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T J Keating

Publications and source records attributed to T J Keating.

6 recordsLinked to original sources
PubMed1997

Microtubule release from the centrosome.

Although microtubules (MTs) are generally thought to originate at the centrosome, a number of cell types have significant populations of MTs with no apparent centrosomal connection. The origin of these noncentrosomal MTs has been unclear. We applied kinetic analysis of MT formation in vivo to establish their mode of origin. Time-lapse fluorescence microscopy demonstrated that noncentrosomal MTs in cultured epithelial cells arise primarily by constitutive nucleation at, and release from, the centrosome. After release, MTs moved away from the centrosome and tended to depolymerize. Laser-marking experiments demonstrated that released MTs moved individually with their plus ends leading, suggesting that they were transported by minus end-directed motors. Released MTs were dynamic. The laser marking experiments demonstrated that plus ends of released MTs grew, paused, or shortened while the minus ends were stable or shortened. Microtubule release may serve two kinds of cellular function. Release and transport could generate the noncentrosomal MT arrays observed in epithelial cells, neurons, and other asymmetric, differentiated cells. Release would also contribute to polymer turnover by exposing MT minus ends, thereby providing additional sites for loss of subunits. The noncentrosomal population of MTs may reflect a steady-state of centrosomal nucleation, release, and dynamics.

Animals↗
PubMed1996

Inhibition of protein synthesis in frog (Xenopus laevis) egg extracts by an antibody against phosphatidylinositol 4,5-bisphosphate.

The antibody kt10, which is directed against the phospholipid PtdIns(4,5)P2, inhibits protein synthesis when added to cytosolic extracts prepared from frog eggs. Addition of stable analogues of diacylglycerol and Ins(1,4,5)P3 failed to rescue the inhibition of translation, suggesting that the effect of the antibody was not to block hydrolysis of PtdIns(4,5)P2. Neomycin, which also binds PtdIns(4,5)P2, produced a similar reduction in protein-synthesis levels in the extract system, supporting the idea that it is the interaction of the antibody with PtdIns(4,5)P2 that is producing the effect.

Animals↗
PubMed1994

Inexpensive techniques for measuring [Ca2+]i changes using a photomultiplier tube.

Photomultiplier tubes remain among the most sensitive methods for detecting light. Their cost is one to two orders of magnitude less than that of other comparably sensitive detectors. Advances in the associated electronics have lowered the cost and reduced the size of the instruments. If an investigator is willing to go to the primary suppliers and has access to a machine shop, systems sensitive enough for fluorescence measurements on single cells (or portions of a single cell) can be assembled for remarkably little money. In this chapter, we have emphasized a design using particular suppliers because we have used this system in our laboratory. The reader should not assume that we have explored all possibilities; further, progress in these areas tends to be rapid and new developments may open up additional opportunities.

Aequorin↗
PubMed1994

Intracellular free calcium oscillations in normal and cleavage-blocked embryos and artificially activated eggs of Xenopus laevis.

We have measured levels of intracellular free calcium ([Ca2+]i) in albino Xenopus laevis embryos using recombinant aequorin and a photon-counting system. We observed sinusoidal oscillations in [Ca2+]i that had the same frequency as cleavage, with cleavage occurring when [Ca2+]i was lowest. An increase in calcium was seen to precede first cleavage. The cyclic changes in calcium were superimposed on a secondary pattern that increased, peaked between third and fifth cleavages and then slowly declined to a level similar to that measured before first cleavage. The amplitude of the oscillations was small during the first few cleavages but became larger with each cycle, with the largest oscillations occurring when the secondary pattern peaked (between third and fifth cleavage). As the secondary pattern declined, the amplitude of the oscillations also became smaller. The oscillations are due to release of calcium from intracellular stores, since the signal was the same in calcium-free solution as in normal medium. When cleavage was blocked with the microtubule-disrupting drugs colchicine or nocodazole, the [Ca2+]i oscillations persisted. Calcium oscillations of a similar magnitude and frequency were also present in artificially activated eggs. The secondary pattern was different in cleavage-blocked embryos and artificially activated eggs, the baseline increasing until about the third cycle and then remaining elevated for the rest of the recording (> 8 hours). By fixing embryos at various points in the calcium cycle, we determined that mitosis began shortly after calcium levels reached their peak and was complete before the calcium level dropped to its lowest point.(ABSTRACT TRUNCATED AT 250 WORDS)

Aequorin↗
PubMed1992

Thin-section ratiometric Ca2+ images obtained by optical sectioning of fura-2 loaded mast cells.

The availability of the ratiometric Ca2+ indicator dyes, fura-2, and indo-1, and advances in digital imaging and computer technology have made it possible to detect Ca2+ changes in single cells with high temporal and spatial resolution. However, the optical properties of the conventional epifluorescence microscope do not produce a perfect image of the specimen. Instead, the observed image is a spatial low pass filtered version of the object and is contaminated with out of focus information. As a result, the image has reduced contrast and an increased depth of field. This problem is especially important for measurements of localized Ca2+ concentrations. One solution to this problem is to use a scanning confocal microscope which only detects in focus information, but this approach has several disadvantages for low light fluorescence measurements in living cells. An alternative approach is to use digital image processing and a deblurring algorithm to remove the out of focus information by using a knowledge of the point spread function of the microscope. All of these algorithms require a stack of two-dimensional images taken at different focal planes, although the "nearest neighbor deblurring" algorithm only requires one image above and below the image plane. We have used a modification of this scheme to construct a simple inverse filter, which extracts optical sections comparable to those of the nearest neighbors scheme, but without the need for adjacent image sections. We have used this "no neighbors" processing scheme to deblur images of fura-2-loaded mast cells from beige mice and generate high resolution ratiometric Ca2+ images of thin sections through the cell. The shallow depth of field of these images is demonstrated by taking pairs of images at different focal planes, 0.5-microns apart. The secretory granules, which exclude the fura-2, appear in focus in all sections and distinct changes in their size and shape can be seen in adjacent sections. In addition, we show, with the aid of model objects, how the combination of inverse filtering and ratiometric imaging corrects for some of the inherent limitations of using an inverse filter and can be used for quantitative measurements of localized Ca2+ gradients. With this technique, we can observe Ca2+ transients in narrow regions of cytosol between the secretory granules and plasma membrane that can be less than 0.5-microns wide. Moreover, these Ca2+ increases can be seen to coincide with the swelling of the secretory granules that follows exocytotic fusion.

Algorithms↗