Figure 4

Analysis of the behavioural significance of the CLL shape change. Panel A. Time-lapse photomicroscopy of cells within culture illustrating movement of CLL lymphocytes (phase contrast image series). Cells make a transition between round and polar form during typical amoeboid motility. The extension and retraction of processes during motility are clearly seen in cells marked 1 and 2 as they move toward cell 3, note that cell 2 makes a characteristic change from round form to elongated form during the 21 second observation period. Panel B. Time-lapse photomicroscopy illustrating transient cell-group formation between CLL lymphocytes (phase contrast image series of 120 second observation). The cell marked with *migrates across consecutively forming groups of 2, 3, 4 cells then back to a single cell. Groups interact by polarised extended projections, and cells show typical transition from elongated to round forms. Panel C. Complex filopodial contact during homotypic interactions (confocal microscopy of F-actin, TR-phalloidin probe) Main panel: basal attachment point to the finronectin-coated Plate. F-actin processes (Texas Red phallacidin probe) extend horizontally linking adjacent cells. Inset panel: enrichment for VLA-family integrin receptors CD29-FITC. Panel D. Rho kinase inhibition reveals extensive interaction between CLL lymphocytes (TR-phalloidin probe for F-actin). Filopodial retraction is prevented using rho-kinase inhibition (Y27536), a branched network of filopodial connections is revealed (arrows indicate points of intercellular contact where F-actin is specifically enriched).