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Correlative Light and Electron Microscopy Reveals the HAS3-Induced Dorsal Plasma Membrane Ruffles.

Rilla K, Koistinen A - Int J Cell Biol (2015)

Bottom Line: In this study, correlative light and electron microscopy (CLEM) was utilized to correlate the GFP-HAS3 signal and the surface ultrastructure of cells in order to study in detail the morphological changes induced by HAS3 overexpression.Surprisingly, this method revealed that GFP-HAS3 not only localizes to ruffles but in fact induces dorsal ruffle formation.Dorsal ruffles regulate diverse cellular functions, such as motility, regulation of glucose metabolism, spreading, adhesion, and matrix degradation, the same functions driven by active hyaluronan synthesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine and SIB Labs, University of Eastern Finland, 70211 Kuopio, Finland.

ABSTRACT
Hyaluronan is a linear sugar polymer synthesized by three isoforms of hyaluronan synthases (HAS1, 2, and 3) that forms a hydrated scaffold around cells and is an essential component of the extracellular matrix. The morphological changes of cells induced by active hyaluronan synthesis are well recognized but not studied in detail with high resolution before. We have previously found that overexpression of HAS3 induces growth of long plasma membrane protrusions that act as platforms for hyaluronan synthesis. The study of these thin and fragile protrusions is challenging, and they are difficult to preserve by fixation unless they are adherent to the substrate. Thus their structure and regulation are still partly unclear despite careful imaging with different microscopic methods in several cell types. In this study, correlative light and electron microscopy (CLEM) was utilized to correlate the GFP-HAS3 signal and the surface ultrastructure of cells in order to study in detail the morphological changes induced by HAS3 overexpression. Surprisingly, this method revealed that GFP-HAS3 not only localizes to ruffles but in fact induces dorsal ruffle formation. Dorsal ruffles regulate diverse cellular functions, such as motility, regulation of glucose metabolism, spreading, adhesion, and matrix degradation, the same functions driven by active hyaluronan synthesis.

No MeSH data available.


Related in: MedlinePlus

GFP-HAS3-induced plasma membrane protrusions imaged by CLEM. A GFP-HAS3 expressing MCF-7 cell imaged by 3D confocal microscopy in (a), (d), and (g) and by SEM in (b), (e), and (h). Merged images are presented in (c), (f), and (i). High magnification images from selected areas of the same cell presented in (a)–(c) are shown in (d)–(i). Arrows in (d), (e), (g), and (h) indicate the GFP-HAS3-positive bulbous expansions in both the body and tips of protrusions. Color depth coding was used to demonstrate the variable length of protrusions in (d). Scale bars 10 μm in (a), 2 μm in (d) and (g).
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fig2: GFP-HAS3-induced plasma membrane protrusions imaged by CLEM. A GFP-HAS3 expressing MCF-7 cell imaged by 3D confocal microscopy in (a), (d), and (g) and by SEM in (b), (e), and (h). Merged images are presented in (c), (f), and (i). High magnification images from selected areas of the same cell presented in (a)–(c) are shown in (d)–(i). Arrows in (d), (e), (g), and (h) indicate the GFP-HAS3-positive bulbous expansions in both the body and tips of protrusions. Color depth coding was used to demonstrate the variable length of protrusions in (d). Scale bars 10 μm in (a), 2 μm in (d) and (g).

Mentions: More detailed visualization of GFP-HAS3-positive MCF-7 cells was performed with higher magnification. A typical example of single GFP-HAS3-positive MCF-7 cell is shown in Figure 2. The morphology was relatively unchanged after sample processing for SEM (Figure 2) and GFP-HAS3-induced protrusions were preserved. As shown before, many of the HAS-induced protrusions have a dilated tip complex [14, 15], but it has been unclear if this dilation is an artefact resulting from high level of GFP-HAS3 fluorescence and light scattering. SEM confirmed that many of the HAS3-induced protrusions have a dilated tip complex (arrows in Figures 2(d) and 2(e)) and dilated areas are occasionally found also in the body of the protrusions (arrows in Figures 2(g) and 2(h)). The protrusions with dilated tips were similarly formed upon overexpression of HAS3 without GFP-tag (data not shown). Thickness of protrusions expressing high levels of GFP-HAS3 signal is overamplified in confocal microscopy because of light scattering. Protrusions also shrink and collapse during fixation and drying for SEM, which increases the differences in thickness observed between CLSM and SEM. As obtained by SEM, the HAS3-induced protrusions are extremely thin, typically between 70 and 130 nm in diameter.


