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During directional locomotion, cells must reorient themselves in response to attractive or repulsive cues. Filopodia are narrow actin-based protrusions whose prevalence at the leading edge of a migrating cell is related to the persistence of locomotion. Although there is a marked absence of calcium-binding components in their structure, they responded to store-operated calcium entry (SOCE). Here, we used a two-phase protocol to determine how they responded. In the first phase, extracellular calcium was removed and ER calcium lowered by blocking reuptake through the calcium pump. This was known to activate stromal interacting molecule (STIM) and cause its microtubule-mediated translocation to the cell surface. In the second phase, extracellular calcium and calcium influx into the ER were restored. ER depletion caused filopodia to increase, followed by a spontaneous decrease that was blocked by inhibiting endocytosis. The intracellular calcium concentration increased during depletion, while the size of the exchangeable compartment of vesicles, measured by fluid-phase marker uptake, shrank. When SOCE mediators and the aquaporin, AQP4, were localized, STIM and transient receptor potential canonical (TPRC) channels occupied vesicular profiles side-by-side in linear arrays. STIM1 was translocated, as expected. TRPC1 was initially in a rapidly recycling pool (RRP) where it partially colocalized with Vamp2. Calcium restoration caused TRPC1 exocytosis, while STIM1 reverted toward its original pattern associated with the ER. The exchangeable compartment was restored and this enabled filopodia extension, which was blocked by inhibitors of TRPC1/4/5 and endocytosis. That vesicle recycling was essential for extension during calcium readdition was indicated by reversal of the effect of endocytosis inhibitors in the depletion and readdition phases. The results suggest that SOCE regulates the size of the RRP in epithelial cells, and vesicle recycling is the immediate mechanism affecting filopodia extension. The conclusions are discussed in light of factors regulating protrusion formation, namely surface tension and vesicle trafficking.

Different protein kinase C (PKC) isoforms have distinct roles in regulating cell functions. The conventional (α, β, γ) and novel (δ, e, η, θ) classes are targets of phorbol ester tumor promoters, which are surrogates of endogenous second messenger, diacylglycerol. The promoter-stimulated disappearance of filopodia was investigated by use of blocking peptides (BPs) that inhibit PKC maturation and/or docking. Filopodia were partially rescued by a peptide representing PKC e hydrophobic sequence, but also by a myristoylated PKC α/β pseudosubstrate sequence, and an inhibitor of T-cell protein tyrosine phosphatase (TC-PTP). The ability to turn over filopodia was widely distributed among PKC isoforms. PKC α and η hydrophobic sequences enhanced filopodia in cells in the absence of tumor promoter treatment. With transcriptional knockdown of PKC α, the content of PKC e predominated over other isoforms. PKC e could decrease filopodia significantly in promoter-treated cells, and this was attributed to ruffling. The presence of PKC α counteracted the PKC e-mediated enhancement of ruffling. The results showed that there were two mechanisms of filopodia downregulation. One operated in the steady-state and relied on PKC α and η. The other was stimulated by tumor promoters and relied on PKC e. Cycles of protrusion and retraction are characteristic of filopodia and are essential for the cell to orient itself during chemotaxis and haptotaxis. By suppressing filopodia, PKC e can create a long-term 'memory' of an environmental signal that may act in nature as a mnemonic device to mark the direction of a repulsive signal. This article is protected by copyright. All rights reserved.

