 1, 1984
Tubulins from different higher plant species are immunologically nonidentical and bind colchicine... more Tubulins from different higher plant species are immunologically nonidentical and bind colchicine differentially (microtubules/cultured plant cells/tubulin antibodies/immunoautoradiography/antimicrotubule drugs)
We have initiated immunological and drug- binding studies on the tubulins from different higher p... more We have initiated immunological and drug- binding studies on the tubulins from different higher plant species. Antibodies were raised against electrophoretically sep- arated rose (Rosa sp.) tubulin a- and 13-subunits and charac- terized by immunoblot autoradiographic assays. Each IgG preparation bound to its antigen and cross-reacted differen- tially with the respective tubulin subunits from an alga, sea urchin, rabbit, and cow. Antigenic determinants were shared more among the ,f-subunits than among the a-subunits from these organisms. Tubulins were isolated from cultured cells of carrot (Daucus carota) and hibiscus (Hibiscus rosa-senensis). Immunoautoradiography and quantitation of cross-reactivity on blots showed nonidentity among homologous subunits from rose, carrot, hibiscus, and alga tubulins, with more antigenic differences among a-subunits than among P-subunits. Com- parative colchicine-binding assays showed that rose and hibis- cus tubulins bound 33% and 65%, respectively,...
Plant Physiology, 1994
Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in ... more Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicofiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds ['4C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (&) = 117 nM (pH 6.9; 23'C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharmacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of ["Cloryzalin to tubulin with an inhi-' Supported by grants to L.C.
Plant Physiology, 1993
Microtubules are hollow, filamentous polymers composed principally of the globular protein tubuli... more Microtubules are hollow, filamentous polymers composed principally of the globular protein tubulin, a 100-kD heterodimer having similar (Y-and P-subunits. Tubulin dimers polyme-ize in a head-to-tail manner to form 4-to 5-nm linear protofilaments, typically 13 of which are laterally associated in the wall of the 24-nm diameter microtubule (A~~~, 1979; Fosket and Morejohn, 1992). During the plant cell cycle,
Cell Biology International Reports, 1985
The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cu... more The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose (Rosa sp* cv. Paul's scarlet) was examined by NadodecylSOqpolyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of a-subunit to B-subunit, and low molecular weight polypeptides, one (-32 Ed) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C-and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual a-and B-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.
Cell and Molecular Biology of the Cytoskeleton, 1986
Because brain tissue is a readily available source of abundant and easily purified microtubule pr... more Because brain tissue is a readily available source of abundant and easily purified microtubule protein, much of our knowledge of the biochemistry of microtubules and tubulin came first from studies on animal tubulin. Subsequently tubulin was isolated and characterized from extracts of numerous types of vertebrate and invertebrate animal tissues. The aggregate of this work has given us a general model for the biochemistry of tubulin. In recent years investigators have begun to purify and characterize the tubulins from plants, fungi, and protists, and we have summarized the methodologies and results herein with some historical perspective. Apparently the tubulins from these nonanimal sources exhibit the general structural characteristics observed with animal tubulins. However, recent immunological, pharmacological, electrophoretic, peptide mapping, and sequencing data indicate that nonanimal tubulins possess several interesting structural properties that are not fully shared with tubulins from animal sources. These results suggest more divergence of tubulins than was previously thought to exist. Our purpose in this review is to catalogue the recent body of work that has come from studies on tubulins from plants, fungi, and protists and to provide some perspective on the differences and similarities between these nonanimal tubulins and their animal counterparts.
Proceedings in Life Sciences, 1981
Cytokinins are characterized as hormones which control cell proliferation in cultured plant tissu... more Cytokinins are characterized as hormones which control cell proliferation in cultured plant tissues (Skoog et al., 1965). The best currently available evidence suggests that cytokinins regulate the passage of cells through their division cycle by regulating events which are necessary for the transition from G2 to mitosis (Jouanneau, 1971; Fosket, 1977). Cytokinins also have been shown to bring about both qualitative and quantitative changes in protein synthesis in various plant systems (Jouanneau, 1971; Short et al., 1974; Tepfer and Fosket, 1978), and, in the case of cultured soybean cells, these changes were shown to precede cytokinin-induced cell division (Fosket and Tepfer, 1978). If these quantitative and qualitative effects on protein synthesis are necessary for cytokinin-induced cell division, then it should be possible to identify and determine the function of those proteins which are required for cell divisions and whose synthesis is dependent upon cytokinin. One of the proteins whose synthesis was strongly promoted by cytokinins in cultured soybean cells had a molecular weight near 55,000 daltons, approximately the molecular weight of the subunits of tubulin (Fosket et al., 1977). Since tubulin is the principle structural component of the microtubules which comprise the mitotic spindle (Hepler and Palevitz, 1974), we have begun an investigation to determine whether or not tubulin synthesis is regulated by cytokinin.
