Organization Of Cytoskeletal Elements Research Articles

Functional Analysis of Phospholipid Signaling and Actin Dynamics: The Use of Apical Growing Tobacco Pollen Tubes in a Case Study.

Signaling molecules are crucial to perceive and translate intra- and extracellular cues. Phosphoinositides and the proteins responsible for their biosynthesis (e.g., lipid kinases) are known to influence the (re)organization of cytoskeletal elements, namely, through interaction with actin and actin-binding proteins. Here we describe methods to functionally characterize lipid kinases and their phosphoinositide metabolites in relation to actin dynamics. These methods include GFP-tagged protein expression followed by time-resolved live imaging and quantitative image analysis. When combined with biochemical and interaction studies, these methods can be used to correlate signaling with actin dynamics, microfilament assembly, and intracellular trafficking, linking structure and function.

Notch-dependent Abl signaling regulates cell motility during ommatidial rotation in Drosophila.

A collective cell motility event that occurs during Drosophila eye development, ommatidial rotation (OR), serves as a paradigm for signaling-pathway-regulated directed movement of cell clusters. OR is instructed by the EGFR and Notch pathways and Frizzled/planar cell polarity (Fz/PCP) signaling, all of which are associated with photoreceptor R3 and R4 specification. Here, we show that Abl kinase negatively regulates OR through its activity in the R3/R4 pair. Abl is localized to apical junctional regions in R4, but not in R3, during OR, and this apical localization requires Notch signaling. We demonstrate that Abl and Notch interact genetically during OR, and Abl co-immunoprecipitates in complexes with Notch in eye discs. Perturbations of Abl interfere with adherens junctional organization of ommatidial preclusters, which mediate the OR process. Together, our data suggest that Abl kinase acts directly downstream of Notch in R4 to fine-tune OR via its effect on adherens junctions.

An unconventional road for the secretion of transglutaminase in pollen tubes?

The transglutaminase (TGase) is present in the pollen tube where it most likely participates in the regulation of different activities including the organization of cytoskeletal elements (microtubules and actin filaments). In addition to a cytosolic form of TGase, new data suggest the existence of TGase forms associated with the internal membranes and with the cell wall of pollen tubes. This different localization extends the functional range of pollen TGase but also raises the question how TGase can be precisely (and in harmony with the pollen tube growth) redistributed in different cellular compartments. The discovery that TGase exists as different isoforms may suggest a pathway to achieve this result.

Efficient induction of apoptosis in HeLa cells by a novel cationic porphycene photosensitizer

In the present study we analyze the photobiological properties of 2,7,12-tris(α-pyridinio-p-tolyl)–17-(p-(methoxymethyl)phenyl) porphycene (Py3MeO-TBPo) in Hela cells, in order to assess its potential as a new photosensitizer for photodynamic therapy of cultured tumor cells. Using 0.5 μM Py3MeO-TBPo, flow cytometry studies demonstrated an increase of intracellular drug levels related to the incubation time, reaching a maximum at 18 h. LysoTracker® Green (LTG) and MitoTracker® Green (MTG) probes were used to identify the subcellular localization. Upon exposure to ultraviolet excitation, red porphycene fluorescence was detected as red granules in the cytoplasm that colocalized with LTG. No significant toxic effects were detected for Py3MeO-TBPo in the dark at concentrations below 1 μM. In contrast, Py3MeO-TBPo combined with red-light irradiation induced concentration- and fluence-dependent HeLa cells inactivation. Besides, all photodynamic protocols assayed induced a clear effect of cell detachment inhibition after trypsin treatment. Both apoptotic and necrotic cell death mechanisms can occur in HeLa cells depending on the experimental protocol. After 18 h incubation with 0.5 μM Py3MeO-TBPo and subsequent red light irradiation (3.6 J/cm2), a high number of cells die by apoptosis, as evaluated by morphological alterations, immunofluorescent relocalization of Bax from cytosol to mitochondria, and TUNEL assay. Likewise, immunofluorescence techniques showed that cytochrome c is released from mitochondria into cytosol in cells undergoing apoptosis, which occurs immediately after relocation of Bax in mitochondria. The highest amount of apoptosis appeared 24 h after treatment (70%) and this cell death occurred without cell detachment to the substrate. In contrast, with 0.75 μM Py3MeO-TBPo and 3.6 J/cm2 irradiation, morphological changes showed a preferential necrotic cell death.Singlet oxygen was identified as the cytotoxic agent involved in cell photoinactivation. Moreover, cell cultures pre-exposed to the singlet oxygen scavenger sodium azide showed pronounced protection against the loss of viability induced by Py3MeO-TBPo and light.Different changes in distribution and organization of cytoskeletal elements (microtubules and actin microfilaments) as well as the protein vinculin, after apoptotic and necrotic photodynamic treatments have been analyzed. Neither of these two cell death mechanisms (apoptosis or necrosis) induced cell detachment.In summary, Py3MeO-TBPo appears to meet the requirements for further scrutiny as a very good photosensitizer for photodynamic therapy: it is water soluble, has a high absorption in the red spectral region (where light penetration in tissue is higher), and is able to induce effective high apoptotic rate (70%) related to the more widely studied photosensitizers.

