Rapid Changes In Cell Shape Research Articles

Microglia in Aging and Alzheimer's Disease: A Comparative Species Review.

Microglia are the primary immune cells of the central nervous system that help nourish and support neurons, clear debris, and respond to foreign stimuli. Greatly impacted by their environment, microglia go through rapid changes in cell shape, gene expression, and functional behavior during states of infection, trauma, and neurodegeneration. Aging also has a profound effect on microglia, leading to chronic inflammation and an increase in the brain’s susceptibility to neurodegenerative processes that occur in Alzheimer’s disease. Despite the scientific community’s growing knowledge in the field of neuroinflammation, the overall success rate of drug treatment for age-related and neurodegenerative diseases remains incredibly low. Potential reasons for the lack of translation from animal models to the clinic include the use of a single species model, an assumption of similarity in humans, and ignoring contradictory data or information from other species. To aid in the selection of validated and predictive animal models and to bridge the translational gap, this review evaluates similarities and differences among species in microglial activation and density, morphology and phenotype, cytokine expression, phagocytosis, and production of oxidative species in aging and Alzheimer’s disease.

Single cell measurement of calpain activity in neutrophils reveals link to cytosolic Ca2+ elevation and individual phagocytotic events

It has been proposed that Ca2+ activation of calpain-1 is important for the rapid cell shape changes which accompany phagocytosis. In this paper, we use a fluorogenic calpain substrate, (CBZ-Ala Ala)2 R110, and find that there was a low calpain activity measureable in resting (ie without intentional activation) neutrophils, but that it was accelerated by an elevation of cytosolic free Ca2+ (ionomycin -induced) and inhibited by calpeptin (an established calpain-1 inhibitor). The fluorescence signal was sufficiently bright for detection in individual neutrophils that enabled the quantification of dynamic changes in calpain activity to be related to elevations in cytosolic Ca2+ within individual neutrophils. It was found that during phagocytosis of C3bi-opsonised zymosan particles, calpain activity was elevated incrementally, each step increase corresponding to the phagocytosis of an individual particle. The sub-cellular source of the fluorescent product of calpain activity was the phagocytic site itself and originated at the phagocytic cup. It was thus concluded that calpain was activated locally during the formation of the phagocytic cup. These data were consistent with central role of Ca2+ activated calpain activation in controlling phagocytosis.

Neutrophil Cell Shape Change: Mechanism and Signalling during Cell Spreading and Phagocytosis.

Perhaps the most important feature of neutrophils is their ability to rapidly change shape. In the bloodstream, the neutrophils circulate as almost spherical cells, with the ability to deform in order to pass along narrower capillaries. Upon receiving the signal to extravasate, they are able to transform their morphology and flatten onto the endothelium surface. This transition, from a spherical to a flattened morphology, is the first key step which neutrophils undergo before moving out of the blood and into the extravascular tissue space. Once they have migrated through tissues towards sites of infection, neutrophils carry out their primary role—killing infecting microbes by performing phagocytosis and producing toxic reactive oxygen species within the microbe-containing phagosome. Phagocytosis involves the second key morphology change that neutrophils undergo, with the formation of pseudopodia which capture the microbe within an internal vesicle. Both the spherical to flattened stage and the phagocytic capture stage are rapid, each being completed within 100 s. Knowing how these rapid cell shape changes occur in neutrophils is thus fundamental to understanding neutrophil behaviour. This article will discuss advances in our current knowledge of this process, and also identify an important regulated molecular event which may represent an important target for anti-inflammatory therapy.

Phagocytosis and Motility in Human Neutrophils is Competent but Compromised by Pharmacological Inhibition of Ezrin Phosphorylation.

Ezrin links the cortical cytoskeleton to the plasma membrane and plays a role in regulating changes in cell shape. Recently, NSC668394 has been shown to inhibit a key step for its activity, i.e. phosphorylation at threonine 567. In neutrophils, another key regulatory step is the Ca2+-mediated cleavage of ezrin by calpain. In this paper, we use NSC668394 as a pharmacological inhibitor to investigate the interplay between these two steps in regulating changes in neutrophil shape. NSC668394 reduced the amount of peripherally located ezrin in neutrophils, and increased Ca2+-dependent ezrin cleavage. Neutrophils with NSC668394-inhibited ezrin phosphorylation remained both phagocytic and chemotactically competent. However, phagocytosis was slightly impaired and chemotaxis could not be maintained over longer periods. The characteristic chemotactic morphology which neutrophils adopt was also aberrant. Although phosphorylation of ezrin plays a minor role in limiting the rapid changes in cell shape in neutrophils, inhibition of ezrin phosphorylation by NSC668394 prevented multiple and prolonged shape changes during extended chemotaxis. The susceptibility of prolonged chemotaxis to inhibition by NSC668394 may point to a useful target for anti-inflammatory therapy. Inhibition of neutrophil chemotaxis towards chronically inflamed sites without compromising their ability to undergo phagocytosis is a much sought after the effect of anti-neutrophil therapy.

