Honeycomb structure of the apical surface of thyroid epithelium during involution of the hyperplastic thyroid gland

A heretofore undescribed feature consisting of closely packed pits 0.5-1 micron in diameter was observed on the apical surface of thyroid epithelial cells. The walls of the pits were generally smooth, except at the base where there was a high incidence of irregularities looking like sites of recent fusion of apical vesicles with the pits. The matrix of the partition between pits was similar to the matrix of pseudopods in these cells in being free of membrane-bounded organelles and containing a low concentration of ribosomes. The pits were observed early in the involution of the hyperplastic gland, most prominently between 14 h and 8 days of involution. This is a time when thyroglobulin is accumulating in follicular lumens and the apical end of the epithelial cells usually contains a high concentration of apical vesicles ordinarily considered to be exocytic in character. It is important to recognize the existence of this feature to avoid confusing profiles of it with structures involved in macropinocytosis.     

Microtubule-Mediated Inositol Lipid Signaling Plays Critical Roles in Regulation of Blebbing

Cells migrate by extending pseudopods such as lamellipodia and blebs. Although the signals leading to lamellipodia extension have been extensively investigated, those for bleb extension remain unclear. Here, we investigated signals for blebbing in Dictyostelium cells using a newly developed assay to induce blebbing. When cells were cut into two pieces with a microneedle, the anucleate fragments vigorously extended blebs. This assay enabled us to induce blebbing reproducibly, and analyses of knockout mutants and specific inhibitors identified candidate molecules that regulate blebbing. Blebs were also induced in anucleate fragments of leukocytes, indicating that this assay is generally applicable to animal cells. After cutting, microtubules in the anucleate fragments promptly depolymerized, followed by the extension of blebs. Furthermore, when intact cells were treated with a microtubule inhibitor, they frequently extended blebs. The depolymerization of microtubules induced the delocalization of inositol lipid phosphatidylinositol 3,4,5-trisphosphate from the cell membrane. PI3 kinase-null cells frequently extended blebs, whereas PTEN-null cells extended fewer blebs. From these observations, we propose a model in which microtubules play a critical role in bleb regulation via inositol lipid metabolism.     

Absence of fodrin (spectrin-like protein) under the pseudopod membrane in stimulated thyroid cells

A fodrin-like protein purified from porcine thyroid cells and characterized by its properties identical to those of pig brain spectrin (F. Regnouf et al., Eur. J. Biochem. 153, 313-319 (1985)) has been localized by immunofluorescence and electron immunocytochemistry in porcine and rat thyroid. Fodrin-like polypeptides were detected in subplasmalemmal meshworks of microfilaments attached to isolated or in situ plasma membranes. In resting cells, fodrin was found under apical and basolateral membrane domains, whereas it was always absent under the pseudopod membrane domain induced by acute TSH stimulation in vitro, using monolayers of porcine cultured cells attached to collagen permeable substrates, as well as in vivo, using rats intravenously treated with TSH. Thyroid fodrin could be involved in exocytosis and membrane stabilization which occurs during the formation of pseudopods induced by TSH stimulation.     

A stochastic model for chemotaxis based on the ordered extension of pseudopods

Many amoeboid cells move by extending pseudopods. Here I present a new stochastic model for chemotaxis that is based on pseudopod extensions by Dictyostelium cells. In the absence of external cues, pseudopod extension is highly ordered with two types of pseudopods: de novo formation of a pseudopod at the cell body in random directions, and alternating right/left splitting of an existing pseudopod that leads to a persistent zig-zag trajectory. We measured the directional probabilities of the extension of splitting and de novo pseudopods in chemoattractant gradients with different steepness. Very shallow cAMP gradients can bias the direction of splitting pseudopods, but the bias is not perfect. Orientation of de novo pseudopods require much steeper cAMP gradients and can be more precise. These measured probabilities of pseudopod directions were used to obtain an analytical model for chemotaxis of cell populations. Measured chemotaxis of wild-type cells and mutants with specific defects in these stochastic pseudopod properties are similar to predictions of the model. These results show that combining splitting and de novo pseudopods is a very effective way for cells to obtain very high sensitivity to stable gradient and still be responsive to changes in the direction of the gradient.     