Effect of Vitrification of Feline Ovarian Cortex on Follicular and Oocyte Quality and Competence

Cryopreservation of ovarian cortex has important implications in the preservation of fertility and biodiversity in animal species. Slow freezing of cat ovarian tissue resulted in the preservation of follicular morphology and in the follicular development after xenografting. Vitrification has been recently applied to ovarian tissues of different species, but no information is available on the effect of this method on feline ovarian cortex. Moreover, meiotic competence of fully grown oocytes isolated from cryopreserved tissue has not been reported. The aim of this study was to evaluate the effect of vitrification of feline ovarian cortex on follicular morphology and oocyte integrity, as well as meiotic competence. A total of 352 fragments (1.5-2 mm(3) ) were obtained from ovarian cortical tissues: 176 were vitrified and 176 were used fresh as control. Histological evaluation of fresh and vitrified fragments showed intact follicles after cryopreservation procedures with no statistically significant destructive effect from primordial to antral follicles. After IVM, oocytes collected from vitrified ovarian fragment showed a higher proportion of gametes arrested at germinal vesicle (GV) stage compared to those isolated from fresh control tissue (33.8% vs 2.9%; p < 0.001). However, oocytes isolated from vitrified tissues were able to resume meiosis, albeit at lower rate than those collected from fresh tissues (39.8% vs 85.9%; p < 0.00001). Vitrification induced changes in the organization of cytoskeletal elements (actin microfilaments and microtubules) of oocytes, but significantly only for actin network (p < 0.001). Finally, chromatin configuration within the GV was not affected by the cryopreservation procedure. Our study demonstrated that vitrification preserves the integrity of ovarian follicles and that oocytes retrieved from cryopreserved tissue maintain the capability of resuming meiosis. To our knowledge, this has not previously been reported in the cat.

Rho GTPases: regulation of cell polarity and growth in yeasts

Eukaryotic cells display a wide range of morphologies important for cellular function and development. A particular cell shape is made via the generation of asymmetry in the organization of cytoskeletal elements, usually leading to actin localization at sites of growth. The Rho family of GTPases is present in all eukaryotic cells, from yeast to mammals, and their role as key regulators in the signalling pathways that control actin organization and morphogenetic processes is well known. In the present review we will discuss the role of Rho GTPases as regulators of yeasts' polarized growth, their mechanism of activation and signalling pathways in Saccharomyces cerevisiae and Schizosaccharomyces pombe. These two model yeasts have been very useful in the study of the molecular mechanisms responsible for cell polarity. As in other organisms with cell walls, yeast's polarized growth is closely related to cell-wall biosynthesis, and Rho GTPases are critical modulators of this process. They provide the co-ordinated regulation of cell-wall biosynthetic enzymes and actin organization required to maintain cell integrity during vegetative growth.

Membrane-mediated interactions drive the condensation and coalescence of FtsZ rings

The role of the coupling between the shape of membrane-bound filaments and the membrane is demonstrated for the dynamics of FtsZ rings on cylindrical membranes. Filaments with an arc-like spontaneous curvature, and a possible added active contractile force, are shown to spontaneously condense into tight rings, associated with a local inward deformation of the membrane. The long-range membrane-mediated interactions are attractive at short ring–ring separations, inducing further coarsening dynamics, whereby smaller rings merge to form larger and fewer rings that deform the membrane more strongly. At the same time, these interactions induce a potential barrier that can suppress further ring coalescence at a separation of about seven times the cylinder radius. These results of the model are in very good agreement with recent in-vitro experiments on the dynamics of FtsZ filaments in cylindrical liposomes. These results emphasize the important role of long-range membrane-mediated interactions in the organization of cytoskeletal elements at the membrane.

