Cell Perimeter Research Articles

Topological Properties and Variations of Cells in Proliferating Epithelia: Impact of Mechanical Force and Division Orientation

Proliferating epithelia are sheets of dividing cells that tightly adhere to each other in many metazoans. Regulation of cell division is important for both normal and diseased epithelia. Quantitative analysis of epithelia shows significant conservation in the dominance of hexagonal cells in both animal and plant. However, there exist species with significant varied amount of hexagonal cells. The mechanism controlling these distributions is poorly understood. In this study, we use a computational method called cellGPS (cell global pattern simulator) to study the dynamic process of proliferating epithelia based on a mechanical off-lattice cellular model. This method keeps track of full geometric and biomechanical properties of epithelia. It models cell shape, area, perimeters and surface tension. Two biologically relevant division rules, namely, the orthogonal orientation found in plants and the largest-side orientation found in the mitotic spindle in human cells, are also incorporated in the division process. With this method, we found that regardless of division rules, our mechanical model can produce the common epithelia pattern of the dominance of hexagonal cells. In addition, we found that division rule of largest-side orientation lead to the formation of cell pattern with around 45% hexagons, which is observed in many animal epithelia and some plant tissues. This suggests that reducing stress on the cell wall by dividing the largest side is widely used in animal epithelia. The percentage of hexagons from division following orthogonal orientation is found to be about 60%, consistent with observations found in certain plant tissues. We conclude that both mechanical force and division orientation play important roles during epithelial proliferation. Mechanical model itself leads to topological dominance of hexagonal cells, and different rules of division orientation lead to varying amount of hexagonal cells.

Effect of Microstructural Parameters on the Relative Densities of Metal Foams

A detailed quantitative microstructural analyses of primarily open cell FeCrAlY and 314 stainless steel metal foams with different relative densities and pores per inch (p.p.i.) were undertaken in the present investigation to determine the effect of microstructural parameters on the relative densities of metal foams. Several elements of the microstructure, such as major and minor cell sizes, cell areas and perimeters, ligament dimensions, cell shapes, and area fractions of closed and open cells, were measured. The cross-sections of the foam ligaments showed numerous pores, and their circularity factors and average sizes were determined. The area fractions of the open cells and ligaments decreased, whereas that of the closed cells increased linearly with increasing relative density. The relative densities and p.p.i. were not significantly dependent on cell size, cell perimeter, and ligament dimensions within the limits of experimental scatter. A phenomenological model is proposed to rationalize the present microstructural observations.

Abstract P1-02-05: EPLIN? Can Negatively Impact on Angiogenesis and Is Associated with ERK Signalling

Abstract Background: EPLIN (epithelial protein lost in neoplasm) is a cytoskeletal associated protein involved in regulating actin dynamics and cell motility. EPLIN expression is frequently lost in cells as they progress to a cancer state and this loss may account for enhanced cancer cell motility. EPLIN has also been shown to be lost in clinical breast cancer, which is associated with patients clinical outcome. Recently, EPLIN has been linked to ERK signalling. This study examined the potential of EPLIN to influence the angiogenesis process and its links to ERK in endothelial cells Methods: Over-expression of EPLIN α was achieved through transfection of the vascular endothelial cell, HECV, with a mammalian expression construct containing the full coding sequence of EPLINα. Enhanced levels of EPLINα were verified through RT-PCR, Q-PCR and Western blot analysis before assessing the angiogenic potential of control and transfected cells using angiogenesis and cell migration models. Treatment of transfected and control cells with ERK inhibitor was used to assess the potential interaction between EPLINα and ERK in endothelial cells. Results: Substantial enhancement in EPLINα expression was seen in transfected cells compared to control cells at both transcript and protein level. Over-expression of EPLINα in HECV endothelial cells resulted in a reduced capacity, compared to control, of these cells to form tubule like structures in an in vitro angiogenesis tubule formation assay (6566 +/− 856µm tubule perimeter of control cells vs 2881 +/− 546µm EPLINα overexpressing HECV cells, p = 0.007). This was seen together with a reduced cell migration following overexpression of EPLINα in the cells. Treatment of control cells with ERK inhibitor significantly reduced tubule formation (3537 +/− 277µm tubule perimeter of ERK inhibitor treated control cells vs 6566 +/− 856µm total perimeter of untreated control cells, p = 0.01), however treatment of HECV cells transfected with EPLINα expression construct caused tubule formation levels to return to control levels (6566 +/− 856µm tubule perimeter of untreated control cells vs 5326 +/− 784µm tubule perimeter of ERK inhibitor treated EPLINα overexpressing HECV cells, p = 0.317). Discussion: EPLINα can negatively influence tubule formation in vitro and thus may impact on the angiogenesis process. Furthermore, its impact on tubule formation appears to be linked to ERK signalling. Together, this study suggests a role for EPLINα in angiogenesis, which may be linked with ERK signalling. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P1-02-05.