Trefoil factor family peptides enhance cell migration by increasing cellular osmotic permeability and aquaporin 3 levels.

Trefoil factor family (TFF) peptides are produced rapidly at sites of injury, stimulating epithelial migration, a process involving rapid changes in cell shape and volume, requiring rapid flow of water into and out of the cell. We examined the effect of TFFs on fluidity of cells by measuring their sensitivity to osmotic challenges and cell migration, and determined whether those results were mediated through altering the levels of aquaporins (AQPs), a family of transmembrane water channels involved in cellular water homeostasis. Gastric (AGS) and colonic (Caco-2) cell lines had intrinsic TFF levels determined and the predominant TFF peptide knocked down (RNA interference). Knockdown caused lessened responsiveness to changes in external osmotic challenge (by 51 and 69% in AGS and Caco-2 cells, respectively) and reduced cell migration and transepithelial permeability but did not influence proliferation. Exogenous TFF increased several AQPs, particularly AQP3, and those were reciprocally reduced in knockdown cells. TFF-induced, but not fetal calf serum-induced, cell migration was inhibited by the presence of AQP3 blocker (CuSO4). We summarize that TFF peptides promptly produced at sites of injury increase AQP levels, most notably AQP3, thereby enhancing the cells' ability to rapidly change their shape as part of the restitutive process. TFF peptides also require functioning AQP3 channels to induce cell migration.-Marchbank, T., Playford, R. J. Trefoil factor family peptides enhance cell migration by increasing cellular osmotic permeability and aquaporin 3 levels.

Topographical interrogation of the living cell surface reveals its role in rapid cell shape changes during phagocytosis and spreading

Dramatic and rapid changes in cell shape are perhaps best exemplified by phagocytes, such as neutrophils. These cells complete the processes of spreading onto surfaces, and phagocytosis within 100 s of stimulation. Although these cell shape changes are accompanied by an apparent large increase in cell surface area, the nature of the membrane “reservoir” for the additional area is unclear. One proposal is that the wrinkled cell surface topography (which forms micro-ridges on the neutrophil surface) provides the resource for neutrophils to expand their available surface area. However, it has been problematic to test this proposal in living cells because these surface structures are sub-light microscopic. In this paper, we report the development of a novel approach, a variant of FRAP (fluorescent recovery after photo-bleaching) modified to interrogate the diffusion path-lengths of membrane associated molecules. This approach provides clear evidence that the cell surface topography changes dramatically during neutrophil shape change (both locally and globally) and can be triggered by elevating cytosolic Ca2+.

Neurokinin 1 Receptor Mediates Membrane Blebbing and Sheer Stress-Induced Microparticle Formation in HEK293 Cells

Cell-derived microparticles participate in intercellular communication similar to the classical messenger systems of small and macro-molecules that bind to specialized membrane receptors. Microparticles have been implicated in the regulation of a variety of complex physiopathologic processes, such as thrombosis, the control of innate and adaptive immunity, and cancer. The neurokinin 1 receptor (NK1R) is a Gq-coupled receptor present on the membrane of a variety of tissues, including neurons in the central and peripheral nervous system, immune cells, endocrine and exocrine glands, and smooth muscle. The endogenous agonist of NK1R is the undecapeptide substance P (SP). We have previously described intracellular signaling mechanisms that regulate NK1R-mediated rapid cell shape changes in HEK293 cells and U373MG cells. In the present study, we show that the activation of NK1R in HEK293 cells, but not in U373MG cells, leads to formation of sheer-stress induced microparticles that stain positive with the membrane-selective fluorescent dye FM 2–10. SP-induced microparticle formation is independent of elevated intracellular calcium concentrations and activation of NK1R present on HEK293-derived microparticles triggers detectable calcium increase in SP-induced microparticles. The ROCK inhibitor Y27632 and the dynamin inhibitor dynasore inhibited membrane blebbing and microparticle formation in HEK293 cells, strongly suggesting that microparticle formation in this cell type is dependent on membrane blebbing.

