Spatial organization of the cytoskeleton enhances cargo delivery to specific target areas on the plasma membrane of spherical cells.

Intracellular transport is vital for the proper functioning and survival of a cell. Cargo (proteins, vesicles, organelles, etc) is transferred from its place of creation to its target locations via molecular motor assisted transport along cytoskeletal filaments. The transport efficiency is strongly affected by the spatial organization of the cytoskeleton, which constitutes an inhomogeneous, complex network. In cells with a centrosome microtubules grow radially from the central microtubule organizing center towards the cell periphery whereas actin filaments form a dense meshwork, the actin cortex, underneath the cell membrane with a broad range of orientations. The emerging ballistic motion along filaments is frequently interrupted due to constricting intersection nodes or cycles of detachment and reattachment processes in the crowded cytoplasm. In order to investigate the efficiency of search strategies established by the cell's specific spatial organization of the cytoskeleton we formulate a random velocity model with intermittent arrest states. With extensive computer simulations we analyze the dependence of the mean first passage times for narrow escape problems on the structural characteristics of the cytoskeleton, the motor properties and the fraction of time spent in each state. We find that an inhomogeneous architecture with a small width of the actin cortex constitutes an efficient intracellular search strategy.

In Vitro Assembly Kinetics of Cytoplasmic Intermediate Filaments: A Correlative Monte Carlo Simulation Study.

Intermediate filament (IF) elongation proceeds via full-width "mini-filaments", referred to as "unit-length" filaments (ULFs), which instantaneously form by lateral association of extended coiled-coil complexes after assembly is initiated. In a comparatively much slower process, ULFs longitudinally interact end-to-end with other ULFs to form short filaments, which further anneal with ULFs and with each other to increasingly longer filaments. This assembly concept was derived from time-lapse electron and atomic force microscopy data. We previously have quantitatively verified this concept through the generation of time-dependent filament length-profiles and an analytical model that describes assembly kinetics well for about the first ten minutes. In this time frame, filaments are shorter than one persistence length, i.e. ~1 µm, and thus filaments were treated as stiff rods associating via their ends. However, when filaments grow several µm in length over hours, their flexibility becomes a significant factor for the kinetics of the longitudinal annealing process. Incorporating now additional filament length distributions that we have recorded after extended assembly times by total internal reflection fluorescence microscopy (TIRFM), we developed a Monte Carlo simulation procedure that accurately describes the underlying assembly kinetics for large time scales.

Three-dimensional structured illumination microscopy using Lukosz bound apodization reduces pixel negativity at no resolution cost.

The quality of the reconstructed image in structured illumination microscopy (SIM) depends on various aspects of the image filtering process. To optimize the trade-off between resolution and ringing artifacts, which lead to negative intensities, we extend Lukosz-bound filtering to 3D SIM and derive the parametrization of the 3D SIM cut-off. We compare the use of the Lukosz-bound as apodization filter to triangular apodization and find a tenfold reduction in the most negative pixel value with a minimal resolution loss. We test this algorithm on experimental SIM images of tubulin filaments and DAPI stained DNA structure in cancer cells and find a substantial reduction in the most negative pixel value and the percentage of pixels with a negative value. This means that there is no longer a need to clip the final image to avoid these negative pixel values.

Downregulation of T­cell lymphoma invasion and metastasis­inducing factor 1 induces cytoskeletal rearrangement and inhibits the invasive capacity of gastric cancer cells.

T-cell lymphoma invasion and metastasis-inducing factor 1 (Tiam-1) is an important member of the diffuse B-cell lymphoma (Dbl) oncogene family. In a previous study, the overexpression of Tiam-1 protein was identified by immunohistochemistry in human gastric cancer tissues, indicating that Tiam-1 may represent a candidate biomarker of the invasive and metastatic capacity of gastric cancer and for patient prognosis. In the present study, in vitro adhesion selection was used to separate two subpopulations with high (MH) or low (ML) invasive and metastatic potential from the MKN-45 human gastric cancer cell line (M0). A positive correlation was observed between Tiam-l mRNA and protein expression levels and the invasive capacity of the cells using RT-PCR and quantitative cellular-ELISA, respectively. To determine the mechanism by which Tiam-1 affects the invasive capacities of gastric cancer cells, Tiam-1 expression was downregulated in the MH subclone by liposomal transfection of antisense oligodeoxynucleotides (ASODNs). Following 48 h of treatment with ASODNs (0.43 µM), Tiam-1 mRNA transcription and protein expression levels in MH cells was decreased by 80 and 24%, respectively, compared with untreated controls. In addition, the in vitro invasive potential of MH cells was suppressed by 60%. Morphological and ultrastructural observations also demonstrated that ASODN-treated MH cells exhibited a smooth surface with markedly reduced filopodia and microspikes, which resembled M0 and ML cells. In addition, cytoskeletal distribution was markedly altered from disordered to regular with reduced long filament-like structures, projections, pseudopodia on the cell surface and decreased actin bodies in the cytoplasm. Results of the current study indicate that the overexpression of Tiam-1 contributes to the invasive phenotypes of gastric cancer cells. These observations are likely to provide an improved insight into the biological mechanisms of Tiam-1 and promote the development of novel treatment strategies in gastric cancer.

