GSBS Dissertations and Theses

Title

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

Approval Date

August 1991

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Physiology

Subjects

Chemotactic Factors, Eosinophil; Calcium; Salamandridae; Academic Dissertations; Dissertations, UMMS

Abstract

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's 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.

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