Bleb formation in granulosa cells of rat pre-ovulatory follicles in vivo and in vitro

To determine if hormone-induced events leading to ovulation an granulosa cell luteinization might be associated with changes in the surface configuration of granulosa cells we have studied the morphology of granulosa cells from the preovulatory follicles both in vivo and in vitro. In vivo, granulosa cells in follicles from rats primed with estradiol and FSH developed bulbous protrusions termed blebs in response to injected hCG. The blebs were restricted to the adluminal granulosa cells which possess the least number of receptors for hCG. When granulosa cells from follicles of rats primed with estradiol and FSH were cultured in vitro, in the absence of serum, approximately 10% of the cells formed blebs. In the presence of 10% rat or fetal calf serum, nearly 90% of the cells formed blebs by 18 hr. Serum-induced bleb formation was prevented by 1 mM dibutyryl cycle-AMP plus 0.5 mM methyl isobutyl xanthine and by cytochalasin B (25 mug/ml), while 0.1 muM colchicine had no effect. Fibronectin at 25 mug/ml increased bleb formation three-fold over control values in serum-free medium. When hCG was included in serum containing medium, the majority of the cells remained smooth without any blebs. Thus, in contrast to its action in vivo, hCG inhibited the formation of blebs in vitro. When the cells incubated in the presence of dbcAMP plus methyl isobutyl xanthine in serum-containing medium, none of the cells formed blebs. One explanation for the seemingly opposite actions of hCG in vivo and in vitro is that hCG might act to alter the permeability of the pre-ovulatory follicles, and thereby allow the admission of serum. The admitted serum component(s) could then induce the formation of blebs on receptor-deficient adluminal cells that did not have elevated cAMP concentrations. The results suggest that fibronectin and/or other serum components, act to induce microfilament-dependent, cAMP-inhibited bleb formation on granulosa cells in vivo and in vitro.     

Membrane tether formation from blebbing cells

Membrane tension has been proposed to be important in regulating cell functions such as endocytosis and cell motility. The apparent membrane tension has been calculated from tether forces measured with laser tweezers. Both membrane-cytoskeleton adhesion and membrane tension contribute to the tether force. Separation of the plasma membrane from the cytoskeleton occurs in membrane blebs, which could remove the membrane-cytoskeleton adhesion term. In renal epithelial cells, tether forces are significantly lower on blebs than on membranes that are supported by cytoskeleton. Furthermore, the tether forces are equal on apical and basolateral blebs. In contrast, tether forces from membranes supported by the cytoskeleton are greater in apical than in basolateral regions, which is consistent with the greater apparent cytoskeletal density in the apical region. We suggest that the tether force on blebs primarily contains only the membrane tension term and that the membrane tension may be uniform over the cell surface. Additional support for this hypothesis comes from observations of melanoma cells that spontaneously bleb. In melanoma cells, tether forces on blebs are proportional to the radius of the bleb, and as large blebs form, there are spikes in the tether force in other cell regions. We suggest that an internal osmotic pressure inflates the blebs, and the pressure calculated from the Law of Laplace is similar to independent measurements of intracellular pressures. When the membrane tension term is subtracted from the apparent membrane tension over the cytoskeleton, the membrane-cytoskeleton adhesion term can be estimated. In both cell systems, membrane-cytoskeleton adhesion was the major factor in generating the tether force.     

Fine structural aspects of phagocytotic activity in inverted follicle cells of cultured porcine thyroids

Inversion of thyroid follicles took place when they were isolated by collagenase and trypsin and cultured in suspension in Eagle's medium supplemented with 10% fetal calf serum without TSH. The apical surface facing the culture medium contained numerous microvilli and a central cilium, while the luminal surface became flattened. Phagocytotic activity by pseudopods was promoted after addition of TSH to the culture medium. When the inverted follicles were incubated in culture medium containing TSH (50 mU/ml) and human red blood cells, or TSH and polystyrene latex beads (2.02 micron in diameter) for 1-3 h, numerous red blood cells or latex beads respectively were observed to be taken up by the epithelial follicle cells by scanning electron microscopy, as well as conventional thin-section electron microscopy. These results show that the apical surface (culture medium side) of the epithelial cell of the cultured thyroid follicle whose polarity is reversed phagocytoses red blood cells and latex beads.     

Competition of contacting heterotypic epithelial sheets for the territory in culture

Co-cultured epithelial cells of various lines formed mixed cohesive sheets. Contact interactions of different cells in the sheets were studied by phase contrast and interference reflection microscopy, time-lapse microcinematography, transmission and scanning electron microscopy. It was found that heterotypic cells in the combined sheets were locked together by the complexes of specialized contacts and were metabolically coupled, as shown by the dye transfer. At the same time heterotypic cells continued to extend pseudopods at the contacting lateral surfaces and to attach these pseudopodies to substratum. Due to competition of pseudopod attachments one group of cells in the sheet often progressively pushed another group from the substratum. This competition of cells for substratum may provide an important mechanism for morphogenetic reorganisation of epithelial sheets and of other groups of interacting cells in vivo.     