Assessment of actin cytoskeleton and nuclei in bovine blastocysts developed under different culture conditions using a novel computer program.

This study was performed to investigate the effects, in terms of nuclear material and actin cytoskeleton quantities (fluorescent pixel counts), of four different bovine blastocyst culturing techniques (in vitro, stepwise in vitro-to-in vivo, or purely in vivo). Cumulus oocyte complexes from abattoir-sourced ovaries were matured in vitro and allocated to four groups: IVP-group embryos developed up to blastocyst stage in vitro. Gamete intra-fallopian transfer (GIFT)-group oocytes were co-incubated with semen for 4 h before transfer to oviducts of heifers. Following in vitro fertilization, cleaved embryos (day 2 of embryo development, day 2-7 group) were transferred into oviducts on day 2. Multiple ovulation embryo transfer (MOET)-group embryos were obtained by superovulating and inseminating heifers; the heifers' genital tracts were flushed at day 7 of blastocyst development. Within each group, ten blastocysts were selected to be differentially dyed (for nuclei and actin cytoskeleton) with fluorescent stains. A novel computer program (ColorAnalyzer) provided differential pixel counts representing organelle quantities. Blastocysts developed only in vivo (MOET group) showed significantly more nuclear material than did blastocysts produced by any other technique. In terms of actin cytoskeleton quantity, blastocysts produced by IVP and by day 2-7 transfer did not differ significantly from each other. Gamete intra-fallopian transfer- and MOET-group embryos showed significantly larger quantities of actin cytoskeleton when compared with any other group and differed significantly from each other. The results of this study indicate that culturing under in vitro conditions, even with part time in vivo techniques, may adversely affect the quantity of blastocyst nuclear material and actin cytoskeleton. The software employed may be useful for culture environment evaluation/developmental competence assessment.

Single cells spreading on a protein lattice adopt an energy minimizing shape.

When spreading onto a protein microlattice living cells spontaneously acquire simple shapes determined by the lattice geometry. This suggests that, on a lattice, living cells' shapes are in thermodynamic metastable states. Using a model at thermodynamic equilibrium we are able to reproduce the observed shapes. We build a phase diagram based on two adimensional parameters characterizing essential cellular properties involved in spreading: the cell's compressibility and fluctuations.

A novel terminal web-like structure in cortical lens fibers: architecture and functional assessment.

This study describes a novel cytoskeletal array in fiber cells of the ocular lens of the rat and shows its relationship to the classical terminal web of other epithelial tissues. Naive adult Sprague-Dawley rats (n = 28) were utilized. F-actin, fodrin, myosin IIA, and CP49 distribution was assessed in anterior and posterior polar sections. For functional analysis, lenses were cultured with or without cytochalasin-D for 3 hr, then processed for confocal microscopy or assessed by laser scan analysis along sutures. Phalloidin labeling demonstrated a dense mesh of F-actin adjacent to posterior sutural domains to a subcapsular depth of 400 µm. Anterior polar sections revealed a comparable actin structure adjacent to anterior suture branches however, it was not developed in superficial fibers. Fodrin and myosin were localized within the web-like actin apparatus. The data was used to construct a model showing that the cytoskeletal array is located within the blunt, variable-width fiber ends that abut at sutures such that the "terminal web" flanks the suture on either side. Treatment with cytochalasin-D resulted in partial disassembly of the "terminal web" and perturbed cellular organization. Laser scan analysis revealed that cytochalasin-D treated lenses had significantly greater focal variability than control lenses (P = 0.020). We conclude that cortical fibers of rat lenses contain a bipolar structure that is structurally and compositionally analogous to classical terminal webs. The results indicate that the lens "terminal web" functions to stabilize lens fiber ends at sutures thus minimizing structural disorder, which in turn, promotes the establishment and maintenance of lens transparency.

Structural alterations provoked by narrow-band ultraviolet B in immortalized keratinocytes: assessment by atomic force microscopy.