Biochemistry and molecular biology international, 1994
To obtain information on the functional domains of tubulin from a dicot plant, we investigated th... more To obtain information on the functional domains of tubulin from a dicot plant, we investigated the interactions of tobacco tubulin with MAP2 from bovine brain supernatant. Taxol-stabilized tobacco and bovine brain microtubules had similar binding capacities for MAP2 (1 mol MAP2 per 8-9 mol tubulin). However, MAP2 dissociated from tobacco microtubules more readily than from bovine brain microtubules and induced the polymerization of tobacco tubulin into aberrant helical ribbon polymers, rather than microtubules. Ribbon assembly was partially suppressed by 50 mM KCl. Abundant tobacco microtubules formed when MAP2-induced nucleation was by-passed with microtubule seeds. Thus, deficient nucleation of tobacco tubulin assembly by MAP2 reflects distinct properties of the polymerization and regulatory domains of plant and animal tubulins.
THE PLANT CELL ONLINE, 1994
The Plant Cell, 1995
The p86 subunit of eukaryotic initiation factor-(iso)4F from wheat germ exhibits saturable and su... more The p86 subunit of eukaryotic initiation factor-(iso)4F from wheat germ exhibits saturable and substoichiometric binding to maize microtubules, induces microtubule bundling in vitro, and is colocalized or closely associated with cortical microtubule bundles in maize root cells, indicating its function as a microtubule-associated protein (MAP). The effects of p86 on the growth of short, taxol-stabilized maize microtubules were investigated. Pure microtubules underwent a gradual length redistribution, an increase in mean length, anda decrease in number concentration consistent with an end-to-end annealing mechanism of microtubule growth. Saturating p86 enhanced the microtubule length distribution and produced significantly longer and fewer microtubules than the control, indicating a facilitation of annealing by p86. Confirmation of endwise annealing rather than of dynamic instability as the mechanism for microtubule growth was made using mammalian MAPS, which also promoted the redistribution of length, increase in mean length, and decrease in number concentration of taxol-stabilized maize microtubules. Enhancement of microtubule growth occurred concomitant with bundling by p86, indicating that an alignment of microtubules in bundles facilitated endwise annealing kinetics. The results demonstrate that nonfacile plant microtubules can spontaneously elongate by endwise annealing and that MAPs enhance the rate of annealing. The p86 subunit of eukaryotic initiation factor-(iso)4F may be an important regulator of microtubule dynamics in plant cells.
The Plant Cell, 1993
An understanding of the regulation of microtubule polymerization and dynamics in plant cells requ... more An understanding of the regulation of microtubule polymerization and dynamics in plant cells requires biochemical information on the structures, functions, and molecular interactions of plant tubulin and microtubule-associated proteins (MAPs) that regulate microtubule function. We have probed the regulatory domain and polymerization domain of purified maize tubulin using MAP2, an extensively characterized mammalian neumnal MAP. MAP2 bound to the surface of preformed, taxol-stabilized maize microtubules, with binding saturation occurring with one MAP2 molecule per five to six tubulin dimers, as it does with mammalian microtubules. MAP2 binding and dissociation analyses revealed two affinity classes of binding sites on maize microtubules: a high-affinity site 12 dimers apart that may be homologous to the cognate mammalian MAP2 binding site and an additional low-affinity site also 12 dimers apart that may be homologous to the mammalian tau binding site. MAPS corrected in vitro folding errors in taxol-stabilized maize microtubules and reduced the critical concentration of maize tubulin polymerization eightfold, from 8.3 to 1.0 pM. However, MAPS dissociated much more readily from maize microtubules than from mammalian microtubules and induced the assembly of maize tubulin into aberrant helical ribbon polymers that remained stable for prolonged periods. Our results indicated that MAPS binds to maize tubulin via a partially specific, low-fidelity interaction that reflects unique structural and functional properties of the polymerization and regulatory domains of plant tubulin and possibly of the tubulin binding domains of undocumented MAPs that regulate microtubule function in plant cells.