Paradoxical Dose-Dependent Chemoattractive and Chemorepellent Effects of Kit Ligand (KL) In Mast Cells Revealed In Novel Microfluidic Biochambers.

AbstractAbstract 1496Besides its cooperating effects on stem cell proliferation and survival, KL (stem cell factor) is a potent chemotactic protein; much of the Kit (-) phenotype is related to loss of chemotaxis of progenitor cells. While transwell assays permit studies of the frequency of migrating cells, the lack of direct visualization during chemotaxis precludes dynamic or detailed studies of cell behavior. We have developed microfluidic biochambers fabricated from optically clear plastic with microcapillaries continuously perfused from source and sink channels to create stable ligand gradients of desired concentration and steepness without mechanical shear forces. We have analyzed KL-induced chemotaxis, using Kit+ cultured murine bone marrow-derived mast cells (BMMC) as a model. BMMC were plated onto fibronectin coated chambers and two linear KL equilibrium concentration profiles were tested (gradients of 4 and 8 ng/ml/mm across a concentration range of 1.5–11 ng/ml) for their ability to induce BMMC chemotaxis. Cell movement and morphology was captured by time-lapse microscopy and quantitatively analyzed for migration rate, frequency and duration of pauses, directional persistence, filopodia extension rate, filopodia asymmetry, and cell perimeter asymmetry. Our results indicate a minimum activating threshold of ∼3 ng/ml is required to induce BMMC chemoattraction. The chemotactic index (CI) is a ratio of distance travelled towards a gradient to total distance travelled (e.g., CI=0 indicates random migration, CI=1 indicates movement in a straight line towards the gradient). Between 3–8 ng/ml, higher KL concentrations resulted in faster cell speed (means 1.0–1.9 μm/min; maximum up to 2.9 μ/min), higher migratory persistence (means 0.2–0.5), higher CI (means 0.05 to 0.42), and greater filopodial formation. Analysis of cells at the lowest KL concentrations revealed a paradoxical chemorepulsive activity, which has not been previously described. The majority of the cells undergo chemorepulsion at KL concentrations 1.5–3 ng/ml. Unlike chemoattraction which was continuous, chemorepulsion only occurs during the first 30–40 minutes of KL stimulation, followed by predominantly random or no migration, i.e., chemorepulsion is followed by a chemotaxis-refractory state. Both chemoattractive and chemorepulsive movements were characterized by filopodia formation on the leading edge of movement. The results with the Kit receptor tyrosine kinase differ significantly from previous studies of G-protein coupled receptors, where chemorepulsion only occurred at high ligand concentrations. The results indicate that Kit-mediated chemotaxis is mainly concentration-dependent; involves complex dose-response relationships; and includes both chemoattractive and chemorepellent phases. Studies of Kit+ hematopoietic stem cells are being performed to determine whether KL chemotactic responses similar to those seen in BMMC are a general phenomenon, which would have major implications for understanding HSC migration, homing and niche relationships, as well as mast cell biology and inflammation. Disclosures:No relevant conflicts of interest to declare.