Previously, we found that oncogenically transformed cells had fewer filopodia and more large, p21-activated kinase (PAK)-dependent features than normal cells. These large protrusions (LPs) were increased in cells expressing RhoA(N19) with Cdc42-associated kinase (ACK). Here, we determine how GTPase-mediated mechanisms of focal contact (FC) regulation affect these protrusions. Constructs encoding various proteins were introduced into cells which were then studied by microscopy and computerized image processing and analysis. Constructs that prevented PAK recruitment by PAK-interacting exchange factor (PIX) or restricted PAK residence time on FCs decreased both protrusions. Thus, filopodia were also PAK-dependent. A comparison of FC distribution in cells expressing PAK in the presence or absence of PAK kinase inhibitor domain (KID) suggested that PAK enlarged FCs without affecting the prevalence of either protrusion. KID or Nck expression increased LPs but not filopodia. Nck failed to synergize with KID or ACK and RhoA(N19) in enhancing LPs. Nck and KID synergistically enhanced filopodia, possibly because Nck recruited PAK to FCs while KID prevented their dissociation by PAK-mediated autophosphorylation. Coexpression of Nck, ACK, and RhoA(N19) abrogated filopodia and replicated the transformed phenotype. Since Nck recruitment of PAK is implicated in persistence of directional movement, we studied the PAK-Nck interface. Filopodia were eliminated by the Nck PAK-binding domain and LPs by the PAK Nck-binding domain. The results suggested that filopodia formation has more stringent requirements than LP formation, and Nck and PAK are used differently in the protrusions. Loss of filopodia in transformed cells may reflect defective regulation of GTPase mechanisms.

As all microscopists are taught, resolution can be measured by knowing the closest distance between two points that can be discriminated in the image. There are some occasions when we want to measure something in a digital image, and the nominal resolution isn't sufficient for the task. One of these, extracting a contour from the image, was summarized in a previous research report. The principles would be valid for any filled area, such as a particle or structure from an X-ray dot map. The ground rule was set that the outermost pixel would be selected at every point on the contour. It was clear from the geometry shown in Fig. 1 that only values of 0°, 45°, 90°, 135° and 180° could be measured. Because of the stair step defect, tracing the sequence of pixel locations only gave offsets to 8 pixels, one directly below, two on the corners below, three pixels in the same location above, and one pixel on either side of the subject pixel. Therefore it was impossible to measure the true curvature of the contour.

A computerized morphometric classification technique based on latent factors reveals major protrusion classes: factors 4, 5, and 7. Previous work showed that factor 4 represented filopodia, 5 the distribution of lamellar cytoplasm, and 7 a blunt protrusion. We explore the relationship of focal contact (FC) characteristics and their integrated actin cables to factors values. The results show that FC maturation/cytoskeletal integration affects factor 5, because FC elongation/integration was correlated with its values. On the contrary, 7 values decreased with maturation, so cable or FC size or their integration must be restricted to form these protrusions. Where integration did occur, the cables showed distinctive size and orientation, as indicated by correlation of 7 values with FC shape. Results obtained with myosin inhibitors support the interpretation that a central, isometric, contractile network puts constraints on both factor 5 and 7 protrusions. We conclude that cells establish functional domains by rearranging the cytoskeleton.

Although growth factor-initiated cascades in cells are networked with mechanisms such as “inside-out signaling”, it is not known how these pathways are integrated. Earlier studies reported that ruffling was enhanced and filopodia reduced in transformed cells. Since dissecting relationships among features was impossible if subjective recognition was relied upon, features in two epithelial cell lines were recognized by latent factor analysis. Factor-based classification revealed four protrusion classes, but none of them corresponded to ruffles. Loss of filopodia, defined by factor 4 (F4) values, accounted for the greatest change in features of oncogenically transformed cells. Factor 5 (F5, lamella) was unchanged during transformation of an airway epithelium cell line. The tumor promoter, phorbol 12-myristate 13-acetate (PMA), increased ruffling but decreased filopodia. F4 retained this relationship to ruffling in untreated cells and at multiple times after treatment. F5 values decreased but were positively correlated with measures of ruffling. Because factors are created as mutually orthogonal variables, this suggested that ruffles were not flagged in factor analysis because they originate from other features. Actin filament capping with sub-micromolar cytochalasin D (Cyto D) suppressed ruffling without affecting F4 or F5. Cyto D increased factor 7 (F7) values, thus showing specificity for this feature. However, cytochalasin treatment of PMA-treated cells that had developed stress fibers increased F4 and decreased F5. The results suggest that PMA changes the state of the cytoskeleton, causing protrusions to show novel responses to Cyto D compared to untreated cells. Results suggest that the factors identify physiologically distinct features.