We applied atomic force microscopy (AFM) to visualize ultrastructural changes of the keratinocyte morphology after narrow-band ultraviolet B (NB-UVB) irradiation. Immortalized human keratinocytes were cultured under standard conditions, irradiated with NB-UVB light at doses ranging from 50 to 800 mJ/cm2 and imaged by AFM mounted on an inverted optical microscope. It was observed, that NB-UVB irradiation provoked dose-dependent alterations of the keratinocyte morphology. While the surface of non-irradiated cells exhibited homogenously distributed crest-like shaped protrusions (height 0.16 +/- 0.05 microm), cells irradiated with a dose of 800 mJ/cm2 in addition showed round shaped protrusions (height 0.14 +/- 0.06 microm) distributed predominantly around the nucleus and bleb-like protrusions irregularly distributed on the cell surface (height 0.95 +/- 0.29 microm). These irradiated cells easily detached from the supporting glass surface, showed impaired contact with adjacent keratinocytes and significantly rearranged their cytoskeleton network. We hypothesize that these structural and functional alterations reflect ongoing apoptosis in UVB treated cells.

Comparison of environmental scanning electron microscopy with high vacuum scanning electron microscopy as applied to the assessment of cell morphology.

The efficacy of conventional high vacuum scanning electron microscopy (SEM), environmental SEM (ESEM), and confocal laser scanning microscopy techniques in the assessment of cell-material interactions is compared. Specific attention is given to the application of these techniques in the assessment of the early morphological response of human osteoblast-like cells cultured on titanium dioxide. The processing of cells cultured for conventional high vacuum SEM leads to the loss of morphological features that are retained when using ESEM. The use of cytoskeletal labeling, viewed with confocal laser scanning microscopy, in conjunction with ESEM gives an indication of the changes to cell morphology as a consequence of incubation time in response to interactions at the biological/material interface.

Quantitative assessment of the response of primary derived human osteoblasts and macrophages to a range of nanotopography surfaces in a single culture model in vitro.

The effect of nanotopography on a range of Ti oxide surfaces was determined. Flat Ti, 3%, 19%, 30% and 43% topography densities of 110 nm high hemispherical protrusions were cultured in contact with primary derived human macrophages and osteoblasts in single culture models. Prior to introduction of the test substrate the phenotype and optimum conditions for in vitro cell culture were established. The cellular response was investigated and quantified by assessments of cytoskeletal development and orientation, viable cell adhesion, cytokine production and release and RT-PCR analysis of osteogenic markers. The tested nanotopographies did not have a statistically significant effect on viable cell adhesion and subsequent cytoskeletal formation. Surface chemistry was the dominant factor as established via incorporation of a tissue culture polystyrene, TCPS, control. The topography surfaces induced a release of chemotactic macrophage activation agents at 1 day in conjunction with stress fibre formation and a subsequent fibronectin network formation. Osteoblasts migrated away from the topography surfaces to the exposed TCPS within the wells during the 7-day period.

Mechanical assessment by magnetocytometry of the cytosolic and cortical cytoskeletal compartments in adherent epithelial cells.

This study aims at quantifying the cellular mechanical properties based on a partitioning of the cytoskeleton in a cortical and a cytosolic compartments. The mechanical response of epithelial cells obtained by magnetocytometry - a micromanipulation technique which uses twisted ferromagnetic beads specifically linked to integrin receptors - was purposely analysed using a series of two Voigt bodies. Results showed that the cortical cytoskeleton has a faster response ( approximately 1 s) than the cytosolic compartment ( approximately 30 s). Moreover, the two cytoskeletal compartments have specific mechanical properties, i.e., the cortical (resp. cytosolic) cytoskeleton has a rigidity in the range: 49-85 Pa (resp.: 74-159 Pa) and a viscosity in the range 5-14 Pa.s (resp.: 593-1534 Pa.s), depending on the level of applied stress. Depolymerising actin-filaments strongly modified these values and especially those of the cytosolic compartment. The structural relevance of this two-compartment partitioning was supported by images of F-actin structure obtained on the same cells.

Endocytic internalization of adenovirus, nonspecific phagocytosis, and cytoskeletal organization are coordinately regulated in alveolar macrophages by GM-CSF and PU.1.