A cell-based model of Nematostella vectensis gastrulation including bottle cell formation, invagination and zippering

The gastrulation of Nematostella vectensis, the starlet sea anemone, is morphologically simple yet involves many conserved cell behaviors such as apical constriction, invagination, bottle cell formation, cell migration and zippering found during gastrulation in a wide range of more morphologically complex animals.In this article we study Nematostella gastrulation using a combination of morphometrics and computational modeling. Through this analysis we frame gastrulation as a non-trivial problem, in which two distinct cell domains must change shape to match each other geometrically, while maintaining the integrity of the embryo. Using a detailed cell-based model capable of representing arbitrary cell-shapes such as bottle cells, as well as filopodia, localized adhesion and constriction, we are able to simulate gastrulation and associate emergent macroscopic changes in embryo shape to individual cell behaviors.We have developed a number of testable hypotheses based on the model. First, we hypothesize that the blastomeres need to be stiffer at their apical ends, relative to the rest of the cell perimeter, in order to be able to hold their wedge shape and the dimensions of the blastula, regardless of whether the blastula is sealed or leaky. We also postulate that bottle cells are a consequence of cell strain and low cell–cell adhesion, and can be produced within an epithelium even without apical constriction. Finally, we postulate that apical constriction, filopodia and de-epithelialization are necessary and sufficient for gastrulation based on parameter variation studies.

Morphometry of Corneal Epithelial Cells on Normal Eyes and After Anterior Lamellar Keratoplasty

To analyze morphometry of corneal epithelial cells in normal eyes and after anterior lamellar keratoplasty (ALK) using confocal microscopy. This retrospective comparative case series included 29 eyes of patients who had undergone ALK (ALK group) and 29 subjects before refractive surgery with normal corneas (control group). The Heidelberg Retina Tomograph II with the Cornea Module was used. Images were recorded and analyzed with Image J software. Morphometric parameters used to analyze epithelial cells were cell area, coefficient of variation of cell area, perimeter, circularity, and Feret diameter. Confocal microscopy was performed between 3 and 70 months after surgery. For basal epithelial cells, no significant differences between right eyes and left eyes were found in normal eyes for all morphometric parameters (P < 0.05 and rs > 0.40). The mean epithelial cell area increased during central and vertical migration (migration of epithelial cells from the basal stratum to the outermost layer) (P < 0.001). The mean basal cell area in the central zone was significantly higher (P < 0.001) after ALK in the ALK group than that in the control group. After ALK, the mean basal cell area decreased with postoperative time (rS = -0.78; P < 0.001) and returned to normal 24 months after transplantation (P = 0.27). Comparison of both groups showed significant differences in cell perimeter, circularity, and Feret diameter of basal cells and intermediate cells (P ≤ 0.001). Morphometric analysis of the corneal basal epithelium seems to be a relevant tool to assess the epithelial condition.

Toward a Fully Automated High-Throughput Phototransfection System

We have designed and implemented a framework for creating a fully automated high-throughput phototransfection system. Integrated image processing, laser target position calculation, and stage movements show a throughput increase of > 23X over the current manual phototransfection method while the potential for even greater throughput improvements (> 110X) is described. A software tool for automated off-line single cell morphological measurements, as well as real-time image segmentation analysis, has also been constructed and shown to be able quantify changes in the cell before and after the process, successfully characterizing them, using metrics such as cell perimeter, area, major and minor axis length, and eccentricity values.

Effects of Sirolimus on Human Mesangial Cells

Mesangial cell (MC) proliferation and production of extracellular matrix or loss of MC are both central findings in a number of renal proteinuric diseases. However, the role of MC as components of the glomerular filtration barrier and whether MC alterations induce changes in the glomerular filtration barrier leading to proteinuria are still matters of debate. The effects of Sirolimus (SRL) in proteinuric nephropathies is controversial: some papers have indicated a reduction and others, an increase in proteinuria after sirolimus treatment. Considering the pivotal role of MC in the pathogenesis of many chronic nephropathies, we evaluated the effect of SRL on cultured human MC. We treated primary human MC cultures with SRL, or platelet-derived growth factor (PDGF) or SRL + PDGF, or dimethylsulfoxide, the SRL vehicle, as a control. PDGF was used to activate MC. After 48 hours treatment, MC showed a significant growth increase that was significantly reduced by SRL ( P < .01). Apoptosis, determined by the TUNEL assay and flow cytometry, was not modified by the treatments at 24 hours. SRL treatment increased significantly the number of α-smooth muscle actin–positive cells compared with controls ( P < .05). Cells treated with SRL and SRL + PDGF showed significant changes in morphology with increased mean cell surface, perimeter, and maximum diameter ( P < .01) but not protein content. Furthermore, MC treated with SRL showed decreased migration through polycarbonate membranes. The changes induced by SRL may help to explain some of the in vivo effects observed in SRL-treated patients.

