Publications (14)106.16 Total impact
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Article: Laser ablation of the microtubule cytoskeleton: setting up and working with an ablation system.
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ABSTRACT: Laser ablation is a powerful tool that can be used to study a variety of biological mechanisms. Microscopes with high optical performances are nowadays available, and lasers that could be used to perform ablations have become accessible to every laboratory. Setting up a laser ablation system is a relatively straightforward task; however, it requires some basic knowledge of optics. We illustrate the fundamental components of the experimental setup and describe the most common pitfalls and difficulties encountered when designing, setting up, and working with a laser ablation system.Methods in molecular biology (Clifton, N.J.) 01/2011; 777:261-71. -
Article: Cell polarity: which way to grow in an electric field?
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ABSTRACT: Cell polarity can be influenced by an electric field, but the mechanisms behind this response are poorly understood. A new paper shows that fission yeast cells change their direction of growth in an external electric field and suggests mechanisms based on the cortical pH gradient and on electrophoresis of membrane proteins.Current biology: CB 04/2010; 20(8):R355-6. · 10.99 Impact Factor -
Article: Axon extension occurs independently of centrosomal microtubule nucleation.
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ABSTRACT: Microtubules are polymeric protein structures and components of the cytoskeleton. Their dynamic polymerization is important for diverse cellular functions. The centrosome is the classical site of microtubule nucleation and is thought to be essential for axon growth and neuronal differentiation--processes that require microtubule assembly. We found that the centrosome loses its function as a microtubule organizing center during development of rodent hippocampal neurons. Axons still extended and regenerated through acentrosomal microtubule nucleation, and axons continued to grow after laser ablation of the centrosome in early neuronal development. Thus, decentralized microtubule assembly enables axon extension and regeneration, and, after axon initiation, acentrosomal microtubule nucleation arranges the cytoskeleton, which is the source of the sophisticated morphology of neurons.Science 02/2010; 327(5966):704-7. · 31.20 Impact Factor -
Article: Force and length regulation in the microtubule cytoskeleton: lessons from fission yeast.
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ABSTRACT: How does a living cell deal with basic concepts of physics such as length and force? The cell has to measure distances and regulate forces to dynamically organize its interior. This is to a large extent based on microtubules (MTs) and motor proteins. Two concepts are emerging from recent studies as key to the positioning of cell components: preferred disassembly of longer MTs and preferred detachment of motors under high load force. The role of these concepts in nuclear centering and nuclear oscillations is coming to light from experimental and theoretical studies in fission yeast. These universal concepts are likely crucial for a variety of cell processes, including nuclear and mitotic spindle positioning, control of spindle length, and chromosome congression on the metaphase plate.Current opinion in cell biology 02/2010; 22(1):21-8. · 14.15 Impact Factor -
Article: Optical trapping and laser ablation of microtubules in fission yeast.
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ABSTRACT: Manipulation has been used as a powerful investigation technique since the early history of biology. Every technical advance resulted in more refined instruments that led to the discovery of new phenomena and to the solution of old problems. The invention of laser in 1960 gave birth to what is now called optical manipulation: the use of light to interact with matter. Since then, the tremendous progress of laser technology made optical manipulation not only an affordable, reliable alternative to traditional manipulation techniques but disclosed also new, intriguing applications that were previously impossible, such as contact-free manipulation. Currently, optical manipulation is used in many fields, yet has the potential of becoming an everyday technique in a broader variety of contexts. Here, we focus on two main optical manipulation techniques: optical trapping and laser ablation. We illustrate with selected applications in fission yeast how in vivo optical manipulation can be used to study organelle positioning and the force balance in the microtubule cytoskeleton.Methods in cell biology 01/2010; 97:173-83. · 2.05 Impact Factor -
Article: Growth pattern of single fission yeast cells is bilinear and depends on temperature and DNA synthesis.
