Microvilli Structures Research Articles

Novel In Vitro Models for Cell Differentiation and Drug Transport Studies of the Human Intestine.

The most common in vitro model for absorption, distribution, metabolism, and excretion (ADME) purposes is currently the Caco-2 cell line. However, clear differences in gene and protein expression towards the small intestine and an, at best, fair prediction accuracy of intestinal drug absorption restrict the usefulness of a model for intestinal epithelial cells. To overcome these limitations, we evaluated a panel of low-passaged patient-derived colorectal cancer cell lines of the HROC collection concerning similarities to small intestinal epithelial cells and their potential to predict intestinal drug absorption. After initial screening of a larger panel, ten cell lines with confluent outgrowth and long-lasting barrier-forming potential were further characterized in close detail. Tight junctional complexes and microvilli structures were detected in all lines, anda higher degree of differentiation was observed in 5/10 cell lines. All lines expressed multiple transporter molecules, with the expression levels in three lines being close to those of small intestinal epithelial cells. Compared with the Caco-2 model, three HROC lines demonstrated both higher similarity to jejunal epithelial tissue cells and higher regulatory potential of relevant drug transporters. In summary, these lines would be better-suited human small intestinal epithelium models for basic and translational research, especially for ADME studies.

White feces syndrome in Penaeus vannamei is potentially an Enterocytozoon hepatopenaei (EHP) associated pathobiome origin of Vibrio spp

White feces syndrome (WFS) is a commercially important disease in Penaeus vannamei (whiteleg shrimp) farming. The aetiology beyond the white or golden white midgut with mediocre growth performance producing a floating mass of white fecal strings in WFS-affected shrimp farms remains uncharted. To give WFS a perception of pathobiome, healthy P. vannamei shrimps were subjected to an enteric microsporidian Enterocytozoon hepatopenaei (EHP) infection along with Vibrio harveyi and V. alginolyticus in different combinations. Immune responses in haemolymph (total haemocyte count (THC), prophenoloxidase activity (proPO), respiratory burst activity (RBA), superoxide dismutase activity (SOD) and catalase activity (CAT)), plasma biochemical changes (aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP)) and digestive enzymes activity (alpha-amylase (AMY), lipase (LIP) and protease (PRO)) were assessed in the challenged shrimps at 5, 10 and 15 days post-infection (dpi). The microbial interactions between the EHP and Vibrio spp. have led to the formation of WFS in the challenged shrimps. The histological sections of the hepatopancreas revealed the presence of EHP along with colonized bacterial masses, leading to the formation of aggregated transformed microvilli (ATM) structures and increased sloughing of lipid vacuoles into the tubule lumen. A significantly decreased THC and increased proPO levels, dysregulated antioxidant system, prominent hepatic damage, reduced energy metabolism and higher lipid production were the key records supporting that EHP-associated WFS in P. vannamei is due to the pathobiome.

Abstract 3613: Effect of thrombomodulin and plasminogen interaction on melanoma spheroid invasion