GFP‐actin dynamics during histamine‐induced endothelial barrier dysfunction

We tested the hypothesis that histamine‐induced endothelial barrier dysfunction is associated with disruption of normal actin dynamics at the endothelial cell periphery. Human umbilical vein endothelial cells (HUVEC) were transfected with GFP‐actin (500 ng vector/5 × 105 cells). Transendothelial resistance (TER) served as an index of barrier function. Time‐lapse image sets were acquired before and after the addition of 10 μM histamine. GFP‐actin expression slightly enhanced histamine‐induced endothelial barrier dysfunction, but did not affect the time course. GFP‐actin was found in cortical fibers moving centrally at a mean rate of 63 nm/min, and in membrane ruffles protruding outward at a mean rate of 1462 nm/min. New cortical fibers formed at a frequency of 0.59/min., and membrane ruffling occurred at a frequency of 0.61 events/min. GFP‐actin also localized in small lamellipodia on the cell periphery. Histamine treatment caused a sudden, coordinated formation of lamellipodia around the cell perimeter, followed by inactivity during the time frame of histamine‐induced barrier dysfunction. Shortly after, lamellepodia and membrane ruffling were restored. The data show that histamine‐indcued barrier dysfunction is associated with a loss of normal cortical actin dynamics in endothelial cells. Supported by NIH RR‐018766 and a grant from the American Heart Association.

Cardiac enlargement during pregnancy may involve new myocyte formation

Cardiac enlargement that develops during pregnancy is an important yet under‐studied adaptation to physiologic stress. In light of recent evidence of resident stem cell pools in the heart, we tested the hypothesis that pregnancy‐induced cardiac enlargement may involve both myocyte hypertrophy and new myocyte formation. 6 nonpregenant age matched controls (C) and 6 mice in late pregnancy (LP, 17–19 days) were studied. Hearts were formalin fixed and 5μ sections were examined. Laminin staining was used to identify cell perimeter and determine myocyte cross‐sectional area (MCSA). Wet weights of LP hearts were 23% greater than C (240±9 vs 195±13mg). Increases in MCSA were not proportional to increases in heart mass. MCSA of LP (262±3μ2, n=225) was 11% greater than of C (236± 3μ2, n=225). Analysis of sections from different regions of the heart showed different degrees of MCSA hypertrophy. Apex, mid and basal sections were analyzed from all hearts. No difference in MCSA among sections was found in C. MCSA of all sections were greater in LP than C, but MCSA of the mid‐heart in LP (275 ±4μ2, n=75) were greater than MCSA of apex (252± 5μ2, n=75) and base (259 ±4μ2, n=75). Myocyte hypertrophy during pregnancy is not sufficient to account for the whole‐heart enlargement suggesting that new myocytes are generated during pregnancy. Hypertrophy is not evenly distributed during pregnancy suggesting an unequal distribution of wall stress.

Adenoviral Transduction of Enterocytes and M-Cells Using in Vitro Models Based on Caco-2 Cells: The Coxsackievirus and Adenovirus Receptor (CAR) Mediates Both Apical and Basolateral Transduction

Understanding virus-cell interaction is a key to the design of successful gene delivery vectors. In the present study we investigated Ad5 transduction of enterocytes and M-cells utilizing differentiated Caco-2 cells and cocultures of Caco-2 cells with lymphocytes. Transduction inhibition studies showed that CAR is the major receptor mediating apical and basolateral virus entry in differentiated Caco-2 cells. Integrins and heparan sulfate glycosaminoglycans do not appear to play a significant role. Immunofluorescence localized CAR to sites of cell-cell contact, with staining mostly observed on the cell perimeter. Staining was observed even in nonpermeabilized monolayers, suggesting apical accessibility of the receptor. Cocultures with mouse Peyer's patch lymphocytes or Raji B human lymphocytes were more susceptible to transduction than Caco-2 cells, and the effects were dose-dependent. Similar to Caco-2 cells, CAR and not integrins mediated apical transduction. In conclusion, contrary to other epithelial cell lines, both apical and basolateral transduction of absorptive enterocytes and M-cells is mediated by binding to CAR. The coculture system can be used to study the interactions between M-cells and gene delivery vectors.