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ABSTRACT: Cell growth and division have to be tightly coordinated to keep the cell size constant over generations. Changes in cell size can be easily studied in the fission yeast Schizosaccharomyces pombe because these cells have a cylindrical shape and grow only at the cell ends. However, the growth pattern of single cells is currently unclear. Linear, exponential, and bilinear growth models have been proposed. Here we measured the length of single fission yeast cells with high spatial precision and temporal resolution over the whole cell cycle by using time-lapse confocal microscopy of cells with green fluorescent protein-labeled plasma membrane. We show that the growth profile between cell separation and the subsequent mitosis is bilinear, consisting of two linear segments separated by a rate-change point (RCP). The change in growth rate occurred at the same relative time during the cell cycle and at the same relative extension for different temperatures. The growth rate before the RCP was independent of temperature, whereas the growth rate after the RCP increased with an increase in temperature, leading to clear bilinear growth profiles at higher temperatures. The RCP was not directly related to the initiation of growth at the new end (new end take-off). When DNA synthesis was inhibited by hydroxyurea, the RCP was not detected. This result suggests that completion of DNA synthesis is required for the increase in growth rate. We conclude that the growth of fission yeast cells is not a simple exponential growth, but a complex process with precise rates regulated by the events during the cell cycle.Biophysical Journal 06/2009; 96(10):4336-47. · 3.65 Impact Factor -
Article: Self-organization of dynein motors generates meiotic nuclear oscillations.
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ABSTRACT: Meiotic nuclear oscillations in the fission yeast Schizosaccharomyces pombe are crucial for proper chromosome pairing and recombination. We report a mechanism of these oscillations on the basis of collective behavior of dynein motors linking the cell cortex and dynamic microtubules that extend from the spindle pole body in opposite directions. By combining quantitative live cell imaging and laser ablation with a theoretical description, we show that dynein dynamically redistributes in the cell in response to load forces, resulting in more dynein attached to the leading than to the trailing microtubules. The redistribution of motors introduces an asymmetry of motor forces pulling in opposite directions, leading to the generation of oscillations. Our work provides the first direct in vivo observation of self-organized dynamic dynein distributions, which, owing to the intrinsic motor properties, generate regular large-scale movements in the cell.PLoS Biology 05/2009; 7(4):e1000087. · 11.45 Impact Factor -
Article: Versatile laser-based cell manipulator.
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ABSTRACT: Here we describe a two-photon microscope and laser ablation setup combined with optical tweezers. We tested the setup on the fission yeast Schizosaccharomyces pombe, a commonly used model organism. We show that long-term imaging can be achieved without significant photo-bleaching or damage of the sample. The setup can precisely ablate sub-micrometer structures, such as microtubules and mitotic spindles, inside living cells, which remain viable after the manipulation. Longer exposure times lead to ablation, while shorter exposures lead to photo-bleaching of the target structure. We used optical tweezers to trap intracellular particles and to displace the cell nucleus. Two-photon fluorescence imaging of the manipulated cell can be performed simultaneously with trapping. The combination of techniques described here may help to solve a variety of problems in cell biology, such as positioning of organelles and the forces exerted by the cytoskeleton.Journal of Biophotonics 10/2008; 1(4):299-309. · 4.34 Impact Factor -
Article: Association of mitochondria with spindle poles facilitates spindle alignment.
Current Biology 09/2008; 18(15):R646-R647. · 9.65 Impact Factor -
Article: Bundling, sliding, and pulling microtubules in cells and in silico.
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ABSTRACT: Microtubules and other proteins self-organize into complex dynamic structures such as the mitotic spindle, which separates the chromosomes during cell division. Much is known about the individual molecular players involved in assembly and positioning of the mitotic spindle, but how they act together to generate the often unexpected behavior of the whole microtubule system is not understood. Two recent papers use a combination of experimental (imaging) and theoretical (computer simulation) methods to explore the formation of bipolar linear microtubule arrays in fission yeast and the oscillatory movement of the mitotic spindle in the nematode worm. In the simulation approach, the rules for the interactions of the components (microtubules and microtubule-associated proteins) are specified and the evolution of the system is followed, with the aim of identifying the minimal set of components that can mimic the real system. The work on fission yeast concludes that bipolar microtubule structures can arise from self-organization of microtubules through nucleators, bundlers, and sliders, without a requirement for a special microtubule-organizing center. The work on the worm embryo suggests that both the positive feedback that drives oscillations and the centering force that limits their amplitude may arise from microtubule pulling forces. The systems approach exemplified by these papers should stimulate new experiments aimed at discovering the principles of cellular organization.Advanced Online Publication Articles for HFSP Journal 06/2007; 1(1):11-4. · 2.32 Impact Factor -
Article: Hypergravity speeds up the development of T-lymphocyte motility.
