title: Minimal impact electro-injection of cells undergoing dynamic shape change reveals calpain activation.
abstract: The ability of neutrophils to rapidly change shape underlies their physiological functions of phagocytosis and spreading. A major problem in establishing the mechanism is that conventional microinjection of substances and indicators interferes with this dynamic cell behaviour. Here we show that electroinjection, a "no-touch" point-and-shoot means of introducing material into the cell, is sufficiently gentle to allow neutrophils to be injected whilst undergoing chemokinesis and spreading without disturbing cell shape change behaviour. Using this approach, a fluorogenic calpain-1 selective peptide substrate was introduced into the cytosol of individual neutrophils undergoing shape changes. These data showed that (i) physiologically elevated cytosolic Ca(2+) concentrations were sufficient to trigger calpain-1 activation, blockade of Ca(2+) influx preventing calpain activation and (ii) calpain-1 activity was elevated in spreading neutrophil. These findings provide the first direct demonstration of a physiological role for Ca(2+) elevation in calpain-1 activation and rapid cell spreading. Electroinjection of cells undergoing dynamic shape changes thus opens new avenues of investigation for defining the molecular mechanism underlying dynamic cell shape changes.
authors.affiliation: Neutrophil Signalling Group, Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Heath Park, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Kimberley J
authors.initials: KJ
authors.lastname: Lewis
authors.affiliation: Neutrophil Signalling Group, Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Heath Park, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Masterman
authors.affiliation: Neutrophil Signalling Group, Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Heath Park, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Iraj
authors.initials: I
authors.lastname: Laffafian
authors.affiliation: School of Dentistry, Cardiff University, Heath Park, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Sharon
authors.initials: S
authors.lastname: Dewitt
authors.affiliation: Neutrophil Signalling Group, Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Heath Park, Cardiff University, Cardiff CF14 4XN, UK.
authors.forename: Jennie S
authors.initials: JS
authors.lastname: Campbell
authors.affiliation: Neutrophil Signalling Group, Institute of Molecular and Experimental Medicine, School of Medicine, Cardiff University, Heath Park, Cardiff University, Cardiff CF14 4XN, UK. Electronic address: hallettmb@cf.ac.uk.
authors.forename: Maurice B
authors.initials: MB
authors.lastname: Hallett
mesh_terms: D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002468:Cell Physiological Phenomena; D048430:Cell Shape / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D009504:Neutrophils / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010446:Peptide Fragments; D002154:Calpain; D002118:Calcium
keywords: Cell spreading; Chemokinesis; Cytosolic Ca(2+); Neutrophil; Phagocytosis