The Fern Sporangium: A Unique Catapult

Various plants and fungi have evolved ingenious devices to disperse their spores. One such mechanism is the cavitation-triggered catapult of fern sporangia. The spherical sporangia enclosing the spores are equipped with a row of 12 to 13 specialized cells, the annulus. When dehydrating, these cells induce a dramatic change of curvature in the sporangium, which is released abruptly after the cavitation of the annulus cells. The entire ejection process is reminiscent of human-made catapults with one notable exception: The sporangia lack the crossbar that arrests the catapult arm in its returning motion. We show that much of the sophistication and efficiency of the ejection mechanism lies in the two very different time scales associated with the annulus closure.

Characteristics of cultured subepithelial fibroblasts in the rat small intestine. II. Localization and functional analysis of endothelin receptors and cell-shape-independent gap junction permeability

Subepithelial fibroblasts form a cellular network with gap junctions under the epithelium of the gastrointestinal tract. Previously, we have reported their unique characteristics, such as reversible rapid cell-shape changes from a flat to a stellate configuration induced by dBcAMP and endothelins (ETs), and Ca2+ responses to, for example, ETs, ATP, and substance-P. We have now investigated the subtypes of ET receptors both in the rat small intestine and in primary cultured subepithelial fibroblasts isolated from rat duodenal villi. Their properties were compared between wild-type and endothelin-B-receptor-mutant sl/sl rats. Light- and electron-microscopic immunohistochemistry showed intense ETA immunoreactivity in the subepithelial fibroblasts from the small intestine and colon of both wild-type and sl/sl rats. In culture, immunocytochemistry, reverse transcription/polymerase chain reaction analysis, Ca2+ response measurements, and cell-shape change analysis indicated functional ETA and ETB receptors in the wild-type cells, but only ETA in the sl/sl cells. However, wild-type cells were more sensitive to ET-1 than to ET-3 by about one order of magnitude. ETA seemed to be dominant both in vivo and in vitro. The relationship between cell-shape change and gap junction permeability was examined by fluorescence recovery after photobleaching; the gap junctions were usually open but were blocked by carbenoxolone. Permeability did not change significantly with cell-shape change. This network of differentiated subepithelial fibroblasts may maintain intercellular communication via gap junctions to transduce signals evoked in the local network to the whole network. The cell-shape change of the cells through ETA activation may play an important role as a barrier and for intercellular signaling in the intestinal villi.

Cholesterol domains regulate the actin cytoskeleton at the leading edge of moving cells

The plasma membrane must be rigid to form a barrier between the cell interior and the extracellular milieu, while remaining sufficiently flexible to enable rapid changes in cell shape. The concept of the plasma membrane as a mosaic of microdomains can help to understand this combination of properties. A recent report shows that the polarization of cholesterol-enriched domains changes the viscoelastic properties of the membrane at the front of moving cells, permitting efficient formation of the actin network required to push the leading-edge membrane forward.

Cholesterol domains regulate the actin cytoskeleton at the leading edge of moving cells

The plasma membrane must be rigid to form a barrier between the cell interior and the extracellular milieu, while remaining sufficiently flexible to enable rapid changes in cell shape. The concept of the plasma membrane as a mosaic of microdomains can help to understand this combination of properties. A recent report shows that the polarization of cholesterol-enriched domains changes the viscoelastic properties of the membrane at the front of moving cells, permitting efficient formation of the actin network required to push the leading-edge membrane forward.

Novel Association of the Src Family Kinases, Hck and c-Fgr, with CCR3 Receptor Stimulation: A Possible Mechanism for Eotaxin-Induced Human Eosinophil Chemotaxis