Abstract Background: Thrombomodulin (TM), a transmembrane glycoprotein that acts as a plasminogen (Plg) receptor, has been found as a potential molecular and prognostic marker that impacts the spread of various cancers, including malignant melanoma. A previous study showed that TM plays a vital function in angiogenesis activity [1] but its role in the malignant melanoma invasion is unclear. Here, we investigate the role of TM-Plg by using melanoma spheroid (MS) embedded in an extracellular matrix (ECM) modeled to understand how TM-Plg affects the progression of MS invasion. Methods: In this work, A2058 (A2058-GFP and A2058-TMGFP) and MeWo (siC and siTM) MS with different TM expressions were formed, harvested, and embedded in type I collagen gel for invasion assay. TM and Plg expression were determined by western blot. The surface of A2058 MS was observed by SEM imaging. Human Plg, MMPs, and Plg inhibitors, tranexamic acid (TXA) and epsilon-aminocaproic acid (ε-ACA), were utilized to investigate the effect of TM on the plasmin system. Immunofluorescence of TM and F-actin were validated. Results: When the two cell lines expressed with TM, the formed MS invaded to the surrounding matrix more distinctively than that of the TM-silencing ones, with a 2.5-fold difference in the ratio invasive area change. Upon placing the two A2058-TMGFP MS in the vicinity of each other within the ECM, they showed a strong interaction with an observed distance between the two MS from 50 μm to 280 μm. Immunofluorescence of TMGFP assessed the role of TM in MS invasion and interaction in ECM. Microvilli structures were found on the surface of A2058-TMGFP cells that may help the cell have more cellular processes such as adhesion, signal transduction, and metabolic activities. Furthermore, TXA, ε-ACA, and MMP inhibitors inhibited the invasion of A2058-TMGFP MSs in a dose-dependent manner. Human Plg stimulated invasion in TM-expressing MSs while no effect on non-TM MSs. The results suggested that the human Plg only significantly increased the MS invasion with the presence of TM. Conclusion: We concluded that with the presence of TM, the MS invasion and interaction become stronger than MS without TM expression. Moreover, TM enhances MS invasion in the 3D model via interacting Plg. This study provides evidence for the development of TM antagonists in the future to analyze the drug's potential as an anti-metastasis treatment.[1] P. K. Chen et al., “Thrombomodulin functions as a plasminogen receptor to modulate angiogenesis,” FASEB J., vol. 27, no. 11, pp. 4520-4531, 2013, doi: 10.1096/fj.13-227561. Citation Format: Thi Kim Ngan Ngo, Hua-Lin Wu, Cheng-Hsiang Kuo, Ting-Yuan Tu. Effect of thrombomodulin and plasminogen interaction on melanoma spheroid invasion. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3613.

Prebiotics modify host metabolism in rainbow trout (Oncorhynchus mykiss) fed with a total plant-based diet: Potential implications for microbiome-mediated diet optimization

The use of plant-based ingredients in aquafeeds is increasing because marine sources of protein and oil are unsustainable. However, plant-based ingredients cause certain metabolic complications in carnivorous species such as rainbow trout. Here, we examined whether prebiotics have the potential to affect the metabolism of juvenile trout (Average weight: 25.88 ± 0.91 g) via microbially derived short-chain fatty acids (SCFAs). Fructo-oligosaccharides (FOS), inulin or mannan-oligosaccharides (MOS) were used at 1% or 2% in a 12-week feeding experiment. We measured changes in the intestinal microbiome, SCFA levels and metabolic responses in the intestine, liver, muscle, and adipose tissue. In the intestine, gene expression and SCFA production did not change significantly with prebiotics, although the MOS fed groups were clustered differently. Prebiotics had a significant effect on the intestinal abundance of Bacillus, Lactobacillus and Weissella in a dose dependent manner, although the microbial diversity (alpha and beta) did not change significantly after feeding prebiotics. Two operational taxonomic units (OTUs) belonging to the genus Mycoplasma dominated all samples with an average relative abundance of >95% per group. Intestinal microvillar structures were significantly improved in length in the inulin-fed groups.Systemically, overall hepatic gene expression was significantly different from control with inulin-fed groups showing upregulation of several metabolic and the fatty acid receptor genes. MOS fed groups showed a dose-dependent but contrasting response in liver and muscle. In addition, a significant effect of prebiotic and the interaction between the 2 factors (prebiotic and dose) on final weight and SGR respectively, was observed. The relative abundance of OTUs belonging to Lactobacillus and Bacillus correlated with hepatic gene expression and final weight of the fish.Inulin and MOS appear to differentially affect host metabolism, mainly in the liver and muscle. Differential abundance of Lactobacillus and Bacillus in the prebiotic-fed groups and their correlations with hepatic gene expression could indicate a prebiotic-microbiome-host axis, although this was not conclusively shown through the levels of SCFAs. In combination with a total plant-based diet, inulin could be a promising prebiotic for trout but need to be further investigated. These findings could implicate in microbiome-mediated dietary optimization of rainbow trout.

