Large Protrusions Research Articles

Vangl2-dependent regulation of membrane protrusions and directed migration requires a fibronectin extracellular matrix.

During zebrafish gastrulation the planar cell polarity (PCP) protein Vang-like 2 (Vangl2) regulates the polarization of cells that are engaged in directed migration. However, it is unclear whether Vangl2 influences membrane-protrusive activities in migrating gastrula cells and whether these processes require the fibronectin extracellular matrix. Here, we report that Vangl2 modulates the formation and polarization of actin-rich filopodia-like and large lamellipodia-like protrusions in ectodermal cells. By contrast, disrupted Glypican4/PCP signaling affects protrusion polarity but not protrusion number or directed migration. Analysis of fluorescent fusion protein expression suggests that there is widespread Vangl2 symmetry in migrating cells, but there is enrichment at membrane domains that are developing large protrusions compared with non-protrusive domains. We show that the fibronectin extracellular matrix is essential for cell-surface Vangl2 expression, membrane-protrusive activity and directed migration. Manipulation of fibronectin protein levels rescues protrusion and directed migration phenotypes in vangl2 mutant embryos, but it is not sufficient to restore either PCP, or convergence and extension movements. Together, our findings identify distinct roles for Vangl2 and Glypican4/PCP signaling during membrane protrusion formation and demonstrate that cell-matrix interactions underlie Vangl2-dependent regulation of protrusive activities in migrating gastrula cells.

A child with multiple café au lait macules: Rare presentation of plexiform neurofibromatosis with facial dysmorphism, alopecia, proptosis, stridor, and limb length discrepancy

Multiple cafe au lait macules are one of the cornerstones in diagnosing neurofibromatosis Type 1 (NF 1). NF 1 is often a multisystem neurocutaneous disorder, plexiform NF being one of the most important variants of this phacomatosis. Here, a rare case of a 2.5-year-old male child without positive family history has been described who presented with multiple large cafe au lait spots, alopecia, dysmorphic facies, proptosis, protrusion and deviation of tongue, biphasic stridor, bilateral neck swelling, and abnormal gait due to foreshortened left leg. Magnetic resonance imaging revealed abnormal soft-tissue infiltration of cavernous sinus through sphenoid bone, retro-orbital, retropharyngeal, parapharyngeal spaces, floor of mouth, and encasement of unilateral neck vessels and compression over trachea. Indirect laryngoscopy revealed unilateral vocal cord palsy most likely due to recurrent laryngeal nerve involvement. Biopsy confirmed the diagnosis of NF. Pediatricians and dermatologists should be aware of syndromic causes and their varied presentations while encountering a child with multiple large cafe au lait spots.

Electron microscopic analysis of different cell types in human pancreatic cancer spheres.

Cancer stem cells (CSCs), which are pluripotent and self-renewable, contribute to the initiation and metastasis of cancer, and are responsible for resistance to chemotherapy and radiation. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive type of cancer that is associated with a high incidence of distant metastasis and recurrence. Sphere formation reveals cell proliferation under nonadherent conditions and is commonly used to identify CSCs; measurements of the number, area and volume of the spheres are used to estimate stemness of PDAC cells. However, detailed morphological analysis of such spheres has not been performed. The aim of the present study was to examine the morphology of spheres isolated from PANC-1 human pancreatic cancer cells via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). PANC-1 cells formed round to irregular oblong spheres within 1 week following seeding in ultra-low-attachment plates. These spheres exhibited higher levels of expression of CSC markers, including nestin, sex determining region Y-box 2, and CD44 containing variant exon 9, compared with adherent cells. SEM analysis revealed that the spheres exhibited a grape-like appearance, harboring cancer cells with smooth or rough surfaces. Similarly, TEM analysis detected cancer cells with varying surface types within the spheres: Those with smooth surfaces, irregular large protrusions, protrusions and a small number of microvilli, and those with many microvilli throughout the entire cell surface. These morphological differences among cancer cells may be indicative of different stages in the differentiation process, from CSCs to non-CSCs, within the spheres.

Mechanically tuned 3 dimensional hydrogels support human mammary fibroblast growth and viability

BackgroundCarcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs).ResultsHere, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G’). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-β levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-β was significantly greater for HMFs encapsulated in stiff hydrogels.ConclusionsOverall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn’t support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype.