Cells exposed to phorbol 12-myristate 13-acetate (PMA) undergo a choreographed sequence of morphological changes. Some of these, including stimulation of membrane ruffles and the later appearance of stress fibers, rely on remodeling of the actin cytoskeleton. Although this process is poorly understood, it is important, because the same features are affected during oncogenic transformation. PMA also activates protein kinase C (PKC). Enzyme activation is followed by degradation. Either process might affect the remodeling of actin. The present studies determined whether any PKC isozymes were subject to degradation in tracheal epithelial cells by quantifying the amount of each isozyme present after PMA exposure. PKC-epsilon was the only isozyme to show declining content correlated with increased stress fiber accumulation. Stress fibers increased between 5 and 10 h, whereas PKC-epsilon declined to 38% of its starting value (95% confidence interval, 10-68%). The relationship could be fit by the function F(x) = 0.683 x exp[-0.841(x - 0.387)], where F is the frequency of fiber-containing cells and x is PKC-epsilon content. Fiber accumulation was further investigated after knockdown of PKC-epsilon with RNA interference and antisense oligodeoxynucleotide. Knockdown enhanced stress fibers in cells not yet exposed to PMA as well as the final frequency of fiber-containing cells after PMA exposure. With knockdown at both transcriptional and protein levels, approximately 15% of the original content was predicted and achieved, as judged from real-time PCR and PKC-epsilon content measurements. The results suggest that PKC-epsilon negatively regulates stress fibers, either by directly turning over one of their components or by regulating an upstream step affecting fiber organization.

The PKCs comprise 12 isoforms, and their substrate-specificity is regulated in part by binding to receptors for C kinases (RACKs). Certain isoforms are activated by phorbol 12-myristate 13-acetate (PMA), which also affects shape and adhesion. Different cell types express different subsets of isoforms. In tracheal epithelial cells, the activation of beta and epsilon isoforms by PMA was followed by their degradation. We sought to determine whether such activation-degradation patterns affected protrusion formation or turnover. Cells were treated by transcriptional knockdown (KD) and the prevalence of each protrusion class was analyzed by computerized morphometry. Latent factors for filopodia (#4) and nascent neurites (#7) were calculated based on geometric variables determined for known populations. At zero time, factor #7 was elevated by alpha KD. Control samples, into which a random KD sequence had been introduced, showed an increase in neurites throughout the PMA exposure. During the time course, all KD samples differed from controls at one or more times. Epsilon KD followed by PMA drastically downregulated the isoform by 15 h and decreased #7 values relative to control. Alpha KD was similar suggesting that the early KD effect (increasing #7) was reversed later on. Beta KD cells resembled control at 15 h but alpha KD at the 5-h time. Filopodia were eliminated by a 2-h PMA exposure regardless of the KD agent introduced. Control samples remained depressed, but epsilon and alpha KD samples partially recovered by 5 h. Alpha KD declined again dramatically by 15 h. The time courses suggest that the main effects were exerted by the actin-binding PKC epsilon, but alpha, which is not actin-binding, could have similar effects. The data suggest that competitive binding on RACKs may be occurring, complicating the picture of kinase regulation of adhesion and protrusive activities.

During long-term culture, certain lines become neoplastic while accumulating changes in cell shape. Early and late cell populations have characteristic shape phenotypes that have been quantified by computerized assay. Phenotypes are determined from variables describing three-dimensional aspects of the subcellular distribution of mass. The features of cells can be recognized by use of latent factors, which are theoretical variables based on the covariance of the primary variables. Factor #7 represented a cell edge feature different from filopodia. We studied the morphological characteristics and morphogenesis of the feature. Brief exposure of cells from rat tracheal epithelium to phorbol 12-myristate 13-acetate (PMA) enhanced #7 values. The time to reach maximal #7 values was prolonged if PMA was administered with calcium ionophore or lysophosphatidic acid (LPA). Factor #7 was elevated during periods of ruffling suppression and stress fiber reorganization. Cells showing high #7 values were examined by scanning electron microscopy (SEM) and found to exhibit strap-shaped and cupola-shaped projections. Because RhoA regulates stress fiber formation, we sought to perturb #7 features by introducing dominant-acting negative and positive constructs of RhoA, RhoA-N19, and RhoA-V14. Neither affected #7 values. Although overexpression of the kinase inhibitory domain of p21-activated kinase 1 (PAK) had no effect on #7 values, they were affected by overexpression of a domain binding PAK-interacting guanine nucleotide exchange factor (PIX). Because a PAK-PIX complex is implicated in the remodeling of focal complexes (FCs) and recycling of PAK to the cytoplasm, the results implicate a component of FCs in the formation of #7 features. The data suggested that feature formation is driven by activated Cdc42-binding kinase (ACK) and Rac. Moreover, they suggested that the #7 protrusions are neurite-like structures and that their development involves FC regulation.

