Figure 5

Cytoskeletal arrangements of CLL lymphocytes treated with chemokine or undergoing heterotypic interaction have specific characteristics that differ from cells undergoing amoeboid motility. Panel A & B Chemokine treated CLL lymphocytes probed for F-actin (representative confocal microscopic images z planes as indicated, cells probed using TR-phalloidin) (A) and conventional immunofluorescence (B). The representative cells were exposed to CXCL12 (30 minutes). In the confocal images F-actin is particularly enriched within an anterior structures forming clear structural folds (white arrows); these may be seen (upper z plane) to form a single structure with linear actin ruffles (lamellaepodium). At the basal plane there is a well-defined posterior attachment point (yellow arrow). Similar changes may also be seen in the conventional fluorescent images. Panel C. Cell shape distribution now indicates a single population of elongated cells (aspect ratio intensity measurment as described in Figure 3). Panel D. A single distribution of high F-actin intensity is also now present (NBD-phallacidin probe for F-actin assessed by flow cytometry). Panel E. CLL lymphocytes interacting with accessory cells in culture have similar elongated and polarised form to chemokine-treated cells. This image is from a later culture point when NLCs are fully formed (7 days). The CLL lymphocytes attached to the central NLC body show clear polarity with anterior interaction and posterior tail, similar to cells treated with chemokine and no filopodia are evident (Texas-Red phalloidin probe for F-actin).