The supporting role of proteases in tumor progression and invasion is well known; however, the use of proteases as therapeutic agents has also been demonstrated. In this article, the authors report on the differential effects of exogenous serine proteases on the motility of tumor and normal cells. The treatment of normal and tumor cells with a single dose of pancreatic serine proteases, trypsin (TR) and chymotrypsin (CH), leads to a concentration-dependent response by cells, first accelerating and then slowing mobility. Tumor cells are 10 to 20 times more sensitive to exogenous TR/CH, suggesting that a single dose of proteases may cause discordant movements of normal and tumor cells within the tumor environment. The inhibitory effects of TR on cell motility are contradicted by thrombin (TH), particularly in the regulation of normal cells' migration. The purpose of this investigation was to ascertain the role of protease-activated receptors (PARs) in terms of normal and tumor cell motility. Duplicate treatments with proteases resulted in diminished mobility of both normal and tumor cells. Repeated application of TR and TH in 1-hour treatment intervals initially desensitizes cell surface PARs. However, cell surface PARs reappear regardless of subsequent protease treatments in both normal and tumor cells. The resensitization process is retarded in tumor cells when compared with normal cells. This is evidenced by lower expression of PARs as well as by their relocalization at the tumor cell surfaces. Under these conditions, normal cells remain responsive to exogenous proteases in terms of cell motility. Exogenous proteases do not modulate motility of repeatedly stimulated tumor cells, and consequently, the migration of tumor cells appears disconnected from the PAR signaling pathways. The use of activating peptides in lieu of the cognate proteases for a given PAR system indicated that proteases may act through additional targets not regulated by PAR signaling. We hypothesize that the divergent migration patterns of normal and tumor cells due to exposure to proteases is in part mediated by PARs. Thus, treatment with exogenous proteases may cause rearrangement of the tumor and stromal cells within the tumor microenvironment. Such topographical effects may lead to the inhibition of tumor progression and metastasis development.
The SNARE proteins Syntaxin and SNAP25 inhabit the plasma membrane, and VAMP/synaptobrevin the membrane of secretory vesicles. When all three combine in a 1:1:1 complex they are thought to fuse the secretory vesicle to the plasma membrane. To explore the interaction of Syntaxin and SNAP25 with secretory vesicles, we have imaged live cells using TIRF microscopy in two colors. Cells co-expressed a fluorescent granule marker as well as EGFP-tagged t-SNAREs at low copy number. Fluorescence was calibrated by single molecule measurements. Granules formed nanodomains beneath them, each with room for 100 syntaxin molecules. The nanodomains repeatedly and spontaneously emptied of syntaxin and then re-filled. They exchanged their syntaxin with plasma membrane with a half time of a few seconds, and when a granule performed exocytosis its nanodomain disassembled. SNAP25 was concentrated beneath granules but with 10 fold lower affinity than Syntaxin. Most Syntaxin and nearly all SNAP25 molecules were seen to move freely in the plasma membrane, but a minor proportion of each t-SNARE was almost immobile. Single Syntaxin molecules could be observed as they were captured and released from granule sites. We have tracked the recruitment and release of SNAREs at exocytic sites in a time-resolved manner and with single molecule sensitivity.
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