Blebbing of Dictyostelium cells in response to chemoattractant

Stimulation of Dictyostelium cells with a high uniform concentration of the chemoattractant cyclic-AMP induces a series of morphological changes, including cell rounding and subsequent extension of pseudopodia in random directions. Here we report that cyclic-AMP also elicits blebs and analyse their mechanism of formation. The surface area and volume of cells remain constant during blebbing indicating that blebs form by the redistribution of cytoplasm and plasma membrane rather than the exocytosis of internal membrane coupled to a swelling of the cell. Blebbing occurs immediately after a rapid rise and fall in submembraneous F-actin, but the blebs themselves contain little F-actin as they expand. A mutant with a partially inactivated Arp2/3 complex has a greatly reduced rise in F-actin content, yet shows a large increase in blebbing. This suggests that bleb formation is not enhanced by the preceding actin dynamics, but is actually inhibited by them. In contrast, cells that lack myosin-II completely fail to bleb. We conclude that bleb expansion is likely to be driven by hydrostatic pressure produced by cortical contraction involving myosin-II. As blebs are induced by chemoattractant, we speculate that hydrostatic pressure is one of the forces driving pseudopod extension during movement up a gradient of cyclic-AMP.     

A pathogenic bacterium triggers epithelial signals to form a functional bacterial receptor that mediates actin pseudopod formation.

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce actin accumulation beneath adherent bacteria. We found that EPEC adherence to epithelial cells mediates the formation of fingerlike pseudopods (up to 10 microm) beneath bacteria. These actin-rich structures also contain tyrosine phosphorylated host proteins concentrated at the pseudopod tip beneath adherent EPEC. Intimate bacterial adherence (and pseudopod formation) occurred only after prior bacterial induction of tyrosine phosphorylation of an epithelial membrane protein, Hp90, which then associates directly with an EPEC adhesin, intimin. These interactions lead to cytoskeletal nucleation and pseudopod formation. This is the first example of a bacterial pathogen that triggers signals in epithelial cells which activates receptor binding activity to a specific bacterial ligand and subsequent cytoskeletal rearrangement.     

Bleb formation during anoxia is not a prerequisite for eventual cell death in renal tubule cells

Membrane deformations, generally termed blebs, are a hallmark of cells subjected to noxious stimuli or injurious conditions. Although not lethal itself, bleb formation has been used as an indicator of the stage of cell injury. Whether or not bleb formation is an integral part of the sequence of events that lead to irreversible injury, or occurs in all cells within a group subjected to the same conditions is not known. We demonstrate here that renal epithelial cells in culture produce blebs when subjected to sustained periods of anoxia and substrate deprivation. However, the occurrence of blebs does not correlate with eventual cell death. The data suggest that bleb formation may be associated with prolonged survival.     

An excitable cortex and memory model successfully predicts new pseudopod dynamics

Motile eukaryotic cells migrate with directional persistence by alternating left and right turns, even in the absence of external cues. For example, Dictyostelium discoideum cells crawl by extending distinct pseudopods in an alternating right-left pattern. The mechanisms underlying this zig-zag behavior, however, remain unknown. Here we propose a new Excitable Cortex and Memory (EC&M) model for understanding the alternating, zig-zag extension of pseudopods. Incorporating elements of previous models, we consider the cell cortex as an excitable system and include global inhibition of new pseudopods while a pseudopod is active. With the novel hypothesis that pseudopod activity makes the local cortex temporarily more excitable--thus creating a memory of previous pseudopod locations--the model reproduces experimentally observed zig-zag behavior. Furthermore, the EC&M model makes four new predictions concerning pseudopod dynamics. To test these predictions we develop an algorithm that detects pseudopods via hierarchical clustering of individual membrane extensions. Data from cell-tracking experiments agrees with all four predictions of the model, revealing that pseudopod placement is a non-Markovian process affected by the dynamics of previous pseudopods. The model is also compatible with known limits of chemotactic sensitivity. In addition to providing a predictive approach to studying eukaryotic cell motion, the EC&M model provides a general framework for future models, and suggests directions for new research regarding the molecular mechanisms underlying directional persistence.     

