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Directing neuronal signaling through cell-surface glycan engineering.

Pulsipher A, Griffin ME, Stone SE, Brown JM, Hsieh-Wilson LC - J. Am. Chem. Soc. (2014)

Bottom Line: The ability to tailor plasma membranes with specific glycans may enable the control of signaling events that are critical for proper development and function.Neurons engineered to display CS-E-enriched polysaccharides exhibited increased activation of neurotrophin-mediated signaling pathways and enhanced axonal growth.This approach provides a facile, general route to tailor cell membranes with biologically active glycans and demonstrates the potential to direct important cellular events through cell-surface glycan engineering.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology and Howard Hughes Medical Institute , 1200 East California Boulevard, Pasadena, California 91125, United States.

ABSTRACT
The ability to tailor plasma membranes with specific glycans may enable the control of signaling events that are critical for proper development and function. We report a method to modify cell surfaces with specific sulfated chondroitin sulfate (CS) glycosaminoglycans using chemically modified liposomes. Neurons engineered to display CS-E-enriched polysaccharides exhibited increased activation of neurotrophin-mediated signaling pathways and enhanced axonal growth. This approach provides a facile, general route to tailor cell membranes with biologically active glycans and demonstrates the potential to direct important cellular events through cell-surface glycan engineering.

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Controlled cell-surface display of CSpolysaccharides and fluorophores.(A) FACS analysis of PC12 cells treated with liposomes presentingvarying amounts (0–20%) of AF488-hyd. (B) Immunofluorescencedetection of CS-E (green) on PC12 cells treated with or without chondroitinase(ChABC) and CS-E-functionalized liposomes as indicated.
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fig2: Controlled cell-surface display of CSpolysaccharides and fluorophores.(A) FACS analysis of PC12 cells treated with liposomes presentingvarying amounts (0–20%) of AF488-hyd. (B) Immunofluorescencedetection of CS-E (green) on PC12 cells treated with or without chondroitinase(ChABC) and CS-E-functionalized liposomes as indicated.

Mentions: Preliminary optimizationof liposomal membrane fusion was performedon rat pheochromocytoma (PC12) cells using liposomes functionalizedwith a hydrazide-conjugated fluorophore (AF488-hyd). We found thata 2:1 ratio of DOPE:DOTAP was optimalfor membrane fusion, as visualized by fluorescence microscopy (Figure S1). To approximate the relative levelsof fluorophore incorporation at the cell surface, we incubated liposomescontaining varying concentrations of AF488-hyd with PC12 cells onice for 30 min. Cells labeled with liposomes containing 10 mol % AF488-hyddisplayed similar fluorescence signal profiles by fluorescence-assistedcell sorting (FACS) analysis as cells labeled with an anti-CS-E monoclonalantibody4d that detected endogenous CS-Elevels (Figure 2A). These results suggest thatthis liposomal strategy can incorporate exogenous molecules into cellmembranes at levels roughly similar to those of endogenous CS polysaccharides.


Directing neuronal signaling through cell-surface glycan engineering.

Pulsipher A, Griffin ME, Stone SE, Brown JM, Hsieh-Wilson LC - J. Am. Chem. Soc. (2014)

Controlled cell-surface display of CSpolysaccharides and fluorophores.(A) FACS analysis of PC12 cells treated with liposomes presentingvarying amounts (0–20%) of AF488-hyd. (B) Immunofluorescencedetection of CS-E (green) on PC12 cells treated with or without chondroitinase(ChABC) and CS-E-functionalized liposomes as indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120997&req=5

fig2: Controlled cell-surface display of CSpolysaccharides and fluorophores.(A) FACS analysis of PC12 cells treated with liposomes presentingvarying amounts (0–20%) of AF488-hyd. (B) Immunofluorescencedetection of CS-E (green) on PC12 cells treated with or without chondroitinase(ChABC) and CS-E-functionalized liposomes as indicated.
Mentions: Preliminary optimizationof liposomal membrane fusion was performedon rat pheochromocytoma (PC12) cells using liposomes functionalizedwith a hydrazide-conjugated fluorophore (AF488-hyd). We found thata 2:1 ratio of DOPE:DOTAP was optimalfor membrane fusion, as visualized by fluorescence microscopy (Figure S1). To approximate the relative levelsof fluorophore incorporation at the cell surface, we incubated liposomescontaining varying concentrations of AF488-hyd with PC12 cells onice for 30 min. Cells labeled with liposomes containing 10 mol % AF488-hyddisplayed similar fluorescence signal profiles by fluorescence-assistedcell sorting (FACS) analysis as cells labeled with an anti-CS-E monoclonalantibody4d that detected endogenous CS-Elevels (Figure 2A). These results suggest thatthis liposomal strategy can incorporate exogenous molecules into cellmembranes at levels roughly similar to those of endogenous CS polysaccharides.

Bottom Line: The ability to tailor plasma membranes with specific glycans may enable the control of signaling events that are critical for proper development and function.Neurons engineered to display CS-E-enriched polysaccharides exhibited increased activation of neurotrophin-mediated signaling pathways and enhanced axonal growth.This approach provides a facile, general route to tailor cell membranes with biologically active glycans and demonstrates the potential to direct important cellular events through cell-surface glycan engineering.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology and Howard Hughes Medical Institute , 1200 East California Boulevard, Pasadena, California 91125, United States.

ABSTRACT
The ability to tailor plasma membranes with specific glycans may enable the control of signaling events that are critical for proper development and function. We report a method to modify cell surfaces with specific sulfated chondroitin sulfate (CS) glycosaminoglycans using chemically modified liposomes. Neurons engineered to display CS-E-enriched polysaccharides exhibited increased activation of neurotrophin-mediated signaling pathways and enhanced axonal growth. This approach provides a facile, general route to tailor cell membranes with biologically active glycans and demonstrates the potential to direct important cellular events through cell-surface glycan engineering.

Show MeSH
Related in: MedlinePlus