The chemokine eotaxin is a potent and relatively eosinophil-specific chemoattractant implicated in the cell migration to inflammatory sites in allergic diseases. Eotaxin exerts its activity solely through the CCR3 receptor, but the signaling pathways are poorly defined. In this study, we show that eotaxin induces an increase in tyrosine phosphorylation of multiple cellular proteins in normal human eosinophils. Eotaxin-dependent tyrosine phosphorylation was detected 1 min after stimulation and increased for at least 15 min with kinetics similar to those of eotaxin-induced cell shape changes. Herbimycin A, a tyrosine kinase inhibitor, blocked both eotaxin-induced tyrosine phosphorylation and cell shape changes as well as chemotaxis. Immunofluorescence microscopy analyses showed that eotaxin-induced cell shape changes were accompanied by redistribution of tyrosine-phosphorylated proteins and F-actin reorganization that were sensitive to herbimycin A. Coimmunoprecipitation studies revealed that binding of eotaxin to CCR3 greatly enhanced association of the Src family kinases, Hck and c-Fgr, with CCR3 after internalization of CCR3. These results may indicate that recruitment of Hck and c-Fgr to CCR3 in a compartment triggers tyrosine phosphorylation, leading to rapid cell shape changes required for cell migration.

V-src-Induced Cell Shape Changes in Rat Fibroblasts Require New Gene Transcription and Precede Loss of Focal Adhesions

The mechanism of v-src-induced morphological transformation is still obscure. We compared LA29 rat fibroblasts, which express a temperature-sensitive (ts) v-src mutant, with D1025 rat fibroblasts, transfected with a ts mutant of v-fps. Upon transformation, LA29 cells adopted an elongated shape with reduced focal adhesions and loss of actin stress fibers. In contrast, activation of v-fps in D1025 cells had little effect on morphology. In both cells, paxillin was strongly tyrosine phosphorylated upon activation of the kinases. This indicates that paxillin phosphorylation is not required, or not sufficient, for the v-src-induced disruption of focal adhesions. As previously described by others, v-src activated the ras–MAP kinase (MAPK) pathway, as indicated by tyrosine phosphorylation of the rasGAP-associated proteins p62 and p190 and MAPK phosphorylation. Since MAPK affects transcription, this suggested that novel gene transcription was required. This notion was confirmed using actinomycin D and cycloheximide, which did not impair activation of v-src kinase activity, but completely blocked v-src-induced morphological changes, as demonstrated using image analysis. Furthermore, we observed that v-src-induced changes in cell shape occurred before the reduction in number and size of focal adhesions. We conclude that v-src-induced transformation of rat fibroblasts depends on synthesis of a protein, which induces rapid changes in cell shape that precede the loss of focal adhesions.

Effects of phenothiazine neuroleptic drugs on the microtubular-membrane complex in bloodstream forms of Trypanosoma brucei.

African trypanosomes are parasitic protozoa causing sleeping sickness in humans and related diseases in domestic animals against which no entirely satisfactory forms of chemotherapy are yet available. It was previously shown that related species of trypanosomes, as well as procyclic (insect) forms of Trypanosoma brucei are extremely sensitive to the action of phenothiazine neuroleptic drugs in vitro. In this work, we have carried out a more detailed investigation of the effects of thioridazine, one of the most potent neuroleptic phenothiazine drugs known, on the morphology of the infective bloodstream forms of T. brucei, with particular reference to the parasite's prominent pellicular membrane complex. Our data show that this drug induces rapid changes in cell shape that appear to involve some reorganization of the microtubular membrane skeleton, but does not affect the structural integrity of the microtubular complex. Another early consequence of drug action involved damage to nuclear and cytoplasmic membranes and the appearance of tubular arrays of coated membrane within the flagellar pocket. It was also revealed that the drug induces a rapid release of the variant-specific glycoprotein (VSG) which makes up the surface coat protecting bloodstream forms of the parasite against the host immune system. Our evidence suggests that this release of VSG involves cleavage of the protein's glycosyl-phosphatidylinositol (GPI) membrane anchor by endogenous GPI-specific phospholipase C, probably as a consequence of minor damage to the parasite plasma membrane induced by the drug.

Structure-activity relationships of phenothiazines in inhibiting lymphocyte motility as determined by a novel flow cytometric assay

Lymphocyte motility is highly dependent on rapid changes in cell shape. The human T-lymphoma cell line, MOLT-4, is constitutively shape-changing and motile, and both of these properties can be inhibited by the phenothiazine, chlorpromazine, as assessed by video analysis and migration across polycarbonate filters. In this paper, the light-scattering facility of a flow cytometer has been used to establish a simpler and more quantitative means of measuring changes in shape. By this method, the structure activity relationship (SAR) of phenothiazines and related compounds has been determined. The most active compounds had the tricyclic phenothiazine nucleus with a constrained dialkylaminoalkyl substituent at the nitrogen. The SAR for inhibition of lymphocyte motility differs from those reported for neuroleptic effects and for inhibition of PKC or calmodulin. Phenothiazine concentrations that inhibited lymphocyte shape-changing resulted in reduced F-actin concentrations. This indicates that the probable mode of action is disruption of mechanisms regulating actin polymerisation.

