Preservation and visualization of molecular structure in detergent‐extracted whole mounts of cultured cells

Lindroth, Margaretha; Bell, Paul B.; Fredriksson, Bengt-Arne; Liu, Xiaodong

doi:10.1002/jemt.1070220203

Cited by 26 publications

(21 citation statements)

References 71 publications

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“…One of the most straightforward ways to observe the complex architecture of the cytoskeleton is to examine whole mounts of cells by TEM [28]. This technique, allows the visualization of the cytoskeleton at the supramolecular level with a minimal disruption to the cells.…”

Section: Resultsmentioning

confidence: 99%

The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

Braet¹,

Spector

Shochet

et al. 2002

BMC Cell Biol

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BackgroundLiver sinusoidal endothelial cells (LSECs) react to different anti-actin agents by increasing their number of fenestrae. A new structure related to fenestrae formation could be observed when LSECs were treated with misakinolide. In this study, we investigated the effects of two new actin-binding agents on fenestrae dynamics. High-resolution microscopy, including immunocytochemistry and a combination of fluorescence- and scanning electron microscopy was applied.ResultsHalichondramide and dihydrohalichondramide disrupt microfilaments within 10 minutes and double the number of fenestrae in 30 minutes. Dihydrohalichondramide induces fenestrae-forming centers, whereas halichondramide only revealed fenestrae-forming centers without attached rows of fenestrae with increasing diameter. Correlative microscopy showed the absence of actin filaments (F-actin) in sieve plates and fenestrae-forming centers. Comparable experiments on umbilical vein endothelial cells and bone marrow sinusoidal endothelial cells revealed cell contraction without the appearance of fenestrae or fenestrae-forming centers.Conclusion(I) A comparison of all anti-actin agents tested so far, revealed that the only activity that misakinolide and dihydrohalichondramide have in common is their barbed end capping activity; (II) this activity seems to slow down the process of fenestrae formation to such extent that it becomes possible to resolve fenestrae-forming centers; (III) fenestrae formation resulting from microfilament disruption is probably unique to LSECs.

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Section: Resultsmentioning

confidence: 99%

The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

Braet¹,

Spector

Shochet

et al. 2002

BMC Cell Biol

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“…microfilaments, intermediate filaments and microtubules. Their reported thickness with electron microscopy in detergent‐extracted cells is 5–10, 7–15 and 22–25 nm, respectively ( Lindroth et al ., 1992 ). Considering these data, in combination with our measurements on the cytoskeleton of fibroblasts, we cannot judge the nature of the depicted filaments.…”

Section: Discussionmentioning

confidence: 99%

Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages

Braet¹,

Seynaeve²,

Zanger³

et al. 1998

Journal of Microscopy

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Atomic force microscopy (AFM) has been used to image a wide variety of cells. Fixed and dried-coated, wet-fixed or living cells were investigated. The major advantage of AFM over SEM is the avoidance of vacuum and electrons, whereas imaging can be done at environmental pressure and in aqueous conditions. Evidence of the successful application of AFM in biological imaging is provided by comparing results of AFM with SEM and/or TEM. In this study, we investigated surface and submembranous structures of living and glutaraldehyde-fixed colon carcinoma cells, skin fibroblasts and liver macrophages by AFM. Special attention was paid to the correct conditions for the acquisition of images of the surface of these cells, because quality SEM examinations have already been abundantly presented. AFM imaging of living cells revealed specific structures, such as the cytoskeleton, which were not observed by SEM. Membrane structures, such as ruffles, lamellipodia, microspikes and microvilli, could only clearly be observed after fixing the cells with 0.1% glutaraldehyde. AFM images of living cells were comparable to SEM images of fixed, dried and coated cells, but contained a number of artefacts due to tip-sample interaction. In addition, AFM imaging allowed the visualization of cytoplasmic submembranous structures without the necessity for further preparative steps, allowing us: (i) to follow cytoskeletal changes in fibroblasts under the influence of the microfilament disrupting agent latrunculin A; (ii) to study particle phagocytosis in macrophages. Therefore, in spite of the slow image acquisition of the AFM, the instrument can be used for high-resolution real-time studies of dynamic changes in submembranous structures.

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“…One of the most straightforward ways to observe the overall complex organization of the cytoskeleton at high resolution is to examine whole mounts of cells by TEM (Lindroth et al, 1992). This technique allows the visualization of the cytoskeleton with minimal disruption of the cells after slight prefixation and brief extraction with detergent.…”

Section: Whole Mountsmentioning

confidence: 99%

Contribution of high‐resolution correlative imaging techniques in the study of the liver sieve in three‐dimensions

Braet

Wisse

Bomans

et al. 2007

Microscopy Res & Technique

101

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Correlative microscopy has become increasingly important for the analysis of the structure, function, and dynamics of cells. This is largely due to the result of recent advances in light-, probe-, laser- and various electron microscopy techniques that facilitate three-dimensional studies. Furthermore, the improved understanding in the past decade of imaging cell compartments in the third dimension has resulted largely from the availability of powerful computers, fast high-resolution CCD cameras, specifically developed imaging analysis software, and various probes designed for labeling living and or fixed cells. In this paper, we review different correlative high-resolution imaging methodologies and how these microscopy techniques facilitated the accumulation of new insights in the morpho-functional and structural organization of the hepatic sieve. Various aspects of hepatic endothelial fenestrae regarding their structure, origin, dynamics, and formation will be explored throughout this paper by comparing the results of confocal laser scanning-, correlative fluorescence and scanning electron-, atomic force-, and whole-mount electron microscopy. Furthermore, the recent advances of vitrifying cells with the vitrobot in combination with the glove box for the preparation of cells for cryo-electron microscopic investigation will be discussed. Finally, the first transmission electron tomography data of the liver sieve in three-dimensions are presented. The obtained data unambiguously show the involvement of special domains in the de novo formation and disappearance of hepatic fenestrae, and focuses future research into the (supra)molecular structure of the fenestrae-forming center, defenestration center and fenestrae-, and sieve plate cytoskeleton ring by using advanced cryo-electron tomography.

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Preservation and visualization of molecular structure in detergent‐extracted whole mounts of cultured cells

Cited by 26 publications

References 71 publications

The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

The new anti-actin agent dihydrohalichondramide reveals fenestrae-forming centers in hepatic endothelial cells

Imaging surface and submembranous structures with the atomic force microscope: a study on living cancer cells, fibroblasts and macrophages

Contribution of high‐resolution correlative imaging techniques in the study of the liver sieve in three‐dimensions

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