The contractility of guinea pig ileum and frog skeletal muscle is inhibited in solutions containing 0.4 to 2.5 M formamide (FMD). Contrary to mammalian vis ceral muscle, this blocking action is not reversed when frog muscles are transferred back to isotonic Ringer's after FMD treatment. Under these conditions the water content of the skeletal muscles is markedly increased and electronmicrographs show a swellingof the transverse tubules. These changes are not observed when frog muscles are transferred to ethylene glycol solutions that are isosmotic with the FMD containing Ringer's solution. In addition, over 50% of the contractility is recovered in these muscles. These observations provide direct evidence of the occurrence of an osmotic shock in frog muscles transferred from FMD solutions to isotonic Ringer's. It is concluded that the resulting alterations in the triad structure and function are re sponsible for the irreversibility of the FMD uncoupling action in these muscles.

When immature oocytes of the starfish, Asterina pectinifera, were treated with calcium-free seawater for 1 hr and then inseminated in normal seawater, they formed several blisters, indicative of polyspermy, and raised fertilization membranes. These oocytes continued to have intact germinal vesicles. Electron microscopic study revealed that the egg surface remained virtually unchanged after the treatment with calcium-free seawater. Upon insemination, however, the cortical granules broke down and the fertilization membrane was formed. These immature oocytes with ferilization membranes underwent maturation (germinal vesicle breakdown) after treatment with 1-methyladenine. The treatment with calcium-free seawater seems to bring about some physiological change on the surface of immature oocyte, which bestows some attributes of maturation but is insufficient to mature the oocytes completely.

Septate junctions have been studied in the hind-gut of Periplaneta americana, Incisitermes schwartzi and Thermobia domestica. The topographical disposition of intercellular septa revealed by lanthanum impregnation corresponds well with that of particle rows seen in freeze fracture preparations. However, there is no precise correspondence between the undulations of septa and the disposition of particles within a single row. The spacing of particles is variable and generally less than that of the undulation periodicity of septa. On the other hand, the disposition of septa, and of the rows of particles that correspond to them, appears to be variable: sometimes rectilinear and in close parallel, these may curve or form series of 'finger-print' loops. Moreover, the septa are evidently not deployed as continuous ribbons around the cell since intrruptions are freuqently observed. In addition to their mechanical role in intercellular cohesion, septate junctions apparently ensure a more or less complete closure of the intercellular space (i.e. provide a permeability barrier) and thus play a role comparable with that of tight junctions in epithelia of vertebrates.

Spine catch ligaments of a sea urchin Arbacia punctulata were extended under constant load. Ligaments from an undisturbed animal may show any extension rate from zero (catch state) to rapid extension to failure. Replacing the preparation bath with CA(2)- and Mg(2+)-free sea water reversibly abolishes the catch state. The fine structure of the outermuscle layer and inner ligament cone associated with the spine base is described. The unstriated paramyosin muscles bear thin flanges and form compact interlocking rows. Subsurface cisternae are associated with the plasma membrane. The muscles are innervated by glia-free axons ending in bulbous terminals containing lucent synaptic vesicles. The ligament comprises cylindrical bundles of collagen fibrils: one or more minute muscle fibers (paramyosin) lie parallel with and closely adjoining each bundle. The mean diameter of these muscles is 0.3 micrometers and they occupy 2-3% of the ligament's cross-sectional area. Axons containing electron-opaque secretory droplets accompany the muscles between the collagen bundles: the cell bodies of these neurones generally lie on the outer surface of the ligament. When an urchin points a spine, the ligament on the side of the contracting spine muscle shortens but does not buckle. A function of the intraligamental muscles is to effect this non-buckled shortening. The catch mechanism (which reside entirely within the ligament) may be due either to the intraligamental muscles and/or to a locked polymer mechanism in which matrix molecules between collagen fibrils are reversibly cross-linked by divalent cations.