Contractile units in stress fibers of fetal human astroglia in tissue culture

The interaction between myosin and F-actin requires the enzyme, myosin light chain kinase (MLCK), as well as Ca2 +-calmodulin and the calmodulin binding protein, caldesmon, which also binds to F-actin. Using immunofluorescence staining, we have demonstrated that in human fetal astroglia as in mouse astroglia (Abd-El-Basset et al., 1991) the stress fibers contain these contractile elements: F-actin, myosin, tropomyosin and caldesmon. F-actin extends continuously along the stress fibers, whereas myosin, tropomyosin and caldesmon are localized discontinuously in a periodic pattern. In addition, we have demonstrated that fetal human astroglia have the enzyme MLCK and calmodulin. The association of the contractile elements listed above together with calmodulin and MLCK constitutes what may be termed ‘contractile units’, suggesting that the stress fibers in astroglia may be contractile. Contractile stress fibers would enable astroglia to exert tension on the matrix surrounding them, thus facilitating rapid changes in cell shape.

Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations

The ventral furrow and posterior midgut invaginations bring mesodermal and endodermal precursor cells into the interior of the Drosophila embryo during gastrulation. Both invaginations proceed through a similar sequence of rapid cell shape changes, which include apical flattening, constriction of the apical diameter, cell elongation and subsequent shortening. Based on the time course of apical constriction in the ventral furrow and posterior midgut, we identify two phases in this process: first, a slow stochastic phase in which some individual cells begin to constrict and, second, a rapid phase in which the remaining unconstricted cells constrict. Mutations in the concertina or folded gastrulation genes appear to block the transition to the second phase in both the ventral furrow and the posterior midgut invaginations.

Animal cell shape changes and gene expression.

Cell shape and cell contacts are determined by transmembrane receptor-mediated associations of the cytoskeleton with specific extracellular matrix proteins and with ligands on the surface of adjacent cells. The cytoplasmic domains of these microfilament-membrane associations at the adherens junction sites, also localize a variety of regulatory molecules involved in signal transduction and gene regulation. The stimulation of cells with soluble polypeptide factors leads to rapid changes in cell shape and microfilament component organization. In addition, this stimulation also activates the phosphoinositide signaling pathway. Recently, a linkage between actin-binding proteins and the phosphoinositide signaling pathway, was discovered. It is suggested that by the association with the second messenger system, and/or by controlling the localization of regulatory molecules, the cytoskeleton may regulate gene expression.

Cyclic AMP-induced shape changes of astrocytes are accompanied by rapid depolymerization of actin

Agents that increase intracellular cyclic AMP produce a process-bearing morphology in astrocytes. We have examined short-term re-arrangements of the astrocyte cytoskeleton during this shape conversion. Primary cultures of astrocytes from neonatal rat forebrain were grown at low density as polygonal shaped cells. Treatment with 1 mM dibutyryl cAMP in the absence of serum produced rapid changes in cell shape (100% of cells as flat to 90% showing cytoplasmic contraction and processes in 60 min). In the presence of serum, similar changes took place, but more slowly. No changes in total cellular levels of GFAP, vimentin, tubulin or actin were observed over a 2-h period of treatment. There was a shift in actin from a Triton X-100-insoluble pool to a soluble pool, with a 40% reduction in insoluble actin. The kinetics of this shift paralleled kinetics of shape change. The shift also corresponded to a loss of stress fibers, visualized with rhodamine-phalloidin. Intermediate stages of stress fiber loss were observed as short, wavy or small ring profiles. Colchicine prevented the dBcAMP-induced changes in shape. If cells were first treated with taxol, however, subsequent exposure to colchicine did not inhibit contraction. Thus, dBcAMP, presumably through a cAMP-dependent kinase, depolymerizes actin in stress fiber forms as cells contract. In addition, an intact microtubule system may be required for the changes in shape. Treatment with dBcAMP also caused the disappearance of vinculin-containing attachment sites, indicating that adhesion plaques, or at least the association of vinculin with them, are lost during the time of microfilament bundle dissociation.