Cytosolic calcium ions regulate lipid mobility in the plasma membrane of the human megakaryoblastic cell line MEG-01.
The fluidity of the plasma membrane is thought to play a role in the activation of blood platelets. We investigated the lateral diffusion of the lipophilic probe 1,1'-ditetradecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiIC14) and derivatives in the plasma membrane of the megakaryoblast MEG-01 by fluorescence recovery after photobleaching. The lateral diffusion coefficient (D) of DiIC14 in an unstimulated cell was (3.53 +/- 0.06) x 10(-9) cm2/s with a mobile fraction of 75%. Similar data were found with DiIC12 and DiIC18, but lipophilic probes specific for the outer leaflet showed a slower diffusion with a D value of (2.99 +/- 0.31) x 10(-9) cm2/s and a mobile fraction of 58%. Stimulation with platelet-activating agents decreased the diffusion of DiIC14 within 2 min, but left the mobile fraction unchanged. Signal processing was required for the decrease in D as D-Phenylalanyl-L-prolyl-L-arginyl-chloromethane-treated thrombin, which binds normally to the thrombin receptor but fails to activate the cell, had no effect. The decrease in D was accompanied by an increase in cytosolic Ca2+ content, [Ca2+]i, and studies using different concentrations of thrombin, the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester and the Ca2+ ionophore ionomycin revealed that lipid mobilty in the plasma membrane is regulated by Ca2+. In contrast, treatments thought to interfere with the mobility of membrane proteins had little effect. We conclude that the rigidification of the plasma membrane during cell activation is caused by an increase in [Ca2+]i and is therefore a late event and might only contribute to signal transduction at steps downstream of the mobilization/influx of Ca2+.
Schootemeijer
A
A
Department of Haematology, University Hospital Utrecht, The Netherlands.
Van Beekhuizen
A E
AE
Tertoolen
L G
LG
De Laat
S W
SW
Akkerman
J W
JW
D000244:Adenosine Diphosphate / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002232:Carbocyanines; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008533:Megakaryocytes; D008560:Membrane Fluidity / Q000187:drug effects / Q000502:physiology*; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000494:pharmacology
D002232:Carbocyanines; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D008565:Membrane Proteins; D004533:Egtazic Acid; D015759:Ionomycin; D000244:Adenosine Diphosphate; C072024:carbocyanine dye DiIC12(3); C055316:ditetradecylindocarbocyanine; D013917:Thrombin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium