Document Detail


Rac1-induced endocytosis is associated with intracellular proteolysis during migration through a three-dimensional matrix.
MedLine Citation:
PMID:  11035924     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
M Ahram; M Sameni; R G Qiu; B Linebaugh; D Kirn; B F Sloane
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Experimental cell research     Volume:  260     ISSN:  0014-4827     ISO Abbreviation:  Exp. Cell Res.     Publication Date:  2000 Nov 
Date Detail:
Created Date:  2000-12-12     Completed Date:  2000-12-12     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0373226     Medline TA:  Exp Cell Res     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  292-303     Citation Subset:  IM    
Copyright Information:
Copyright 2000 Academic Press.
Affiliation:
Department of Pharmacology, Barbara Ann Karmanos Cancer Institute, Detroit, Michigan, 48201, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Biological Markers
Cathepsin B / antagonists & inhibitors,  metabolism*
Cell Line
Cell Movement / physiology*
Cysteine Proteinase Inhibitors / pharmacology
Dextrans / metabolism
Dipeptides / pharmacology
Endocytosis / physiology*
Enzyme Activation
Gelatin
Humans
Intracellular Membranes / enzymology
Leucine / analogs & derivatives,  pharmacology
Lysosomes / metabolism
Phagocytosis / physiology
Pinocytosis / physiology
Rats
Serum Albumin, Bovine / metabolism
rac1 GTP-Binding Protein / genetics,  metabolism*
Grant Support
ID/Acronym/Agency:
NIH 56586//PHS HHS; P30ES06639/ES/NIEHS NIH HHS; P30ES22453/ES/NIEHS NIH HHS
Chemical
Reg. No./Substance:
0/Biological Markers; 0/CA 074 methyl ester; 0/Cysteine Proteinase Inhibitors; 0/Dipeptides; 0/Serum Albumin, Bovine; 61-90-5/Leucine; 88321-09-9/aloxistatin; 9000-70-8/Gelatin; 9004-54-0/Dextrans; EC 3.4.22.1/Cathepsin B; EC 3.6.5.2/rac1 GTP-Binding Protein

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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