Chapter 13 Fluorescence Microscopy in Three Dimensions

Agard, David A.; Hiraoka, Yasushi; Shaw, Peter; Sedat, John W.

doi:10.1016/s0091-679x(08)60986-3

Cited by 628 publications

(438 citation statements)

References 20 publications

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“…The nuclei of P. falciparum-infected RBCs were stained with 5 μM DRAQ5 (Alexis) and live imaging was carried out by a custom-built, wide-field fluorescence microscope with a 100× oil immersion objective (1.4 NA; Olympus). Z-sections of 0.2 μm were taken and three-dimensional stacks were subjected to constrained iterative deconvolution according to the previous method [23].…”

Section: Plasmid Construction Transfection and Microscopy Of Pfemp2mentioning

confidence: 99%

N-terminal processing of proteins exported by malaria parasites

Chang

Falick

Carlton

et al. 2008

Molecular and Biochemical Parasitology

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171

177

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Malaria parasites utilize a short N-terminal amino acid motif termed the Plasmodium export element (PEXEL) to export an array of proteins to the host erythrocyte during blood stage infection. Using immunoaffinity chromatography and mass spectrometry, insight into this signalmediated trafficking mechanism was gained by discovering that the PEXEL motif is cleaved and N-acetylated. PfHRPII and PfEMP2 are two soluble proteins exported by Plasmodium falciparum that were demonstrated to undergo PEXEL cleavage and N-acetylation, thus indicating that this Nterminal processing may be general to many exported soluble proteins. It was established that PEXEL processing occurs upstream of the brefeldin A-sensitive trafficking step in the P. falciparum secretory pathway, therefore cleavage and N-acetylation of the PEXEL motif occurs in the endoplasmic reticulum (ER) of the parasite. Furthermore, it was shown that the recognition of the processed N-terminus of exported proteins within the parasitophorous vacuole may be crucial for protein transport to the host erythrocyte. It appears that the PEXEL may be defined as a novel ER peptidase cleavage site and a classical N-acetyltransferase substrate sequence.

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Section: Plasmid Construction Transfection and Microscopy Of Pfemp2mentioning

confidence: 99%

N-terminal processing of proteins exported by malaria parasites

Chang

Falick

Carlton

et al. 2008

Molecular and Biochemical Parasitology

Self Cite

171

177

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“…An objective produces a diffraction pattern in the form of an Airy disk in the image plane of a point source of light from the specimen such as a single fluorophore (Agard et al, 1989;Inoue and Springer, 1997;Taylor and Salmon, 1989). The intensity distribution in the XY plane at focus along the Z axis is given by:…”

Section: A Characterization Of the Point Spread Function Of The Objementioning

confidence: 99%

Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins

Joglekar

Salmon

Bloom

2008

Fluorescent Proteins

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Genetically encoded fluorescent proteins are an essential tool in cell biology, widely used for investigating cellular processes with molecular specificity. Direct uses of fluorescent proteins include studies of the in vivo cellular localization and dynamics of a protein, as well as measurement of its in vivo concentration. In this chapter, we focus on the use of genetically encoded fluorescent protein as an accurate reporter of in vivo protein numbers. Using the challenge of counting the number of copies of kinetochore proteins in budding yeast as a case study, we discuss the basic considerations in developing a technique for the accurate evaluation of intracellular fluorescence signal. This discussion includes criteria for the selection of a fluorescent protein with optimal characteristics, selection of microscope and image acquisition system components, the design of a fluorescence signal quantification technique, and possible sources of measurement errors. We also include a brief survey of available calibration standards for converting the fluorescence measurements into a number of molecules, since the availability of such a standard usually determines the design of the signal measurement technique as well as the accuracy of final measurements. Finally, we show that, as in the case of budding yeast kinetochore proteins, the in vivo intracellular protein numbers determined from fluorescence measurements can also be employed to elucidate structural details of cellular structures.

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“…Stacks of 25-30 images, taken with a 0.2-m plane to plane distance in each fluorescence channel of interest, were submitted to threedimensional (3D) projection, and the resulting composite images were used for quantitative analysis of the fluorescence intensity/cell, using MetaMorph software. Some of these stacks were deconvolved with the MetaMorph Fast Iterative Constrained PSF-based algorithm (Agard et al, 1989), to improve the resolution of the fluorescent signals in the multifluorescent images. More precisely, 3D stacks of images were acquired for the green fluorescent protein (GFP) and Cy3 or Cy5 fluorescent signals, by using a fixed exposure time for each channel (100 ms for GFP and 200 ms for Cy3 or Cy5).…”

Section: Immunofluorescence Confocal Microscopy and Image Analysismentioning

confidence: 99%

Rab11A Controls the Biogenesis of Birbeck Granules by Regulating Langerin Recycling and Stability

Uzan-Gafsou

Bausinger²,

Proamer³

et al. 2007

MBoC

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The extent to which Rab GTPases, Rab-interacting proteins, and cargo molecules cooperate in the dynamic organization of membrane architecture remains to be clarified. Langerin, a recycling protein accumulating in the Rab11-positive compartments of Langerhans cells, induces the formation of Birbeck granules (BGs), which are membrane subdomains of the endosomal recycling network. We investigated the role of Rab11A and two members of the Rab11 family of interacting proteins, Rip11 and RCP, in Langerin traffic and the biogenesis of BGs. The overexpression of a dominant-negative Rab11A mutant or Rab11A depletion strongly influenced Langerin traffic and stability and the formation of BGs, whereas modulation of other Rab proteins involved in dynamic regulation of the endocytic-recycling pathway had no effect. Impairment of Rab11A function led to a missorting of Langerin to lysosomal compartments, but inhibition of Langerin degradation by chloroquine did not restore the formation of BGs. Loss of RCP, but not of Rip11, also had a modest, but reproducible effect on Langerin stability and BG biogenesis, pointing to a role for Rab11A-RCP complexes in these events. Our results show that Rab11A and Langerin are required for BG biogenesis, and they illustrate the role played by a Rab GTPase in the formation of a specialized subcompartment within the endocytic-recycling system.

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Chapter 13 Fluorescence Microscopy in Three Dimensions

Cited by 628 publications

References 20 publications

N-terminal processing of proteins exported by malaria parasites

N-terminal processing of proteins exported by malaria parasites

Counting Kinetochore Protein Numbers in Budding Yeast Using Genetically Encoded Fluorescent Proteins

Rab11A Controls the Biogenesis of Birbeck Granules by Regulating Langerin Recycling and Stability

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