The Journal of cell biology, 1984
Tubulin was isolated from cultured cells of rose (Rosa, sp.cv. Paul's scarlet) by DEAE-Sephad... more Tubulin was isolated from cultured cells of rose (Rosa, sp.cv. Paul's scarlet) by DEAE-Sephadex A50 chromatography, and the taxol-induced polymerization of microtubules in vitro was characterized at 24 degrees C by turbidity development, sedimentation analysis, and electron microscopy. Numerous, short microtubules were formed in the presence of taxol, and maximum levels of turbidity and polymer yield were obtained at approximately 2:1 molar ratios of taxol to tubulin. The critical concentration of rose tubulin for polymerization in saturating taxol was estimated to be 0.21 mg/ml. Colchicine inhibited the taxol-induced polymerization of tubulin as shown by sedimentation assays; however, much higher concentrations of colchicine were required for the inhibition of taxol-induced rose tubulin assembly than for inhibition of taxol-induced mammalian brain tubulin assembly. On the basis of the relative sensitivity of rose tubulin assembly to taxol and its insensitivity to colchicine, we...
Science, 1984
oxidized surficial layer of the finegrained ooze. Chondrites is a facies breaker because it occur... more oxidized surficial layer of the finegrained ooze. Chondrites is a facies breaker because it occurs in situations where there is a gradual redox boundary within burrowing reach of the sea floor and in sediment that is rich in organic (nutritious) material; this situation underlies many sorts of sea floors. The burrow has a distinctive and elaborate morphology; it does not reflect the sort of generalized pattern that might be expected in a trace-making animal with broad environmental tolerances. The occurrence of Chondrites is related to chemically reducing conditions deep within the sediment and is only indirectly dependent on sea-floor conditions.
Proceedings of the National Academy of Sciences, 1984
We have initiated immunological and drug-binding studies on the tubulins from different higher pl... more We have initiated immunological and drug-binding studies on the tubulins from different higher plant species. Antibodies were raised against electrophoretically separated rose ( Rosa sp.) tubulin α- and β-subunits and characterized by immunoblot autoradiographic assays. Each IgG preparation bound to its antigen and cross-reacted differentially with the respective tubulin subunits from an alga, sea urchin, rabbit, and cow. Antigenic determinants were shared more among the β-subunits than among the α-subunits from these organisms. Tubulins were isolated from cultured cells of carrot ( Daucus carota ) and hibiscus ( Hibiscus rosa-senensis ). Immunoautoradiography and quantitation of cross-reactivity on blots showed nonidentity among homologous subunits from rose, carrot, hibiscus, and alga tubulins, with more antigenic differences among α-subunits than among β-subunits. Comparative colchicine-binding assays showed that rose and hibiscus tubulins bound 33% and 65%, respectively, of the ...
Proceedings of the National Academy of Sciences, 1995
The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of ... more The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cel...