Tau gene alternative splicing: expression patterns, regulation and modulation of function in normal brain and neurodegenerative diseases

Organization of cytoskeletal elements is critical for cellular migration and maintenance of morphology. Tau protein, which binds to and organizes microtubules, is instrumental in forming and maintaining the neuronal axon. Disturbances in tau expression result in disruption of the neuronal cytoskeleton and formation of pathological tau structures (neurofibrillary tangles, NFTs) found in brains of dementia sufferers. Null tau mice, although viable, exhibit developmental and cognitive defects and transgenic mice which overexpress tau develop severe neuropathies. The neuron-specific tau transcript produces multiple isoforms by intricately regulated alternative splicing. These isoforms modulate tau function in normal brain. Moreover, aberrations in tau splicing regulation directly cause several neurodegenerative diseases. Thus, tau splicing regulation is vital to neuronal health and correct brain function. This review briefly presents our cumulative knowledge of tau splicing-cis elements and trans factors which influence it at the RNA level, its effect on the structure and roles of the tau protein and its repercussions on neuronal morphology and neurodegeneration.

Isoflurane alters angiotensin II-induced Ca2+ mobilization in aortic smooth muscle cells from hypertensive rats: implication of cytoskeleton.

Angiotensin II (AngII) is a potent vasoconstrictor involved in the short-term control of arterial blood pressure. Isoflurane was reported to decrease vascular tone through an alteration of vascular smooth muscle cell vasomotor response to several agonists, but its effect on AngII signaling is not known. On the other hand, vascular response to AngII is altered in hypertension. In this study, the authors tested the hypothesis that (1) isoflurane alters AngII-induced intracellular Ca mobilization in aortic vascular smooth muscle cell from Wistar Kyoto and spontaneously hypertensive rats, and (2) this effect could be associated with an alteration of the organization of microtubular network, reported to be involved in AngII signaling. The effect of 0.5-3% isoflurane was studied (1) on AngII (10 m)-induced intracellular Ca mobilization, intracellular Ca release from internal stores, and Ca influx in Fura-2 loaded cultured aortic vascular smooth muscle cell isolated from 6-week-old Wistar Kyoto and spontaneously hypertensive rats, using fluorescent imaging microscopy; and (2) on the organization of cytoskeletal elements, using immunofluorescence labeling. In both stains, isoflurane decreased in a concentration-dependent manner AngII-induced intracellular Ca mobilization, Ca release from internal stores, and Ca influx through nifedipine-insensitive Ca channels. This effect occurred at a lower concentrations of isoflurane in Wistar Kyoto rats than in spontaneously hypertensive rats. In both strains, the effect of isoflurane on AngII- Ca mobilization was abolished by impairment with nocodazole, vinblastine, or paclitaxel of microtubules polymerization. Isoflurane directly altered tubular network organization in a concentration-dependent and reversible manner. Isoflurane decreased AngII-induced Ca mobilization at clinically relevant concentrations, suggesting that vascular response to AngII could be altered during isoflurane anesthesia. The hypertensive strain was found less sensitive than the normotensive one. In both strains, the isoflurane effect was associated with a microtubular network interaction.

Intracellular P-glycoprotein expression is associated with the intrinsic multidrug resistance phenotype in human colon adenocarcinoma cells

