Cellular Shape Changes Research Articles

Heart and soul/PRKCi and nagie oko/Mpp5 regulate myocardial coherence and remodeling during cardiac morphogenesis

Organ morphogenesis requires cellular shape changes and tissue rearrangements that occur in a precisely timed manner. Here, we show that zebrafish heart and soul (Has)/protein kinase C iota (PRKCi) is required tissue-autonomously within the myocardium for normal heart morphogenesis and that this function depends on its catalytic activity. In addition, we demonstrate that nagie oko (Nok) is the functional homolog of mammalian protein associated with Lin-seven 1 (Pals1)/MAGUK p55 subfamily member 5 (Mpp5), and we dissect its earlier and later functions during myocardial morphogenesis. Has/PRKCi and Nok/Mpp5 are required early for the polarized epithelial organization and coherence of myocardial cells during heart cone formation. Zygotic nok/mpp5 mutants have later myocardial defects, including an incomplete heart tube elongation corresponding with a failure of myocardial cells to correctly expand in size. Furthermore, we show that nok/mpp5 acts within myocardial cells during heart tube elongation. Together, these results demonstrate that cardiac morphogenesis depends on the polarized organization and coherence of the myocardium, and that the expansion of myocardial cell size contributes to the transformation of the heart cone into an elongated tube.

Hematological Changes in Cold-Acclimated Xenopus laevis.

While hematopoiesis in individuals is strictly regulated for maintenance of homeostasis, it has been reported that the numbers of peripheral blood cells are modulated in response to environmental temperature in vertebrates including hamsters, rats, squirrels, dogs, and bullfrogs. To date, the physiological and molecular mechanisms have not been elucidated yet. Amphibians are poikilothermic vertebrates exposed to various fluctuations of environmental conditions; therefore they need to exert their capacities to acclimate to such changes. Additionally numerous studies have demonstrated a measurable metabolic reduction in the metabolic rate of cold-acclimated frogs. We examined hematological changes in response to environmental temperature in an aquatic amphibian, Xenopus laevis. Frogs initially maintained at 25°C were acclimated to 10°C, and hematological changes were observed. The numbers of erythrocytes, leukocytes and thrombocytes gradually reduced as the transient phase by 4 weeks, and subsequently reached the steady-state that sustained for more than 4 months. Whereas the reduction in the numbers of white blood cells and thrombocytes were moderate, the number of erythrocytes and the level of hemoglobin at nadir were remarkably low (approximately 40% of the initial values). It is known that oxygen levels may reduce in ice-cold water, and cold-acclimated animals therefore tolerate prolonged severe hypoxia; nevertheless cold-acclimated Xenopus exhibited severe erythrocytopenia. In addition, morphological change of peripheral blood cells and hematopoietic tissues (liver, spleen, kidney, and bone marrow) were examined. There were no remarkable cellular changes in cellular size and shape. However, increased numbers of mature erythrocytes were observed in the bone marrow of the steady state cold-acclimated Xenopus, while mature erythrocytes were not found in the bone marrow in Xenopus at 25 °C. This cold-temperature-induced pancytopenia was reversible when the temperature was put back to 25°C, as all of blood cell counts returned to the normal levels within 4 weeks in a reverse fashion as the transient phase of cold-acclimation. During the recovery phase, immature erythrocytes that were scarcely existed in the normal peripheral blood appeared in the circulation, suggesting that erythrocytes were newly produced at 25 °C after prolonged exposure to cold temperature. The possible explanations for the reduction in the numbers of circulating peripheral blood cells might be due to a number of various reasons such as reduced productions of hamatopoietic progenitors and/or related cytokines, alternation in the storage capacities and/or the life span of blood cells, and systemic suspension of normal activities. To compare the lifespan of erythrocytes between normal and cold-acclimated Xenopus, erythrocytes were covalently labeled with biotin. The surviving biotinylated erythrocytes in the circulation were quantitatively detected as avidin-biotin complex by microscopy and flowcytometry. Furthermore expression levels of several genes responsible for the hematopoietic regulation were comparatively examined. The cold-acclimated Xenopus model developed here may allow for a valuable approach aiming at exploring undiscovered systems in hematopoietic regulation.

