Local Photorelease of Caged Thymosin β4 in Locomoting Keratocytes Causes Cell Turning

The broad aim of this work was to explore the feasibility of using light-directed perturbation techniques to study cell locomotion. Specifically, a caged form of thymosin β4 (Tβ4) was photoactivated in a defined local region of locomoting fish scale keratocytes and the resulting perturbation of locomotion was studied. Purified Tβ4 was produced in an inactive form by “caging” with ([n-nitroveratryl]oxy)chlorocarbamate. In vitro spectrophotofluorometric assays indicated that caged Tβ4 did not change the normal actin polymerization kinetics, whereas photoactivated Tβ4 significantly inhibited actin polymerization. With an a priori knowledge of the cytoplasmic diffusion coefficient of Tβ4 as measured by fluorescence recovery after photobleaching experiments, the rapid sequestration of actin monomers by uncaged Tβ4 and the consequent reduction in the diffusional spread of the Tβ4–actin complex were predicted using Virtual Cell software (developed at the Center for Biomedical Imaging Technology, University of Connecticut Health Center). These simulations demonstrated that locally photoactivating Tβ4 in keratocytes could potentially elicit a regional locomotory response. Indeed, when caged Tβ4 was locally photoactivated at the wings of locomoting keratocytes, specific turning about the irradiated region was observed, whereas various controls were negative. Additionally, loading of exogenous Tβ4 into both keratocytes and fibroblasts caused very rapid disassembly of actin filaments and reduction of cellular contractility. Based on these results, a mechanical model is proposed for the turning behavior of keratocytes in response to photoreleased Tβ4.

Cumulative pattern in pH change alters response to food in the crayfish Cambarus bartoni

In the relatively acid-tolerant crayfish Cambarus bartoni, locomotory response to food was tested (as delay in response, or latency) at both a circumneutral and a sublethal acid pH. Crayfish moved through a Y-maze toward a food source. Sequential tests were at pH 7.5, 4.5, and 7.5, and then again at pH 4.5 and 7.5. One group of 21 crayfish experienced a series of smaller pH shifts (7.5 to 6.5 to 5.5 to 4.5 at 5-day intervals), immediately followed by a larger pH shift (7.5 to 4.5). A second group of 20 crayfish experienced the large shift first, and then the series of smaller shifts. Both groups showed gradually decreasing latency through the first cycle, but then a large latency increase with the second shift to pH 4.5 (P < 0.005 and P < 0.03), and subsequent recovery at pH 7.5 by the first group (P < 0.006) but not the second (P < 0.32). This effect would seem to be due to the previous pH shift, which apparently exceeded their tolerance within our 45-day testing period. An initial tendency to approach the food (P < 0.01 and P < 0.12) became food avoidance (P < 0.01) and (or) no preference during either the first or second exposure to acidic pH.

Low pH alters response to food in the crayfish Cambarus bartoni

In a relatively acid-tolerant crayfish, Cambarus bartoni, locomotory response to food, and associated antennular flicking, were tested at both a circumneutral and a sublethal acid pH. Crayfish were run through a Y maze at pH 7.5, 4.5, and again at 7.5. One arm of the Y maze contained food; the other did not. The time to complete the maze at pH 4.5 increased significantly compared with the time taken at pH 7.5 (P < 0.001). Once pH was restored to 7.5, the time to complete the maze was greater than the initial control at pH 7.5 (P < 0.001) but less than pH 4.5 (P < 0.05). At pH 7.5, 73% of the crayfish chose the food arm; at pH 4.5, 33% chose the food arm; and when pH was returned to 7.5, 46% chose the food arm. Antennular flicking frequency was greatest at pH 7.5 and significantly less at 4.0 (P < 0.0001). Once pH was restored to 7.5, flicking rates were significantly lower than those in the first trial at pH 7.5 (P < 0.01), but significantly greater than those at pH 4.0. Decreased antennular flicking and possible avoidance of food at a sublethal pH (4.0) imply that either chemoreception of food cues or normal central processing of chemical input is seriously impaired.

