scholarly journals 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 ◽  
Vol 99 (6) ◽  
pp. 2157-2164 ◽  
Author(s):  
E D Salmon ◽  
W M Saxton ◽  
R J Leslie ◽  
M L Karow ◽  
J R McIntosh
Keyword(s):  
Diffusion Coefficient ◽  
Sea Urchin ◽  
Video Camera ◽  
Laser Fluorescence ◽  
Lytechinus Variegatus ◽  
Embryonic Cells ◽  
Ion Laser ◽  
Argon Ion ◽  
Brain Tubulin ◽  
Small Oligomer

The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.

scholarly journals Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching.

1984 ◽  
Vol 99 (6) ◽  
pp. 2165-2174 ◽  
Author(s):  
E D Salmon ◽  
R J Leslie ◽  
W M Saxton ◽  
M L Karow ◽  
J R McIntosh
Keyword(s):  
Sea Urchin ◽  
Argon Laser ◽  
Video Camera ◽  
Bovine Brain ◽  
Lytechinus Variegatus ◽  
Laser Bleaching ◽  
Spindle Function ◽  
Spindle Structure ◽  
Brain Tubulin

The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)--fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.

scholarly journals Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching.

1984 ◽  
Vol 99 (6) ◽  
pp. 2146-2156 ◽  
Author(s):  
R J Leslie ◽  
W M Saxton ◽  
T J Mitchison ◽  
B Neighbors ◽  
E D Salmon ◽  
...  
Keyword(s):  
Living Cells ◽  
Before And After ◽  
Ion Laser ◽  
Cross Links ◽  
Protein Incorporation ◽  
And Control ◽  
Argon Ion ◽  
Brain Tubulin ◽  
Control Protein

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.

Laser-fluorescence experiments with a continuous argon-ion laser in an argon plasma

1988 ◽  
Vol 40 (2) ◽  
pp. 135-141 ◽  
Author(s):  
K. Yuasa ◽  
H.M.J. Willems ◽  
B. van der Sijde ◽  
D.C. Schram
Keyword(s):  
Argon Plasma ◽  
Laser Fluorescence ◽  
Argon Ion Laser ◽  
Ion Laser ◽  
Argon Ion

Sea urchin morphogenesis and cell-hyalin adhesion are perturbed by a monoclonal antibody specific for hyalin

Development ◽  
1988 ◽  
Vol 104 (3) ◽  
pp. 391-402 ◽  
Author(s):  
D.L. Adelson ◽  
T. Humphreys
Keyword(s):  
Monoclonal Antibody ◽  
Sea Urchin ◽  
Protein Band ◽  
Lytechinus Variegatus ◽  
Adhesion Assay ◽  
Embryonic Cells ◽  
Lytechinus Pictus ◽  
Arbacia Punctulata ◽  
Evolutionarily Conserved

We have generated and characterized a monoclonal antibody (McA Tg-HYL) that recognizes sea urchin hyalin as evidenced by immunofluorescence staining of the hyaline layer (HL) and immunoblot staining of the hyalin protein band. On immunoblots of HL extracts only the hyalin protein reacted with McA Tg-HYL. Immunoprecipitates of radioactive proteins from embryos incubated with [35S]methionine yielded radioactive hyalin and 190, 140 and 105 × 10(3) Mr proteins associated with hyalin. McA Tg-HYL was generated against Tripneustes gratilla embryos but reacts with hyalin from the distantly related sea urchin species, Colobocentrotus atratus, Strongylocentrotus purpuratus, Arbacia punctulata, Lytechinus variegatus and Lytechinus pictus. Developing embryos of the above-mentioned six species were treated with McA Tg-HYL and did not gastrulate or form arms. Observations of treated embryos revealed areas of separation of the hyaline layer from the underlying embryonic cells, suggesting that McA Tg-HYL was interfering with binding of the cells to the HL. Using the centrifugation-based adhesion assay of McClay et al. (Proc. natn. Acad. Sci. U.S.A. 78, 4975–4979, 1981), Fab' fragments of McA Tg-HYL were found to inhibit cell-hyalin binding. McA Tg-HYL did not inhibit hyalin gelation in vitro or the reaggregation of dissociated blastula cells. We postulate that McA Tg-HYL recognizes an evolutionarily conserved hyalin domain involved in cell-hyalin binding and required for normal epithelial folding.