Lack of Telopeptides in Fibrillar Collagen I Promotes the Invasion of a Metastatic Breast Tumor Cell Line

Defective fibrillar collagen polymerization in primary tumors has been correlated with increased metastasis. However, it is unclear how collagen organization influences tumor invasion. In this study, we show that collagen I polymerized without telopeptides (the flanking regions of collagen molecules) can differentially affect the three-dimensional migration of mammary carcinoma cells. MDA-MB-231 cells capable of proteolytic degradation and mesenchymal motion, invaded telopeptide-intact and telopeptide-free collagen gels to the same extent. In contrast, MDA-MB-435S cells, with typical features of amoeboid cells (poor collagenolytic activity, rounded cell morphology), were 5-fold more invasive in telopeptide-free than telopeptide-intact collagen. A fraction of the MDA-MB-435S cells that invaded telopeptide-intact or telopeptide-free collagen had a rounded morphology; however, in telopeptide-free collagen, a significant fraction of the cells switched from a rounded to elongated morphology (protrusion formation). The dynamic changes in cellular shape facilitated MDA-MB-435S locomotion through the narrow interfiber gaps, which were smaller than cell diameters. Based on the spherical morphology of MDA-MB-435S cells, we tested if the changes in cell shape and invasion were related to RhoA-ROCK activity; GTP-bound RhoA was measured in pull-down assays. RhoA activity was 1.8-fold higher for MDA-MB-435S cells seeded on telopeptide-free than telopeptide-intact collagen. Y27632 inhibition of ROCK, a Rho effector, significantly reduced the changes in cellular morphodynamics and the invasion of MDA-MB-435S cells but did not alter the invasion of MDA-MB-231 cells. Thus, the higher RhoA activity of MDA-MB-435S cells in telopeptide-free collagen enhances the changes in cellular morphodynamics associated with motility and invasion.

Red cell storage lesion: The potential impact of storage-induced CD47 decline on immunomodulation and the survival of leucofiltered red cells

Red blood cells undergo major biochemical and biomechanical changes during storage that could effect their post transfusion performance [Hogman CF, Meryman T. Storage parameters affecting red blood cell survival and function after transfusion. Transfus Med Rev 1999;13:275–96; Tinmouth A, Chin-Yee I. The clinical consequences of the red cell storage lesion. Transfus Med Rev 2001;15:91–107]. Biochemical effects include changes in 2,3-diphosphoglycerate (2,3-DPG), ATP, and calcium levels, as well as metabolic modulation and release of Annexin V, a cytosolic component of blood cells, as a global marker of cellular injury and fragmentation [Krailadsiri P, Seghatchian J, Amiral J, Vissac AM, Contreras M. Annexin V, a new marker of platelet storage lesion: correlation with dMPV. Transfus Sci 1997;18:223–26; Seghatchian J, Krailadsiri P. Red cell storage lesion assessed by the levels of potassium, haemoglobin and Annexin V in supernatants. Transfus Apher Sci 2002;26:139–43]. Biomechanical changes include alterations in cellular membrane, shape changes, phospholipid content, phospholipid asymmetry, and antigenic markers. Although the extent of these changes under various storage conditions has been well documented, their clinical effects remain unclear [McLellan S, Walsh TS, McClelland DBL. Should we demand fresh red blood cells for perioperative and critically ill patients? Br J Anaesth 2002;89:537–39; Mclellan S, Walsh TS, McClelland DBL. Anaemia and red blood cell transfusion in the critically ill patient. Blood Rev 2003;17(4):195–208]. In the current era of universal leucodepletion, the immunomodulatory effects of some essential markers such as CD47 and phosphatidyl serine become the focus of interest as highlighted in this manuscript.

