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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 expression induces plasma membrane ruffling. Confocal 3D projection of transiently transfected MCF-7 cells imaged by CLSM (a) and the corresponding area by SEM (b). Arrows in (b) indicate GFP-HAS3-positive cells with plasma membrane ruffling, while negative cells (asterisks) have smoother dorsal surface. Arrowheads in (a) and (b) indicate a cell that was lost during SEM processing. Stable, inducible transfections were utilized to quantify the dorsal ruffling of MCF-7 cells, which was significantly increased upon induction of GFP-HAS3. Significant difference (P < 0.05) in quantified parameters of ruffling is indicated by an asterisk (*) in table (c). N = 20 in both groups. A GFP-HAS3-positive cell treated with hyaluronidase before fixation is shown with CLSM and SEM in (d) and (e), respectively. Scale bars 10 μm in (a) and 5 μm in (d).
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fig3: GFP-HAS3 expression induces plasma membrane ruffling. Confocal 3D projection of transiently transfected MCF-7 cells imaged by CLSM (a) and the corresponding area by SEM (b). Arrows in (b) indicate GFP-HAS3-positive cells with plasma membrane ruffling, while negative cells (asterisks) have smoother dorsal surface. Arrowheads in (a) and (b) indicate a cell that was lost during SEM processing. Stable, inducible transfections were utilized to quantify the dorsal ruffling of MCF-7 cells, which was significantly increased upon induction of GFP-HAS3. Significant difference (P < 0.05) in quantified parameters of ruffling is indicated by an asterisk (*) in table (c). N = 20 in both groups. A GFP-HAS3-positive cell treated with hyaluronidase before fixation is shown with CLSM and SEM in (d) and (e), respectively. Scale bars 10 μm in (a) and 5 μm in (d).

Mentions: A typical morphology of cells with GFP-HAS3 overexpression was spindle-shaped, with no clear, single lamellipodia or distinguishable “front and rear” (arrows in Figure 3). Another typical feature of GFP-HAS3-positive cells was ruffling of the plasma membrane, appearing mainly on the apical faces of the plasma membrane. Comparison of negative cells (asterisks in Figures 3(a) and 3(b)) and GFP-HAS3-positive cells (arrows in Figures 3(a) and 3(b)) in transiently transfected cultures suggested that overexpression of HAS3 induces dorsal ruffling of the plasma membrane. To control if the ruffles are sensitive to hyaluronidase treatment, samples were treated with streptomyces hyaluronidase (5 TRU/mL, 30 min at 37°C) prior to fixation. The results showed that removal of hyaluronan did not completely destroy them (Figures 3(d) and 3(e)). This indicates that their structure is not completely dependent on pericellular hyaluronan.


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

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

GFP-HAS3 expression induces plasma membrane ruffling. Confocal 3D projection of transiently transfected MCF-7 cells imaged by CLSM (a) and the corresponding area by SEM (b). Arrows in (b) indicate GFP-HAS3-positive cells with plasma membrane ruffling, while negative cells (asterisks) have smoother dorsal surface. Arrowheads in (a) and (b) indicate a cell that was lost during SEM processing. Stable, inducible transfections were utilized to quantify the dorsal ruffling of MCF-7 cells, which was significantly increased upon induction of GFP-HAS3. Significant difference (P < 0.05) in quantified parameters of ruffling is indicated by an asterisk (*) in table (c). N = 20 in both groups. A GFP-HAS3-positive cell treated with hyaluronidase before fixation is shown with CLSM and SEM in (d) and (e), respectively. Scale bars 10 μm in (a) and 5 μm in (d).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: GFP-HAS3 expression induces plasma membrane ruffling. Confocal 3D projection of transiently transfected MCF-7 cells imaged by CLSM (a) and the corresponding area by SEM (b). Arrows in (b) indicate GFP-HAS3-positive cells with plasma membrane ruffling, while negative cells (asterisks) have smoother dorsal surface. Arrowheads in (a) and (b) indicate a cell that was lost during SEM processing. Stable, inducible transfections were utilized to quantify the dorsal ruffling of MCF-7 cells, which was significantly increased upon induction of GFP-HAS3. Significant difference (P < 0.05) in quantified parameters of ruffling is indicated by an asterisk (*) in table (c). N = 20 in both groups. A GFP-HAS3-positive cell treated with hyaluronidase before fixation is shown with CLSM and SEM in (d) and (e), respectively. Scale bars 10 μm in (a) and 5 μm in (d).
Mentions: A typical morphology of cells with GFP-HAS3 overexpression was spindle-shaped, with no clear, single lamellipodia or distinguishable “front and rear” (arrows in Figure 3). Another typical feature of GFP-HAS3-positive cells was ruffling of the plasma membrane, appearing mainly on the apical faces of the plasma membrane. Comparison of negative cells (asterisks in Figures 3(a) and 3(b)) and GFP-HAS3-positive cells (arrows in Figures 3(a) and 3(b)) in transiently transfected cultures suggested that overexpression of HAS3 induces dorsal ruffling of the plasma membrane. To control if the ruffles are sensitive to hyaluronidase treatment, samples were treated with streptomyces hyaluronidase (5 TRU/mL, 30 min at 37°C) prior to fixation. The results showed that removal of hyaluronan did not completely destroy them (Figures 3(d) and 3(e)). This indicates that their structure is not completely dependent on pericellular hyaluronan.

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