ECB-ART-34772
J Cell Biol
1984 Dec 01;996:2146-56. doi: 10.1083/jcb.99.6.2146.
Show Gene links
Show Anatomy links
Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching.
Leslie RJ
,
Saxton WM
,
Mitchison TJ
,
Neighbors B
,
Salmon ED
,
McIntosh JR
.
Abstract
Brain
tubulin has been conjugated with dichlorotriazinyl-aminofluorescein (DTAF) to form a visualizable complex for the study of
tubulin dynamics in living cells. By using several assays we confirm the finding of Keith et al. (Keith, C. H., J. R. Feramisco, and M. Shelanski, 1981, J. Cell Biol., 88:234-240) that DTAF-
tubulin polymerizes like control
tubulin in vitro. The fluorescein moiety of the complex is readily bleached by the 488-nm line from an argon ion laser. When irradiations are performed over short times (less than 1 s) and in the presence of 2 mM glutathione, a mixture of DTAF-
tubulin and control protein (as occurs after microinjection of the fluorescent conjugate into living cells) will retain full polymerization activity. Slow bleaching (approximately 5 min) or bleaching without glutathione promotes formation of covalent cross-links between neighboring polypeptides and kills the polymerization activity of DTAF-
tubulin , including some molecules that are neither cross-linked nor bleached. Even under conditions that damage DTAF-
tubulin , however, DTAF-microtubules are not destroyed by bleaching. They will continue to elongate by addition of DTAF-
tubulin subunits to their free ends, and they neither bind nor exchange subunits along their lateral surfaces. These results suggest that DTAF-
tubulin is a suitable analog for
tubulin , both in studies of protein incorporation and for investigations of fluorescence redistribution after photobleaching.
PubMed ID:
6438113
PMC ID:
PMC2113541
Article link:
J Cell Biol
Grant support:
[+]
Genes referenced:
LOC115919910
tubgcp2
References [+] :
Allen,
Structural polarity and directional growth of microtubules of Chlamydomonas flagella.
1974,
Pubmed
Allen,
Structural polarity and directional growth of microtubules of Chlamydomonas flagella.
1974,
Pubmed
Batteiger,
The use of Tween 20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose membranes.
1982,
Pubmed
Bergen,
Head-to-tail polymerization of microtubules in vitro. Electron microscope analysis of seeded assembly.
1980,
Pubmed
Bradford,
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
1976,
Pubmed
Bryan,
A quantitative analysis of microtubule elongation.
1976,
Pubmed
Cooper,
Insolubilization of hydroxyproline-rich cell wall glycoprotein in aerated carrot root slices.
1983,
Pubmed
Erickson,
Quantitative electrophoretic transfer of polypeptides from SDS polyacrylamide gels to nitrocellulose sheets: a method for their re-use in immunoautoradiographic detection of antigens.
1982,
Pubmed
Gibbons,
Chemical dissection of cilia.
1965,
Pubmed
Giloh,
Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate.
1982,
Pubmed
Hamel,
Glutamate-induced polymerization of tubulin: characteristics of the reaction and application to the large-scale purification of tubulin.
1981,
Pubmed
Heidemann,
Evidence for microtubule subunit addition to the distal end of mitotic structures in vitro.
1980,
Pubmed
Inoué,
Cell motility by labile association of molecules. The nature of mitotic spindle fibers and their role in chromosome movement.
1967,
Pubmed
Izant,
A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells.
1983,
Pubmed
Izant,
The role of calcium ions during mitosis. Calcium participates in the anaphase trigger.
1983,
Pubmed
Izant,
Microtubule-associated proteins: a monoclonal antibody to MAP2 binds to differentiated neurons.
1980,
Pubmed
Jacobson,
Measurement of the lateral mobility of cell surface components in single, living cells by fluorescence recovery after photobleaching.
1976,
Pubmed
Jacobson,
International workshop on the application of fluorescence photobleaching techniques to problems in cell biology.
