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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

Utilization of gridded coverslips for localization of cultured cells for correlative imaging. Low magnification images showing an overview of transiently transfected cells imaged by CLSM and DIC (a) and the corresponding area imaged by SEM (b). Arrows in (a) indicate cells that were detached in the subsequent preparation steps for scanning electron microscopy. Scale bar 100 μm.
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fig1: Utilization of gridded coverslips for localization of cultured cells for correlative imaging. Low magnification images showing an overview of transiently transfected cells imaged by CLSM and DIC (a) and the corresponding area imaged by SEM (b). Arrows in (a) indicate cells that were detached in the subsequent preparation steps for scanning electron microscopy. Scale bar 100 μm.

Mentions: This study presents a simple and easy process to image live or fixed cells by high resolution CLSM and SEM. The area imaged by CLSM was easily relocalized in SEM by utilizing gridded coverslips (Figure 1). A simultaneous DIC imaging made recognition of the same cells easy via CLSM (Figure 1(a)) and SEM (Figure 1(b)). Some shrinking during fixation and dehydration was detected, but the overall morphology of the cells was well preserved after fixation and processing for SEM. The GFP-HAS3 overexpressing cells were easily detached during processing (arrows in Figure 1), which indicates a decreased adhesion as a result of HAS overexpression, a finding in line with previous results [22].


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

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

Utilization of gridded coverslips for localization of cultured cells for correlative imaging. Low magnification images showing an overview of transiently transfected cells imaged by CLSM and DIC (a) and the corresponding area imaged by SEM (b). Arrows in (a) indicate cells that were detached in the subsequent preparation steps for scanning electron microscopy. Scale bar 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: Utilization of gridded coverslips for localization of cultured cells for correlative imaging. Low magnification images showing an overview of transiently transfected cells imaged by CLSM and DIC (a) and the corresponding area imaged by SEM (b). Arrows in (a) indicate cells that were detached in the subsequent preparation steps for scanning electron microscopy. Scale bar 100 μm.
Mentions: This study presents a simple and easy process to image live or fixed cells by high resolution CLSM and SEM. The area imaged by CLSM was easily relocalized in SEM by utilizing gridded coverslips (Figure 1). A simultaneous DIC imaging made recognition of the same cells easy via CLSM (Figure 1(a)) and SEM (Figure 1(b)). Some shrinking during fixation and dehydration was detected, but the overall morphology of the cells was well preserved after fixation and processing for SEM. The GFP-HAS3 overexpressing cells were easily detached during processing (arrows in Figure 1), which indicates a decreased adhesion as a result of HAS overexpression, a finding in line with previous results [22].

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