GM-CSF gene-targeted (GM(-/-)) mice have impaired pulmonary clearance of bacterial and fungal pathogens by alveolar macrophages (AMs). Because AMs also clear adenovirus from the lung, the role of GM-CSF in endocytic internalization of adenovirus by AMs was evaluated. Pulmonary clearance of adenovirus was severely impaired in GM(-/-) mice compared to wild-type (GM(+/+)) mice as determined by Southern analysis of viral DNA. Internalization of adenovirus by AMs was deficient in GM(-/-) mice in vivo and in vitro as determined by uptake of fluorescently labeled adenovirus or by PCR quantification of adenoviral DNA internalized within AMs. An AM cell line previously established from GM(-/-) mice (mAM) had impaired internalization of adenovirus and transferrin-coated 100-nm latex beads compared to MH-S, a GM(+/+) AM cell line. Phagocytosis of 4- micro m latex beads was also impaired in mAM cells as determined by confocal and fluorescence microscopy. Retroviral vector-mediated reconstitution of PU.1 expression in cultured GM(-/-) AMs restored phagocytosis of 4- micro m beads, endocytosis of adenovirus, and transferrin-coated 100-nm beads (independent of integrin alpha(V) and transferrin receptors, respectively), and restored normal cytoskeletal organization, filamentous actin distribution, and stimulated formation of filopodia. Interestingly, mRNA for the phosphoinositide 3 kinase p110gamma isoform, important in macrophage phagocytic function, was absent in GM(-/-) AMs and was restored by PU.1 expression. These data show that GM-CSF, via PU.1, regulates endocytosis of small ( approximately 100 nm) pathogens/inert particles and phagocytosis of very large inert particles and suggests regulation of cytoskeletal organization by GM-CSF/PU.1 as the molecular basis of this control.

The myofibroblast: an assessment of controversial issues and a definition useful in diagnosis and research.

Some interpretational problems associated with the myofibroblast, which affect how this cell is identified, are discussed. Questions addressed include distinguishing between "external" lamina ("basement membrane") and the fibronectin fibril of the fibronexus; the nature of stress fibers (bundles of smooth-muscle myofilaments with focal densities); the utility of some of these features to distinguish between myofibroblastic and smooth-muscle cell surfaces; and cytoskeletal immunophenotype. The following points are emphasized. Myofibroblasts have a surface characterized by prominent fibronectin fibrils and fibronexus junctions, which are distinct from lamina ("basement membrane"). This can permit a distinction to be made between smooth-muscle and myofibroblastic lesions and tumors. Myofibroblasts are typically positive for vimentin and alpha-smooth-muscle actin, but desmin is not a useful discriminant between smooth-muscle and myofibroblastic lesions. The main features for defining the myofibroblast are abundant rough endoplasmic reticulum; modestly developed peripheral myofilaments with focal densities (stress fibers); fibronexus junctions; vimentin and smooth-muscle actin immunostaining. Other features include a Golgi apparatus and collagen secretion granules, gap junctions, and actin-associated nondesmosomal junctions. Illustrations of the usefulness of these criteria in the diagnosis of soft-tissue lesions (myofibrosarcoma, so-called myofibroblastoma) are given.

Actin protofilament orientation in deformation of the erythrocyte membrane skeleton.

The red cell's spectrin-actin network is known to sustain local states of shear, dilation, and condensation, and yet the short actin filaments are found to maintain membrane-tangent and near-random azimuthal orientations. When calibrated with polarization results for single actin filaments, imaging of micropipette-deformed red cell ghosts has allowed an assessment of actin orientations and possible reorientations in the network. At the hemispherical cap of the aspirated projection, where the network can be dilated severalfold, filaments have the same membrane-tangent orientation as on a relatively unstrained portion of membrane. Likewise, over the length of the network projection pulled into the micropipette, where the network is strongly sheared in axial extension and circumferential contraction, actin maintains its tangent orientation and is only very weakly aligned with network extension. Similar results are found for the integral membrane protein Band 3. Allowing for thermal fluctuations, we deduce a bound for the effective coupling constant, alpha, between network shear and azimuthal orientation of the protofilament. The finding that alpha must be about an order of magnitude or more below its tight-coupling value illustrates how nanostructural kinematics can decouple from more macroscopic responses. Monte Carlo simulations of spectrin-actin networks at approximately 10-nm resolution further support this conclusion and substantiate an image of protofilaments as elements of a high-temperature spin glass.

Assessment of strain field in endothelial cells subjected to uniaxial deformation of their substrate.