Microfilament reorganization in normal and cytochalasin B treated adherent thrombocytes

Thrombocyte adhesion following activation by a Formvar surface involves a morphologic transition resulting in a fully spread cell. Correlative SEM and whole mount TEM were used to study the cytoskeletal alterations that accompany changes in surface morphology during adhesion. Following initial adhesion, thrombocytes extend slender pseudopods containing longitudinally oriented bundles of filaments that are 13–22 nm in diameter. Concomitant with pseudopod extension, a cytoplasmic hyalomere, consisting of a dense filamentous network, extends between the pseudopods and ultimately results in a fully spread cell. Treatment of thromboyctes with cytochalasin B (10^?5 M) caused clumping of the hyalomere filament network and retraction of the hyalomere. Examination of partially retracted cells revealed that pseudopod filament bundles were continuous with the contracting filamentous network. It is concluded that pseudopod filament bundles and cytoplasmic hyalomere filaments are interconvertible and that their organizational relationship changes in accordance with gross morphologic changes.     

Chemotaxis: a feedback-based computational model robustly predicts multiple aspects of real cell behaviour

The mechanism of eukaryotic chemotaxis remains unclear despite intensive study. The most frequently described mechanism acts through attractants causing actin polymerization, in turn leading to pseudopod formation and cell movement. We recently proposed an alternative mechanism, supported by several lines of data, in which pseudopods are made by a self-generated cycle. If chemoattractants are present, they modulate the cycle rather than directly causing actin polymerization. The aim of this work is to test the explanatory and predictive powers of such pseudopod-based models to predict the complex behaviour of cells in chemotaxis. We have now tested the effectiveness of this mechanism using a computational model of cell movement and chemotaxis based on pseudopod autocatalysis. The model reproduces a surprisingly wide range of existing data about cell movement and chemotaxis. It simulates cell polarization and persistence without stimuli and selection of accurate pseudopods when chemoattractant gradients are present. It predicts both bias of pseudopod position in low chemoattractant gradients and--unexpectedly--lateral pseudopod initiation in high gradients. To test the predictive ability of the model, we looked for untested and novel predictions. One prediction from the model is that the angle between successive pseudopods at the front of the cell will increase in proportion to the difference between the cell's direction and the direction of the gradient. We measured the angles between pseudopods in chemotaxing Dictyostelium cells under different conditions and found the results agreed with the model extremely well. Our model and data together suggest that in rapidly moving cells like Dictyostelium and neutrophils an intrinsic pseudopod cycle lies at the heart of cell motility. This implies that the mechanism behind chemotaxis relies on modification of intrinsic pseudopod behaviour, more than generation of new pseudopods or actin polymerization by chemoattractants.     

Ponticulin plays a role in the positional stabilization of pseudopods

Ponticulin is a 17-kD glycoprotein that represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently generated mutants lacking ponticulin was analyzed using computer- assisted two- and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin- minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than wild-type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin- minus cells extend and retract pseudopods with the same time course as wild-type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild-type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis.     

Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells: alterations underlying surface bleb formation

Several in vitro and in vivo studies have suggested that surface bleb formation during oxidative cell injury is related to alteration in cytoskeleton organization. Various cell lines different in origin and growth characteristics were exposed to 2-methyl-1,4-naphthoquinone (menadione) which is known to induce bleb formation and cytotoxicity by generating considerable amounts of oxygen-reactive species. Treated cells were analyzed by means of immunocytochemistry and electron microscopy in order to investigate the morphological and molecular features underlying bleb generation. The results obtained indicate that menadione-induced bleb formation is a widely observed phenomenon present mainly in round or mitotic cells. Surface blebs appear free of organelles and contain only few ribosomes and amorphous material. Occasionally, they undergo detachment from the cell surface as large cytoplasmic vesicles. Bleb surfaces with protein clusters as well as bald blisters with an almost exclusive localization of intramembrane particles on their narrow base were detected using freeze-fracture techniques. Immunocytochemical investigations performed on menadione-exposed cells revealed that some surface proteins (collagen IV, sialo-proteins, beta 2 microglobulin and fibronectin) and adhesion molecules (vinculin) underwent changes in their expression over the bleb surface. Moreover, different behavioural characteristics of actin microfilaments, vimentin and keratin intermediate filaments and microtubules was observed. Alpha-actinin, vimentin and microtubular proteins (tubulin, MAPs and tau) were detected within the blebs. On the other hand, actin and keratin filaments appeared to be absent. The results presented here demonstrate that cytoskeletal structures and the microfilament system in particular, represent important targets in menadione-induced morphological changes in cultured cells. These changes appear to lead to the redistribution of several cytoskeletal and membrane proteins as well as dissociation of the cytoskeleton network from its anchoring domains in the plasma membrane thus generating sites of structural weakness where blebs would arise and progressively grow. Experimental evidence supporting a crucial role of thiol oxidation and elevation of cytoplasmic calcium concentration in bleb formation is also provided.     

Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions

Current models of eukaryotic chemotaxis propose that directional sensing causes localized generation of new pseudopods. However, quantitative analysis of pseudopod generation suggests a fundamentally different mechanism for chemotaxis in shallow gradients: first, pseudopods in multiple cell types are usually generated when existing ones bifurcate and are rarely made de novo; second, in Dictyostelium cells in shallow chemoattractant gradients, pseudopods are made at the same rate whether cells are moving up or down gradients. The location and direction of new pseudopods are random within the range allowed by bifurcation and are not oriented by chemoattractants. Thus, pseudopod generation is controlled independently of chemotactic signalling. Third, directional sensing is mediated by maintaining the most accurate existing pseudopod, rather than through the generation of new ones. Finally, the phosphatidylinositol 3-kinase (PI(3)K) inhibitor LY294002 affects the frequency of pseudopod generation, but not the accuracy of selection, suggesting that PI(3)K regulates the underlying mechanism of cell movement, rather than control of direction.     

Directional budding of human immunodeficiency virus from monocytes.

Time-lapse cinematography revealed that activated human immunodeficiency virus (HIV)-infected monocytes crawl along surfaces, putting forward a leading pseudopod. Scanning electron micrographs showed monocyte pseudopods associated with spherical structures the size of HIV virions, and transmission electron micrographs revealed HIV virions budding from pseudopods. Filamentous actin (F-actin) was localized by electron microscopy in the pseudopod by heavy meromyosin decoration. Colocalization of F-actin and p24 viral antigen by light microscopy immunofluorescence indicated that F-actin and virus were present on the same pseudopod. These observations indicate that monocytes produce virus from a leading pseudopod. We suggest that HIV secretion at the leading edges of donor monocytes/macrophages may be an efficient way for HIV to infect target cells.     

Irreversible injury in anoxic hepatocytes precipitated by an abrupt increase in plasma membrane permeability

Using low-light digitized video microscopy, the onset, progression, and reversibility of anoxic injury were assessed in single hepatocytes isolated from fasted rats. Cell-surface bleb formation occurred in three stages over 1-3 h after anoxia. Stage I was characterized by formation of numerous small blebs. In stage II, small blebs enlarged by coalescence and fusion to form a few large terminal blebs. Near the end of stage II, cells began to swell rapidly, ending with the apparent breakdown of one of the terminal blebs. Breakdown of the bleb membrane initiated stage III of injury and was coincident with a rapid increase of nonspecific permeability to organic cationic and anionic molecules. On reoxygenation, stages I and II were fully reversible, and plasma membrane blebs were resorbed completely within 6 min of reoxygenation without loss of viability. Stage III, however, was not reversible, and no morphological changes occurred on reoxygenation. The results indicate that onset of cell death owing to anoxia is a rapid event initiated by a sudden increase of nonspecific plasma membrane permeability caused by rupture of a terminal bleb. Anoxic injury is reversible until this event occurs.     

Navigation of Chemotactic Cells by Parallel Signaling to Pseudopod Persistence and Orientation

The mechanism of chemotaxis is one of the most interesting issues in modern cell biology. Recent work shows that shallow chemoattractant gradients do not induce the generation of pseudopods, as has been predicted in many models. This poses the question of how else cells can steer towards chemoattractants. Here we use a new computational algorithm to analyze the extension of pseudopods by Dictyostelium cells. We show that a shallow gradient of cAMP induces a small bias in the direction of pseudopod extension, without significantly affecting parameters such as pseudopod frequency or size. Persistent movement, caused by alternating left/right splitting of existing pseudopodia, amplifies the effects of this bias by up to 5-fold. Known players in chemotactic pathways play contrasting parts in this mechanism; PLA2 and cGMP signal to the cytoskeleton to regulate the splitting process, while PI 3-kinase and soluble guanylyl cyclase mediate the directional bias. The coordinated regulation of pseudopod generation, orientation and persistence by multiple signaling pathways allows eukaryotic cells to detect extremely shallow gradients.