The distribution of microtubules and mitochondria in central axons of an insect (Periplaneta americana) is assessed by comparison between counts on micrographs and computed axon random ‘models’. These studies show that the observed multiple association of microtubules with individual mitochondria is statistically highly significant. Electron micrographs of thin sections show that linkage is effected by physical cross-bridges, possibly comprising components from the microtubule and mitochondrion. Linear particle arrays are described on the outer mitochondrial membrane in freeze-fracture replicas, and tentatively related to the bridges seen in thin sections. The results are discussed in terms of proposed roles of microtubules in neurons and other cells.

Disulfide-reducing agents such as DTT and 2,3-dimercapto-1-propanol (BAL) are known to induce starfish oocyte maturation [16]. However, microinjection of DTT into the immature oocytes of the starfish, Asterina pectinifera, failed to induce oocyte maturation, suggesting that the substance acts on the oocyte surface from the outside and indirectly induces oocyte maturation. Microinjection of the cytoplasm taken from the DTT-treated oocytes into immature oocytes was found to induce oocyte maturation, indicating that the cytoplasmic factor triggering the germinal vesicle breakdown and subsequent meiotic process is produced under the influence of DTT, as well as 1-methyladenine (1-MeAde). The pattern of changes in maturation-inducing capacity of the maturing cytoplasm of the DTT-treated oocytes was similar to that of the 1-MeAde-treated oocytes. Morphological changes in the oocyte surface and the vitelline coat of the DTT-treated oocytes were similar to those in the 1-MeAde-treated oocytes. The action of DTT in inducing maturation is considered to be the same as that of 1-MeAde. Sulfhydryl content of oocyte-cortex protein was found to increase after the treatment with 1-MeAde, suggesting that the action of 1-MeAde in inducing oocyte maturation is to be ascribed to its action of reducing disulfide bond.

The complement system of the nurse shark was investigated. Six functionally pure components were isolated from a single serum sample. Sequential reactions of the components with sensitized sheep erythrocytes resulted in membrane lesions indistinguishable from the "holes" caused by guinea pig complement.

In termites and roaches the well-defined rectal papillae each comprise a layer of columnar principal cells specialized for active transport and a layer of basal cells. The whole cell group is entirely surrounded by several series of flattened 'sheath cells' (formerly termed 'junctional cells') which abut onto the basal components of the papilla. The sheath cells secrete a specialized sclerified cuticle which forms the framework of the papilla. Their regularly pleated apical membrane is closely apposed to the cuticle and contains parallel and closely spaced rows of intramembranous particles. at this level, no subcuticular space is present and hence the space associated with the apical surface of the principal cells is defined as an isolated compartment. Typical septate junctions are present between the sheath and basal cells; however those linking adjacent sheath cells are structurally unusual: they extend to the basal surface rather than being restricted to the apical zone, are frequently interrupted and in replicas are represented by relatively short and irregularly oriented particle rows. Moreover, lateral sheath cell contacts display two further peculiarities: absence of an apical desmosomal ring and paucity of gap junctions. Structural observations suggest that the sheath cells isolate the principal cells from communication with the hemolymph, consequently enhancing their efficiency in water and ionic regulation. Comparable cells have been described in a number of insects, but the 'isolation' system presents varying degrees of complexity, for which an evolutionary scheme is proposed.

