Microquantification of cellular and in vitro F-actin by rhodamine phalloidin fluorescence enhancement

被引:18
作者
Katanaev, VL [1 ]
Wymann, MP [1 ]
机构
[1] Inst Biochem, CH-1700 Fribourg, Switzerland
关键词
D O I
10.1006/abio.1998.2837
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Based on the enhancement of rhodamine phalloidin fluorescence after its binding to actin filaments we have developed a technique to quantify F-actin, drastically (much greater than 100 times) reducing consumption of the expensive fluorescent dye and sample material in comparison to previous methods. Depolymerization of F-actin is prevented by utilizing short incubation times and stabilization of the filaments by actin-binding proteins or formaldehyde. Equilibrium and kinetic mathematical models relating rhodamine fluorescence with F-actin concentrations were used to predict the optimal assay conditions. The method has been applied to measure relative and absolute F-actin concentrations in cytosolic fractions and stimulus-induced actin polymerization in neutrophils. The cells were lysed with octyl-beta-D-glucopyranoside, which is compatible with the assay due to its high critical micelle concentration. As the assay takes less than 1 h and eliminates all previously required washing or extraction steps, it is faster and much simpler than any other presented up to now for quantification of filamentous actin. Moreover, the method is unique for reliable and easy F-actin measurements in cell-free systems. (C) 1998 Academic Press.
引用
收藏
页码:185 / 190
页数:6
相关论文
共 30 条
[1]   SELECTIVE ASSAY OF MONOMERIC AND FILAMENTOUS ACTIN IN CELL-EXTRACTS, USING INHIBITION OF DEOXYRIBONUCLEASE-I [J].
BLIKSTAD, I ;
MARKEY, F ;
CARLSSON, L ;
PERSSON, T ;
LINDBERG, U .
CELL, 1978, 15 (03) :935-943
[2]  
BOYUM A, 1968, SCAND J CLIN LAB INV, VS 21, P77
[3]  
*CALB CORP, 1997, GUID PROP US DET BIO
[4]   KINETIC-ANALYSIS OF F-ACTIN DEPOLYMERIZATION IN POLYMORPHONUCLEAR LEUKOCYTE LYSATES INDICATES THAT CHEMOATTRACTANT STIMULATION INCREASES ACTIN FILAMENT NUMBER WITHOUT ALTERING THE FILAMENT LENGTH DISTRIBUTION [J].
CANO, ML ;
LAUFFENBURGER, DA ;
ZIGMOND, SH .
JOURNAL OF CELL BIOLOGY, 1991, 115 (03) :677-687
[5]   CHARACTERIZATION OF TETRAMETHYLRHODAMINYL-PHALLOIDIN BINDING TO CELLULAR F-ACTIN [J].
CANO, ML ;
CASSIMERIS, L ;
JOYCE, M ;
ZIGMOND, SH .
CELL MOTILITY AND THE CYTOSKELETON, 1992, 21 (02) :147-158
[6]   MECHANISMS RESPONSIBLE FOR F-ACTIN STABILIZATION AFTER LYSIS OF POLYMORPHONUCLEAR LEUKOCYTES [J].
CANO, ML ;
CASSIMERIS, L ;
FECHHEIMER, M ;
ZIGMOND, SH .
JOURNAL OF CELL BIOLOGY, 1992, 116 (05) :1123-1134
[7]   LIFE AT THE LEADING-EDGE - THE FORMATION OF CELL PROTRUSIONS [J].
CONDEELIS, J .
ANNUAL REVIEW OF CELL BIOLOGY, 1993, 9 :411-444
[8]   EFFECTS OF CYTOCHALASIN AND PHALLOIDIN ON ACTIN [J].
COOPER, JA .
JOURNAL OF CELL BIOLOGY, 1987, 105 (04) :1473-1478
[9]   PYRENE ACTIN - DOCUMENTATION OF THE VALIDITY OF A SENSITIVE ASSAY FOR ACTIN POLYMERIZATION [J].
COOPER, JA ;
WALKER, SB ;
POLLARD, TD .
JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY, 1983, 4 (02) :253-262
[10]   INTERACTION OF ACTIN WITH PHALLOIDIN - POLYMERIZATION AND STABILIZATION OF F-ACTIN [J].
DANCKER, P ;
LOW, I ;
HASSELBACH, W ;
WIELAND, T .
BIOCHIMICA ET BIOPHYSICA ACTA, 1975, 400 (02) :407-404