Planta, Feb 1, 1987
The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's... more The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24 ~ C with an apparent inhibition constant (Ki) of 1.4.10-4 M for rose tubulin and an apparent Ki= 8.8" 10-v M for brain tubulin. The binding of [3H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2-3 h to reach equilibrium at 24 ~ C, and was not affected by vinblastine sulfate. The binding of [3H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7.102 M-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24 ~ C. The values for brain tubulin were K=2.46" 106 M-1 and r=0.45 at 37 ~ C. The binding of [3H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to
The Plant Cell, Sep 1, 1993
An understanding of the regulation of microtubule polymerization and dynamics in plant cells requ... more An understanding of the regulation of microtubule polymerization and dynamics in plant cells requires biochemical information on the structures, functions, and molecular interactions of plant tubulin and microtubule-associated proteins (MAPs) that regulate microtubule function. We have probed the regulatory domain and polymerization domain of purified maize tubulin using MAP2, an extensively characterized mammalian neumnal MAP. MAP2 bound to the surface of preformed, taxol-stabilized maize microtubules, with binding saturation occurring with one MAP2 molecule per five to six tubulin dimers, as it does with mammalian microtubules. MAP2 binding and dissociation analyses revealed two affinity classes of binding sites on maize microtubules: a high-affinity site 12 dimers apart that may be homologous to the cognate mammalian MAP2 binding site and an additional low-affinity site also 12 dimers apart that may be homologous to the mammalian tau binding site. MAPS corrected in vitro folding errors in taxol-stabilized maize microtubules and reduced the critical concentration of maize tubulin polymerization eightfold, from 8.3 to 1.0 pM. However, MAPS dissociated much more readily from maize microtubules than from mammalian microtubules and induced the assembly of maize tubulin into aberrant helical ribbon polymers that remained stable for prolonged periods. Our results indicated that MAPS binds to maize tubulin via a partially specific, low-fidelity interaction that reflects unique structural and functional properties of the polymerization and regulatory domains of plant tubulin and possibly of the tubulin binding domains of undocumented MAPs that regulate microtubule function in plant cells.
The Plant Cell, Dec 1, 1994
Cell and Tissue Research, May 1, 1979
The filamentous cytoskeletons of epidermal cells of the bullfrog (Rana catesbeiana) were investig... more The filamentous cytoskeletons of epidermal cells of the bullfrog (Rana catesbeiana) were investigated by electron microscopy. Following treatment with trypsin, sheets of epithelium were removed from swatches of abdominal skin. Trypsinization produces differential effects on the ultrastructure of the various cell layers. The desmosomes of all layers, except those of the stratum corneum, are split by trypsinization and the resulting desmosomal plaques fastened to tonofilaments are retracted into cells to form deep "inpouchings" of the plasma membranes, while tonofilament bundles become diffuse. Epidermal sheets were gently homogenized to form a suspension of cell remnants with damaged plasma membranes as indicated by vital dye exclusion tests and electron microscopy. Cytoskeletons retain their shapes, yet the lateral distances between individual tonofilaments within bundles appear to increase, thus forming diffuse lacelike structures. These observations support the suggestion that tonofilament bundles, when fastened to desmosomes, have elastic properties. The possible role of the cytoskeletons in the maintenance of cell size and shape in an ion-transporting epithelium is discussed.
Proceedings of the National Academy of Sciences of the United States of America, Mar 1, 1984
Tubulins from different higher plant species are immunologically nonidentical and bind colchicine... more Tubulins from different higher plant species are immunologically nonidentical and bind colchicine differentially (microtubules/cultured plant cells/tubulin antibodies/immunoautoradiography/antimicrotubule drugs)
We have initiated immunological and drug- binding studies on the tubulins from different higher p... more We have initiated immunological and drug- binding studies on the tubulins from different higher plant species. Antibodies were raised against electrophoretically sep- arated rose (Rosa sp.) tubulin a- and 13-subunits and charac- terized by immunoblot autoradiographic assays. Each IgG preparation bound to its antigen and cross-reacted differen- tially with the respective tubulin subunits from an alga, sea urchin, rabbit, and cow. Antigenic determinants were shared more among the ,f-subunits than among the a-subunits from these organisms. Tubulins were isolated from cultured cells of carrot (Daucus carota) and hibiscus (Hibiscus rosa-senensis). Immunoautoradiography and quantitation of cross-reactivity on blots showed nonidentity among homologous subunits from rose, carrot, hibiscus, and alga tubulins, with more antigenic differences among a-subunits than among P-subunits. Com- parative colchicine-binding assays showed that rose and hibis- cus tubulins bound 33% and 65%, respectively,...
