Biochemical, electron microscopic and immunohistological observations of cationic detergent-extracted cells: detection and improved preservation of microextensions and ultramicroextensions

Nakamura, Fumihiko

doi:10.1186/1471-2121-2-10

Cited by 25 publications

(6 citation statements)

References 57 publications

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“…GA fixation, in particular the optimized two-step GA approach, was again shown to preserve MT architecture even better than PFA. This is in line with EM and SMLM studies that have investigated the effects of PFA and GA fixation on actin, another cytoskeletal structure, and also found that PFA, but not GA, introduces subdiffraction artifacts . Permeabilization was observed to strongly influence the ratio of polymeric to dimeric tubulin localizations and demands further investigation combining SMLM and IR with assays for cytosolic content postfixation.…”

Section: Discussionsupporting

confidence: 87%

Correlative Synchrotron Fourier Transform Infrared Spectroscopy and Single Molecule Super Resolution Microscopy for the Detection of Composition and Ultrastructure Alterations in Single Cells

Whelan

Bell

2015

ACS Chem. Biol.

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Single molecule localization microscopy (SMLM) and synchrotron Fourier transform infrared (S-FTIR) spectroscopy are two techniques capable of elucidating unique and valuable biological detail. SMLM provides images of the structures and distributions of targeted biomolecules at spatial resolutions up to an order of magnitude better than the diffraction limit, whereas IR spectroscopy objectively measures the holistic biochemistry of an entire sample, thereby revealing any variations in overall composition. Both tools are currently applied extensively to detect cellular response to disease, chemical treatment, and environmental change. Here, these two techniques have been applied correlatively at the single cell level to probe the biochemistry of common fixation methods and have detected various fixation-induced losses of biomolecular composition and cellular ultrastructure. Furthermore, by extensive honing and optimizing of fixation protocols, many fixation artifacts previously considered pervasive and regularly identified using IR spectroscopy and fluorescence techniques have been avoided. Both paraformaldehyde and two-step glutaraldehyde fixation were identified as best preserving biochemistry for both SMLM and IR studies while other glutaraldehyde and methanol fixation protocols were demonstrated to cause significant biochemical changes and higher variability between samples. Moreover, the potential complementarity of the two techniques was strikingly demonstrated in the correlated detection of biochemical changes as well as in the detection of fixation-induced damage that was only revealed by one of the two techniques.

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Section: Discussionsupporting

confidence: 87%

Correlative Synchrotron Fourier Transform Infrared Spectroscopy and Single Molecule Super Resolution Microscopy for the Detection of Composition and Ultrastructure Alterations in Single Cells

Whelan

Bell

2015

ACS Chem. Biol.

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“…No other such pits were found in t = 0 samples and where cells were absent in the t = 72 samples. Under the SEM, most cells were seen to have spherical morphologies; this is most likely due to the glutaraldehyde fixing/ethanol dehydration and CO 2 critical point drying steps, rendering the cell culture environment unfavorable and causing the cells to retract their micro-extensions 41 . Furthermore, it appeared that the fibroblasts migrated deeper and became more settled into the nanoscaffolds as compared with the keratinocytes.…”

Section: Discussionmentioning

confidence: 99%

Designer self-assembling hydrogel scaffolds can impact skin cell proliferation and migration

Bradshaw

Fear

et al. 2014

Sci Rep

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There is a need to develop economical, efficient and widely available therapeutic approaches to enhance the rate of skin wound healing. The optimal outcome of wound healing is restoration to the pre-wound quality of health. In this study we investigate the cellular response to biological stimuli using functionalized nanofibers from the self-assembling peptide, RADA16. We demonstrate that adding different functional motifs to the RADA16 base peptide can influence the rate of proliferation and migration of keratinocytes and dermal fibroblasts. Relative to unmodified RADA16; the Collagen I motif significantly promotes cell migration, and reduces proliferation.

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“…Immunocytochemistry was performed on glass coverslips, coated with PLL alone (Sigma-Aldrich), 0.1 mg/ml, or followed by coating with 10 μg/ml vitronectin or laminin 1 (Sigma-Aldrich). Purified mouse SCs or DRG were fixed in 4% PFA or in dodecyl-trimethylammonium chloride + 1% PFA ( Nakamura, 2001 ) and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) or 100% methanol. To reveal active Rac1 localization, recombinant PAK1 PBD-GST protein was detected with an anti-GST antibody.…”

Section: Methodsmentioning

confidence: 99%

β1 integrin activates Rac1 in Schwann cells to generate radial lamellae during axonal sorting and myelination

Nodari¹,

Zambroni²,

Quattrini

et al. 2007

The Journal of Cell Biology

174

235

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Myelin is a multispiraled extension of glial membrane that surrounds axons. How glia extend a surface many-fold larger than their body is poorly understood. Schwann cells are peripheral glia and insert radial cytoplasmic extensions into bundles of axons to sort, ensheath, and myelinate them. Laminins and β1 integrins are required for axonal sorting, but the downstream signals are largely unknown. We show that Schwann cells devoid of β1 integrin migrate to and elongate on axons but cannot extend radial lamellae of cytoplasm, similar to cells with low Rac1 activation. Accordingly, active Rac1 is decreased in β1 integrin–null nerves, inhibiting Rac1 activity decreases radial lamellae in Schwann cells, and ablating Rac1 in Schwann cells of transgenic mice delays axonal sorting and impairs myelination. Finally, expressing active Rac1 in β1 integrin–null nerves improves sorting. Thus, increased activation of Rac1 by β1 integrins allows Schwann cells to switch from migration/elongation to the extension of radial membranes required for axonal sorting and myelination.

show abstract

Biochemical, electron microscopic and immunohistological observations of cationic detergent-extracted cells: detection and improved preservation of microextensions and ultramicroextensions

Cited by 25 publications

References 57 publications

Correlative Synchrotron Fourier Transform Infrared Spectroscopy and Single Molecule Super Resolution Microscopy for the Detection of Composition and Ultrastructure Alterations in Single Cells

Correlative Synchrotron Fourier Transform Infrared Spectroscopy and Single Molecule Super Resolution Microscopy for the Detection of Composition and Ultrastructure Alterations in Single Cells

Designer self-assembling hydrogel scaffolds can impact skin cell proliferation and migration

β1 integrin activates Rac1 in Schwann cells to generate radial lamellae during axonal sorting and myelination

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