Pure preparations of Trypanosoma brucei , free from plasma and cellular components were isolated from rat blood, and concentrated into loose pellets by low-speed centrifugation. Pellets were either processed for thin sectioning as a control for general morphology, or glycerol-treated after glutaraldehyde fixation for preparation of freeze-fracture replicas. Concentration of cells of 50,000–100,000/mm 2 of sectioned or fractured surface facilitated identification of fracture faces of the cell body, invaginated flagellar pocket and flagellum. Particle distribution and A and B faces of these regions of the cell are described. A collar of B face particles occurs around the neck of the flagellar pocket, possibly associated with a junction controlling ingress of ingested materials to coated vesicles formed along the membrane defining the pocket. A and B faces of the flagellum and adjoining surface of the cell body have shown that the only intra-membrane specialization corresponding to the miniature ‘maculae adherentes’ described previously in thin sections is probably an uninterrupted series of small clusters (3–6) of 80 A particles on the A face of the flagellar membrane. It is proposed that these arrays represent attachment points for strands linking the axoneme and paraxial rod to the flagellar surface, and are not directly concerned with the physical adhesion of the flagellum to the cell body surface—a linkage that appears to be established within the extracellular gap between these apposed surfaces of the cell. The potential use of freeze-etching in further study of the external antigens of the infective cell is discussed.

We devised a method to enhance delivery of vinblastine to macrophages, the cells believed to be responsible for platelet destruction in idiopathic thrombocytopenic purpura. Our strategy was based on the ability of platelets to bind vinca alkaloids such as vinblastine. Platelets were incubated with an excess of vinblastine, concentrated and then, after excess alkaloid had been removed, given to 11 patients with idiopathic thrombocytopenia refractory to other treatment (including intravenous injections of vinca alkaloids). Platelet antibodies in the patients' plasma led to ingestion of the vinca-laden platelets by macrophages. There were six complete remissions, three partial remissions and two failures. Side effects in a few patients, reversible but annoying, were minimized as technics were refined. We conclude that in patients with idiopathic thrombocytopenia refractory to all other measures, including the use of vinca alkaloids, platelet-vinca complex may be effective.

A highly purified preparation of horse erythrocyte glycoprotein was prepared from an aqueous ethanolic extract of hemoglobin-free membranes. The subunit apparent mol. wt was 30,000. In aqueous solution the glycoprotein formed globular aggregates of 93 ± 16 A diameter. The glycoprotein had a receptor for the Paul-Bunnell antibody of infectious mononucleosis which was associated with an O -glycosidically linked oligosaccharide and dependent on the presence of N -glycolylneuraminic acid. In addition the glycoprotein had a neuraminidase-sensitive receptor for human peripheral blood lymphocytes. Fifty per cent inhibition of the rosetting of sheep red cells by 4 × 10 5 lymphocytes was caused by 30 μg of glycoprotein.

Self-assembly of actin-myosin filamentous complexes was assayed by polymerizing rabbit G-ADP actin on formed filaments of lobster myosin. The resulting contractile units indicate a 12-member actin orbital rather than the six-member orbital obtained previously using rabbit myosin and actin. Furthermore, the pattern of actin distribution surrounding the myosin filament is similar to that of the lobster tonic muscle sarcomere rather than the trigonal actin position characteristic of vertebrate muscle. The results show that the pattern and mode of actin complexing is determined by the specific myosin and the arrangement of the cross-bridges on the organized filament.

Tonic muscle of the crusher claw of the American lobster (Homarus americanus) was investigated with respect to sarcomeric organization and the capacity for self-assembly of extracted myosin for comparison with the same properties of rabbit muscle. Native myosin filaments in the lobster muscle are much longer than in rabbit skeletal fibers, and differ further in sarcomeric organization in showing an actin-to-myosin relationship in which two actin filaments are shared between adjacent myosins in a 12-membered orbital. The self-assembly of lobster myosin into filaments comparable in length and the fine structure to the natural filament was achieved in the presence of excess Mg2+, a condition not required for rabbit myosin self-assembly. Results of in situ and self-assembly studies indicate a difference in molecular organization between lobster and rabbit myosin filaments and of the inferred presence of regulatory factors in the formation of these ultrastructural elements. These studies represent the groundwork for an investigation of in vitro polymerization of actin in association with the synthetic lobster myosin filament.