1983,
Pubmed
Keith,
Direct visualization of fluorescein-labeled microtubules in vitro and in microinjected fibroblasts.
1981,
Pubmed
Kiehart,
Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system.
1981,
Pubmed
,
Echinobase
Kilmartin,
Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line.
1982,
Pubmed
Kim,
The periodic association of MAP2 with brain microtubules in vitro.
1979,
Pubmed
Kosower,
The glutathione status of cells.
1978,
Pubmed
Kreis,
Mobility of microinjected rhodamine actin within living chicken gizzard cells determined by fluorescence photobleaching recovery.
1982,
Pubmed
Kreis,
Microinjection of fluorescently labeled proteins into living cells with emphasis on cytoskeletal proteins.
1982,
Pubmed
Laemmli,
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
1970,
Pubmed
Lanni,
Fluorescence photobleaching recovery in solutions of labeled actin.
1981,
Pubmed
Margolis,
Opposite end assembly and disassembly of microtubules at steady state in vitro.
1978,
Pubmed
Meister,
Glutathione and related gamma-glutamyl compounds: biosynthesis and utilization.
1976,
Pubmed
Murphy,
Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro.
1975,
Pubmed
O'Farrell,
High resolution two-dimensional electrophoresis of proteins.
1975,
Pubmed
Rebhun,
Cyclic nucleotides, thioldisulfide status of proteins, and cellular control processes.
1976,
Pubmed
,
Echinobase
Salmon,
Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching.
1984,
Pubmed
,
Echinobase
Salmon,
Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.
1984,
Pubmed
,
Echinobase
Salmon,
Pressure-induced depolymerization of spindle microtubules. I. Changes in birefringence and spindle length.
1975,
Pubmed
Saxton,
Tubulin dynamics in cultured mammalian cells.
1984,
Pubmed
Scholey,
Isolation of microtubules and a dynein-like MgATPase from unfertilized sea urchin eggs.
1984,
Pubmed
,
Echinobase
Sheetz,
Membrane damage caused by irradiation of fluorescent concanavalin A.
1979,
Pubmed
Sloboda,
Microtubule-associated proteins and the stimulation of tubulin assembly in vitro.
1976,
Pubmed
Snell,
Assembly of chick brain tubulin onto isolated basal bodies of Chlamydomonas reinhardi.
1974,
Pubmed
Taylor,
Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells.
1978,
Pubmed
Towbin,
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
1979,
Pubmed
Travis,
Preparation and characterization of native, fluorescently labelled brain tubulin and microtubule-associated proteins (MAPs).
1980,
Pubmed
Wadsworth,
Microinjection of fluorescent tubulin into dividing sea urchin cells.
1983,
Pubmed
,
Echinobase
Wadsworth,
Characterization of tubulin modified with the sulfhydryl-reactive fluorochrome monobromo(trimethylammonio)bimane.
1982,
Pubmed
Wang,
Fluorescent localization of contractile proteins in tissue culture cells.
1982,
Pubmed
Wang,
Fluorescent analog cytochemistry of contractile proteins.
1982,
Pubmed
Wang,
Mobility of cytoplasmic and membrane-associated actin in living cells.
1982,
Pubmed
Webb,
Molecular mobility on the cell surface.
1981,
Pubmed
Weingarten,
A protein factor essential for microtubule assembly.
1975,
Pubmed
Weingarten,
Properties of the depolymerization products of microtubules from mammalian brain.
1974,
Pubmed
Weisenberg,
Microtubule formation in vitro in solutions containing low calcium concentrations.
1972,
Pubmed
Willingham,
The visualization of fluorescent proteins in living cells by video intensification microscopy (VIM).
1978,
Pubmed
Wilson,
A rapid filtration assay for analysis of microtubule assembly, disassembly, and steady-state tubulin flux.
1982,
Pubmed
Wojcieszyn,
Diffusion of injected macromolecules within the cytoplasm of living cells.
1981,
Pubmed