The effect of endothelial leukocyte-adhesion molecule-1 on the shape and cytoskeleton of porcine trabecular meshwork cells

To study the effects of endothelial leukocyte-adhesion molecule-1 (ELAM-1) on the shape and actin cytoskeleton of cultured porcine trabecular meshwork cells (PTCs). The cultured PTCs were stimulated by interleukin-1 (IL-1) and conjugated by ELAM-1 antibody or further cross-linked by IgG. Immunocytochemical procedures were used to study the expression of ELAM-1 and FITC-phalloidin was used to stain the actin filaments on PTCs; phase contrast microscope was employed to observe the dynamic changes of cellular shape and the intercellular conjunction and confocal microscope was used to observe the changes of actin and cellular thickness. IL-1 treatment increased the expression of ELAM-1 in PTCs which resulted in enlarged intercellular space in PTCs with more rounded and stereoscopic morphology after specific antibody conjugation or IgG cross linking. In addition, actin filaments became sparse, cellular rigidity decreased, and the cellular thickness increased after these treatments. Our results demonstrated that IL-1 activated PTCs can express ELAM-1 and interaction of ELAM-1 with specific antibodies and cross linking with IgG, induced a rounding-up of PTCs. This change in cell shape may be due to its action on the actin cytoskeleton.

Photosensitization with protoporphyrin IX inhibits attachment of cancer cells to a substratum

Effects of photodynamic therapy (PDT) on adhesion of human adenocarcinoma cells of the line WiDr to a plastic substratum were investigated. Protoporphyrin IX induced by 5-aminolevulinic acid (ALA) was used as a photosensitizer. Light exposure inhibited attachment of suspended cells to a substratum. The adhesion was most strongly pronounced for light exposures around 200mJ/cm2 causing cell death. However, sub-lethal exposures (42mJ/cm2, 97% survival) inhibited cell adhesion as well. Sub-lethal ALA-PDT increased the intracellular space in dense colonies of WiDr cells. This was attributed to formation of lamellipodia between the cells and to increased numbers of focal contacts containing αVβ3 integrin in some of the cells. The E-cadherin distribution was not changed by the treatment. Complex processes, including changes in cellular shape and reorganization of the cytoskeleton, are suggested to participate in the observed ALA-PDT effect on the cell adhesion.

Human neutrophil-derived elastase induces airway smooth muscle cell proliferation

Neutrophils and their derived elastase are abundant in chronic inflammatory responses of asthma. This study aimed to investigate the mitogenic effect of elastase on airway smooth muscle (ASM) cells and the implicated signal transduction pathway. Near confluent cultured human ASM cells were treated with human neutrophil elastase (HNE, 0.01 to 0.5 μg/ml) or vehicle for 24 hours with or without extracellular signal-regulated kinase (ERK) inhibitor (PD98059, 30 μM), p38 kinase inhibitor (SB203580, 10 μM) or elastase inhibitor II (100 μg/ml). The ASM cell numbers were counted by a hemocytometer and DNA synthesis was assessed by flowcytometry. Western blots analysis for the expression of ERK, p38 and cyclin D1 was determined. HNE dose-dependently increased ASM cell numbers and the percentage of cells entering S-phase of cell cycle. This response was abolished by neutrophil elastase inhibitors and attenuated by PD98059, but not SB203580. HNE increased ERK phosphorylation and cyclin D 1 expression. Pretreatment with PD98059 significantly inhibited elastase-induced cyclin D1 activity. The increased ASM cellular gap and cell shape change by proteolytic activity of HNE may be contributory to ERK activation and therefore cell proliferation. Our results demonstrate that HNE is mitogenic for ASM cells by increasing cyclin D1 activity through ERK signaling pathway.

Bacterial DNA activates human neutrophils by a CpG-independent pathway.

Bacterial DNA stimulates macrophages, monocytes, B lymphocytes, NK cells, and dendritic cells in a CpG-dependent manner. In this work we demonstrate that bacterial DNA, but not mammalian DNA, induces human neutrophil activation as assessed by L-selectin shedding, CD11b upregulation, and stimulation of cellular shape change, IL-8 secretion, and cell migration. Induction of these responses is not dependent on the presence of unmethylated CpG motifs, as neutrophil stimulatory properties were neither modified by CpG-methylation of bacterial DNA nor reproduced by oligonucleotides bearing CpG motifs. We found that human neutrophils express Toll-like receptor (TLR) 9 mRNA. However, as expected for a CpG-independent mechanism, activation does not involve a TLR9-dependent signaling pathway; neutrophil stimulation was not prevented by immobilization of bacterial DNA or by wortmannin or chloroquine, two agents that inhibit TLR9 signaling. Of note, both single-stranded and double-stranded DNA were able to induce activation, suggesting that neutrophils might be activated by bacterial DNA at inflammatory foci even in the absence of conditions required to induce DNA denaturation. Our findings provide the first evidence that neutrophils might be alerted to the presence of invading bacteria through recognition of its DNA via a novel mechanism not involving CpG motifs.

