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Katia Cortese Alberto Diaspro Carlo Tacchetti 《The journal of histochemistry and cytochemistry》2009,57(12):1103-1112
Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers. Sample preparation is easy and relatively inexpensive, and the use of appropriate tags provides the ability to track specific proteins of interest. Of course, only electron microscopy (EM) achieves the highest definition in terms of ultrastructure and protein labeling. To fill the gap between light microscopy and EM, correlative light and electron microscopy (CLEM) strategies have been developed. In particular, hybrid techniques based upon immuno-EM provide sensitive protein detection combined with high-resolution information on cell structures and protein localization. By adding the third dimension to EM with electron tomography (ET) combined with rapid freezing, CLEM techniques now provide additional tools for quantitative 3D analysis. Here, we overview the major methods applied and highlight the latest advances in the field of CLEM. We then focus on two selected techniques that use cryosections as substrate for combined biomolecular imaging. Finally, we provide a perspective of future developments in the field. (J Histochem Cytochem 57:1103–1112, 2009) 相似文献
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Sabine Schipper-Krom Katrin Juenemann Anne H. Jansen Anne Wiemhoefer Rianne van den Nieuwendijk Donna L. Smith Mark A. Hink Gillian P. Bates Hermen Overkleeft Huib Ovaa Eric Reits 《FEBS letters》2014
Neurodegenerative disorders such as Huntington’s disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin–proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington’s disease, and support the reported absence of proteasome impairment in mouse models of Huntington’s disease. 相似文献
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Daniela Boassa Joell L. Solan Adrian Papas Perry Thornton Paul D. Lampe Gina E. Sosinsky 《Traffic (Copenhagen, Denmark)》2010,11(11):1471-1486
During the cell cycle, gap junction communication, morphology and distribution of connexin43 (Cx43)‐containing structures change dramatically. As cells round up in mitosis, Cx43 labeling is mostly intracellular and intercellular coupling is reduced. We investigated Cx43 distributions during mitosis both in endogenous and exogenous expressing cells using optical pulse‐chase labeling, correlated light and electron microscopy, immunocytochemistry and biochemical analysis. Time‐lapse imaging of green fluorescent protein (GFP)/tetracysteine tagged Cx43 (Cx43‐GFP‐4C) expressing cells revealed an early disappearance of gap junctions, progressive accumulation of Cx43 in cytoplasmic structures, and an unexpected subset pool of protein concentrated in the plasma membrane surrounding the midbody region in telophase followed by rapid reappearance of punctate plaques upon mitotic exit. These distributions were also observed in immuno‐labeled endogenous Cx43‐expressing cells. Photo‐oxidation of ReAsH‐labeled Cx43‐GFP‐4C cells in telophase confirmed that Cx43 is distributed in the plasma membrane surrounding the midbody as apparent connexons and in cytoplasmic vesicles. We performed optical pulse‐chase labeling and single label time‐lapse imaging of synchronized cells stably expressing Cx43 with internal tetracysteine domains through mitosis. In late telophase, older Cx43 is segregated mainly to the plasma membrane while newer Cx43 is intracellular. This older population nucleates new gap junctions permitting rapid resumption of communication upon mitotic exit. 相似文献
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