Agrobacterium type IV secretion system and its substrates form helical arrays around the circumference of virulence -induced cells

The genetic transformation of plant cells by Agrobacterium tumefaciens results from the transfer of DNA and proteins via a specific virulence (vir) -induced type IV secretion system (T4SS). To better understand T4SS function, we analyzed the localization of its structural components and substrates by deconvolution fluorescence microscopy. GFP fusions to T4SS proteins with cytoplasmic tails, VirB8 and VirD4, or cytoplasmic T4SS substrate proteins, VirD2, VirE2, and VirF, localize in a helical pattern of fluorescent foci around the perimeter of the bacterial cell. All fusion proteins were expressed at native levels of vir induction. Importantly, most fusion proteins are functional and do not exhibit dominant-negative effects on DNA transfer to plant cells. Further, GFP-VirB8 complements a virB8 deletion strain. We also detect native VirB8 localization as a helical array of foci by immunofluorescence microscopy. T4SS foci likely use an existing helical scaffold during their assembly. Indeed, the bacterial cytoskeletal component MinD colocalizes with GFP-VirB8. Helical arrays of foci are found at all times investigated between 12 and 48 h post vir induction at 19 degrees C. These data lead to a model with multiple T4SSs around the bacterial cell that likely facilitate host cell attachment and DNA transfer. In support, we find multiple T pili around vir-induced bacterial cells.

Endometrial cytology and computerized morphometric analysis of epithelial nuclei: A useful tool for reproductive diagnosis in the bitch

New diagnostic approaches are required to recognize early canine hypofertility or infertility. We suggest that the identification of different cytologic types, cellular aspects, and nuclear features of the endometrial epithelial cells may be suitable for this purpose. This study was performed on the bitch ( Canis familiaris) during the physiologic reproductive cycle and in uterine diseases. We also applied computerized cytomorphometry to evaluate nuclear area, perimeter, diameter, density, aspect, and roundness of endometrial epithelial cells in healthy dogs (N = 35) at different stages of the reproductive cycle (before puberty, during proestrus, estrus, diestrus, and anestrus) and in bitches affected by uterine disorders (N = 10). The stage of the estrous cycle was determined by vaginal cytology and progesterone evaluation and also confirmed by clinical and histologic observations. Samples for endometrial cytology were collected in vivo by uterine flushing with transcervical uterine cannulation. After uterine sampling, each dog underwent OHE or uterine stump revision. Cytologic analyses were compared with histologic examinations to verify the uterine condition. The uterine cellular population was represented by endometrial epithelial cells, erythrocytes, neutrophils, lymphocytes, eosinophils, macrophages, plasma cells, and cervical or incidental vaginal cells. Bacteria and amorphous material were observed. The proportion of different cells and nuclear features in the cytologic samples varied throughout the stages of the reproductive cycle and between normal and pathologic uterine conditions. The computer-assisted nuclear morphometry, performed in cytologic specimens by means of the six nuclear parameters chosen to evaluate the endometrial epithelial cell population, proved to be useful for determining the stage of the reproductive cycle. Furthermore, this system was demonstrated to be a valid support to diagnose and distinguish uterine disorders.