Cell Invasion In Vivo via Rapid Exocytosis of a Transient Lysosome-Derived Membrane Domain

Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

Effect of laser fluence and ambient gas pressure on surface morphology and chemical composition of hydroxyapatite thin films deposited using pulsed laser deposition

The dependence of the surface morphology and chemical composition of hydroxyapatite (HA) thin films on the laser fluence and ambient gas pressure during their formation by pulsed laser deposition was studied as the first step to investigate the effect of physical and chemical interactions between the ablated chemical species and ambient gas molecules on HA film formation. It was found that a higher fluence could decrease the number of large protrusions on the surface of HA thin films. However, too high a fluence caused a phosphorus deficiency from the stoichiometric value, particularly in the case of lower ambient gas pressure. It was also found that for lower fluences, the atomic species among the ablated chemical species were easily scattered by collision processes with ambient gas molecules. This was caused by the lower velocity of the ablated chemical species and higher ambient gas pressure, which induced a shorter mean free path. In addition, these collision processes played an important role in the adsorption, migration, and re-evaporation of the ablated chemical species on the substrate via chemical reactions.

Optimisation of injection-related parameters using the non-linear programming by quadratic Lagrangian algorithm for a marine medium-speed diesel engine

The match of an injector with the combustion chamber was studied through computational fluid dynamics simulation under four different engine loads. Four design parameters including the start of injection, spray angle, injector protrusion length and swirl ratio were examined. The Latin hypercube together with a non-linear programming by quadratic Lagrangian algorithm were used in the optimisation. Comparisons were made in the engine loads in terms of the optimisation history, objectives, sub-objectives and design parameters. The commonalities of the design parameters of the optimums were summarised. Additionally, a detailed combustion process comparison was conducted on the same engine loads (100% and 25% engine loads) between the optimum and baseline designs, respectively. Finally, the effects of the design parameters on the objective were investigated by the response surface methodology. The results indicate that the non-linear programming by quadratic Lagrangian method is an effective algorithm to spot the optimums with the best trade-off between nitrogen oxides and soot emissions. The optimisation process presents better qualities at 100% and 75% engine loads than in the case of 50% and 25% engine loads. The design parameters of the optimum under each engine load have something in common, namely, that they all prefer late injection, low swirl, large injection angle and slightly smaller nozzle protrusion length. Besides, start of injection and swirl ratio have larger influence on the objective as opposed to the nozzle protrusion length and spray angle. The large start of injection together with the small swirl ratio can reduce the objective significantly and vice versa. A large nozzle protrusion length with a small spray angle contributes to the reduction of the objective and so does the combination of a small nozzle protrusion length with a large spray angle.

Quantitative 3D Ultrastructure of Thalamocortical Synapses from the "Lemniscal" Ventral Posteromedial Nucleus in Mouse Barrel Cortex.

Thalamocortical synapses from “lemniscal” neurons of the dorsomedial portion of the rodent ventral posteromedial nucleus (VPMdm) are able to induce with remarkable efficacy, despite their relative low numbers, the firing of primary somatosensory cortex (S1) layer 4 (L4) neurons. To which extent this high efficacy depends on structural synaptic features remains unclear. Using both serial transmission (TEM) and focused ion beam milling scanning electron microscopy (FIB/SEM), we 3D-reconstructed and quantitatively analyzed anterogradely labeled VPMdm axons in L4 of adult mouse S1. All VPMdm synapses are asymmetric. Virtually all are established by axonal boutons, 53% of which contact multiple (2–4) elements (overall synapse/bouton ratio = 1.6). Most boutons are large (mean 0.47 μm3), and contain 1–3 mitochondria. Vesicle pools and postsynaptic density (PSD) surface areas are large compared to others in rodent cortex. Most PSDs are complex. Most synapses (83%) are established on dendritic spine heads. Furthermore, 15% of the postsynaptic spines receive a second, symmetric synapse. In addition, 13% of the spine heads have a large protrusion inserted into a membrane pouch of the VPMdm bouton. The unusual combination of structural features in VPMdm synapses is likely to contribute significantly to the high efficacy, strength, and plasticity of these thalamocortical synapses.