Polymer nano manufacturing of a biomimicking surface for kidney stone crystallization studies

Detected kidney stone cases are increasing globally, yet knowledge on the conditions for stone formation is lacking. Experimental approaches mimicking the micro-environmental conditions present in vivo can help scientists untangle intertwined physiochemical and biological phenomena leading to kidney stone formation. As crystal nucleation often initiates at liquid-solid interfaces, the interface morphology plays a significant role in the rate of nucleation. Within the nephron, the functional unit of the kidney, four segments can be distinguished that contain different surface morphologies. Particularly, the cells lining these segments contain protrusions in the shape of nanopillars that vary in length, diameter and spacing. Exploiting the opportunities provided by organ-on-a-chip technology, we designed and manufactured a proof-of-principle microfluidic device proposed to increase our understanding of the relation between kidney surface morphology and kidney stone crystallization. We used two-photon polymerization to fabricate biocompatible surfaces that mimic the nephron morphologies with materials properties similar to those of biological structures. The fabricated cilia were incorporated in the microfluidic device, which was designed to observe in vitro crystallization of calcium oxalate under flow. • Free-standing polymer wires of aspect ratio ~ 100 using 2-photon-lithography. • High-density polymer nanopillars down to 150 nm diameter and 150 nm spacing. • Mimicking the cilia and microvilli structures and topography in the kidney nephron. • Combining with microfluidic device to study Ca crystallization.

Clinical manifestations of White feces syndrome (WFS), and its association with Enterocytozoon hepatopenaei in Penaeus vannamei grow-out farms: A pathobiological investigation

White feces syndrome (WFS) has recently been reported as a serious problem for shrimp aquaculture in major shrimp farming nations. WFS affected shrimp show white/golden brown intestine, reduced feed intake, stunted growth and is often associated with loose shell, while WFS affected pond show masses of floating white feces on the surface of the pond. Accumulation of aggregated transformed microvilli (ATM) structures in hepatopancreas (HP) lumen that superficially resemble gregarine are reported to cause WFS. But the causal association of ATM and WFS is unknown. Also, WFS occurrences are significantly associated with incidences of the emerging microsporidian Enterocytozoon hepatopenaei (EHP) in the Penaeus vannamei grow-out system. The present study unveiled the occurrences of EHP spores along with ATM structures in the WFS affected shrimps. The histology section stained with calcofluor white stain (CFW) and semithin section revealed the presence of EHP spores in and around the transformed microvilli. Further, EHP spores were detected in all the individual WFS affected shrimps and white fecal threads by light microscopy and SWP-PCR. All WFS samples tested negative for WSSV, IHHNV, IMNV, and AHPND. None of the Vibrio isolates were found predominant in WFS affected shrimps. In bioassay studies, by feeding with WFS affected HP tissue, white fecal threads were successfully reproduced along with ATM structures and EHP spores. However, purified EHP spores (106 ml−1) failed to elicit WFS in challenged animals. In the clinical experiment, when WFS affected shrimp were reared under optimal laboratory settings, white feces were found to resolve, and the animals excreted normal feces in about 7–10 days, without any interventions. Further, histological studies showed the epithelial cells (E cells or embryonic cells) regeneration ability in WFS resolved shrimp. However, the WFS recovery observed was not a complete recovery since the resolved shrimps continue to shed EHP spores in normal feces. It is observed that EHP is the major pathogen excreted/disseminated via white feces in shrimp. With this study, we postulate that WFS is a clinical condition/shrimp diarrhea associated with severe EHP infection that may be caused by EHP in combination with an unknown agent.