Target Affinities of Faropenem to and Its Impact on the Morphology of Gram-Positive and Gram-Negative Bacteria

Faropenem is a new oral β-lactam antibiotic unique from carbapenems and other available β-lactams. Determinants of the in vitro activity of β-lactam antibiotics include affinity to penicillin-binding proteins (PBPs) and β-lactamase stability. In this study, the binding affinity of faropenem to various PBPs and its impact on the morphology of Staphylococcus aureus and Escherichia coli were evaluated. In general, faropenem demonstrated high binding affinity to high-molecular-weight PBPs but low affinity to low-molecular-weight PBPs. In S. aureus and Streptococcus pneumoniae, faropenem exhibited high binding affinity to PBP1, followed by PBP3 and PBP2. In E. coli, faropenem showed the highest affinity for PBP2, followed by PBP1A, PBP1B, PBP3 and PBP4. In Proteus vulgaris, binding was highest to PBP4, followed by PBP1A, PBP2 and PBP3. In Serratia marcescens, faropenem bound preferentially to PBP2 and PBP4. Exposure of S. aureus to faropenem at minimum inhibitory concentrations (MICs) of 1/8 or 1/4 resulted in irregular septum formation. At 1× MIC or higher, a larger number of lysed cells were observed. Exposure of E. coli to 1/8× MIC or 1/4× MIC also induced changes in cellular shape; the normal rod-shaped form changed to a spherical form in a time-dependent manner. After exposure of E. coli to 1× MIC for 2 h, bulging-shaped E. coli cells were observed and after 4 h of exposure cell lysis was demonstrated. In the presence of 4× MIC, spheroplast-like forms and cell lysis were observed. The morphological changes triggered by faropenem are in agreement with the PBP binding affinities reported. Thus, the high binding affinities of faropenem to PBPs from gram-negative and gram-positive bacteria are mirrored by its pronounced and concentration-dependent bactericidal effect.

Proteoglycans are potent modulators of the biological responses of eosinophils to chemokines

Chemokines play critical roles in governing the recruitment and activation of eosinophils at sites of allergic inflammation, particularly the asthmatic lung. However, we know little of how chemokine function is regulated post-translationally. Proteoglycans, consisting of a core protein and glycosaminoglycan (GAG) side chains, are cell surface molecules and components of the extracellular matrix that are able to bind chemokines, whilst heparin is a GAG with therapeutic value in asthma. We examined whether soluble GAG could alter the actions of chemokines in assays of eosinophil activation. Heparin inhibited intracellular calcium flux, respiratory burst and chemotactic responses of eosinophils to CCL11, but not to the chemoattractant C5a, and inhibited binding of CCL11 to CCR3. Heparin also inhibited eosinophil stimulation by CCL11, CCL24, CCL7, CCL13 and CCL5 to differing degrees, which broadly correlated with their relative affinities for heparin. Heparan sulfate and dermatan sulfate, but not chondroitin sulfate, also inhibited the actions of CCL11 and CCL13 in assays of cellular shape change and chemotaxis. Following treatment with the sulfation inhibitor chlorate or proteoglycanases, no inhibition of CCL11-induced activity was observed using either eosinophils or a CCR3-expressing transfectant cell line. This suggests that cell surface proteoglycans are not necessary for signaling via CCR3. However, the GAG context in which chemokines are expressed is likely to represent an important level of regulation of allergic inflammation.

Generation of Ci-Brachyury-GFP stable transgenic lines in the ascidian Ciona savignyi.

