Metastatic tumor cells that actively migrate and invade surrounding tissues rely 
on invadopodia to degrade extracellular matrix (ECM) barriers. Invadopodia are 
membrane protrusions that localize enzymes required for ECM degradation. Little 
is known about the formation, function, and regulation of invadopodia. Here, we 
show that invadopodia have two distinct aspects: (a) structural for organizing 
the cellular actin cytoskeleton to form membrane protrusions and (b) functional 
for using proteolytic enzyme(s) for ECM degradation. Small interfering RNA 
(siRNA) inhibition established that organization of invadopodia structure 
requires cortactin, whereas protease inhibitor studies identified membrane type 
1 matrix metalloproteinase (MT1-MMP) as the key invadopodial enzyme responsible 
for gelatin matrix degradation in the breast carcinoma cell line MDA-MB-231. The 
inhibition of invadopodial structure assembly by cortactin depletion resulted in 
a block of matrix degradation due to failure of invadopodia formation. Either 
protease inhibition or MT1-MMP siRNA depletion moderately decreased the 
formation of invadopodial structures that were identified as actin-cortactin 
accumulations at the ventral cell membrane adherent to matrix. The invadopodia 
that were able to form upon MT1-MMP inhibition or depletion retained 
actin-cortactin accumulations but were unable to degrade matrix. Examination of 
cells at different time points as well as live-cell imaging revealed four 
distinct invadopodial stages: membrane cortactin aggregation at membranes 
adherent to matrix, MT1-MMP accumulation at the region of cortactin 
accumulation, matrix degradation at the invadopodia region, and subsequent 
cortactin dissociation from the area of continued MT1-MMP accumulation 
associated with foci of degraded matrix. Based on these results, we propose a 
stepwise model of invadopodia formation and function.