Distinct labelling of fusion events in rat lactotrophs by FM 1-43 and FM 4-64 is associated with conformational differences.
OBJECTIVE
Conformational analysis of fluorescent styryl dyes FM 1-43 and FM 4-64 was undertaken to clarify if distinct activity-dependent labelling of single lactotrophs vesicles and plasma membrane by two dyes is associated with their structural differences.


METHODS
The activity-dependent labelling of single vesicles and plasma membrane by FM 1-43 and FM 4-64 was studied using confocal microscopy. The fluorescence intensity of vesicles fused with the plasma membrane, and the plasma membrane alone was measured; the ratio of their respective peak amplitudes was calculated. The conformational analysis of FM 1-43 and FM 4-64 was further undertaken by employing the Monte Carlo approach to search the conformational space of these molecules.


RESULTS
In FM 1-43 staining of vesicles and plasma membrane, the ratio of the fluorescence peak amplitudes (vesicle vs. plasma membrane) was 2.6 times higher in comparison with FM 4-64 staining. In FM 4-64 molecule the low-energy conformations are distributed in three conformational states (consisting of 3, 4 and 2 conformers respectively) in which the proportion of the molecules residing in a given state is 62%, 28% and 9% respectively. In FM 1-43 the conformation distribution is limited to just one conformational state with three approximately equally populated conformers what can be explained by greater intrinsic rigidity of the molecule.


CONCLUSIONS
The observed structural characteristics of FM 1-43 molecules may account for a higher increase in quantum yield and/or binding affinity upon incorporation of the dye into the vesicle matrix and therefore stronger fluorescence emission in comparison with FM 4-64.
Stenovec
M
M
Celica Biomedical Center, Ljubljana, Slovenia.
Solmajer
T
T
Perdih
A
A
Vardjan
N
N
Kreft
M
M
Zorec
R
R
D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D002478:Cells, Cultured; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology*; D052682:Lactotrophs / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D008297:Male; D008561:Membrane Fusion; D018613:Microscopy, Confocal; D008968:Molecular Conformation; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D011726:Pyridinium Compounds / Q000737:chemistry / Q000494:pharmacology*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D022142:Secretory Vesicles / Q000648:ultrastructure; D013194:Staining and Labeling
C092350:FM 4-64; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011188:Potassium