Limits...
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

Ultrastructure of GFP-HAS3-positive dorsal ruffles. 2D confocal images showing the dorsal GFP-HAS3 signal are shown in (a) and (d) and corresponding SEM images in (b) and (e). Merged images are shown in (c) and (f), respectively. Many of the dorsal ruffles were linear or curved in shape (a) and provided a basis for several thinner protrusions (arrows in (b)). Occasionally, circular ruffles were detected (arrows in (d)-(e)). Scale bar 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4581547&req=5

fig4: Ultrastructure of GFP-HAS3-positive dorsal ruffles. 2D confocal images showing the dorsal GFP-HAS3 signal are shown in (a) and (d) and corresponding SEM images in (b) and (e). Merged images are shown in (c) and (f), respectively. Many of the dorsal ruffles were linear or curved in shape (a) and provided a basis for several thinner protrusions (arrows in (b)). Occasionally, circular ruffles were detected (arrows in (d)-(e)). Scale bar 2 μm.

Mentions: Next high resolution SEM images were utilized to analyze the detailed structure of the HAS3-induced ruffles. Most of the ruffles appeared on the dorsal surface of the cell (Figures 2(b), 3, and 4) rather than on peripheral areas. GFP-HAS3 signal was detected on the apical plasma membrane of cells and accumulated on the ruffles. Furthermore, SEM revealed that many of the HAS-positive protrusions were embedded in the ruffle, suggesting that ruffles provide a basis for thinner protrusions. There was typically 1–5 or even higher number of protrusions arising from a single ruffle (arrows in Figure 4(b)). This indicates specific modeling of plasma membrane dynamics and the underlying actin network by HAS activity. Most of the ruffles were linear or curved, sheet-like protrusions of variable size, but some circular structures were also found (arrows in Figures 4(a), 4(b), 4(d), and 4(e)), which in line with the suggested dynamic formation of ruffles followed by constriction into circular structures before disappearing [5].


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

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

Ultrastructure of GFP-HAS3-positive dorsal ruffles. 2D confocal images showing the dorsal GFP-HAS3 signal are shown in (a) and (d) and corresponding SEM images in (b) and (e). Merged images are shown in (c) and (f), respectively. Many of the dorsal ruffles were linear or curved in shape (a) and provided a basis for several thinner protrusions (arrows in (b)). Occasionally, circular ruffles were detected (arrows in (d)-(e)). Scale bar 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4581547&req=5

fig4: Ultrastructure of GFP-HAS3-positive dorsal ruffles. 2D confocal images showing the dorsal GFP-HAS3 signal are shown in (a) and (d) and corresponding SEM images in (b) and (e). Merged images are shown in (c) and (f), respectively. Many of the dorsal ruffles were linear or curved in shape (a) and provided a basis for several thinner protrusions (arrows in (b)). Occasionally, circular ruffles were detected (arrows in (d)-(e)). Scale bar 2 μm.
Mentions: Next high resolution SEM images were utilized to analyze the detailed structure of the HAS3-induced ruffles. Most of the ruffles appeared on the dorsal surface of the cell (Figures 2(b), 3, and 4) rather than on peripheral areas. GFP-HAS3 signal was detected on the apical plasma membrane of cells and accumulated on the ruffles. Furthermore, SEM revealed that many of the HAS-positive protrusions were embedded in the ruffle, suggesting that ruffles provide a basis for thinner protrusions. There was typically 1–5 or even higher number of protrusions arising from a single ruffle (arrows in Figure 4(b)). This indicates specific modeling of plasma membrane dynamics and the underlying actin network by HAS activity. Most of the ruffles were linear or curved, sheet-like protrusions of variable size, but some circular structures were also found (arrows in Figures 4(a), 4(b), 4(d), and 4(e)), which in line with the suggested dynamic formation of ruffles followed by constriction into circular structures before disappearing [5].

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