A stretch chamber has been developed in order to visualize the deformation of cells subjected to controlled uniaxial stretch of their substrate. A rectangular, custom-made, transparent silicone channel is used as a deformable substrate. Bovine aortic endothelial cells are plated at the bottom of the channel whose lateral deformation is controlled by two piezoelectric translators. The system is mounted on the stage of a confocal microscope where three-dimensional (3D) images of the cells can be acquired simultaneously in the three RGB channels. The first channel provides images of 216 nm fluorescent beads embedded in the cytoskeleton (used as internal markers). The second is used to image the shape of the nucleus revealed by live cell nucleic acid staining. The third one provides a transmitted light image of the cell outline. 3D images of the cell are taken before deformation, after uniaxial deformation of the substrate (up to 25%) and after relaxation. Results indicate that: (a) the cell closely follows the deformation imposed by the substrate with no measurable residual strain after relaxation, and (b) there is a clear mechanical coupling between the extracellular matrix and the nucleus, which deforms significantly under the applied substrate stretch. Suggesting that the nucleus can directly sense the mechanical environment of the cell, the latter result has potentially important implications for signal transduction.

Assessment of f-actin organization and apical-basal polarity during in vivo cat endothelial wound healing.

PURPOSE: To assess the relationships between cytoskeletal changes and apical-basal polarity during healing of mechanical scrape injuries in the cat corneal endothelium. METHODS: Ten cats (20 eyes) were used in this study. One mechanical scrape injury was created in the corneal endothelium of each eye using a blunt olive tip cannula. Tandem scanning confocal microscopy (TSCM) was performed at sequential time points after injury for in vivo assessment of cell morphology and wound healing rates. In two eyes, scanning electron microscopy was performed to allow verification of TSCM observations. Ten eyes were collected between 6 and 48 hours after wounding for in situ labeling of f-actin, ZO-1, or both. RESULTS: Cat endothelial cell morphology observed using in vivo microscopy was identical to that shown using scanning electron microscopy. During healing, endothelial cells always remained attached to the endothelial sheet, although some showed extensions of lamellipodia into the open wound area. The in situ localization of f-actin also correlated with the TSCM in vivo wound morphology. Quantitative analysis showed that there was a decrease in the intensity of phalloidin-fluorescein isothiocyanate staining at the leading edge of the wound, suggesting a decrease in f-actin; a significant correlation was found between the relative intensity of f-actin staining and the distance from the wound margin (R = 0.98, P < 0.01). At 24 and 48 hours after injury, both ZO-1 and f-actin maintained an apical localization within cells immediately adjacent to the leading edge, despite the considerable distance of movement and dramatic decrease in the intensity of f-actin staining. CONCLUSIONS: Overall, these data demonstrate that after scrape injury in the cat, endothelial cells exhibit a pattern of healing in which total intracellular f-actin is reduced, but normal cell connectivity and apical-basal polarity are maintained throughout.

Computer simulation of a model network for the erythrocyte cytoskeleton.

The geometry and mechanical properties of the human erythrocyte membrane cytoskeleton are investigated by a computer simulation in which the cytoskeleton is represented by a network of polymer chains. Four elastic moduli as well as the area and thickness are predicted for the chain network as a function of temperature and the number of segments in each chain. Comparisons are made with mean field arguments to examine the importance of steric interactions in determining network properties. Applied to the red blood cell, the simulation predicts that in the bilayer plane the membrane cytoskeleton has a shear modulus of 10 +/- 2 x 10(-6) J/m2 and an areal compression modulus of 17 +/- 2 x 10(-6) J/m2. The volume compression modulus and the transverse Young's modulus of the cytoskeleton are predicted to be 1.2 +/- 0.1 x 10(3) J/m3 and 2.0 +/- 0.1 x 10(3) J/m3, respectively. Elements of the cytoskeleton are predicted to have a mean displacement from the bilayer plane of 15 nm. The simulation agrees with some, but not all, of the shear modulus measurements. The other predicted moduli have not been measured.

Cytoskeletal alterations as a parameter for assessment of toxicity.

1. Some environmental substances, drugs and pollutants affect the growth of cultured cells, and produce cytoskeletal alterations. 2. These have been used as parameters for toxicity assessment of cholera toxin and pertussis toxin in Chinese Hamster Ovary cells. 3. Cholera toxin stabilized microtubules and had no effect on microfilaments and intermediate filaments. 4. Pertussis toxin affected microfilaments but appeared to have no effect on microtubules and intermediate filaments.

Cytologic diagnosis and ultrastructure of fine-needle aspirates of ganglion cysts.

This report describes 28 ganglion cysts in 21 patients. The presence of a colorless to pale-yellow gelatinous material in the aspirate is pathognomonic of ganglion cyst. The smears are fairly monotonous, and show abundant mucoid material, single cells resembling histiocytes, a few tight clusters of cells, some collagen fibers, and some red blood cells with altered shapes. Ultrastructural studies performed on five specimens reveal the fibroblastic and/or histiocytic nature of the cells in the aspirates.