Plant Physiology, 1994
Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in ... more Amiprophos-methyl (APM), a phosphoric amide herbicide, was previously reported to inhibit the in vitro polymerization of isolated plant tubulin (L.C. Morejohn, D.E. Fosket [1984] Science 224: 874-876), yet little other biochemical information exists concerning this compound. To characterize further the mechanism of action of APM, its interactions with tubulin and microtubules purified from cultured cells of tobacco (Nicofiana tabacum cv Bright Yellow-2) were investigated. Low micromolar concentrations of APM depolymerized preformed, taxol-stabilized tobacco microtubules. Remarkably, at the lowest APM concentration examined, many short microtubules were redistributed into fewer but 2.7-fold longer microtubules without a substantial decrease in total polymer mass, a result consistent with an end-to-end annealing of microtubules with enhanced kinetic properties. Quasi-equilibrium binding measurements showed that tobacco tubulin binds ['4C]oryzalin with high affinity to produce a tubulin-oryzalin complex having a dissociation constant (&) = 117 nM (pH 6.9; 23'C). Also, an estimated maximum molar binding stoichiometry of 0.32 indicates pharmacological heterogeneity of tobacco dimers and may be related to structural heterogeneity of tobacco tubulin subunits. APM inhibits competitively the binding of ["Cloryzalin to tubulin with an inhi-' Supported by grants to L.C.
Plant Physiology, 1993
Microtubules are hollow, filamentous polymers composed principally of the globular protein tubuli... more Microtubules are hollow, filamentous polymers composed principally of the globular protein tubulin, a 100-kD heterodimer having similar (Y-and P-subunits. Tubulin dimers polyme-ize in a head-to-tail manner to form 4-to 5-nm linear protofilaments, typically 13 of which are laterally associated in the wall of the 24-nm diameter microtubule (A~~~, 1979; Fosket and Morejohn, 1992). During the plant cell cycle,
Cell Biology International Reports, 1985
The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cu... more The requirement for proteinase inhibitors during the chromatographic isolation of tubulin from cultured cells of rose (Rosa sp* cv. Paul's scarlet) was examined by NadodecylSOqpolyacrylamide gel electrophoresis, electron microscopy and immunoblotting. Tubulin fractions isolated in the absence of proteinase inhibitors showed substoichiometric ratios of a-subunit to B-subunit, and low molecular weight polypeptides, one (-32 Ed) of which coassembled with polymers. Electron microscopy revealed polymorphic structures, including C-and S-shaped ribbons and free protofilaments. Immunoblotting experiments with IgGs to the individual a-and B-subunits showed that some of the low molecular weight polypeptides were fragments of proteolytically degraded subunits. The use of low micromolar concentrations of the synthetic proteinase inhibitors leupeptin hemisulfate and pepstatin A protected tubulin from endogenous proteolytic activities during the isolation procedure and resulted in increased tubulin purity.
Cell and Molecular Biology of the Cytoskeleton, 1986
Because brain tissue is a readily available source of abundant and easily purified microtubule pr... more Because brain tissue is a readily available source of abundant and easily purified microtubule protein, much of our knowledge of the biochemistry of microtubules and tubulin came first from studies on animal tubulin. Subsequently tubulin was isolated and characterized from extracts of numerous types of vertebrate and invertebrate animal tissues. The aggregate of this work has given us a general model for the biochemistry of tubulin. In recent years investigators have begun to purify and characterize the tubulins from plants, fungi, and protists, and we have summarized the methodologies and results herein with some historical perspective. Apparently the tubulins from these nonanimal sources exhibit the general structural characteristics observed with animal tubulins. However, recent immunological, pharmacological, electrophoretic, peptide mapping, and sequencing data indicate that nonanimal tubulins possess several interesting structural properties that are not fully shared with tubulins from animal sources. These results suggest more divergence of tubulins than was previously thought to exist. Our purpose in this review is to catalogue the recent body of work that has come from studies on tubulins from plants, fungi, and protists and to provide some perspective on the differences and similarities between these nonanimal tubulins and their animal counterparts.
Proceedings in Life Sciences, 1981
Cytokinins are characterized as hormones which control cell proliferation in cultured plant tissu... more Cytokinins are characterized as hormones which control cell proliferation in cultured plant tissues (Skoog et al., 1965). The best currently available evidence suggests that cytokinins regulate the passage of cells through their division cycle by regulating events which are necessary for the transition from G2 to mitosis (Jouanneau, 1971; Fosket, 1977). Cytokinins also have been shown to bring about both qualitative and quantitative changes in protein synthesis in various plant systems (Jouanneau, 1971; Short et al., 1974; Tepfer and Fosket, 1978), and, in the case of cultured soybean cells, these changes were shown to precede cytokinin-induced cell division (Fosket and Tepfer, 1978). If these quantitative and qualitative effects on protein synthesis are necessary for cytokinin-induced cell division, then it should be possible to identify and determine the function of those proteins which are required for cell divisions and whose synthesis is dependent upon cytokinin. One of the proteins whose synthesis was strongly promoted by cytokinins in cultured soybean cells had a molecular weight near 55,000 daltons, approximately the molecular weight of the subunits of tubulin (Fosket et al., 1977). Since tubulin is the principle structural component of the microtubules which comprise the mitotic spindle (Hepler and Palevitz, 1974), we have begun an investigation to determine whether or not tubulin synthesis is regulated by cytokinin.