We report generation of stable transgenic lines of the ascidian Ciona savignyi carrying a Ciona intestinalis-Brachyury-promoter/Green Fluorescent Protein-reporter (Ci-Bra-GFP) construct. The transgenic lines were made using a technique in which the endonuclease I-SceI was coinjected into fertilized eggs with a transgene construct containing flanking recognition sites for I-SceI. Two founder animals, out of 12 F(0) adults tested, were found to transmit the transgene to their offspring (F(1)s) at frequencies of 42% and 23%. The transgene was further inherited by the F(2) in a Mendelian fashion and displayed nonmosaic expression, indicating integration into the genome. The Mendelian inheritance and the absence of mosaicism persisted through the F(3) and F(4) generations. Southern blot analyses showed that the transgene was organized in tandem arrays of no more than 10 copies. Using these Ci-Bra-GFP transgenics, we describe cellular movements and shape changes involved in notochord morphogenesis in both wildtype and mutant embryos.

Structure of the Cryptosporidium parvum microneme: a metabolically and osmotically labile apicomplexan organelle

From an EM study of thin sections, the rod-like microneme organelles within conventionally glutaraldehyde fixed Cryptosporidium parvum sporozoites have been shown to undergo a shape change to a more spherical structure when the sporozoites age in vitro for a period of ∼12 to 24 h. This correlates with the shape change of intact sporozoites, from motile hence viable thin banana-shaped cells to swollen pear-shaped cells, shown by differential interference contrast light microscopy of unstained unfixed and glutaraldehyde-fixed samples, as well as by thin section EM of fixed sporozoites. From negatively stained EM specimens of unfixed and fixed sporozoites the cellular shape change has been confirmed as has the rod to sphere micronemal shape change. Intact micronemes released directly from sporozoites exclude negative stain and appear as smooth-surfaced electron transparent particles. Biochemically purified rod-shaped C. parvum micronemes are shown to be fragile organelles that inevitably undergo variable damage during isolation, storage and subsequent specimen preparation for EM study. In the absence of glutaraldehyde fixation, damaged micronemes allow the negative stain to enter and loose their contents and during storage undergo a rod-to-sphere shape transformation. Glutaraldehyde-fixed micronemes maintain the rod shape; intact fixed micronemes still exclude negative stain but damaged micronemes reveal a complex quasi-helical arrangement of internal protein within the rod-like micronemes. Loss of this internal organized structure appears to be responsible for the micronemal shape change. This interpretation has been advanced from mutually supportive data obtained from cryoelectron microscopy of unstained vitrified samples, conventional air-dry negative staining and cryo-negative staining. Attempts to biochemically solubilize the micronemal content by lysis and ultrasonication, and separate it from the micronemal membranes, have so far met with limited success as the internal material tends to remain as a disorganized cluster of particles upon release.

Receptor internalization is required for eotaxin-induced responses in human eosinophils

Background: CC chemokine receptor 3 (CCR3) is a major chemokine receptor involved in regulating eosinophil trafficking, and therefore the elucidation of ligand-induced CCR3 events has important implications in understanding the biologic and pathologic properties of eosinophils. After ligand binding to CCR3, cellular signals include stimulatory (ie, calcium mobilization, actin polymerization, shape change, and chemotaxis) and inhibitory (ie, desensitization of the receptor) events. We have previously demonstrated that CCR3 undergoes rapid and prolonged ligand-induced internalization. Objective: Here we explore the role of internalization in downstream cellular processes, including shape change, actin polymerization, calcium mobilization, and desensitization. Methods: Peripheral blood–derived human eosinophils were pretreated with 2 mechanistically distinct inhibitors of internalization, sucrose and phenylarsine oxide, and functional responses were monitored. Results: We first demonstrate that ligand-induced internalization is required for chemokine-induced eosinophil shape change. To define which signaling components upstream of eosinophil shape change required internalization, we next studied the role of internalization in calcium mobilization and actin polymerization. Sucrose and phenylarsine oxide pretreatment inhibited actin polymerization, implicating receptor internalization in this early response. In contrast, calcium mobilization was not inhibited by blockade of internalization. Finally, we were interested in testing the role of internalization in receptor desensitization. We first demonstrated that preincubation with eotaxin induced a dose-dependent desensitization in eotaxin-induced eosinophil transepithelial migration. However, this phenomenon was not inhibited by blockade of internalization. Conclusion: These results establish that CCR3 internalization is critically involved in select eosinophil functional responses (ie, cellular shape change and actin polymerization) but not desensitization and calcium mobilization. (J Allergy Clin Immunol 2003;111:97-105.)