Laser Excited Fluorescence Spectra of Gaseous Te2

1975 ◽  
Vol 53 (19) ◽  
pp. 1976-1982 ◽  
Author(s):  
T. J. Stone ◽  
R. F. Barrow
Keyword(s):  
Ground State ◽  
Fluorescence Spectra ◽  
Single Mode ◽  
Laser Fluorescence ◽  
Fixed Frequency ◽  
Moderate Resolution ◽  
Ion Laser ◽  
Argon Ion ◽  
Emission Studies ◽  
Laser Fluorescence Spectroscopy

Three lines of the argon ion laser have been used to excite spectra of 128Te2 and 130Te2 which have been photographed with moderate resolution (up to about 300 000). Excitation is critically dependent on the operation of the laser as between multimode or single mode. The development of rotational structure can be varied by control of the pressure of Te2. In addition to the systems A 0u+, B 0u+–X 0g+, well known in the absorption spectrum, four series assigned to B 0u+–X 1g have been analyzed and a new system ascribed to B 1u±–X 1g± has been discovered. Constants (in cm−1) for the ground state of 130Te2 are as follows:[Formula: see text]It is concluded that laser fluorescence spectroscopy, even with fixed frequency lasers, has some advantages over conventional absorption and emission studies.

Antimitotic Activity on Sea Urchin Embryonic Cells of Seven Antiparasitic Morita-Baylis-Hillman Adducts: A Potential New Class of Anticancer Drugs

2012 ◽  
Vol 8 (6) ◽  
pp. 1003-1011
Author(s):  
Jocelmo C. A. Leite ◽  
Claudio G. L. Junior ◽  
Fabio P. L. Silva ◽  
Suervy C.O. Sousa ◽  
Mario L. A. A. Vasconcellos ◽  
...  
Keyword(s):  
Anticancer Drugs ◽  
Sea Urchin ◽  
Embryonic Cells ◽  
New Class ◽  
Antimitotic Activity

The effect of dietary protein on consumption, survival, growth and production of the sea urchin Lytechinus variegatus

Aquaculture ◽  
2006 ◽  
Vol 254 (1-4) ◽  
pp. 483-495 ◽  
Author(s):  
Hugh Hammer ◽  
Stephen Watts ◽  
Addison Lawrence ◽  
John Lawrence ◽  
Renee Desmond
Keyword(s):  
Sea Urchin ◽  
Dietary Protein ◽  
Lytechinus Variegatus

scholarly journals Spicule formation by cultured embryonic cells from the sea urchin.

1981 ◽  
Vol 256 (24) ◽  
pp. 13105-13111
Author(s):  
G.R. Mintz ◽  
S DeFrancesco ◽  
W.J. Lennarz
Keyword(s):  
Sea Urchin ◽  
Embryonic Cells ◽  
Spicule Formation

Laser Induced Etching for Generation of Si Nanocrystals and Spectroscopic Investigation of Morphology

2005 ◽  
Vol 23 ◽  
pp. 43-46
Author(s):  
H.S. Mavi ◽  
S. Rath ◽  
Arun Shukla
Keyword(s):  
Quantum Confinement ◽  
Silicon Nanocrystals ◽  
Laser Wavelength ◽  
Interconnected Network ◽  
Substrate Type ◽  
Argon Ion Laser ◽  
Si Nanocrystals ◽  
Ion Laser ◽  
Laser Induced Etching ◽  
Argon Ion

Laser-induced etching of silicon is used to generate silicon nanocrystals. The pore structure depends on the substrate type and etching laser wavelength. Porous silicon (PS) samples prepared by Nd:YAG laser (1.16 eV) etching of n-type substrate showed a fairly uniform and highly interconnected network of nearly circular pores separated by thin columnar boundaries, while no circular pits were produced by argon- ion laser (2.41 eV) etching under similar conditions. The size and size distribution of the nanocrystals are investigated by Raman and photoluminescence spectroscopies and analyzed within the framework of quantum confinement models.

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