Biochemistry and molecular biology international, 1994
To obtain information on the functional domains of tubulin from a dicot plant, we investigated th... more To obtain information on the functional domains of tubulin from a dicot plant, we investigated the interactions of tobacco tubulin with MAP2 from bovine brain supernatant. Taxol-stabilized tobacco and bovine brain microtubules had similar binding capacities for MAP2 (1 mol MAP2 per 8-9 mol tubulin). However, MAP2 dissociated from tobacco microtubules more readily than from bovine brain microtubules and induced the polymerization of tobacco tubulin into aberrant helical ribbon polymers, rather than microtubules. Ribbon assembly was partially suppressed by 50 mM KCl. Abundant tobacco microtubules formed when MAP2-induced nucleation was by-passed with microtubule seeds. Thus, deficient nucleation of tobacco tubulin assembly by MAP2 reflects distinct properties of the polymerization and regulatory domains of plant and animal tubulins.
THE PLANT CELL ONLINE, 1994
The Plant Cell, 1995
The p86 subunit of eukaryotic initiation factor-(iso)4F from wheat germ exhibits saturable and su... more The p86 subunit of eukaryotic initiation factor-(iso)4F from wheat germ exhibits saturable and substoichiometric binding to maize microtubules, induces microtubule bundling in vitro, and is colocalized or closely associated with cortical microtubule bundles in maize root cells, indicating its function as a microtubule-associated protein (MAP). The effects of p86 on the growth of short, taxol-stabilized maize microtubules were investigated. Pure microtubules underwent a gradual length redistribution, an increase in mean length, anda decrease in number concentration consistent with an end-to-end annealing mechanism of microtubule growth. Saturating p86 enhanced the microtubule length distribution and produced significantly longer and fewer microtubules than the control, indicating a facilitation of annealing by p86. Confirmation of endwise annealing rather than of dynamic instability as the mechanism for microtubule growth was made using mammalian MAPS, which also promoted the redistribution of length, increase in mean length, and decrease in number concentration of taxol-stabilized maize microtubules. Enhancement of microtubule growth occurred concomitant with bundling by p86, indicating that an alignment of microtubules in bundles facilitated endwise annealing kinetics. The results demonstrate that nonfacile plant microtubules can spontaneously elongate by endwise annealing and that MAPs enhance the rate of annealing. The p86 subunit of eukaryotic initiation factor-(iso)4F may be an important regulator of microtubule dynamics in plant cells.
The Plant Cell, 1993
An understanding of the regulation of microtubule polymerization and dynamics in plant cells requ... more An understanding of the regulation of microtubule polymerization and dynamics in plant cells requires biochemical information on the structures, functions, and molecular interactions of plant tubulin and microtubule-associated proteins (MAPs) that regulate microtubule function. We have probed the regulatory domain and polymerization domain of purified maize tubulin using MAP2, an extensively characterized mammalian neumnal MAP. MAP2 bound to the surface of preformed, taxol-stabilized maize microtubules, with binding saturation occurring with one MAP2 molecule per five to six tubulin dimers, as it does with mammalian microtubules. MAP2 binding and dissociation analyses revealed two affinity classes of binding sites on maize microtubules: a high-affinity site 12 dimers apart that may be homologous to the cognate mammalian MAP2 binding site and an additional low-affinity site also 12 dimers apart that may be homologous to the mammalian tau binding site. MAPS corrected in vitro folding errors in taxol-stabilized maize microtubules and reduced the critical concentration of maize tubulin polymerization eightfold, from 8.3 to 1.0 pM. However, MAPS dissociated much more readily from maize microtubules than from mammalian microtubules and induced the assembly of maize tubulin into aberrant helical ribbon polymers that remained stable for prolonged periods. Our results indicated that MAPS binds to maize tubulin via a partially specific, low-fidelity interaction that reflects unique structural and functional properties of the polymerization and regulatory domains of plant tubulin and possibly of the tubulin binding domains of undocumented MAPs that regulate microtubule function in plant cells.