Interleukin-5 selectively enhances the chemotactic response of eosinophils obtained from normal but not eosinophilic subjects

We have attempted to determine whether interleukin-5 (IL-5), a cytokine that selectively affects eosinophil (as opposed to neutrophil) differentiation and activation, also modulates eosinophil migrational responses. Using a modified Boyden chemotaxis assay, IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gave a weak locomotory response for eosinophils from normal nonatopic subjects (optimal at 10(-11), 10(-8), and 10(-9) mol/L, respectively), but not for eosinophils from subjects with an eosinophilia associated with asthma and/or allergic rhinitis. In contrast, IL-5 and IL-3 had no effect on neutrophils, while GM-CSF was chemotactic for neutrophils over a limited concentration range, optimal at 10(-8) mol/L. When eosinophils from normal subjects were incubated with IL-5 (10(-9) mol/L), the locomotory response to platelet-activating factor (PAF; 10(- 8) mol/L, P less than .05), leukotriene B4 (LTB4; 10(-6) mol/L, P less than .01), and N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10(-8) mol/L, P less than .01) was significantly enhanced. The percentage enhancement of eosinophil locomotion by IL-5 was greater for eosinophils from normal as compared with subjects with an eosinophilia associated with asthma (P less than .05 for PAF and LTB4; P less than .01 for FMLP). Preincubation of eosinophils from normal subjects with IL-5 (10(-9) mol/L) attenuated the subsequent locomotory response to IL- 5 (10(-12) and 10(-11) mol/L, P less than .05). Therefore, the observed refractoriness of eosinophils from eosinophilic subjects to both directional migratory and priming effects of IL-5 in vitro, may reflect a deactivation process resulting from prior exposure in vivo. The selective priming of eosinophil but not neutrophil locomotion by IL-5 suggests that this cytokine may play a significant role in the preferential accumulation of eosinophils at sites of allergic inflammation.

Interleukin-5 selectively enhances the chemotactic response of eosinophils obtained from normal but not eosinophilic subjects

We have attempted to determine whether interleukin-5 (IL-5), a cytokine that selectively affects eosinophil (as opposed to neutrophil) differentiation and activation, also modulates eosinophil migrational responses. Using a modified Boyden chemotaxis assay, IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gave a weak locomotory response for eosinophils from normal nonatopic subjects (optimal at 10(-11), 10(-8), and 10(-9) mol/L, respectively), but not for eosinophils from subjects with an eosinophilia associated with asthma and/or allergic rhinitis. In contrast, IL-5 and IL-3 had no effect on neutrophils, while GM-CSF was chemotactic for neutrophils over a limited concentration range, optimal at 10(-8) mol/L. When eosinophils from normal subjects were incubated with IL-5 (10(-9) mol/L), the locomotory response to platelet-activating factor (PAF; 10(- 8) mol/L, P less than .05), leukotriene B4 (LTB4; 10(-6) mol/L, P less than .01), and N-formyl-methionyl-leucyl-phenylalanine (FMLP; 10(-8) mol/L, P less than .01) was significantly enhanced. The percentage enhancement of eosinophil locomotion by IL-5 was greater for eosinophils from normal as compared with subjects with an eosinophilia associated with asthma (P less than .05 for PAF and LTB4; P less than .01 for FMLP). Preincubation of eosinophils from normal subjects with IL-5 (10(-9) mol/L) attenuated the subsequent locomotory response to IL- 5 (10(-12) and 10(-11) mol/L, P less than .05). Therefore, the observed refractoriness of eosinophils from eosinophilic subjects to both directional migratory and priming effects of IL-5 in vitro, may reflect a deactivation process resulting from prior exposure in vivo. The selective priming of eosinophil but not neutrophil locomotion by IL-5 suggests that this cytokine may play a significant role in the preferential accumulation of eosinophils at sites of allergic inflammation.

Locomotory competence and laminin-specific cell surface binding sites are lost during myoblast differentiation

The specific interaction of embryonal cells with the extracellular matrix (ECM) is one of the principal forces influencing embryonal development (Hay, 1984; Trinkaus, 1984). We used a muscle satellite cell line (MM14dy) to determine the relationship between locomotory response to laminin and the expression of specific cell surface binding sites for it. Time lapse videomicroscopic analysis was used to study the locomotory response and radioligand binding assays and cell attachment assays were used to follow the expression levels of binding sites for laminin and its subfragments E8 and E1–4. We report here the novel finding that the ability of MM14dy to locomote over laminin diminishes and finally vanishes as the cells differentiate. The simultaneous drop in expression of binding sites for laminin is interpreted as being of potential significance during development and repair.