Cellular Conditioning and Activation of β-Catenin Signaling by the FPB Prostanoid Receptor

FP prostanoid receptors have been identified as two isoforms named FP(A) and FP(B). We have shown that the FP(B) isoform, but not the FP(A), activates beta-catenin-mediated transcription. We now report that the mechanism of this FP(B)-specific activation of beta-catenin signaling occurs in two steps. The first is a conditioning step that involves an agonist-independent association of the FP(B) receptor with phosphatidylinositol 3-kinase followed by constitutive internalization of a receptor complex containing E-cadherin and beta-catenin. This constitutive internalization conditions the cell for subsequent beta-catenin signaling by increasing the cellular content of cytosolic beta-catenin. The second step involves agonist-dependent activation of Rho followed by cell rounding. Because of the conditioning step, this agonist-dependent step results in a stabilization of beta-catenin and activation of transcription. Although stimulation of the FP(A) isoform activates Rho and induces cellular shape change, it does not activate beta-catenin signaling, because the FP(A) does not undergo constitutive internalization and does not condition the cell for beta-catenin signaling. The cellular conditioning described here for the FP(B) illustrates the potential of the receptor to alter the signaling environment of a cell even in the absence of agonist and has general significance for understanding G-protein-coupled receptor signaling.

Flow cytometric testing of immunological effects of a phytomedicinal combination (equimun) and its compounds on bovine leucocytes.

One of the major goals of this study was to establish fast, reliable and sensitive assays for the quality control of immunomodulating phytopreparations and to determine whether pharmacological compounds or phytopreparations have effects on bovine immune cells. Flow cytometric methods were chosen because they are very sensitive in the detection of even subtle effects on cells. In this study, we addressed the question of whether these methods are useful in monitoring the effects of EquiMun and its compounds on bovine leucocytes in vitro. EquiMun is a fixed combination of Echinacea purpurea (Ec), Thuja occidentalis (Th) and elemental phosphorus (Ph) in different starting concentrations. Separated blood mononuclear cells (MNC) and polymorphonuclear cells (PMN, mainly neutrophils) were cultured for up to 44 h in vitro in the presence or absence of the tested substances. Whereas MNC were not affected by any of the compounds, EquiMun, Ec, Th and Ph significantly reduced the forward scatter (size) of cultured PMN without affecting their side scatter (granularity). The size effects were paralleled by a significantly enhanced viability of PMN after 20 h in culture. The observed effects were constant over wide concentration ranges and indicate a very similar reaction of leucocytes from individual cows. Whereas spontaneous generation of reactive oxygen species (ROS) by neutrophils was up-regulated by Ph and EquiMun, EquiMun down-regulated the phorbol ester-stimulated ROS production. However, ROS generation by neutrophils displayed a large inter-individual variation with less apparent, down-regulatory effects of EquiMun. The ability of PMN to kill target cells via antibody-independent cellular cytotoxicity showed small inter-individual variations and was enhanced by Ec and Th but not by Ph and EquiMun, probably due to dose-dependent effects. In summary, the flow cytometric characterization of cellular viability and shape changes of neutrophils seem to be a suitable and reliable approach for the quality test of immunomodulating phytomedicines based on bioassays.

Cellular Processes Associated with Germ Band Retraction in Drosophila

Large-scale movements of epithelial sheets are necessary for most embryonic and regenerative morphogenetic events. We have characterized the cellular processes associated with germ band retraction (GBR) in the Drosophila embryo. During GBR, the caudal end of the embryo retracts to its final posterior position. We show using time-lapse recordings that, in contrast to germ band extension, cells within the lateral germ band do not intercalate. In addition, the germ band and amnioserosa move as one coherent sheet, and the amnioserosa strongly shortens along its dorsal–ventral axis. Furthermore, during GBR, the amnioserosa adheres to and migrates over the caudal end of the germ band via lamellipodia. Expression of both dominant-negative and constitutively active RhoA in the amnioserosa disrupts GBR. As RhoA acts on both actomyosin contractility and cell–matrix adhesion, it suggests a role for such processes in the amnioserosa during GBR. The results establish the cellular movements and shape changes occurring during GBR and provide the basis for an analysis of the forces acting during GBR.

