Imaging green fluorescent protein fusions in living fission yeast cells

Tran, Paulina

doi:10.1016/s1046-2023(03)00316-5

Cited by 21 publications

(24 citation statements)

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“…Cells were mounted on agarose pads following protocols by Tran et al (2004) and imaged at 24–25°C with a confocal spinning disc microscope, comprising a confocal scanner unit (CSU22; Yokogawa Electric Corp.) attached to an inverted microscope (DMIRB; Leica) equipped with a × 100/1.3 NA oil immersion lens (PL FLUOTAR; Leica) and a built‐in × 1.5 magnification changer lens. The sample was illuminated using a 488 nm laser (Sapphire 488‐30; Coherent Inc.).…”

Section: Methodsmentioning

confidence: 99%

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Tischer

Brunner

Dogterom

2009

Molecular Systems Biology

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Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length-dependent increase in the catastrophe frequency that is mediated by kinesin-8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin-8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.

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Section: Methodsmentioning

confidence: 99%

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Tischer

Brunner

Dogterom

2009

Molecular Systems Biology

100

150

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“…NotI fragments were ligated into pSLF172, pSLF173 and pSLF373. Cells were washed twice and transferred onto 1% agarose pads prepared as described by Tran et al (2004). All inserts were checked for correctness by sequencing.…”

Section: Expression Of Epitope-tagged and Fusion Proteins; Localizatimentioning

confidence: 99%

Loss of Zhf and the tightly regulated zinc-uptake system SpZrt1 inSchizosaccharomyces pombereveals the delicacy of cellular zinc balance

Boch

Trampczynska

Simm

et al. 2008

FEMS Yeast Research

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Zinc is an essential micronutrient, and yet it can be toxic when present in excess. Zinc acquisition and distribution are dependent on tightly controlled transport of Zn(2+) ions. Schizosaccharomyces pombe represents a second eukaryotic model to study cellular metal homeostasis. In several ways its micronutrient metabolism is fundamentally different from Saccharomyces cerevisiae. We identified the first Zn(2+)-uptake system in S. pombe and named it SpZrt1. Knock-out strains for all three ZIP (Zrt, Irt-like protein) transporters in fission yeast were constructed. Only zrt1Delta cells were unable to grow at low Zn(2+) and showed reduced (65)Zn(2+) uptake. Elemental profiles revealed a strong decrease in zinc accumulation. Cd(2+) ions inhibited uptake but Fe(2+) or Mn(2+) did not. Both mRNA abundance and protein amount are tightly regulated. Zrt1 activity is rapidly shut down upon transfer of zinc-deficient cells to zinc-replete conditions. In cells lacking Zhf, a transporter mediating endoplasmic reticulum storage of zinc, this response is about 100-fold more sensitive. Thus, removal of excess of zinc from the cytosol is largely Zhf dependent. Moreover, cells deficient for both transporters are no longer able to adjust to changing external Zn(2+) concentrations. Optimal growth is restricted to a narrow range of Zn(2+) concentrations, illustrating the fine balance between micronutrient deficiency and toxicity.

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“…For these reasons, a variety of micro-environments based on hydrogels are being explored for tissue engineering [3,23]. So far, the most commonly used hydrogel for the study of micro-organisms as well as multicellular organisms is agarose, which is commonly used as an ‘agar pad’, a single monolayer of hydrogel, to support the growth of bacteria [24], yeast [25] or nematodes [26,27] in the imaging plane for live microscopy imaging. Simple layers of agarose have also recently been used as membranes to control bacterial medium in time [28-30].…”

Section: Introductionmentioning

confidence: 99%

Microfabricated Polyacrylamide Devices for the Controlled Culture of Growing Cells and Developing Organisms

et al. 2013

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The ability to spatially confine living cells or small organisms while dynamically controlling their aqueous environment is important for a host of microscopy applications. Here, we show how polyacrylamide layers can be patterned to construct simple microfluidic devices for this purpose. We find that polyacrylamide gels can be molded like PDMS into micron-scale structures that can enclose organisms, while being permeable to liquids, and transparent to allow for microscopic observation. We present a range of chemostat-like devices to observe bacterial and yeast growth, and C. elegans nematode development. The devices can integrate PDMS layers and allow for temporal control of nutrient conditions and the presence of drugs on a minute timescale. We show how spatial confinement of motile C. elegans enables for time-lapse microscopy in a parallel fashion.

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Imaging green fluorescent protein fusions in living fission yeast cells

Cited by 21 publications

References 0 publications

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Loss of Zhf and the tightly regulated zinc-uptake system SpZrt1 inSchizosaccharomyces pombereveals the delicacy of cellular zinc balance

Microfabricated Polyacrylamide Devices for the Controlled Culture of Growing Cells and Developing Organisms

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