Antigen‐responsive regulation of Cell motility and migration via the signalobodies based on c‐Fms and c‐Mpl

Abstract

Since cell migration plays critical roles in development and homeostasis of the body, artificial control of cell migration would be promising for the treatment of various diseases related to migration. To this end, we previously developed single-chain Fv (scFv)/receptor chimeras, named signalobodies, which can control cell fates via a specific antigen that is different from natural cytokines. Although a conventional chemotaxis chamber assay revealed that several signalobodies based on receptor tyrosine kinases transduced antigen-dependent migration signals, we have never performed direct observation of the cells to obtain more information on overall properties of cell motility and migration. In this study, we utilized murine pro-B Ba/F3 cells expressing either a scFv-Fms or scFv-Mpl signalobody, and compared their migratory characteristics. We employed a lipid-polyethylene glycol conjugate to softly immobilize the suspension cells on a slide, which facilitated direct observation of chemokinetic activity of the cells. Consequently, both cells markedly exhibited chemokinesis in response to a specific antigen. In addition, the cells were subjected to a stable antigen-concentration gradient to observe horizontal directional cell migration in real time. The results showed that the cells expressing scFv-Fms underwent directional migration toward a positive antigen-concentration gradient. Taken together, we successfully demonstrated antigen-responsive regulation of cell motility and migration via the signalobodies.

High-Throughput Cell Motility Studies on Surface-Bound Protein Nanoparticles with Diverse Structural and Compositional Characteristics

“…A few distinct techniques to produce gradients in controlled laboratory conditions on model substrates have emerged. − The combination of microfluidics and immersion techniques has become a successful approach for gradient generation and production of surface-bound gradients of such soluble molecules. Thus, various types of molecules have been used to form gradients for studying cell motility, such as chemokines, , antigens, drugs, RGD peptides, proteins, or calcium ions . In these studies, the directionality of cell movement can be assessed, , by analyzing the directional persistence time (i.e., the time in which the cell movement persists in the same direction), the random motility coefficient (i.e., a coefficient analogous to a molecular diffusion coefficient), and the chemotaxis index (i.e., a weighted ratio between the distance along a gradient and the total traveled distance).…”

Cell fate‐inducing CARs orthogonally control multiple signaling pathways

“…Our previous study demonstrated that Fms‐CAR enhances not only antigen‐dependent proliferation but also motility with elongated cell shape. [ 38 ] To confirm whether cell death was surely induced by Fas‐CAR, we observed the cells co‐expressing Fms‐CAR and Fas‐CAR and those expressing Fms‐CAR alone as a control. After depleting the cells for 16 h in the absence of IL‐3, the cells were cultured with the ligands, during which the cells were imaged at 3 min intervals, starting from 0.5 h when the suspension cells seemed to settle down on the plate sufficiently and terminating at 6.5 h. Here, we show the movies composed of the time‐lapse images (Supplementary Movies S1–S10).…”

Canonical Wnt signaling activation by chimeric antigen receptors for efficient cardiac differentiation from mouse embryonic stem cells

“…We previously developed proliferation-inducing and migration-inducing chimeric antigen receptors (piCARs and miCARs), which we also called growth signalobodies and migration signalobodies, respectively [12][13][14][15][16][17][18][19][20][21][22]. In most of the piCARs and miCARs, an anti-fluorescein (FL) single-chain Fv (scFv) fused to the extracellular D2 domain of EpoR was joined to the TM and cytoplasmic domains of single membrane-spanning type I cytokine receptors or receptor tyrosine kinases.…”