Live Cell Multicolor Imaging of Lipid Droplets with a New Dye, LD540

Spandl, Johanna; White, Daniel J.; Peychl, Jan; Thiele, Christoph

doi:10.1111/j.1600-0854.2009.00980.x

Cited by 233 publications

(187 citation statements)

References 18 publications

(21 reference statements)

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“…not requiring sophisticated proteomics to quantify lipases) monitoring of lipid esterases using a fluorescent readout. Although the quantitative relationship between fluorescence and protein levels has not been determined in detail in this study, imaging of THL-alk1 adducts in cells with larger dimensions (such a eukaryotic cells) will help understand better the spatial distribution of enzymes involved in the dynamics of lipid droplets (47)(48)(49). In addition, THL-alk1 and its closely related derivatives could be useful in other innovative approaches for the systematic determination of protein-small molecule interactions (50 -52).…”

Section: Discussionmentioning

confidence: 99%

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Ravindran

Rao

Cheng

et al. 2014

Molecular & Cellular Proteomics

104

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Tetrahydrolipstatin (THL) is bactericidal but its precise target spectrum is poorly characterized. Here, we used a THL analog and activity-based protein profiling to identify target proteins after enrichment from whole cell lysates of Mycobacterium bovis Bacillus Calmette-Gué rin cultured under replicating and non-replicating conditions. THL targets ␣/␤-hydrolases, including many lipid esterases (LipD, G, H, I, M, N, O, V, W, and TesA). Target protein concentrations and total esterase activity correlated inversely with cellular triacylglycerol upon entry into and exit from non-replicating conditions. Cellular overexpression of lipH and tesA led to decreased THL susceptibility thus providing functional validation. Our results define the target spectrum of THL in a biological species with particularly diverse lipid metabolic pathways. We furthermore derive a conceptual approach that demonstrates the use of such THL probes for the characterization of substrate recognition by lipases and related enzymes. Molecular & Cellular

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Section: Discussionmentioning

confidence: 99%

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Ravindran

Rao

Cheng

et al. 2014

Molecular & Cellular Proteomics

104

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“…Laurdan generalized polarization Besides lipid probes for the plasma membrane, there are also some tools to probe other lipidic environments in the cell, such as lipid droplets (Thiele and Spandl 2008). New fluorescent lipids were developed to visualize the intracellular and membrane lipids in their native environment without any external fluorescent labels (Kuerschner et al 2005;Spandl et al 2009). …”

Section: Fluorescent Probes To Study Lipid Dynamicsmentioning

confidence: 99%

“…New fluorescent lipids were developed to visualize the intracellular and membrane lipids in their native environment without any external fluorescent labels (Kuerschner et al 2005;Spandl et al 2009). …”

Section: Fluorescent Probes To Study Lipid Dynamicsmentioning

confidence: 99%

Fluorescence Techniques to Study Lipid Dynamics

Sezgin¹,

Schwille²

2011

Cold Spring Harbor Perspectives in Biology

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Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.T o elucidate cellular processes in their native dynamic environment has been one of the main issues in cell biology over the past decades. The lack of appropriate techniques has long been the main limiting step for the research on dynamic systems, because it was impossible to acquire real time information with the well-known biochemical techniques. The key challenge in dynamically observing biological systems is to combine the ability to resolve moderate to very low concentrations of molecules-because they are simply limited in living cells-on relevant timescales. Relevant timescales in cell biology can be minutes and hours, on a systemic level of cell metabolism, down to the microsecond and even nanosecond regime in which molecular and intramolecular rearrangements take place. With respect to lipidic systems, relevant dynamics range from the local movements of lipids by diffusion to the mechanical transformations of whole membranes, spanning several orders of magnitude in time to be covered. Like for other cellular processes, the investigation of lipids and membranes also in general benefited greatly from the introduction of fluorescence microscopy and spectroscopy to biology. After the 1960s, great technological inventions based on the phenomenon of fluorescence were made, such as confocal microscopy, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), total internal reflection fluorescence (TIRF), and two-photon microscopy, that not only revolutionized imaging but also yielded access to dynamics on previously inaccessible timescales. Another very big step was certainly taken after the introduction of fluorescent proteins, which again accelerated the use of these techniques in living cells and organisms. Nowadays, the technical advancements of fluorescence-based methods allow us to explore systems as small as single molecules with temporal resolution down to the nanoseconds regime. Lately, even the resolution limit of optical microscopy, for a long time being one of the fundamental barriers in elucidating cellular processes, has been overcome by smart applications of the phenomenon of fluorescence.This article aims at giving a short overview on mainly fluorescence-based metho...

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“…and in A. thaliana (the current work) as well as in COS7 fibroblasts [11], it has been shown that diacylglycerol acyltransferase 2 (DGAT2), an enzyme involved in the final stage of triacylglycerol synthesis, was present at the surfaces of lipid bodies, which may confirm the suggestion that lipid synthesis also occurs on them. Microtubules and lipid bodies in close contact have been observed in different animal cells in connection with lipid bodies' motion [28][29][30]. On the other hand, the observations of Pacheco et al [19] implicate microtubule contribution to lipid synthesis.…”

Section: Resultsmentioning

confidence: 81%

DGAT2 revealed by the immunogold technique in Arabidopsis thaliana lipid bodies associated with microtubules

Kwiatkowska¹,

Stępiński²,

Popłońska³

et al. 2012

Folia Histochem Cytobiol.

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Abstract:The immunogold technique with anti-diacylglycerol acyltransferase 2 (DGAT2) antibody revealed in A. thaliana embryo and root meristematic cells gold particles manifesting the presence of DGAT2 in ER as well as in lipid bodies. This being so, lipid synthesis could take place both in ER and in the lipid bodies. The presence of microtubules around the lipid bodies was evidenced under transmission EM. Detection of tubulin around the lipid bodies using the immunogold technique with anti-a-tubulin is in agreement with the above observations. Connection of lipid bodies with microtubules was also detected by us in other plants where they probably participated in lipid synthesis. A similar phenomenon may take place in

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Live Cell Multicolor Imaging of Lipid Droplets with a New Dye, LD540

Cited by 233 publications

References 18 publications

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

Fluorescence Techniques to Study Lipid Dynamics

DGAT2 revealed by the immunogold technique in Arabidopsis thaliana lipid bodies associated with microtubules

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