Systematic study of packaging designs on the performance of CMOS thermoresistive micro calorimetric flow sensors

We systematically study the effect of two packaging configurations for the CMOS thermoresistive micro calorimetric flow (TMCF) sensors: S-type with the sensor chip protrusion-mounted on the flow channel wall and E-type with the sensor chip flush-mounted on the flow channel wall. Although the experimental results indicated that the sensitivity of the S-type was increased by more than 30%; the corresponding flow range as compared to the E-type was dramatically reduced by 60% from 0–11 m s−1 to 0–4.5 m s−1. Comprehensive 2D CFD simulation and in-house developed 3D numerical simulations based on the gas-kinetic scheme were applied to study the flow separation of these two packaging designs with the major parameters. Indeed, the S-type design with the large protrusion would change the local convective heat transfer of the TMCF sensor and dramatically decrease the sensors’ performance. In addition, parametric CFD simulations of the packaging designs provide inspiration to propose a novel general flow regime map (FRM), i.e. normalized protrusion d* versus reduced chip Reynolds number Re*, where the critical boundary curve for the flow separation of TMCF sensors was determined at different channel aspect ratios. The proposed FRM can be a useful guideline for the packaging design and manufacturing of different micro thermal flow sensors.

Morphology of the UV aurorae Jupiter during Juno's first perijove observations

AbstractOn 27 August 2016, the NASA Juno spacecraft performed its first close‐up observations of Jupiter during its perijove. Here we present the UV images and color ratio maps from the Juno‐UVS UV imaging spectrograph acquired at that time. Data were acquired during four sequences (three in the north, one in the south) from 5:00 UT to 13:00 UT. From these observations, we produced complete maps of the Jovian aurorae, including the nightside. The sequence shows the development of intense outer emission outside the main oval, first in a localized region (255°–295° System III longitude) and then all around the pole, followed by a large nightside protrusion of auroral emissions from the main emission into the polar region. Some localized features show signs of differential drift with energy, typical of plasma injections in the middle magnetosphere. Finally, the color‐ratio map in the north shows a well‐defined area in the polar region possibly linked to the polar cap.

A new species of Cavernocypris Hartmann, 1964 (Crustacea: Ostracoda) from caves in South Korea.

Surveys of caves in South Korea yielded a new species of the ostracod genus Cavernocypris Hartmann, 1964, herein described as Cavernocypris danielopoli Smith & Kamiya n. sp. This is the fifth described species in this widely distributed genus, and can be discriminated from its congeners by the highly triangular shape of the carapace in lateral view, caused by a large dorsal protrusion on the left valve. A second triangular-shaped morphotype was also collected, differing from the first by a lower carapace and less pronounced dorsal protrusion. As it is not clear at present if this represents a separate species or intraspecific variation, it is left in open nomenclature. The lack of a pigmented eye, a highly triangular carapace and no carapace pigmentation suggests that Cavernocypris danielopoli Smith & Kamiya n. sp. is the most adapted species in the genus to a stygobiotic lifestyle. Another Korean species, Cavernocypris coreana (McKenzie, 1972), found in caves and spring discharges, has a reduced number of antennule segments, common for stygobiotic species, but this feature is lacking in the new species. This suggests that within the genus, species have followed different evolutionary pathways to adapt to the subterranean realm.

Chondroitin Sulfate Impairs Neural Stem Cell Migration Through ROCK Activation

Brain injuries such as trauma and stroke lead to glial scar formation by reactive astrocytes which produce and secret axonal outgrowth inhibitors. Chondroitin sulfate proteoglycans (CSPG) constitute a well-known class of extracellular matrix molecules produced at the glial scar and cause growth cone collapse. The CSPG glycosaminoglycan side chains composed of chondroitin sulfate (CS) are responsible for its inhibitory activity on neurite outgrowth and are dependent on RhoA activation. Here, we hypothesize that CSPG also impairs neural stem cell migration inhibiting their penetration into an injury site. We show that DCX+ neuroblasts do not penetrate a CSPG-rich injured area probably due to Nogo receptor activation and RhoA/ROCK signaling pathway as we demonstrate in vitro with neural stem cells cultured as neurospheres and pull-down for RhoA. Furthermore, CS-impaired cell migration in vitro induced the formation of large mature adhesions and altered cell protrusion dynamics. ROCK inhibition restored migration in vitro as well as decreased adhesion size.

The effect of Rho drugs on mast cell activation and degranulation.