Correlative Light and Electron Microscopy Reveals the HAS3-Induced Dorsal Plasma Membrane Ruffles.

Rilla K, Koistinen A - Int J Cell Biol (2015)

GFP-HAS3-induced plasma membrane protrusions imaged by CLEM. A GFP-HAS3 expressing MCF-7 cell imaged by 3D confocal microscopy in (a), (d), and (g) and by SEM in (b), (e), and (h). Merged images are presented in (c), (f), and (i). High magnification images from selected areas of the same cell presented in (a)–(c) are shown in (d)–(i). Arrows in (d), (e), (g), and (h) indicate the GFP-HAS3-positive bulbous expansions in both the body and tips of protrusions. Color depth coding was used to demonstrate the variable length of protrusions in (d). Scale bars 10 μm in (a), 2 μm in (d) and (g).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: GFP-HAS3-induced plasma membrane protrusions imaged by CLEM. A GFP-HAS3 expressing MCF-7 cell imaged by 3D confocal microscopy in (a), (d), and (g) and by SEM in (b), (e), and (h). Merged images are presented in (c), (f), and (i). High magnification images from selected areas of the same cell presented in (a)–(c) are shown in (d)–(i). Arrows in (d), (e), (g), and (h) indicate the GFP-HAS3-positive bulbous expansions in both the body and tips of protrusions. Color depth coding was used to demonstrate the variable length of protrusions in (d). Scale bars 10 μm in (a), 2 μm in (d) and (g).
Mentions: More detailed visualization of GFP-HAS3-positive MCF-7 cells was performed with higher magnification. A typical example of single GFP-HAS3-positive MCF-7 cell is shown in Figure 2. The morphology was relatively unchanged after sample processing for SEM (Figure 2) and GFP-HAS3-induced protrusions were preserved. As shown before, many of the HAS-induced protrusions have a dilated tip complex [14, 15], but it has been unclear if this dilation is an artefact resulting from high level of GFP-HAS3 fluorescence and light scattering. SEM confirmed that many of the HAS3-induced protrusions have a dilated tip complex (arrows in Figures 2(d) and 2(e)) and dilated areas are occasionally found also in the body of the protrusions (arrows in Figures 2(g) and 2(h)). The protrusions with dilated tips were similarly formed upon overexpression of HAS3 without GFP-tag (data not shown). Thickness of protrusions expressing high levels of GFP-HAS3 signal is overamplified in confocal microscopy because of light scattering. Protrusions also shrink and collapse during fixation and drying for SEM, which increases the differences in thickness observed between CLSM and SEM. As obtained by SEM, the HAS3-induced protrusions are extremely thin, typically between 70 and 130 nm in diameter.

Bottom Line: In this study, correlative light and electron microscopy (CLEM) was utilized to correlate the GFP-HAS3 signal and the surface ultrastructure of cells in order to study in detail the morphological changes induced by HAS3 overexpression.Surprisingly, this method revealed that GFP-HAS3 not only localizes to ruffles but in fact induces dorsal ruffle formation.Dorsal ruffles regulate diverse cellular functions, such as motility, regulation of glucose metabolism, spreading, adhesion, and matrix degradation, the same functions driven by active hyaluronan synthesis.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biomedicine and SIB Labs, University of Eastern Finland, 70211 Kuopio, Finland.

ABSTRACT
Hyaluronan is a linear sugar polymer synthesized by three isoforms of hyaluronan synthases (HAS1, 2, and 3) that forms a hydrated scaffold around cells and is an essential component of the extracellular matrix. The morphological changes of cells induced by active hyaluronan synthesis are well recognized but not studied in detail with high resolution before. We have previously found that overexpression of HAS3 induces growth of long plasma membrane protrusions that act as platforms for hyaluronan synthesis. The study of these thin and fragile protrusions is challenging, and they are difficult to preserve by fixation unless they are adherent to the substrate. Thus their structure and regulation are still partly unclear despite careful imaging with different microscopic methods in several cell types. In this study, correlative light and electron microscopy (CLEM) was utilized to correlate the GFP-HAS3 signal and the surface ultrastructure of cells in order to study in detail the morphological changes induced by HAS3 overexpression. Surprisingly, this method revealed that GFP-HAS3 not only localizes to ruffles but in fact induces dorsal ruffle formation. Dorsal ruffles regulate diverse cellular functions, such as motility, regulation of glucose metabolism, spreading, adhesion, and matrix degradation, the same functions driven by active hyaluronan synthesis.

No MeSH data available.


Related in: MedlinePlus