Oxidative Stress Boosts the Uptake of Cerium Oxide Nanoparticles by Changing Brain Endothelium Microvilli Pattern

Vascular oxidative stress is considered a worsening factor in the progression of Alzheimer’s disease (AD). Increased reactive oxygen species (ROS) levels promote the accumulation of amyloid-β peptide (Aβ), one of the main hallmarks of AD. In turn, Aβ is a potent inducer of oxidative stress. In early stages of AD, the concomitant action of oxidative stress and Aβ on brain capillary endothelial cells was observed to compromise the blood–brain barrier functionality. In this context, antioxidant compounds might provide therapeutic benefits. To this aim, we investigated the antioxidant activity of cerium oxide nanoparticles (CNP) in human cerebral microvascular endothelial cells (hCMEC/D3) exposed to Aβ oligomers. Treatment with CNP (13.9 ± 0.7 nm in diameter) restored basal ROS levels in hCMEC/D3 cells, both after acute or prolonged exposure to Aβ. Moreover, we found that the extent of CNP uptake by hCMEC/D3 was +43% higher in the presence of Aβ. Scanning electron microscopy and western blot analysis suggested that changes in microvilli structures on the cell surface, under pro-oxidant stimuli (Aβ or H2O2), might be involved in the enhancement of CNP uptake. This finding opens the possibility to exploit the modulation of endothelial microvilli pattern to improve the uptake of anti-oxidant particles designed to counteract ROS-mediated cerebrovascular dysfunctions.

The Reliability of Fluorescence Z-Scan Analysis in the Complex Environment of the Living Cell

Fluorescence z-scan analysis aims to fit the intensity traces recorded while moving a two-photon excitation volume vertically through a cell in order to identify the vertical concentration profile of a fluorescent species. Z-scan analysis has proved able to quantify delicate PM-protein binding interactions as well as sub-resolution partitioning of proteins due to the actin cortex. Despite recent progress, questions remain regarding the reliability and applicability of z-scan analysis in the complex environment of the living cell. In high precision z-scan applications, obtaining a good quality of fit is critical to ensuring that experimental results remain uncontaminated by fluorescent features outside the scope of the z-scan modeling. However, a statistical method to robustly assess the quality of fit in z-scan analysis has been lacking. To address these issues, we provide control data validating core aspects of the z-scan method at high precision and demonstrate the potential for error when applying the method without rigorous quality of fit controls. We propose a conceptual framework for estimating the amplitude of errors in z-scan analysis due to fluorescent features that may not be included in the z-scan fit model. We apply this framework to analyze the potential for microvilli structures, abundant in some cell lines, to perturb z-scan measurements and we outline data quality controls that contain the potential for error. This work provides a foundation supporting the use of simple stratified layers to model concentration profiles within the living cell together with checks that identify when such simplified models may be inapplicable for a given level of precision. This work has been supported by grants from the National Institutes of Health (R01 GM064589, R01 GM098550, RO1 GM124279).

Ultrastructural evaluation of oocyte envelopes of zebrafish (Danio Rerio) (Hamilton, 1822) after TiO2 nanoparticle exposure

Titanium dioxide (TiO 2 )is one of the most widely used nanoparticles, and aquatic organisms are especially exposed to it. To examine reproductive toxicology, zebrafish were exposed to different concentrations (1, 2 and 4 mg/L) of TiO 2 nanoparticles. The ultrastructure of the theca cell, zona radiata structure and follicular epithelium were examined in detail by transmission electron microscopy (TEM). No abnormalities were observed in the control group; however, degeneration of pore and microvilli structures of the zona radiata, vacuolization in the ooplasm, mitochondrial swelling and mitotic catastrophe (the mechanism for eliminating mitosis-incompetent cells in eukaryotes) were detected in the exposure groups. These results indicate that TiO 2 nanoparticle exposure causes paraptotic-type cell death in zebrafish oocytes, follicular and theca cells. In light of the observed histopathological changes, it was concluded that TiO 2 exposure inhibited oogenesis and the reproductive capability in zebrafish. https://doi.org/10.2298/ABS170303035A Received: March 3, 2017; Revised: August 8, 2017; Accepted: September 20, 2017; Published online: October 3, 2017 How to cite this article: Akbulut C, Kotil T, Yon DN. Ultrastructural evaluation of oocyte envelopes of zebrafish ( Danio Rerio ) (Hamilton, 1822) after TiO2 nanoparticle exposure. Arch Biol Sci. 2018;70(1):159-65.