The 2 clones, LoVo 5 and LoVo 7, derived from untreated LoVo WT human colon adenocarcinoma cells and exhibiting different sensitivity to doxorubicin (DOX), were compared in order to identify possible determinants of intrinsic drug resistance. A multidrug resistant variant cell line, selected from LoVo WT cells by continuous exposure to DOX (LoVo DX), was also included in the study. Analysis of the expression and organization of cytoskeletal elements by flow cytometry and fluorescence microscopy evidenced a positive correlation between vimentin expression and DOX resistance in LoVo 7 and LoVo DX cells, whereas differences in actin, tubulin or cytokeratin did not seem to relate to drug response. The expression and localization of different drug transporters commonly implicated in drug resistance, i.e., the MDR1 gene product P-glycoprotein (P-gp), the multidrug resistance-related protein MRP and the lung resistance-related protein LRP were also investigated by means of flow cytometry and fluorescence microscopy, following labeling with specific monoclonal antibodies. Surface expression of P-gp was only detected in LoVo DX cells, which also exhibited increased MRP and LRP protein levels. However, significant amounts of P-gp were found at intracellular sites in the intrinsically resistant LoVo 7 clone. Modulation of P-gp function by cyclosporin A was found to alter DOX accumulation and efflux in LoVo 7 cells, indicating that intracellular P-gp plays a functional role in drug trafficking and suggesting possible implications in determining the intrinsic resistance displayed by this clone.

Function of p55 and its nonerythroid homologues.

The human erythrocyte membrane has served as a model for elucidating novel protein-protein and protein-membrane interactions that have broad implications in many nonerythroid cells. A detailed analysis of erythrocyte membrane polypeptides that migrate in the region of band 4.9 led to the cloning and characterization of p55 phosphoprotein. Subsequent studies established that the p55 protein is an obligate component of the protein 4.1-glycophorin C complex, which regulates the stability and mechanical properties of the erythrocyte plasma membrane. p55 is a member of a growing family of signaling and cytoskeletal proteins termed membrane-associated guanylate kinase homologues (MAGUKs). MAGUKs are multidomain proteins consisting of either a single or three copies of the PDZ (PSD-95/Discs large/ZO-1) domain, an SH3 motif, and a guanylate kinaselike domain. Recent studies have implicated MAGUKs in the clustering of ion channels, organization of cytoskeletal elements, cell signaling events, and regulation of cell proliferation and tumor-suppression pathways. The purpose of this review is to summarize recent developments concerning the characterization of two human MAGUKs, erythrocyte p55, and lymphocyte hDIg.

Confocal microscopy of cytoskeletal organization in amphibian oocytes and eggs

Amphibian oocytes, eggs, and early embryos have been increasingly used as models for studying the regulation of microtubule assembly, the regulation and mechanics of spindle assembly, and the generation of cellular and embryonic axes. However, little information has been available regarding the organization of cytoskeletal elements in vivo during oogenesis. In large part, this results from the unique challenges posed by immunofluorescence microscopy of these large cells (up to 2 mm in diameter). Preservation and staining of labile cytoskeletal elements is often difficult in large cells, due to the limited penetration of fixative and antibodies or other dyes. In addition, the accumulation of yolk during oogenesis renders larger oocytes, eggs, and early embryos opaque. Finally, cell size also often limits the magnification and resolution possible, since the details of cytoskeletal organization in large oocytes and eggs often are obscured by fluorescence from outside of the focal plane when objectives with high numerical aperture are used.

Alterations in cytoskeletal protein sulfhydryls and cellular glutathione in cultured cells exposed to cadmium and nickel ions

To understand the mechanisms of Cd 2+ and Ni 2+ cytotoxicity, we have studied the effects of these two metal ions on the organization of cytoskeletal elements, microtubules (MT) and microfilaments (MF), cytoskeletal protein sulfhydryls and cellular glutathione (GSH) in cultured 3T3 cells. At a metal ion dose that caused 95% inhibition of DNA synthesis, Cd 2+ (10 μM, 16 h exposure) induced MT depolymerization whereas Ni 2+ (2 mM, 20 h exposure) elicited MT aggregation and bundling. Under these conditions, Cd 2+ and Ni 2+ also caused MF aggregation and redistribution. Furthermore, exposure of cells to Cd 2+ resulted in a dose-dependent increase in cytoskeletal protein sulfhydryls and cellular GSH levels. In contrast, treatment of cells with Ni 2+ resulted in a dose-dependent decrease in cytoskeletal protein sulfhydryls as well as cellular GSH content. Time course studies showed that cells exposed to 10 μM Cd 2+ exhibited a biphasic response in regulating their cytoskeletal protein sulfhydryls and cellular GSH, e.g. an initial decrease followed by a steady recovery and overshooting upon prolonged incubation. However, restoration of cytoskeletal protein sulfhydryls occured ∼ 2 h after commencement of cellular GSH recovery in Cd 2+-treated cells. These results suggest that cellular GSH may play an important role in regulating cytoskeletal protein sulfhydryls. On the other hand, decrease of cellular GSH induced by Ni 2+ might facilitate oxidation of cytoskeletal protein sulfhydryls and formationn of disulfide bonds between individual MT polymers which would favor MT aggregation in Ni 2+-exposed cells. in addition, we also demonstrated that elevation of cellular GSH in Cd 2+-treated cells probably resulted from new GSH synthesis.

