The Tandem PH Domain-Containing Protein 2 (TAPP2) Regulates Chemokine-Induced Cytoskeletal Reorganization and Malignant B Cell Migration

<div><p>The intracellular signaling processes controlling malignant B cell migration and tissue localization remain largely undefined. Tandem PH domain-containing proteins TAPP1 and TAPP2 are adaptor proteins that specifically bind to phosphatidylinositol-3,4-bisphosphate, or PI(3,4)P2, a product of phosphoinositide 3-kinases (PI3K). While PI3K enzymes have a number of functions in cell biology, including cell migration, the functions of PI(3,4)P2 and its binding proteins are not well understood. Previously we found that TAPP2 is highly expressed in primary leukemic B cells that have strong migratory capacity. Here we find that SDF-1-dependent migration of human malignant B cells requires both PI3K signaling and TAPP2. Migration in a transwell assay is significantly impaired by pan-PI3K and isoform-selective PI3K inhibitors, or by TAPP2 shRNA knockdown (KD). Strikingly, TAPP2 KD in combination with PI3K inhibitor treatment nearly abolished the migration response, suggesting that TAPP2 may contribute some functions independent of the PI3K pathway. In microfluidic chamber cell tracking assays, TAPP2 KD cells show reduction in percentage of migrating cells, migration velocity and directionality. TAPP2 KD led to alterations in chemokine-induced rearrangement of the actin cytoskeleton and failure to form polarized morphology. TAPP2 co-localized with the stable F-actin-binding protein utrophin, with both molecules reciprocally localizing against F-actin accumulated at the leading edge upon SDF-1 stimulation. In TAPP2 KD cells, Rac was over-activated and localized to multiple membrane protrusions, suggesting that TAPP2 may act in concert with utrophin and stable F-actin to spatially restrict Rac activation and reduce formation of multiple membrane protrusions. TAPP2 function in cell migration is also apparent in the more complex context of B cell migration into stromal cell layers – a process that is only partially dependent on PI3K and SDF-1. In summary, this study identified TAPP2 as a novel regulator of malignant B cell migration and a potential therapeutic intervention target.</p> </div

TAPP2 KD leads to decreased migration speed and directionality in a stable SDF-1 gradient.

<p>NALM-6 cells transduced with control or TAPP2 KD lentivirus were loaded onto a microfluidic chemotaxis device and exposed to a 100 nM SDF-1 gradient (represented with triangles). Time-lapse images were taken and cell movement was analyzed using cell-tracking software. <b>A.</b> Panels show the first and last image from a representative experiment. Migrating cell tracks are marked in blue (migrating toward higher SDF-1 concentration) or red (migrating towards lower SDF-1 concentration). <b>B.</b> Migration tracks of control or TAPP2 KD cells in a 1 hour experiment were normalized to a common origin (0, 0) and plotted to visualize patterns of movement in two dimensions (the chemokine gradient is in the y dimension as indicated). Black tracks represent cells migrating toward the gradient and grey tracks are for cells migrating against the gradient. The solid circles indicate the end of the cell tracks. <b>C.</b> Migration tracks of control or TAPP2 KD cells in a 4 hour experiment were plotted as in C. <b>D.</b> Chemotaxis index of control and TAPP2 KD cells, calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057809#s2" target="_blank">Methods</a>. <b>E.</b> Migration speed of control and TAPP2 KD cells, calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057809#s2" target="_blank">Methods</a>. Data are representative of four independent experiments.</p

TAPP2 KD impairs chemokine-induced rearrangement of the actin cytoskeleton.

<p><b>A.</b> TAPP2 KD cells show impaired accumulation of F-actin in response to SDF-1 stimulation. Control or TAPP2 KD NALM-6 cells were stimulated with 100 ng/ml SDF-1 for a series of time points ranging from 15–120 seconds. Control samples without addition of SDF-1 were used for “time point 0”. Cells were fixed immediately following stimulation, permeabilized, stained with fluorescent phalloidin and mean fluorescence intensity (MFI) was determined by flow cytometry. Results were normalized relative to the level at time point 0. The values were shown as mean ± SEM for three experiments based on 2 batches of transductions. Significance for KD cells versus control cells at each stimulation time point was confirmed by Student’s <i>t</i> test: **p < 0.01; *p < 0.05. <b>B.</b> TAPP2 KD leads to altered organization of the F-actin cytoskeleton. Cells plated on fibronectin-coated chambered coverglass were stimulated with or without 100 ng/ml SDF-1 for 1 minute, fixed, permeabilized and stained for F-actin using Alexa Fluor 647 phalloidin. Confocal microscopy was performed using a 60× objective. Representative images are shown indicating polarized F-actin distribution in SDF-1-stimulated control cells and multiple cellular protrusions with less intense accumulations of F-actin in TAPP2 KD cells. <b>C.</b> Frequency of cells having more than 2 protrusions in SDF-1-stimulated control versus TAPP2 KD cells. Results are presented as mean percentage of cells scored as multi-protrusion ± SD from four independent experiments. For each group more than 400 cells were analyzed. The multi-protruding cell frequency is significantly higher in TAPP2 KD cells than in control cells, as indicated by Student’s <i>t</i> test: ***p<0.001.</p

TAPP2 KD impairs leukemic cell migration into bone marrow stromal cell layers.

<p>NALM-6 cells were added to a confluent layer of S17 stromal cells and allowed to migrate for 10 hours. Under-stroma migration was quantified as percentage of total input cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057809#s2" target="_blank">Methods</a>. <b>A.</b> Under-stroma migration is pertussis toxin sensitive and partially blocked by PI3K inhibtion. PT: addition of 0.1 µg/ml pertussis toxin (GPCR inhibitor); GDC: addition of 2 µM GDC-0941 (pan PI3K inhibitor); DMSO, vehicle control for GDC-0941. <b>B.</b> Under-stroma migration is partially dependent on the SDF-1 receptor CXCR4. Control, not treated; AMD, 10 or 100 µM AMD3100 (CXCR4 inhibitor); SDF, 0.1 or 1 µg/ml SDF-1 (to desensitize CXCR4). Results are mean ± SD of three independent experiments. Significant difference in leukemic migration into the stromal layer was determined by Student’s <i>t</i> test: **p < 0.01 or ***p<0.001. <b>C.</b> Phase contrast imaging of the under-stroma migration of control or TAPP2 KD NALM-6 cells into S17 stromal cells. Migrated cells, indicated by black arrows, are characterized by the dark round appearance in contrast to the stromal cells, indicated by white arrows. Images represent two independent experiments. <b>D.</b> TAPP2 KD inhibits under-stroma migration. NALM-6 cells were partially transduced with GFP-expressing pSIH1-H1-copGFP control lentivirus or TAPP2 KD lentivirus. Migration into S17 or M2-10B4 stromal layers was normalized as the migration of GFP+ (transduced) cells relative to that of GFP- cells (internal control) in the same sample. Results from one experiment performed in replicates were shown as mean ± SD, representing two independent experiments confirming migration inhibition by TAPP2 KD. Significant difference between control and KD cells based on each stromal type was calculated with Student <i>t</i> test: *p<0.05.</p