QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 11, 2005
doi: 10.1242/10.1242/jcs.02601

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Churchward, M. A. Articles by Coorssen, J. R. Search for Related Content PubMed PubMed Citation Articles by Churchward, M. A. Articles by Coorssen, J. R.

Cholesterol facilitates the native mechanism of Ca²⁺-triggered membrane fusion

¹ Department of Physiology and Biophysics, Hotchkiss Brain Institute, University of Calgary, Health Sciences Centre, Faculty of Medicine, Calgary, Alberta, T2N 4N1, Canada
² Biochemistry and Molecular Biology, Hotchkiss Brain Institute, University of Calgary, Health Sciences Centre, Faculty of Medicine, Calgary, Alberta, T2N 4N1, Canada
³ Cell Biology and Anatomy, Hotchkiss Brain Institute, University of Calgary, Health Sciences Centre, Faculty of Medicine, Calgary, Alberta, T2N 4N1, Canada

View larger version (23K): [in a new window]   Fig. 1. The dose-dependent effects of mßcd on CV-PM fusion. (A) Ca2+ activity curves (n=3) following treatments with concentrations of mßcd as indicated. (B) Kinetics of fusion of CV-PM preparations treated as in A, triggered with 189±19 µM [Ca2+]free (n=2). (C) Total cholesterol in CV-PM preparations after treatment with the indicated concentration of mßcd, expressed relative to untreated preparations. *Significant difference from control (P<0.05) and other treatments (P<0.005); **, ***significant difference from control and other treatments (P<0.001).

View larger version (45K): [in a new window]   Fig. 2. Effects of mßcd on CV docking. (A) Treatment with 0.5 mM mßcd does not alter CV density at the cortex, but affects fusion of CVs after perfusion of 113±15 µM [Ca2+]free; overlay shows unfused vesicles in white, fused vesicles in green. (B) Lysis of CV-PM preparations and (C) suspensions of isolated CVs after treatment with the indicated concentrations of mßcd (n=3).

View larger version (21K): [in a new window]   Fig. 3. The dose-dependent effects of mßcd on CV-CV fusion. (A) Ca2+ activity curves (n=4) following treatments with various concentrations of mßcd as indicated. (B) Kinetics of CV-CV fusion treated as in A, triggered with 71±21 µM [Ca2+]free (n=3). (C) Total CV cholesterol after treatment with the indicated concentration of mßcd, expressed relative to untreated CVs. *Significant difference from control (P<0.05); **, ***, ****significant difference from control and all other conditions (P<0.001).

View larger version (13K): [in a new window]   Fig. 4. Extrapolation of the number of intact fusion sites (<n>) for CV-CV fusion (black circles) and CV-PM fusion (red squares) to estimate <n>Max (open symbols), the total number of fusion machines per vesicle.

View larger version (37K): [in a new window]   Fig. 5. Effects of cyclodextrins on CV-CV fusion. CVs treated with 0 mM (closed circles) or with (A) concentrations of cd (n=2), or ßcd as indicated (n=2). Dashed lines in A represent the 99% confidence intervals of the fusion plot for untreated CVs. (C) Total CV cholesterol assayed after treatment with the indicated cyclodextrins. Results are expressed as percentage of untreated control CVs. *, **significant difference from control and all other conditions (P<0.001). (D) HPTLC analysis of supernatant extracts following incubation with 0 mM or 2 mM of the indicated cylcodextrin. Lipid standards include cholesteryl esters (CE), triacylglycerol (TAG), free fatty acids (FFA), diacylglycerol (DAG), cholesterol (Chol), monoacylglycerol (MAG), ceramide (Cer), cardiolipin (Card), diacylphosphatidylethanolamine (PE), diacylphosphatidylinositol (PI), lysophosphatidylethanolamine (LPE), diacylphosphatidylserine (PS), diacylphosphatidylcholine (PC), lysophosphatidylserine (LPS), sphingomyelin (Sph), and lysophosphatidylcholine (LPC). The prominent band in the mßcd lane is mßcd. (E) Kinetics of CV-CV fusion following treatments as in A, in response to 87±3 µM [Ca2+]free (n=2).

