Regulation of Polarization and Chemotaxis in Newt Eosinophils: The Role of Calcium: A Dissertation

Chemotaxis, the ability of a cell to migrate towards a directional stimulus, is a basic property of virtually all cells at some stage in their development. Chemotaxis is preceded by the development of a polarized cellular morphology. The region of the cell closest to the attractant forms a broad lamellipod. The contents of the cell flow forward into this lamellipod and the rear of the cell becomes constricted into a narrow uropod. These local differences in cell structure and function presumably reflect local differences in cell chemistry, but the chemical processes involved are poorly understood. Ca+2 is known to play a ubiquitous role as an essential second messenger in many cellular processes, but its role in chemotaxis is unclear. While many chemotactic stimuli cause Ca+2 to rise intracellularly, the relationship between this rise in Ca+2 and local changes in cell behavior has been difficult to understand. In my dissertation work, I directly tested the role of cellular Ca+2 changes in polarization and chemotaxis by simultaneously imaging intracellular Ca+2 and cell morphology. This work was carried out on single eosinophils isolated from the newt, Taricha granulosa, because of their large size (~100 um, when polarized) and rapid responsiveness (~20 um/min) to chemotactic stimuli present in newt serum. An imaging system was developed to simultaneously image cell behavior, and intracellular Ca+2 following microinjection of the Ca+2 sensitive fluorescent probe, Fura-2. Cell behavior was quantified from time lapse video images captured by a SIT video camera, stored on a video optical disk recorder, and later digitized for analysis. Quantitation was accomplished by interactively tracing the cell\u27s outline and determining the position of the geometric centroid. Variation in the radius of the outline from the centroid was used to calculate a polarization index , which could be monitored over time. Cell speed was calculated from the movement of the centroid over time. Agents which are known to interfere with Ca+2 signalling significantly inhibited both the polarization and the movement of cells in response to 10% newt serum. These treatments included: chelation of extracellular Ca+2 with EGTA, the organic Ca+2 channel antagonist, verapamil, the inorganic Ca+2 channel blocker, cobalt, the Ca+2 ionophore, ionomycin, and caffeine, an agent known to release Ca+2 from internal stores. In contrast, the K+ ionophore, valinomycin, and treatment of cells with dibutryl cAMP had no effect on cell behavior. The development of a polarized, motile morphology following stimulation of newt eosinophils with 10% serum was accompanied by a rise in intracellular Ca+2. In addition, Ca+2 in a polarized, moving cell was non-uniformly distributed and periodic elevations in intracellular Ca+2 were seen during changes in cell behavior. In turning cells, Ca+2 was significantly higher than in cells moving in a straight line and there was a clearly detectable gradient of Ca+2 within the cell. The region closest to the new direction of movement had the lowest Ca+2 and the rear of the cell was significantly higher. This gradient persisted following a turn, even though Ca+2 was much lower overall in cells moving in a straight line. A gradient of Ca+2 along the long axis of the cell might be important for the differential regulation of different regions of the moving cell. Loading cells with the cell-permeant, esterified form of Fura-2 revealed a region of high Ca+2 associated with the microtubule organizing center (MTOC). This region was surrounded by a membrane system labeled by the lipid soluble, membrane potential sensitive dye, DiOC6(3). This region of Ca+2 was depleted by caffeine treatment. These observations, coupled with the effects of caffeine on cell behavior, suggest that a Ca+2 storage site associated with the MTOC may play a role in regulating cell polarization and chemotaxis. The effects of releasing caged calcium on cell behavior and [Ca2+]i were examined as a means of directly testing the ability of changes in [Ca2+]i to regulate cell behavior. Although photolysis of the compound inhibited cell polarization and movement, technical problems made it difficult to attribute these effects entirely to the release of Ca2+. The results presented here, particularly the gradients of [Ca2+]i which were observed, suggest that local regulation of the cytoplasmic components involved in cell movement by local differences in [Ca2+]i could, in part, explain the regional specialization seen during this process. This form of regulation will be discussed in detail, as will potential mechanisms to test for its function during cell polarization and chemotaxis

3,4-Dichloropropionanilide (DCPA) inhibits T-cell activation by altering the intracellular calcium concentration following store depletion