Mast cells are tissue-resident immune cells that produce potent proinflammatory mediators, which are stored in cytoplasmic granules. Stimulation triggers degranulation, a process that mobilizes granules to dock and fuse to the plasma membrane, releasing mediators. Mast cell degranulation has an important role in immunity but can also intensify inflammation and contribute to allergic disorders. Hence, it is important to understand signaling pathways that regulate mast cell degranulation. Here, we examined the role of Rho proteins in regulating mast cell activation leading to degranulation. RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) were stimulated through aggregation of FcεRI receptors. Stimulated cells showed a large increase in the levels of activated Rac and, to a lesser extent, RhoA. Drugs were used to acutely inhibit the function of specific Rho proteins. The Rac inhibitor EHT-1864 and the RhoA inhibitor rhosin inhibited degranulation. Microscopic characterization showed that, upon stimulation, RBL-2H3 cells formed surface ridges that grew into large protrusions reminiscent of circular dorsal ruffles, which flattened into large lamellipodia. LysoTracker-labeled cells showed granules stream into peripheral protrusions. EHT-1864 reduced granule motility, whereas rhosin increased motility; both drugs affected the formation of peripheral protrusions. These results showed that, in response to stimuli, Rho proteins control discrete cytoskeletal remodeling processes that are needed for granule exocytosis. Rac is required to stimulate the remodeling of mast cells, triggering actin-mediated flattening of the cell periphery to create an active degranulation zone, whereas RhoA controls the streaming of highly motile granules into the active zone.

Anatomical and Palynological Characters of Celtis L. In Kurdistan-Iraq

Anatomical and palynological characters of Celtis genus growing in Iraq were studied in an attempt to provide useful features to delimit taxa and to refine taxonomic relationships. Results revealed the presence of lithocysts inside the leaf structure of C. tournefortii with few of them forming very small protrusion out of the leaf, while in C. australis lithocysts form large protrusion out from the leaf. The large midrib adaxial hump of C. tournefortii constitute a wide area between the two sides of the leaf blade, while in C. australis the small midrib adaxial hump produce narrow area between the two sides of the leaf blade. Stomata are of anomocytic type in C. australis while, anomocytic and paracytic in C. tourneforti. Furthermore, simple hairs are dense on the adaxial side of the petiole of C. tournefortii var. tournefortii, while absent from the adaxial side of the petiole of C. tournefortii var. glabrata. Leaves of all taxa are distinctly bifacial, hypostomatic, and provided with one layer of palisade parenchyma. Pollen grains are 3-porate, rarely 4-porate. A sunken area in the aperture surrounding a pore is distinct. Pollen is mediae in size and subprolate in shape, the pollen grains of C. australis together with var. glabrata belong to Celtis-type grain, while, var. tournefoertii to the Ulmus-type grain. The high anatomical and palynological differences between varieties of C. tournefoertii support promotion of these two varieties to the specific level.

Virion Structure of Black Queen Cell Virus, a Common Honeybee Pathogen.

ABSTRACTViral diseases are a major threat to honeybee (Apis mellifera) populations worldwide and therefore an important factor in reliable crop pollination and food security. Black queen cell virus (BQCV) is the etiological agent of a fatal disease of honeybee queen larvae and pupae. The virus belongs to the genus Triatovirus from the family Dicistroviridae, which is part of the order Picornavirales. Here we present a crystal structure of BQCV determined to a resolution of 3.4 Å. The virion is formed by 60 copies of each of the major capsid proteins VP1, VP2, and VP3; however, there is no density corresponding to a 75-residue-long minor capsid protein VP4 encoded by the BQCV genome. We show that the VP4 subunits are present in the crystallized virions that are infectious. This aspect of the BQCV virion is similar to that of the previously characterized triatoma virus and supports the recent establishment of the separate genus Triatovirus within the family Dicistroviridae. The C terminus of VP1 and CD loops of capsid proteins VP1 and VP3 of BQCV form 34-Å-tall finger-like protrusions at the virion surface. The protrusions are larger than those of related dicistroviruses.IMPORTANCE The western honeybee is the most important pollinator of all, and it is required to sustain the agricultural production and biodiversity of wild flowering plants. However, honeybee populations worldwide are suffering from virus infections that cause colony losses. One of the most common, and least known, honeybee pathogens is black queen cell virus (BQCV), which at high titers causes queen larvae and pupae to turn black and die. Here we present the three-dimensional virion structure of BQCV, determined by X-ray crystallography. The structure of BQCV reveals large protrusions on the virion surface. Capsid protein VP1 of BQCV does not contain a hydrophobic pocket. Therefore, the BQCV virion structure provides evidence that capsid-binding antiviral compounds that can prevent the replication of vertebrate picornaviruses may be ineffective against honeybee virus infections.