50 Hz magnetic fields of varying flux intensity affect cell shape changes in invertebrate immunocytes: the role of potassium ion channels.

The effect induced by exposure to 50 Hz magnetic fields (MFs) in immunocytes from the mussel Mytilus galloprovincialis is evaluated. The whole animal was exposed for 15 and 30 min to MF intensities ranging from 200 to 1,000 microT. The changes in the cellular shape of immunocytes, expressed as shape factor (SF), were studied at different times after addition of the chemotacting substance N-formyl-Meth-Leu-Phe (fMLP). Results show that MFs provoke differing delays in fMLP-induced cellular shape changes: 200 microT are ineffective, while levels from 300 microT upwards cause a significant increase in immunocyte SF values compared to controls. Reactivation of the cells is possible up to an intensity of 600 microT. The use of PCO 400, an opener of ATP-sensitive K+ channels, shows that potassium channels are involved in the effect of MFs on M. galloprovincialis immunocytes.

Cell adhesion as wetting transition?

Cell adhesion is controlled by a complex interplay of short range (lock-and-key) forces mediated by cell surface receptors, a phalanx of (short and long range) nonspecific (generic) interactions, and last but not least membrane elasticity. The physical basis of cell adhesion is explored by the design of simplified model systems, mimicking cell and tissue surfaces, enabling local measurements of cellular shape changes and adhesion forces by microinterferometry. Cell adhesion can be understood as first-order dewetting transition that results in the formation of adhesion plaques, such as focal adhesion sites of cells, which allow cell adhesion at astonishingly low receptor densities. The repeller molecules of the glycocalix play a key role for the control of the adhesion transition and the mechanical stability of the adhering cells by relaxing the strength of the binding forces. Stress fibers are postulated to be essential for the stabilization of adhesion domains against leverage through bending moments enforced by hydrodynamic shear forces.

Myosin II Light Chain Phosphorylation Regulates Membrane Localization and Apoptotic Signaling of Tumor Necrosis Factor Receptor-1

Activation of myosin II by myosin light chain kinase (MLCK) produces the force for many cellular processes including muscle contraction, mitosis, migration, and other cellular shape changes. The results of this study show that inhibition or potentiation of myosin II activation via over-expression of a dominant negative or wild type MLCK can delay or accelerate tumor necrosis factor-alpha (TNF)-induced apoptotic cell death in cells. Changes in the activation of caspase-8 that parallel changes in regulatory light chain phosphorylation levels reveal that myosin II motor activities regulate TNF receptor-1 (TNFR-1) signaling at an early step in the TNF death signaling pathway. Treatment of cells with either ionomycin or endotoxin (lipopolysaccharide) leads to activation of myosin II and increased translocation of TNFR-1 to the plasma membrane independent of TNF signaling. The results of these studies establish a new role for myosin II motor activity in regulating TNFR-1-mediated apoptosis through the translocation of TNFR-1 to or within the plasma membrane.

Endocrine pancreas: summary of observations generated by surgical fellows.

The present article summarizes scientific observations of the endocrine pancreas anatomy and physiology into a unifying hypothesis. The data were generated over the years by surgical fellows using human and rat isolated perfused pancreas models and in vivo islet microcirculation models. The endocrine pancreas secretes a hormonal milieu regulated by neural, hormonal, and nutritional stimuli and the microcirculation. Variation of the nutrient and hormone concentrations in the islet capillaries generates two levels of response. A first level of response is in the central nervous system (CNS). Cholinergic signals from the CNS regulate the islet microcirculation through internal and external gates. This regulation is mediated by nitric oxide (NO). The gates are endothelial cells found in feeding arterioles and capillaries of the islet capable of directing blood flow through changes in cellular shape. External gates shunt flow to the entire endocrine pancreas, and internal gates divert blood flow within the islet to expose selectively the appropriate cell type required for changes in hormonal milieu. The second level is within the islet. The islet cells respond directly to variations in hormonal and nutrient concentrations. There is extensive communication between the various types of islet cells regulated by NO and hormonal feedback loops. The two levels of response allow an appropriate hormonal milieu, which secreted into the bloodstream contributes to the maintenance of glucose homeostasis.