Optimization of micro-fabricated porous membranes for intestinal epithelial cell culture and in vitro modeling of the human intestinal barrier

In vitro modeling of organs could provide a controlled platform for studying physiological events and has great potential in the field of pharmaceutical development. Here, we describe the characterization of in vitro modeling of the human intestinal barrier mimicked using silicon porous membranes as a substrate. To mimic an intestinal in vivo setup as closely as possible, a porous substrate is required in a dynamic environment for the cells to grow rather than a static setup with an impermeable surface such as a petri dish. In this study, we focus on the detailed characterization of Caco-2 cells cultured on a silicon membrane with different pore sizes as well as the effect of dynamic fluid flow on the model. The porous silicon membrane together with continuous perfusion of liquid applying shear stress on the cells enhances the differentiation of polarized cells by providing access to the both their basal and apical surfaces. Membranes with pore sizes of 0.5–3 µm were used and a shear stress of ~0.03 dyne cm−2 was created by applying a low flow rate of 20 nl s−1. By providing these optimized conditions, cells were able to differentiate with columnar morphology, which developed microvilli structures on their apical side and tight junctions between adjacent cells like those in a healthy human intestinal barrier. In this setup, it is possible to study the important cellular functions of the intestine such as transport, absorption and secretion, and thus this model has great potential in drug screening.

Control of cytoskeletal dynamics during cellular responses to pore forming toxins

ABSTRACTFollowing damage by pore forming toxins (PFTs) host cells engage repair processes and display profound cytoskeletal remodeling and concomitant plasma membrane (PM) blebbing. We have recently demonstrated that host cells utilize similar mechanisms to control cytoskeletal dynamics in response to PFTs and during cell migration. This involves assembly of cortical actomyosin bundles, reorganisation of the endoplasmic reticulum (ER) network, and the interaction between the ER chaperone Gp96 and the molecular motor Non-muscle Myosin Heavy Chain IIA (NMHCIIA). Consequently, Gp96 regulates actomyosin activity, PM blebbing and cell migration, and protects PM integrity against PFTs. In addition, we observed that PFTs increase association of Gp96 and ER vacuoles with the cell surface or within PM blebs loosely attached to the cell body. Similarly, gut epithelial cells damaged by PFTs in vivo were shown to release microvilli structures or directly purge cytoplasmic content. Cytoplasmic purging involves profound cytoskeletal remodeling and ER vacuolation, suggesting that our observations recapitulate recovery processes in vivo. Here, we discuss our findings in light of the current understanding of PM repair mechanisms and in vivo recovery responses to PFTs.

TAGLN2 inhibits Arp2/3 complex-nucleated actin branching and controls microvilli formation in T cells

Abstract Microvilli are abundant on the surfaces of circulating lymphocytes. However, factors that regulate microvilli formation on lymphocytes are not completely understood. Moreover, definitive roles of microvilli have not been fully addressed. Here we identified that TAGLN2—an actin-binding protein predominantly expressed in T cells— specifically localizes at the microvilli on T cells. Ectopic expression of TAGLN2 enhanced microvilli protrusion at the initial development of immunological synapse, and the opposite results were obtained by TAGLN2 depletion. Obviously, T-cell activation was influenced by the microvilli structures on T cells. Of interest was that TAGLN2 blocks Arp2/3 complex binding to actin filaments that leads to the inhibition of actin branch. Electron microscopy-based helical image analysis revealed that the main sites of TAGLN2 binding on actin share the Arp2/3 complex binding sites. These results demonstrate a novel function of TAGLN2 that contributes to enhance T-cell antigen recognition, and hence T-cell activation.