ZnSnP2 thin-film solar cell prepared by phosphidation method under optimized Zn/Sn atomic ratio of its absorbing layer

ZnSnP2 film is a promising absorber for thin-film solar cell due to earth-abandant and nontoxic element. Phosphidation method is utilized for the fabrication of ZnSnP2 films with different Zn/Sn atomic ratios. ZnSnP2 film with the large ZnSnP2 protrusions are demonstrated with Zn/Sn of ∼1 (near stoichiometry), while the film with the relatively smooth surface is presented with Zn/Sn of above 1 (Zn rich). According to grazing incidence X-ray diffraction measurement, Sn4P3 secondary phase is appeared in ZnSnP2 film with Zn/Sn of below 1 (Sn rich), whereas Zn3P2 secondary phase is presented in the film with Zn rich. On the other hand, Sn4P3 and Zn3P2 secondary phases are not observed in ZnSnP2 thin film with near stoichiometry, thus leading to the longest carrier lifetime, implied by time-resolved photoluminescence measurement. Ultimately, the conversion efficiency of 0.021% with short-circuit current density of 5.03 mA/cm2 is reported for ZnSnP2 thin-film solar cell, where its absorbing layer possesses single ZnSnP2 phase and Zn/Sn ratio near stoichiometry. The energy bandgap of ZnSnP2 thin films by phosphidation method is estimated to be 1.38 eV by external quantum efficiency, implying that the ZnSnP2 has sphalerite structure.

Asymmetric separated flow about a Nikolsky wing with a protrusion

Abstract The flow of an inviscid incompressible fluid about a wing of low aspect ratio with a parabolic planform (a Nikolsky wing) is investigated; a protrusion, whose height grows according to a parabolic law, is mounted on the leeward side of the wing in the symmetry plane. It is shown that for symmetric boundary conditions, along with a symmetric solution, an asymmetric solution also exists. The dependence of the asymmetric solution on the wing geometry is examined. It is shown that critical values of the wing curvature and the height of the protrusion exist, for which the asymmetric solution continuously transitions to the symmetric one. In addition, it is shown that a limit asymmetric solution exists which corresponds to an infinitely large protrusion. The stability of the solutions found is discussed.

Large abdominal protrusion.

Large abdominal protrusion.

Tropomodulin 1 Regulation of Actin Is Required for the Formation of Large Paddle Protrusions Between Mature Lens Fiber Cells.

PurposeTo elucidate the proteins required for specialized small interlocking protrusions and large paddle domains at lens fiber cell tricellular junctions (vertices), we developed a novel method to immunostain single lens fibers and studied changes in cell morphology due to loss of tropomodulin 1 (Tmod1), an F-actin pointed end–capping protein.MethodsWe investigated F-actin and F-actin–binding protein localization in interdigitations of Tmod1+/+ and Tmod1−/− single mature lens fibers.ResultsF-actin–rich small protrusions and large paddles were present along cell vertices of Tmod1+/+ mature fibers. In contrast, Tmod1−/− mature fiber cells lack normal paddle domains, while small protrusions were unaffected. In Tmod1+/+ mature fibers, Tmod1, β2-spectrin, and α-actinin are localized in large puncta in valleys between paddles; but in Tmod1−/− mature fibers, β2-spectrin was dispersed while α-actinin was redistributed at the base of small protrusions and rudimentary paddles. Fimbrin and Arp3 (actin-related protein 3) were located in puncta at the base of small protrusions, while N-cadherin and ezrin outlined the cell membrane in both Tmod1+/+ and Tmod1−/− mature fibers.ConclusionsThese results suggest that distinct F-actin organizations are present in small protrusions versus large paddles. Formation and/or maintenance of large paddle domains depends on a β2-spectrin–actin network stabilized by Tmod1. α-Actinin–crosslinked F-actin bundles are enhanced in absence of Tmod1, indicating altered cytoskeleton organization. Formation of small protrusions is likely facilitated by Arp3-branched and fimbrin-bundled F-actin networks, which do not depend on Tmod1. This is the first work to reveal the F-actin–associated proteins required for the formation of paddles between lens fibers.

Front-Rear Polarization by Mechanical Cues: From Single Cells to Tissues.

Directed cell migration is a complex process that involves front-rear polarization, characterized by cell adhesion and cytoskeleton-based protrusion, retraction, and contraction of either a single cell or a cell collective. Single cell polarization depends on a variety of mechanochemical signals including external adhesive cues, substrate stiffness, and confinement. In cell ensembles, coordinated polarization of migrating tissues results not only from the application of traction forces on the extracellular matrix but also from the transmission of mechanical stress through intercellular junctions. We focus here on the impact of mechanical cues on the establishment and maintenance of front-rear polarization from single cell to collective cell behaviors through local or large-scale mechanisms.