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At least 217 records · Page 12Linked to original sources
PubMed2005

Nuclear and division-plane positioning revealed by optical micromanipulation.

The position of the division plane affects cell shape and size, as well as tissue organization. Cells of the fission yeast Schizosaccharomyces pombe have a centrally placed nucleus and divide by fission at the cell center. Microtubules (MTs) are required for the central position of the nucleus. Genetic studies lead to the hypothesis that the position of the nucleus may determine the position of the division plane. Alternatively, the division plane may be positioned by the spindle or by morphogen gradients or reaction diffusion mechanisms. Here, we investigate the role of MTs in nuclear positioning and the role of the nucleus in division-plane positioning by displacing the nucleus with optical tweezers. A displaced nucleus returned to the cell center by MT pushing against the cell tips. Nuclear displacement during interphase or early prophase resulted in asymmetric cell division, whereas displacement during prometaphase resulted in symmetric division as in unmanipulated cells. These results suggest that the division plane is specified by the predividing nucleus. Because the yeast nucleus is centered by MTs during interphase but not in mitosis, we hypothesize that the establishment of the division plane at the beginning of mitosis is an optimal mechanism for accurate symmetric division in these cells.

Cell Division↗
PubMed2004

Micromanipulation of endothelial cells: ultrasound-microbubble-cell interaction.

Ultrasound (US) in combination with contrast microbubbles has been shown to alter the permeability of cell membranes without affecting cell viability. This permeabilisation feature is used to design new drug delivery systems using US and contrast agents. The underlying mechanisms are still unknown. One hypothesis is that oscillating microbubbles cause cell deformation resulting in enhanced cell membrane permeability. This technical note reveals the interaction between oscillating microbubbles and endothelial cells under a microscope recorded with a fast framing camera at 10 million frames per second. A microbubble expansion of 100% resulted a 2.3-mum displacement of the cell membrane. During the insonification, changes of approximately 15% in the cross-sectional distance of the endothelial cells were observed due to microbubble vibrations. In conclusion, the use of such a camera makes it possible to reveal the mechanisms of interactions between ultrasound, microbubbles and cells.

Animals↗
PubMed2003

Hydrodynamic micromanipulation of individual cells onto patterned attachment sites on biomicroelectromechanical system chips.

We report on a simple methodology to move selected single live cells to a desired location on a flat substrate, such as a patterned biomicroelectromechanical system chip. A macroscopic syringe-and-tube-based hydrodynamic manipulation system is used to achieve controlled cell navigation onto hydrophilic sites for cell attachment. Centimeter-per-second flow velocities generated by the system get downgraded to micrometers-per-second flow at the height of settled cells as a result of viscous flow in the medium. By pushing/pulling two syringes that produce two orthogonal flows, fine manipulation in any horizontal direction is feasible. After attachment of the desired cell(s) onto the selected hydrophilic site, all other unwanted cells are washed away from the surrounding hydrophobic surface with faster flow. This simple methodology is applicable for rapid cell pattern formation with high precision.

Animals↗
PubMed1988

Force measurements by micromanipulation of a single actin filament by glass needles.

Single actin filaments (approximately 7 nm in diameter) labelled with fluorescent phalloidin can be clearly seen by video-fluorescence microscopy. This technique has been used to observe motions of single filaments in solution and in several in vitro movement assays. In a further development of the technique, we report here a method to catch and manipulate a single actin filament (F-actin) by glass microneedles under conditions in which external force on the filament can be applied and measured. Using this method, we directly measured the tensile strength of a filament (the force necessary to break the bond between two actin monomers) and the force required for a filament to be moved by myosin or its proteolytic fragment bound to a glass surface in the presence of ATP. The first result shows that the tensile strength of the F-actin-phalloidin complex is comparable with the average force exerted on a single thin filament in muscle fibres during isometric contraction. This force is increased only slightly by tropomyosin. The second measurement shows that the myosin head (subfragment-1) can produce the same ATP-dependent force as intact myosin. The magnitude of this force is comparable with that produced by each head of myosin in muscle during isometric contraction.

Actin Cytoskeleton↗
PubMed2001

Micromanipulation of chloroplasts using optical tweezers.

This paper describes experiments using optical tweezers to probe chloroplast arrangement, shape and consistency in cells of living leaf tissue and in suspension. Dual optical tweezers provided two-point contact on a single chloroplast or two-point contact on two adhered chloroplasts for manipulation in suspension. Alternatively, a microstirrer consisting of a birefringent particle trapped in an elliptically polarized laser trap was used to induce motion and tumbling of a selected chloroplast suspended in a solution. We demonstrate that displacement of chloroplasts inside the cell is extremely difficult, presumably due to chloroplast adhesion to the cytoskeleton and connections between organelles. The study also confirms that the chloroplasts are very thin and extremely cup-shaped with a concave inner surface and a convex outer surface.

Chloroplasts↗
PubMed1997

Elasticity and structure of eukaryote chromosomes studied by micromanipulation and micropipette aspiration.

The structure of mitotic chromosomes in cultured newt lung cells was investigated by a quantitative study of their deformability, using micropipettes. Metaphase chromosomes are highly extensible objects that return to their native shape after being stretched up to 10 times their normal length. Larger deformations of 10 to 100 times irreversibly and progressively transform the chromosomes into a "thin filament," parts of which display a helical organization. Chromosomes break for elongations of the order of 100 times, at which time the applied force is around 100 nanonewtons. We have also observed that as mitosis proceeds from nuclear envelope breakdown to metaphase, the native chromosomes progressively become more flexible. (The elastic Young modulus drops from 5,000 +/- 1,000 to 1,000 +/- 200 Pa.) These observations and measurements are in agreement with a helix-hierarchy model of chromosome structure. Knowing the Young modulus allows us to estimate that the force exerted by the spindle on a newt chromosome at anaphase is roughly one nanonewton.

Anaphase↗
PubMed1976

Cytoplasmic zone analysis. RNA flow studied by micromanipulation.

A technique is described, cytoplasmic zone analysis, by which it is possible to study the flow of different RNA-containing components in the cytoplasm after their release from the nucleus. After a pulse of RNA precursors, the salivary glands of the insect Chironomus tentans are fixed and microdissected for the isolation of three zones of cytoplasm situated at increasing distances from the nucleus. The RNA from each zone is isolated and analyzed by gel electrophoresis. The three ribosomal RNA components, 18 S, 28 S and 5 S RNA, appear in steep, specific radioactivity gradients (exit gradients) during the time interval 2-3 h after a precursor injection, the 18 S RNA gradient lying 30-50 mum peripheral to that of the 28 S or 5 S RNA gradient. Administration of puromycin led to the complete disappearance of the 28 S RNA and most of the 28 S RNA gradient within 45 min, suggesting that the gradients are caused by an engagement of the ribosomal subunits into polysomes close to the nucleus immediately or soon after their exit from the nucleus. The gradients may offer a unique model for the study of polysome formation and maintenance in vivo.

Cytological Techniques↗
PubMed1979

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle.

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.

Animals↗
PubMed2006

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics.

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.

Adenosine Triphosphate↗