The Journal of cell biology, 1984
Tubulin was isolated from cultured cells of rose (Rosa, sp.cv. Paul's scarlet) by DEAE-Sephad... more Tubulin was isolated from cultured cells of rose (Rosa, sp.cv. Paul's scarlet) by DEAE-Sephadex A50 chromatography, and the taxol-induced polymerization of microtubules in vitro was characterized at 24 degrees C by turbidity development, sedimentation analysis, and electron microscopy. Numerous, short microtubules were formed in the presence of taxol, and maximum levels of turbidity and polymer yield were obtained at approximately 2:1 molar ratios of taxol to tubulin. The critical concentration of rose tubulin for polymerization in saturating taxol was estimated to be 0.21 mg/ml. Colchicine inhibited the taxol-induced polymerization of tubulin as shown by sedimentation assays; however, much higher concentrations of colchicine were required for the inhibition of taxol-induced rose tubulin assembly than for inhibition of taxol-induced mammalian brain tubulin assembly. On the basis of the relative sensitivity of rose tubulin assembly to taxol and its insensitivity to colchicine, we...
Science, 1984
oxidized surficial layer of the finegrained ooze. Chondrites is a facies breaker because it occur... more oxidized surficial layer of the finegrained ooze. Chondrites is a facies breaker because it occurs in situations where there is a gradual redox boundary within burrowing reach of the sea floor and in sediment that is rich in organic (nutritious) material; this situation underlies many sorts of sea floors. The burrow has a distinctive and elaborate morphology; it does not reflect the sort of generalized pattern that might be expected in a trace-making animal with broad environmental tolerances. The occurrence of Chondrites is related to chemically reducing conditions deep within the sediment and is only indirectly dependent on sea-floor conditions.
Proceedings of the National Academy of Sciences, 1984
We have initiated immunological and drug-binding studies on the tubulins from different higher pl... more We have initiated immunological and drug-binding studies on the tubulins from different higher plant species. Antibodies were raised against electrophoretically separated rose ( Rosa sp.) tubulin α- and β-subunits and characterized by immunoblot autoradiographic assays. Each IgG preparation bound to its antigen and cross-reacted differentially with the respective tubulin subunits from an alga, sea urchin, rabbit, and cow. Antigenic determinants were shared more among the β-subunits than among the α-subunits from these organisms. Tubulins were isolated from cultured cells of carrot ( Daucus carota ) and hibiscus ( Hibiscus rosa-senensis ). Immunoautoradiography and quantitation of cross-reactivity on blots showed nonidentity among homologous subunits from rose, carrot, hibiscus, and alga tubulins, with more antigenic differences among α-subunits than among β-subunits. Comparative colchicine-binding assays showed that rose and hibiscus tubulins bound 33% and 65%, respectively, of the ...
Proceedings of the National Academy of Sciences, 1995
The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of ... more The isozyme form of eukaryotic initiation factor 4F [eIF-(iso)4F] from wheat germ is composed of a p28 subunit that binds the 7-methylguanine cap of mRNA and a p86 subunit having unknown function. The p86 subunit was found to have limited sequence similarity to a kinesin-like protein encoded by the katA gene of Arabidopsis thaliana. Native wheat germ eIF-(iso)4F and bacterially expressed p86 subunit and p86-p28 complex bound to taxol-stabilized maize microtubules (MTs) in vitro. Binding saturation occurred at 1 mol of p86 per 5-6 mol of polymerized tubulin dimer, demonstrating a substoichiometric interaction of p86 with MTs. No evidence was found for a direct interaction of the p28 subunit with MTs. Unlike kinesin, cosedimentation of eIF-(iso)4F with MTs was neither reduced by MgATP nor enhanced by adenosine 5'-[gamma-imido]triphosphate. Both p86 subunit and p86-p28 complex induced the bundling of MTs in vitro. The p86 subunit was immunolocalized to the cytosol in root maize cel...