Live-cell confocal microscopy and quantitative 4D image analysis of anchor-cell invasion through the basement...

Cell invasion through basement membrane (BM) barriers is crucial in development, leukocyte trafficking and the spread of cancer. The mechanisms that direct invasion, despite their importance in normal and disease states, are poorly understood, largely because of the inability to visualize dynamic cell-BM interactions in vivo. This protocol describes multichannel time-lapse confocal imaging of anchor-cell invasion in live Caenorhabditis elegans. Methods presented include outline-slide preparation and worm growth synchronization (15 min), mounting (20 min), image acquisition (20-180 min), image processing (20 min) and quantitative analysis (variable timing). The acquired images enable direct measurement of invasive dynamics including formation of invadopodia and cell-membrane protrusions, and removal of BM. This protocol can be combined with genetic analysis, molecular-activity probes and optogenetic approaches to uncover the molecular mechanisms underlying cell invasion. These methods can also be readily adapted by any worm laboratory for real-time analysis of cell migration, BM turnover and cell-membrane dynamics.