ASAP3 regulates microvilli structure in parietal cells and presents intervention target for gastric acidity

Gastric acidity-associated disorders such as peptic ulcer and reflux diseases are widespread, and the reported resistance and side effects of currently used medicines suggest an urgent requirement for alternative therapeutic approaches. Here we demonstrate a critical role of ASAP3 in regulating the microvilli structure of parietal cells in vivo, and reveal the feasibility of controlling gastric acidity by targeting ASAP3. Conditional knockout of ASAP3 in mice caused elongation and stacking of microvilli in parietal cells, and substantially decreased gastric acid secretion. These were associated with active assembly of F-actin caused by a higher level of GTP-bound Arf6 GTPase. Consistently, a small molecular compound QS11 inhibited ASAP3 function and significantly reduced gastric acidity in vivo. Of note, the expression of ASAP3 was positively correlated with gastric acid secretion in 90 human cases, and high expression of ASAP3 was associated with reflux disease and peptic ulcer. These results reveal for the first time that ASAP3 regulates the microvilli structures in parietal cells. Our data also suggest ASAP3 as a feasible and drugable therapeutic target for gastric acidity-associated diseases.

Mechanistic Basis of Organization of the Harmonin/USH1C-Mediated Brush Border Microvilli Tip-Link Complex

Brush border microvilli are actin-based protrusions lining the apical surface of epithelial cells in intestines and proximal tubules of kidneys. While brush border microvilli resemble the relatively well-characterized stereocilia of hair cells, the mechanistic basis of tip-link complex organization in microvilli is poorly understood. Here, we have biochemically and structurally characterized the following pairs of interactions: protocadherin 24 and Harmonin (also known as USH1C or AIE-75), Harmonin and myosin VIIb (MYO7B), Harmonin and ANKS4B, and ANKS4B and MYO7B. We show that Harmonin, ANKS4B, and MYO7B form a stable ternary complex for anchoring microvilli tip-link cadherins. Despite having only Harmonin in common, the microvilli and the stereocilia tip-link complexes are formed via strikingly similar interaction modes. These results not only provide insight into the mechanistic bases of brush border microvilli formation and maintenance but may also be valuable for understanding some gut and/or kidney diseases caused by perturbations of brush border microvilli structures.

PeakForce Tapping resolves individual microvilli on living cells.

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip–sample interactions in time (microseconds) and controlling force in the low pico‐Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM‐Live Cell probe, having a short cantilever with a 17‐µm‐long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico‐Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

Lymphatic Endothelium Forms Integrin-Engaging 3D Structures during DC Transit across Inflamed Lymphatic Vessels

Dendritic cell (DC) transmigration across the lymphatic endothelium is critical for the initiation and sustenance of immune responses. Under noninflammatory conditions, DC transit across the lymphatic endothelial cell (LEC) has been shown to be integrin independent. In contrast, there is increasing evidence for the participation of integrins and their ligands in DC transit across lymphatic endothelium under inflammation. In this sense, we describe the formation of ICAM-1 (CD54)-enriched three-dimensional structures on LEC/DC contacts, as these DCs adhere to inflamed skin lymphatic vessels and transmigrate into them. In vitro imaging revealed that under inflammation ICAM-1 accumulated on microvilli projections surrounding 60% of adhered DCs. In contrast, these structures were scarcely formed in noninflammatory conditions. Furthermore, ICAM-1-enriched microvilli were important in promoting DC transendothelial migration and DC crawling over the LEC surface. Microvilli formation was dependent on the presence of β-integrins on the DC side and on integrin conformational affinity to ligand. Finally, we observed that LEC microvilli structures appeared in close vicinity of CCL21 depots and that their assembly was partially inhibited by CCL21-neutralizing antibodies. Therefore, under inflammatory conditions, integrin ligands form three-dimensional membrane projections around DCs. These structures offer docking sites for DC transit from the tissue toward the lymphatic vessel lumen.