The role of Brn-4 in the regulation of neural stem cell differentiation into neurons

Brn-4, a member of the homeobox family of transcription factors, has previously been implicated in the regeneration and repair of denervated striatum. We investigated the effects of Brn-4 on the differentiation and development of neural stem cells (NSCs) from E16 rat hippocampus. Immunocytochemistry revealed that extracts of deafferented hippocampus promoted neuronal differentiation to a greater extent than extracts from normal hippocampus. Deafferented extracts also promoted maturation of newborn neurons as reflected in changes in cell areas and perimeters, and enhanced Brn-4 expression in MAP-2 positive neurons. Suppression or overexpression of Brn-4 in NSCs markedly decreased or increased neuronal differentiation and maturation of newborn neurons, respectively. These results suggest that Brn-4 expression is required both for neuronal differentiation of NSCs and maturation of newborn neurons, and that there may be some regulatory factors in deafferented hippocampus that can regulate Brn-4 expression in neuronal progenitors. Brn-4 is therefore a potential research target for the development of new therapeutics to promote brain repair.

Short-term molecular polarization of cells on symmetric and asymmetric micropatterns.

The ability of cells to sense geometrical/physical constraints of local environment is important for cell movements during development, immune surveillance, and in cancer invasion. In this paper, we quantify "front-rear" polarization - the crucial step in initiating cell migration - based on cytoskeleton and substrate adhesion anisotropy in micropatterned cells of well-defined shapes. We then show that the general viewpoint that asymmetric cell shape is one of the defining characteristics of polarized cells is incomplete. Specifically, we demonstrate that cells on circular micropatterned islands can exhibit asymmetric distribution of both filamentous actin (f-actin) and focal adhesions (FAs) as well as directional, lamellipodial-like ruffling activity. This asymmetry, however, is transient and persists only for the period of several hours during which actin filaments and adhesion structures reorganize into symmetric peripheral arrangement. Cells on asymmetric tear-drop shape islands also display polarized f-actin and FAs, but polarization axes are oriented towards the wide end of the islands. Polarization of actin filaments on tear-drop islands is short-term, while focal adhesions remain asymmetrically distributed for long times. From a practical perspective, circular cells constitute a convenient experimental system, in which phenomena related to cell polarization are "decoupled" from the effects of cells' local curvature (constant along circular cell's perimeter), while asymmetric (tear-drop) micropatterned cells standardize the organization of motility machinery of polarized/ moving cells. Both systems may prove useful for the design of diagnostic tools with which to probe and quantify ex vivo the motility/invasiveness status of cells from cancer patients.

Electrical Resistivity Tomography in Cylindrical Cells—Guidelines for Hardware Pre-Design

Abstract Electrical resistivity tomography (ERT) allows for fast, non-destructive, and efficient sediment characterization and geotechnical process monitoring in the field as well as in laboratory applications. Besides the spatial distribution of resistivity, specimen geometry and electrode configuration determine the electrical potential distribution and the ensuing spatial resolution in the tomogram. We examine potential and current density distribution in various ERT system configurations using both experimental and numerical methods and explore optimal electrode configurations for cylindrical cells. Results show that optimal ERT configurations must take into consideration the required spatial resolution, sensitivity to anomalies, signal strength, and shunting effects along the cell perimeter. The system characteristics are defined in terms of electrode width Welec and length Lelec, cell diameter Dcell, and the distance from the electrode plane to conductive end-plates δ. The following dimensionless ratios emerge as guidelines for system pre-design: Welec/Dcell≈π/2n, Lelec /D cell ≈0.4, δ/D cell≥1, where n is the number of electrodes around the perimeter.

Morphogenesis and Cell Division of E. Coli Under Mechanical Confinement

Bacteria have characteristic shapes and sizes which are conserved by an elaborate cytoskeletal machinery. Surprisingly, these well-defined shapes are strongly modified in E. coli bacteria in narrow nanofabricated channels. Growth in constrictions where bacteria are squeezed to about twice thinner than their typical diameter leads to flattened cells that laterally are much wider (up to 5 micron) than regular E. coli [1]. We will report on the cell growth, spatial structure and dynamics of cytoskeletal proteins and the nucleoid in this unusual bacterial phenotype. While the physical confinement has a profound effect on the cell shape and the pattern of cell division, it has only a limited effect on the replication rate of the cells. In most cases, broad (multinucleate) cells are still able to segregate chromosomes in roughly equal amounts to two daughter cells. This process typically starts with the formation of a chromosome-free area in the middle of the cell, which propagates asymmetrically to the perimeter of cell. From this asymmetric location, the cell wall starts to constrict and a septum forms. Our results show that the cellular structure of bacteria has a high degree of plasticity in coping with lateral stress and confinement.[1] Bacterial growth and motility in sub-micron constrictions, J. Mannik, R. Driessen, P. Galajda, J.E. Keymer and C. Dekker, Proc. Natl. Acad. Sci. U. S. A. 106 (2009) 14861.