Measurements of cells in culture by scanning acoustic microscopy

A novel method allowing the determination of thickness, attenuation and impedance of both living and fixed cells in culture using a single image recorded with a scanning acoustic microscope is described. The method is based on the recording of the image data locating the surface of the cell far below the geometric focus. In this way only the normal incident acoustic wave is used for image formation. Higher values of impedance and attenuation coefficients were found in the cell periphery than in the central part of the cell. This phenomenon is suggested to be due to the different organization of cytoskeletal elements.

Isolation of Swiss 3T3 cell variants with altered heparan sulfate

The replica filter technique has been used to isolate variants of Swiss mouse 3T3 cells which produce heparan sulfates with altered levels of sulfation. These changes in the extent of sulfation correlate with alterations in cell morphology, in the organization of cytoskeletal elements, focal contacts, and the extracellular matrix, and in the growth regulation of cells, as expressed by saturation density. An increase in the extent of heparan sulfate sulfation occurs concomitantly with a decreased saturation density and enhanced focal contact formation. In contrast, graded decreases in sulfation correlate with graded increases in saturation density and losses of cytoskeletal and extracellular matrix organization. These graded responses appear very similar to those which have been reported for the transformation of cells with fusiform mutants of Rous sarcoma virus or the adenovirus type 2 E1a transforming gene and suggest that the morphological changes observed in the transformed cells can be controlled by cellular systems.

A morphometric study of cytoskeletal reorganization in rat sciatic nerve axons following β,β′-iminodipropionitrile (IDPN) treatment

Electron microscopy and morphometry were used to study changes in the organization of cytoskeletal elements (microtubules (MTs) and neurofilaments (NFs)) in myelinated rat sciatic nerve axons resulting from exposure to β,β′-iminodipropionitrile (IDPN). Adult male rats were injected subepineurially with IDPN and sacrificed at intervals. Electron micrographs of thin sections through injected nerve segments revealed that MTs segregated from NFs in axons and formed prominent clusters. Morphometric measurements of intermicrotubular distances within these clusters showed that adjacent MTs had a fairly uniform spatial separation that appeared to be maintained by granular MT-associated cross-bridges. In addition, MT clusters underwent a progressive, but reversible, compaction during the first 12 h of IDPN treatment.

Extracellular matrix-cytoskeleton interactions in locomoting embryonic cells

Despite the great advances in understanding the composition and organization of the cytoskeleton in recent years, the molecular mechanism of cell locomotion is poorly understood. One of the main reasons for our ignorance is that the biochemical machinery responsible for these movements is not localized precisely to a single structure, but is widely distributed throughout the cell. Furthermore, because the machinery is labile and easily disrupted during biochemical fractionation, it is difficult to isolate in a functional form. So far, most studies on cell locomotion used fibroblasts as a model system. This system appeared particularly suitable for understanding not only the organization of cytoskeletal elements within the cells but, most interestingly, the flow of membrane components throughout the cell surface during extension of the leading lamellipodia and retraction of the trailing edge (for reviews, ABERCROMBIE 1980, BRETCHER 1984, ISHIHARA et al. 1988, RINNERTHALER et al. 1988). However, many of these studies dealt with surface molecules that are not involved in the adhesion of the cell to its substratum and thus that probably exhibit a different behavior within the plane of the membrane. A possible alternative is to examine in molecular terms how the moving cells interact with their substratum. Since cells naturally crawl in a complex network of extracellular matrix molecules, it is necessary to search among these molecules the ones which mediate the anchorage of the

Cell configuration and adhesive properties of metastasizing and non-metastasizing BSp73 rat adenocarcinoma cells

