Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix.
Transfection of Rat1 fibroblasts with an activated form of rac1 (V12rac1) stimulated cell migration in vitro compared to transfection of Rat1 fibroblasts with vector only or with dominant negative rac1 (N17rac1). To investigate the involvement of proteases in this migration, we used a novel confocal assay to evaluate the ability of the Rat1 transfectants to degrade a quenched fluorescent protein substrate (DQ-green bovine serum albumin) embedded in a three-dimensional gelatin matrix. Cleavage of the substrate results in fluorescence, thus enabling one to image extracellular and intracellular proteolysis by living cells. The Rat1 transfectants accumulated degraded substrate intracellularly. V12rac1 increased accumulation of the fluorescent product in vesicles that also labeled with the lysosomal marker LysoTracker. Treatment of the V12rac1-transfected cells with membrane-permeable inhibitors of lysosomal cysteine proteases and a membrane-permeable selective inhibitor of the cysteine protease cathepsin B significantly reduced intracellular accumulation of degraded substrate, indicating that degradation occurred intracellularly. V12rac1 stimulated uptake of dextran 70 (a marker of macropinocytosis) and polystyrene beads (markers of phagocytosis) into vesicles that also labeled for cathepsin B. Thus, stimulation of the endocytic pathways of macropinocytosis and phagocytosis by activated Rac1 may be responsible for the increased internalization and subsequent degradation of extracellular proteins.
Ahram
M
M
Department of Pharmacology, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan, 48201, USA.
Sameni
M
M
Qiu
R G
RG
Linebaugh
B
B
Kirn
D
D
Sloane
B F
BF
D000818:Animals; D015415:Biomarkers; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003911:Dextrans / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004705:Endocytosis / Q000502:physiology*; D004789:Enzyme Activation; D005780:Gelatin; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D010587:Phagocytosis / Q000502:physiology; D010873:Pinocytosis / Q000502:physiology; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D015415:Biomarkers; C400541:CA 074 methyl ester; D015853:Cysteine Proteinase Inhibitors; D003911:Dextrans; D004151:Dipeptides; D012710:Serum Albumin, Bovine; D005780:Gelatin; D002401:Cathepsin B; D020830:rac1 GTP-Binding Protein; D007930:Leucine; C108192:aloxistatin