Anatomy of different forage cacti with contrasting insect resistance

Anatomical aspects of four cacti with contrasting insect resistance using morpho-anatomical analysis and morphometry were evaluated. The following cacti were studied: F-21 [Nopalea cochenillifera (L.) Salm Dyck; IPA-200021]; Giant [Opuntia ficus-indica (L.) Mill.; IPA-100001]; IPA-20 [O. ficus-indica (L.) Mill.; IPA-100003] and Opuntia undulata Griffiths (IPA-200174). Previous research has indicated O. undulata and F-21 is resistant to Dactylopius opuntiae (Cockerell). Cladode measurements included cuticle thickness, epidermis and xylem cell wall as well as the area and perimeter of mesophyll cells. Morphometric analysis revealed that O. undulata has the largest proportion of epidermis and cuticle (39.24 and 220.41μm, respectively). Regarding the thickness of the vascular bundles, O. undulata differed only from the IPA-20 variety (12.13 and 16.61μm, respectively). In the mesophyll cells, F-21 had the greatest perimeter/area ratio. Morpho-anatomical analysis revealed a thick cuticle and epidermis with the presence of crystals, xylem vessels with ligneous fibers and a thick wall, and a large, thin-walled mesophyll. Epidermis was the main anatomical barrier, providing greater resistance and integrity of the cladode and varied among the studied cultivars. Cultivars with thicker epidermis also presented insect resistance in other studies. Further investigation is needed to prove that this particular trait has a causal influence on insect resistance.

Myosin II Is Essential for the Spatiotemporal Organization of Traction Forces during Cell Motility

Amoeboid motility requires spatiotemporal coordination of biochemical pathways regulating force generation and consists of the quasi-periodic repetition of a motility cycle driven by actin polymerization and actomyosin contraction. Using new analytical tools and statistical methods, we provide, for the first time, a statistically significant quantification of the spatial distribution of the traction forces generated at each phase of the cycle (protrusion, contraction, retraction, and relaxation). We show that cells are constantly under tensional stress and that wild-type cells develop two opposing "pole" forces pulling the front and back toward the center whose strength is modulated up and down periodically in each cycle. We demonstrate that nonmuscular myosin II complex (MyoII) cross-linking and motor functions have different roles in controlling the spatiotemporal distribution of traction forces, the changes in cell shape, and the duration of all the phases. We show that the time required to complete each phase is dramatically increased in cells with altered MyoII motor function, demonstrating that it is required not only for contraction but also for protrusion. Concomitant loss of MyoII actin cross-linking leads to a force redistribution throughout the cell perimeter pulling inward toward the center. However, it does not reduce significantly the magnitude of the traction forces, uncovering a non-MyoII-mediated mechanism for the contractility of the cell.

Loss of Myosin VI No Insert Isoform (NoI) Induces a Defect in Clathrin-mediated Endocytosis and Leads to Caveolar Endocytosis of Transferrin Receptor

Myosin VI is a motor protein that moves toward the minus end of actin filaments. It is involved in clathrin-mediated endocytosis and associates with clathrin-coated pits/vesicles at the plasma membrane. In this article the effect of the loss of myosin VI no insert isoform (NoI) on endocytosis in nonpolarized cells was examined. The absence of myosin VI in fibroblasts derived from the Snell's waltzer mouse (myosin VI knock-out) gives rise to defective clathrin-mediated endocytosis with shallow clathrin-coated pits and a strong reduction in the internalization of clathrin-coated vesicles. To compensate for this defect in clathrin-mediated endocytosis, plasma membrane receptors such as the transferrin receptor (TfR) are internalized by a caveola-dependent pathway. Moreover the clathrin adaptor protein, AP-2, necessary for TfR internalization, follows the receptor and relocalizes in caveolae in Snell's waltzer fibroblasts.