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ABSTRACT: The effect of altered gravity on single cells has been reported in a number of studies. From the investigation of the immune system response to spaceflight conditions, interest has focused on the influence of gravity on single lymphocytes. Microgravity has been shown to decrease lymphocyte activation and to influence motility. On the other hand, the effect of hypergravity on lymphocyte motility has not been explored. We studied the migration of human peripheral blood T lymphocytes cultured in vitro in a hypergravity environment (10g). After hypergravity culture for 1-11 days, T cells were seeded on a fibronectin-coated glass surface, observed by time-lapse bright-field microscopy, and tracked by a computer program. We found that T cells, activated and then cultured in hypergravity, become motile earlier than cells cultured at normal gravity. These results suggest that hypergravity stimulates T cell migration.European Biophysics Journal 06/2006; 35(5):393-400. · 2.14 Impact Factor -
Article: Optical micromanipulations inside yeast cells.
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ABSTRACT: We present a combination of nonlinear microscopy and optical trapping applied to three-dimensional imaging and manipulation of intracellular structures in living cells. We use Titanium-sapphire laser pulses for nonlinear microscopy of the nuclear envelope and the microtubules marked with green fluorescent protein in fission yeast. The same laser source is also used to trap small lipid granules naturally present in the cell. The trapped granule is used as a handle to exert a pushing force on the cell nucleus. The granule is moved in a raster-scanning fashion to cover the area of the nucleus and hence displace the nucleus away from its normal position in the center of the cell. Such indirect manipulations of an organelle (e.g., nucleus) can be useful when direct trapping of the chosen organelle is disadvantageous or inefficient. We show that nonlinear microscopy and optical manipulation can be performed without substantial damage or heating of the cell. We present this method as an important tool in cell biology for manipulation of specific structures, as an alternative to genetic and biochemical methods. This technique can be applied to several fundamental problems in cell biology, including the mechanism of nuclear positioning and the spatial coordination of nuclear and cell division.Applied Optics 05/2005; 44(11):2001-7. · 1.41 Impact Factor -
Article: Positioning and elongation of the fission yeast spindle by microtubule-based pushing.
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ABSTRACT: In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development. We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe. We hypothesized that astral microtubules exert mechanical force on the S. pombe spindle and thus help align the spindle with the major axis of the cell. Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy. Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules. We found that astral microtubules push on the spindle poles in S. pombe, in contrast to the pulling forces observed in a number of other cell types. Further, laser dissection of the spindle midzone induced spindle collapse inward. This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Broken spindles recovered and mitosis completed as usual. We propose a model of spindle centering and elongation by microtubule-based pushing forces.Current Biology 08/2004; 14(13):1181-6. · 9.65 Impact Factor -
Article: Combined intracellular three-dimensional imaging and selective nanosurgery by a nonlinear microscope.
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ABSTRACT: We use near-IR femtosecond laser pulses for a combination of microscopy and nanosurgery on fluorescently labeled structures within living cells. Three-dimensional reconstructions of microtubule structures tagged with green fluorescent protein (GFP) are made during different phases of the cell cycle. Further, the microtubules are dissected using the same laser beam but with a higher laser power than for microscopy. We establish the viability of this technique for the cells of a fission yeast, which is a common model to study the mechanics of cell division. We show that nanosurgery can be performed with submicrometer precision and without visible collateral damage to the cell. The energy is primarily absorbed by the GFP molecules, and not by other native structures in the cell. GFP is particularly suitable for multiphoton excitation, as its excitation wavelength near 900 nm is benign for most cellular structures. The ability to use GFP to label structures for destruction by multiphoton excitation may be a valuable tool in cell biology.Journal of Biomedical Optics 10(1):14002. · 3.16 Impact Factor
Top co-authors
Top Journals
Institutions
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2007–2011
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Max-Planck-Institut für molekulare Zellbiologie und Genetik
Dresden, Saxony, Germany
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2006
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Università degli Studi di Firenze
- European Laboratory for Non-Linear Spectroscopy LENS
Florence, Tuscany, Italy
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2005
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Università degli Studi di Trento
Trento, Trentino-Alto Adige, Italy
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2004
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European Laboratory for Non-Linear Spectroscopy
Florence, Tuscany, Italy
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