View larger version (30K): [in a new window]   Fig. 6. Addition of exogenous cholesterol to cholesterol-depleted CVs. Homotypic CV-CV fusion assays of CVs treated with 0 mM or 2 mM mßcd, and recovered by delivering cholesterol using (A) 2 mM mßcd as a vehicle (n=3), saturated cholesterol solutions (n=4), and (B) with 2 mM hpßcd as a vehicle as indicated(n=4). Vertical dashed lines in A and B indicate the EC50 of each curve. (C) Kinetics of CV-CV fusion triggered with 67±14 µM [Ca2+]free (n=4), after treatments as in A and B. (D) Total CV cholesterol assayed before and after 2 mM mßcd treatment, and with subsequent recovery with the indicated delivery methods. *Significant difference from control and all other conditions (P<0.005); **,***significant differences from control and all other conditions (P<0.05).

View larger version (11K): [in a new window]   Fig. 7. The effects of supplementing native, untreated CVs with cholesterol using hpßcd. (A) Ca2+ activity curves of CVs treated with 0 mM or 5 mM hpßcd-cholesterol (n=3). Inset: total CV cholesterol of samples: 1, untreated CVs; 2, treated with 5 mM hpßcd-cholesterol. Results are presented as percentage of control cholesterol; *significant differences from control (P<0.001). (B) Kinetics of CV-CV fusion in response to 96±11 µM [Ca2+]free (n=3) as in A.

View larger version (15K): [in a new window]   Fig. 8. The effects of cholesterol oxidase activity on CV-CV fusion. (A) Ca2+ activity curves of CVs treated with 0 U/ml (n=3), 0.01 U/ml (n=3), 0.1 U/ml (n=3), or 1.0 U/ml cholesterol oxidase (n=3). Vertical dashed lines indicate the respective EC50 values. (B) Total cholesterol of CVs treated as indicated. Results are expressed as percent of untreated CVs; *, **significant difference from control and all other samples (P<0.005).

View larger version (32K): [in a new window]   Fig. 9. The effects of sterol binding antibiotics on CV-CV fusion. (A) Ca2+ activity curves of CVs treated with concentrations of filipin III as indicated (n=4). (B) Ca2+ activity curves of CVs treated with concentrations of pimaricin as indicated. (C) Ca2+ activity curves of CVs treated with concentrations of amphotericin B as indicated. Vertical dashed lines in A-C indicate the EC50 of each curve. (D) Fusion kinetics of CVs treated as in A and B, or CVs treated with 50 µM or 100 µM pimaricin in response to 104±10 µM [Ca2+]free (n=2-3).

View larger version (15K): [in a new window]   Fig. 10. Effects of a positive curvature agent, LPC, on CV-CV fusion. Ca2+ activity curves of untreated CVs and CVs treated with (A) 10 µM LPC, or 2 mM mßcd, or treated first with 2 mM mßcd followed by treatment with 10 µM LPC (n=3) as indicated. Vertical dashed lines indicate the EC50 of each curve. (B) Samples paralled to A first treated with 10 µM LPC and subsequently with 1 mM hpßcd (n=3). Following sequential treatments with 2 mM mßcd and 10 µM LPC, samples were treated with 1 mM hpßcd, or 1 mM hpßcd-cholesterol (n=3). Blue line indicates the 2 mM mßcd-treated sample as in A.

View larger version (14K): [in a new window]   Fig. 11. Addition of negative curvature lipids to cholesterol-depleted CVs. (A) Ca2+-activity curves of untreated CVs (n=3), CVs treated with 2 mM mßcd (n=3), or CVs treated with 2 mM mßcd and then with 10 µM -tocopherol (n=3) or 10 µM DOPE (n=3). Vertical dashed lines indicate the EC50 of each curve. (B) Fusion kinetics of CVs treated as in A, in response to 111±14 µM [Ca2+]free (n=3).