Gaining access to the cytosol: the technique and some applications of electropermeabilization

Knight, D. E.; Scrutton, Michael C.

doi:10.1042/bj2340497

Cited by 271 publications

(107 citation statements)

References 69 publications

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“…The major problem with the permeabilized cell system is the very high levels of Ca2+ required to elicit protein discharge. We observe maximal release at 10 /tM-Ca2+, as has been widely observed in a variety of other exocytotic systems (Knight & Scrutton, 1986), which is at least 10-fold higher than is observed by fluorimetric measurements with fura-2 or quin-2. Williams and co-workers have reported that SLO-permeabilized mouse pancreatic acini undergo maximal release at 1 /tM-Ca2+ (Kitagawa etal., 1990).…”

Section: Discussionsupporting

confidence: 77%

“…We have demonstrated that activation of PKC by PMA potentiates amylase release by increasing the sensitivity of the system to Ca2+, as is observed in a wide variety of other exocytotic systems (Knight & Scrutton, 1986). In pancreatic acini, increasing doses of Ca2+ and potentiation of Ca2+-dependent release by PMA appear to have little effect on the initial rate of release, but increase the length of time over which cells undergo discharge.…”

Section: Discussionsupporting

confidence: 52%

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Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

O’Sullivan

Jamieson

1992

Biochemical Journal

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The effect of protein kinase C (PKC) on amylase discharge from streptolysin-O-permeabilized rat pancreatic acini was investigated. Addition of phorbol 12-myristate 13-acetate (PMA) to permeabilized cells potentiated Ca2+-stimulated release, but had no effect on discharge at non-stimulatory Ca2+ concentrations. PMA markedly shifted the Ca2+-concentration-dependence of amylase discharge to the left, by enhancing the time over which the permeabilized cells release. This effect was inhibited by both staurosporine and PKC-19-31-amide peptide inhibitor, indicating that the effect of PMA was due to its action on PKC. Staurosporine also partially inhibited amylase release at the optimal concentration of Ca2+; this effect was not replicated by the more specific PKC-19-3 1-amide peptide inhibitor and may be due to an effect on another second-messenger system. PKC appears to be an important modulator of release in pancreatic acini, but its activation is not an absolute requirement for Ca2+-dependent amylase discharge.

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Section: Discussionsupporting

confidence: 77%

Section: Discussionsupporting

confidence: 52%

Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

O’Sullivan

Jamieson

1992

Biochemical Journal

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“…The data were normalized to the control conditions with the low and high [Ca 2+ ] free plateaus defining 0% and 100% fusion, respectively. The resulting Ca 2+ -activity curves were fit using the sigmoidal cumulative log-normal model to establish fusion extent and Ca 2+ sensitivity; the applicability of a log-normal cumulative distribution function to exocytotic survival and thus Ca 2+ -activity curves has been clearly demonstrated [8,49,84]-the fraction of CV that fuse at a given [Ca 2+ ] free is solely a function of the average number of active fusion complexes (<n>) at a docking site. As CV-CV fusion proceeds through the same fusion complexes as exocytosis, it is fully described by the same analysis developed for CV-PM fusion, yielding comparable fitting parameters for the sigmoidal relationship between <n> and [Ca 2+ ] free [8,27,31,84].…”

Section: Egg Manipulationsmentioning

confidence: 99%

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Rogasevskaia

Coorssen

2011

J Chem Biol

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Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca 2+ -triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca 2+ -triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.

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“…Similar features to those obtained with these new developments also can be achieved by electroporation, which uses microsecond, high-voltage pulses to transiently permeabilize the plasma membrane (35,36). Similar to the SL-O method about 50% of the treated cells survive and take up the molecule of interest.…”

Section: Transfer By Transient Permeabilization Of the Plasma Membranementioning

confidence: 58%

The many ways to cross the plasma membrane

Stephens

Pepperkok

2001

Proc. Natl. Acad. Sci. U.S.A.

116

106

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Gaining access to the cytosol: the technique and some applications of electropermeabilization

Cited by 271 publications

References 69 publications

Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

Activation of protein kinase C is not an absolute requirement for amylase release from permeabilized rat pancreatic acini

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

The many ways to cross the plasma membrane

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