Abstract
MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion.
Highlights
Some matrix metalloproteinases (MMPs) are proinvasive and employed by motile and invasive cells to degrade extracellular matrix (ECM)
MT1MMP is a membrane protein involved in degradation of ECM that is highly expressed at invadopodia, which are small protrusions of cancer cells
ECM degradation by MT1-MMP at invadopodia is hypothesized as the initial step of cancer cell invasion
Summary
Some matrix metalloproteinases (MMPs) are proinvasive and employed by motile and invasive cells to degrade extracellular matrix (ECM). MT1-MMP must be an important component of the cellular invasion machinery, and active ECM degradation is produced at structures called invadopodia [5,6], which are specialized membrane protrusions extending into the ECM. The accumulation of MT1-MMP in invadopodia was followed by the degradation of ECM at the same sites [8]. These observations indicate that MT1-MMP plays a crucial role in ECM degradation at invadopodia [10]. The ECM-degrading activity of MT1-MMP will not be persistent, because cell-surface MT1-MMP is inactivated by TIMP-2 which is an endogenous MT1-MMP inhibitor that inhibits the proteolytic degradation of ECM. A continuous supply of active MT1-MMP to the surface seems to be critical for maintaining ECM degrading [11]
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