title: Accessible Synthetic Probes for Staining Actin inside Platelets and Megakaryocytes by Employing Lifeact Peptide.
abstract: Lifeact is a 17-residue peptide that can be employed in cell microscopy as a probe for F-actin when fused to fluorescent proteins, but therefore is not suitable for all cell types. We have conjugated fluorescently labelled Lifeact to three different cell-penetrating systems (a myristoylated carrier (myr), the pH low insertion peptide (pHLIP) and the cationic peptide TAT) as a strategy to deliver Lifeact into cells and developed new tools for actin staining with improved synthetic accessibility and low toxicity, focusing on their suitability in platelets and megakaryocytes. Using confocal microscopy, we characterised the cell distribution of the new hybrids in fixed cells, and found that both myr- and pHLIP-Lifeact conjugates provide efficient actin staining upon cleavage of Lifeact from the carriers, without affecting cell spreading. This new approach could facilitate the design of new tools for actin visualisation.
authors.affiliation: School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).|Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).
authors.forename: Lucia
authors.initials: L
authors.lastname: Cardo
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).
authors.forename: Steve G
authors.initials: SG
authors.lastname: Thomas
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).
authors.forename: Alexandra
authors.initials: A
authors.lastname: Mazharian
authors.affiliation: School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).
authors.forename: Zoe
authors.initials: Z
authors.lastname: Pikramenou
authors.affiliation: School of Bioscience, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).
authors.forename: Joshua Z
authors.initials: JZ
authors.lastname: Rappoport
authors.affiliation: School of Chemistry, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK). m.j.hannon@bham.ac.uk.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hannon
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT (UK).
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Watson
mesh_terms: D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism*; D057846:Cell-Penetrating Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D015195:Drug Design; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008533:Megakaryocytes / Q000378:metabolism*; D008969:Molecular Sequence Data; D013194:Staining and Labeling
chemical_list: D000199:Actins; D057846:Cell-Penetrating Peptides; D005456:Fluorescent Dyes
keywords: FRET; Lifeact; actin; cell-penetrating hybrids; fluorescent probes