Stimulation of T cells through the T-cell receptor results in the activation of a series of signaling pathways that leads to the secretion of interleukin (IL)-2 and cell proliferation. Influx of calcium (Ca(2+)) from the extracellular environment, following internal Ca(2+) store depletion, provides the elevated and sustained intracellular calcium concentration ([Ca(2+)](i)) critical for optimal T-cell activation. Our laboratory has documented that exposure to the herbicide 3,4-dichloropropionanilide (DCPA) inhibits intracellular signaling events that have one or more Ca(2+) dependent steps. Herein we report that DCPA attenuates the normal elevated and sustained [Ca(2+)](i) that follows internal store depletion in the human leukemic Jurkat T cell line and primary mouse T cells. DCPA did not alter the depletion of internal Ca(2+) stores when stimulated by anti-CD3 or thapsigargin demonstrating that early inositol 1,4,5-triphosphate-mediated signaling and depletion of Ca(2+) stores were unaffected. 2-Aminoethyldiphenol borate (2-APB) is known to alter the store-operated Ca(2+) (SOC) influx that follows Ca(2+) store depletion. Exposure of Jurkat cells to either DCPA or 50 microM 2-APB attenuated the increase in [Ca(2+)](i) following thapsigargin or anti-CD3 induced store depletion in a similar manner. At low concentrations, 2-APB enhances SOC influx but this enhancement is abrogated in the presence of DCPA. This alteration in [Ca(2+)](i), when exposed to DCPA, significantly reduces nuclear levels of nuclear factor of activated T cells (NFAT) and IL-2 secretion. The plasma membrane polarization profile is not altered by DCPA exposure. Taken together, these data indicate that DCPA inhibits T-cell activation by altering Ca(2+) homeostasis following store depletion

Calcium signalling during chemotaxis

The role of Ca2+ in chemotaxis of eosinophils from the newt Taricha granulosa was investigated using fluorescent indicators and digital imaging microscopy. In response to serum chemoattractant, cytoplasmic Ca2+ concentration ([Ca2+]i) rises prior to polarization. In polarized locomoting cells [Ca2+]i gradients (tail-high-front-low) are always seen, and when cells turn [Ca2+]i rises transiently and falls fastest and furthest in the new direction of cell motion. These Ca2+ signals, which are required for polarization and locomotion, arise from Ca2+ derived from internal stores released in response to inositol 1,4,5-trisphosphate (InsP3) (because microinjected heparin fully blocks them). 1,2-Diacyl-sn-glycerol (DAG), which is co-produced with InsP3, has an inhibitory effect on Ca2+ signals, an effect apparently mediated by protein kinase C. Studies with caged InsP3 reveal that InsP3-responsive stores appear to be concentrated in the nuclear and microtubule-organizing centre regions and that InsP3 moves so rapidly within the cell that it is effectively a global secondary messenger. Thus, stable [Ca2+] gradients observed during unidirectional migration appear to result from the concentration of InsP3-responsive Ca2+ stores in the rear of the cell. By contrast, we propose that reorientation of the [Ca2+] gradient prior to a change in direction of motion results from the joint actions of InsP3 and DAG, with InsP3 acting as a global secondary messenger stimulating Ca2+ release and DAG, through protein kinase C, acting as a spatially restricted secondary messenger inhibiting [Ca2+] increases locally near the site of chemotactic stimulation

Chemotaxis of newt eosinophils: calcium regulation of chemotactic response

Local chemical events underlying chemotaxis were characterized in a new model cell, the newt eosinophil. These cells exhibit a chemotactic response to a trypsin-sensitive component of newt serum. Ca2+ plays a role in this process, since treatments expected to diminish Ca2+ availability from the medium [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N\u27,N\u27-tetraacetic acid, Co2+, and verapamil], to break down transmembrane Ca2+ gradients (ionomycin), or to interfere with the function of intracellular Ca2+ stores (caffeine and neomycin) inhibited cell polarization and movement. Using imaging techniques we found that cytosolic Ca2+ concentration ([Ca2+]i) increased in response to newt serum. Migrating newt eosinophils exhibited a dynamic heterogeneous distribution of [Ca2+]i. [Ca2+]i was elevated in cells undergoing a change of direction relative to cells migrating persistently in one direction. Migrating cells contained gradients of [Ca2+]i along their long axis, with the front of the cell having consistently lower [Ca2+]i than the rear. When cells were loaded with the cell-permeant form of fura 2, fura 2 acetoxymethyl ester, a caffeine-sensitive membrane-delimited region of elevated [Ca2+]i was seen associated with the microtubule organizing center. A model is proposed relating the distribution of [Ca2+]i and the location of the external stimulus to the generation and interaction of substances within the cell that both simulate and inhibit increases in [Ca2+]i

Calcium gradients underlying polarization and chemotaxis of eosinophils

The concentration of intracellular free calcium ([Ca2+]i) in polarized eosinophils was imaged during chemotaxis by monitoring fluorescence of the calcium-sensitive dye Fura-2 with a modified digital imaging microscope. Chemotactic stimuli caused [Ca2+]i to increase in a nonuniform manner that was related to cell activity. In cells moving persistently in one direction, [Ca2+]i was highest at the rear and lowest at the front of the cell. Before cells turned, [Ca2+]i transiently increased. The region of the cell that became the new leading edge had the lowest [Ca2+]i. These changes in [Ca2+]i provide a basis for understanding the organization and local activity of cytoskeletal proteins thought to underlie the directed migration of many cells