The pattern of cell substrate interaction, the cell surface composition and the organization of cytoskeletal elements was studied in tumour cell variants of the BSp73 rat adenocarcinoma displaying different metastatic capabilities and cell configuration. The non-metastasizing AS variant cells adhered to the substrate and spread via vinculin-containing focal contacts. These cells also synthesized, secreted and assembled fibronectin at the pericellular area. The metastasizing ASML variant cells adhered to the substrate at a slower rate via thick cytoplasmic protrusions, but were removed from the substrate by trypsin-EDTA slower than the non-metastasizing AS variant cells. The ASML cells also synthesized very low levels of both vinculin and fibronectin, displayed a diffuse pattern of actin and tubulin organization, and were unable to spread on the substrate. Spreading could not be induced in the ASML cells by seeding the cells on an extracellular matrix derived from bovine corneal endothelial cells or on concanavalin A (conA)-coated substrates, or by the addition of db-cAMP to the medium. The metastasizing cells expressed a unique and abundant cell surface glycoprotein of M r 170 000 which was also shedded into the growth medium. The relationships among the adhesive properties, the organization of cell surface components and of the cytoskeleton in the tumour cell variants, and the expression of their metastatic phenotype is discussed.

Cytoskeletal organization in locomoting cells of the V2 rabbit carcinoma

The relationship between the organization of cytoskeletal elements and locomotory activity was studied in single cells of the V2 rabbit carcinoma. Like migratory fibroblasts, and unlike colony-forming epithelial cells, these cells show a pronounced horizontal polarization, and develop a large lamella at their leading front. With affinity-purified antibodies and a combination of light and electron microscopic techniques, actin and alpha-actinin (but not myosin and tropomyosin) were found highly concentrated within the marginal region of the leading lamella, both in ruffles and in the underlying zone of contacts with the substratum. Close contacts prevailed in the locomotory cells and small focal contacts developed only in cells detaching from others. Focal contacts always contained small microfilament bundles. Reorganization of actin filaments is suggested as the fundamental event for the dynamic contact formation of the leading lamella. Large microfilament bundles (stress fibers) were absent in all stages of locomotion. Since locomotory behavior and shape changes of V2 cells are the same on glass as on the surface of a natural membrane, the rabbit mesentery, organization and distribution of contractile elements of cultured V2 cells probably reflect the in vivo situation.

Effects of free fatty acids on the organization of cytoskeletal elements in lymphocytes.

Treatment of mouse lymphocytes with cis-unsaturated free fatty acids produced alterations in the immunofluorescence patterns of the cytoskeleton and contractile proteins. Saturated free fatty acids and trans-unsaturated free fatty acids had no effect. In untreated cells, the microtubular pattern exhibited radiation from an organizing center, resembling the spokes of an umbrella. The addition of linoleic acid produced a polarized submembranous aggregate. Under control conditions, staining for actin revealed a diffuse pattern over the entire cell, but the addition of linoleic acid caused the formation of a single large patch, or polarized submembranous aggregate. The pattern for alpha-actinin normally revealed intense perinuclear staining on a diffuse background. Linoleic acid caused the loss of this pattern and the formation of a polarized submembranous aggregate. Linoleic acid treatment also caused the pattern for myosin to change from diffuse to uniform submembranous patching around the periphery of the cell. For all of these proteins, calcium (8 mM), but not magnesium, partially reversed the effects of linoleic acid. Sodium azide had little effect on the normal distribution of actin, tubulin, and alpha-actinin; however, myosin staining revealed prominent patch formation. Colchicine treatment caused diffuse staining, some polarized submembranous aggregate formation of tubulin, and some patching of myosin, but not as extensively as did treatment with linoleic acid. Actin and alpha-actinin were unaffected. These results, in view of the previously shown facts that pretreatment of cells with linoleic acid followed by anti-immunoglobulin inhibits capping of surface immunoglobulin (Klausner, et al., Proc. Natl. Acad. Sci. U.S.A. 77:437-441, 1980) and that free fatty acids partition into the surface membrane (Klausner et al., J. Biol. Chem. 255:1286-1295, 1980), suggest that the perturbation of the plasma membrane with unsaturated free fatty acids alters the interaction of surface receptors with the cytoskeleton, which in turn affects cytoplasmic distribution of the proteins.