Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator
dc.contributor.author | Amhad, Imran | en_US |
dc.contributor.author | Haugland, Richard P. | en_US |
dc.contributor.author | Keller, Donald | en_US |
dc.contributor.author | Kindzelskii, Andrei L. | en_US |
dc.contributor.author | Garni-Wagner, Beth Ann | en_US |
dc.contributor.author | Zhou, M. -J. | en_US |
dc.contributor.author | Gyetko, Margaret R. | en_US |
dc.contributor.author | Petty, Howard R. | en_US |
dc.contributor.author | Todd, Robert F. III | en_US |
dc.date.accessioned | 2006-09-11T18:55:51Z | |
dc.date.available | 2006-09-11T18:55:51Z | |
dc.date.issued | 2004-04 | en_US |
dc.identifier.citation | Kindzelskii, Andrei L.; Amhad, Imran; Keller, Donald; Zhou, M.-J.; Haugland, Richard P.; Garni-Wagner, B. A.; Gyetko, Margaret R.; Todd, Robert F.; Petty, Howard R.; (2004). "Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator." Histochemistry and Cell Biology 121(4): 299-310. <http://hdl.handle.net/2027.42/47396> | en_US |
dc.identifier.issn | 0948-6143 | en_US |
dc.identifier.issn | 1432-119X | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/47396 | |
dc.identifier.uri | http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=retrieve&db=pubmed&list_uids=15042374&dopt=citation | en_US |
dc.description.abstract | Previous studies have shown that the urokinase-type plasminogen activator receptor (uPAR) is localized to the adherence sites of leukocytes and tumor cells suggesting that pericellular proteolysis may accompany focal activation of adherence. To assess for focused pericellular proteolytic activity, we prepared two-dimensional substrates coated with FITC-casein or Bodipy FL-BSA. These molecules are poorly fluorescent, but become highly fluorescent after proteolytic degradation. Fluorescent peptide products were observed at adherence sites of stationary human neutrophils and at lamellipodia of polarized neutrophils. During cell migration, multiple regions of proteolysis appeared sequentially beneath the cell. Similarly, proteolytic action was restricted to adherence sites of resting HT1080 tumor cells but localized to the invadopodia of active cells. Using an extracellular fluorescence quenching method, we demonstrate that these fluorescent peptide products are extracellular. The uPA/uPAR system played an important role in the observed proteolytic activation. Plasminogen activator inhibitor-1 significantly reduced focal proteolysis. Sites of focal proteolysis matched the membrane distribution of uPAR. When uPA was dissociated from uPAR by acid washing, substantially reduced pericellular proteolysis was found. uPAR-negative T47D tumor cells did not express significant levels of substrate proteolysis. However, transfectant clones expressing uPAR (for example, T47D-26) displayed high levels of fluorescence indicating proteolysis at adherence sites. To provide further evidence for the role of the uPA/uPAR system in pericellular proteolysis, peritoneal macrophages from uPA knock-out (uPA−/−) and control (uPA+/+) mice were studied. Pericellular proteolysis was dramatically reduced in uPA-negative peritoneal macrophages. Thus, we have: (1) developed a novel methodology to detect pericellular proteolytic function, (2) demonstrated focused activation of proteolytic enzymatic activity in several cell types, (3) demonstrated its usefulness in real-time studies of cell migration, and (4) showed that the uPA/uPAR system is an important contributor to focal pericellular proteolysis. | en_US |
dc.format.extent | 406809 bytes | |
dc.format.extent | 3115 bytes | |
dc.format.mimetype | application/pdf | |
dc.format.mimetype | text/plain | |
dc.language.iso | en_US | |
dc.publisher | Springer-Verlag | en_US |
dc.subject.other | Plasminogen Activator | en_US |
dc.subject.other | Neutrophils | en_US |
dc.subject.other | LifeSciences | en_US |
dc.subject.other | Tumor Cells | en_US |
dc.subject.other | Proteolysis | en_US |
dc.subject.other | Adhesion | en_US |
dc.title | Pericellular proteolysis by leukocytes and tumor cells on substrates: focal activation and the role of urokinase-type plasminogen activator | en_US |
dc.type | Article | en_US |
dc.subject.hlbsecondlevel | Molecular, Cellular and Developmental Biology | en_US |
dc.subject.hlbtoplevel | Science | en_US |
dc.subject.hlbtoplevel | Health Sciences | en_US |
dc.description.peerreviewed | Peer Reviewed | en_US |
dc.contributor.affiliationum | Department of Ophthalmology and Visual Sciences and Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, 48105, USA | en_US |
dc.contributor.affiliationum | Department of Ophthalmology and Visual Sciences and Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, 48105, USA | en_US |
dc.contributor.affiliationum | Department of Ophthalmology and Visual Sciences and Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, 48105, USA | en_US |
dc.contributor.affiliationum | Hematology/Oncology Division, Department of Internal Medicine, University of Michigan Medical School, Michigan, USA | en_US |
dc.contributor.affiliationum | Pulmonary and Critical Care Division, Ann Arbor Veterans Affairs Medical Center and University of Michigan Medical School, Michigan, USA | en_US |
dc.contributor.affiliationum | Hematology/Oncology Division, Department of Internal Medicine, University of Michigan Medical School, Michigan, USA | en_US |
dc.contributor.affiliationum | Department of Ophthalmology and Visual Sciences and Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, 48105, USA | en_US |
dc.contributor.affiliationother | Molecular Probes, Eugene, Oregon, USA | en_US |
dc.contributor.affiliationother | Molecular Probes, Eugene, Oregon, USA | en_US |
dc.contributor.affiliationumcampus | Ann Arbor | en_US |
dc.identifier.pmid | 15042374 | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/47396/1/418_2004_Article_639.pdf | en_US |
dc.identifier.doi | http://dx.doi.org/10.1007/s00418-004-0639-3 | en_US |
dc.identifier.source | Histochemistry and Cell Biology | en_US |
dc.owningcollname | Interdisciplinary and Peer-Reviewed |
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