Dissection and Immunohistochemistry of Larval, Pupal and Adult <em>Drosophila</em> Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8. The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes: In the 'p' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the 'y' subtype, Rh4 in yR7s is associated with Rh6 in yR8s. Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc and initiates the assembly of undifferentiated cells into ommatidia. The 'founder cell' R8 is specified first and recruits R1-6 and then R7. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes, including rhabdomere formation, synaptogenesis and eventually rh expression. In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).

Adsorption of hematite nanoparticles onto Caco-2 cells and the cellular impairments: effectof particle size

The increasing applications of engineered nanomaterials nowadays have elevated thepotential of human exposure through various routes including inhalation, skin penetrationand digestion. To date there is scarce information on a quantitative description of theinteractions between nanoparticles (NPs) and cell surfaces and the detrimental effects from theexposure. The purpose of this work was to study in vitro exposure of Caco-2 cells to hematite (α-Fe2O3) NPs and to determine the particle size effects on the adsorption behaviors. Cellularimpairment was also investigated and compared. Hematite NPs were synthesized as part ofthis study with a discrete size distribution and uniform morphology examined by dynamiclight scattering (DLS) and confirmed by transmission electron microscopy (TEM). Caco-2cells were cultured as a model epithelium to mirror human intestinal cells and used toevaluate the impacts of the exposure to NPs by measuring transepithelial electricalresistance (TEER). Cell surface disruption, localization and translocation of NPs throughthe cells were analyzed with immunocytochemical staining and confocal microscopy.Results showed that hematite NPs had mean diameters of 26, 53, 76 and 98 nm and werepositively charged with minor aggregation in the buffer solution. Adsorption of thefour sizes of NPs on cells reached equilibrium within approximately 5 min butadsorption kinetics were found to be size-dependent. The adsorption rates expressed asmg m − 2 min − 1 were greater for large NPs (76 and 98 nm) than those for small NPs(26 and 53 nm). However, adsorption rates, expressed in units ofm − 2 min − 1, were much greater for small NPs than large ones. After the adsorptionequilibrium was reached, the adsorbed mass of NPs on a unit area of cells wascalculated and showed no significant size dependence. Longer exposure time (>3 h) induced adverse cellular effects as indicated by the drop in TEER compared tothe control cells without the exposure to NPs. NPs initially triggered a dynamicreorganization and detachment of microvilli structures on Caco-2 cell surfaces.Following this impact, the drop in TEER occurred more significantly, particularlyfor the exposure to 26 nm NPs, which was consistent with the observations withconfocal microscopy that the junctions were more severely disrupted by 26 nm NPsthan other sizes. In conclusion, this paper demonstrates the interactions at theultrastructural level from initial surface adsorption of NPs upon cells, to thesubsequent microvilli reorganization, membrane penetration and the disruption ofadherens junction and provides the fundamental information on size effects on NPbehavior which is often poorly addressed for in vitro cytotoxicity studies of NPs.

Modulation Particle Tracking

In this study, we present a novel optical imaging method that makes use of high precision particle tracking of fluorescent particles to obtain images of nanometer size structures in live cells. Particle tracking not only provides the trajectory of the center of mass but also the particle orientation and size can now be observed, in vivo and real time with the nanometer resolution. This method helps in further understanding of the dynamics of the small particles in biological systems, which was hard to achieve by the current optical techniques. The method is based in rapidly modulating the position of the laser beam around small structures on the order of 100nm in size. When the laser spot oscillates in the direction toward the particle surface, the fluorescence of the particle is modulated. The modulation, which is the ratio of the alternating part to the average fluorescence intensity, is a function of the distance of the particle from the center of mass to the oscillation. In order to track the particle, we circularly moved the oscillating laser spot around the moving particle, and at the same time, analyzed the modulation in the frequency spectrum of the intensity along the orbit to perform a feedback loop updating the average laser position to the center of mass of the particle position. The size, shape and orientation information of the fluorescent structure can then be obtained by looking at the higher order modulations components. We explain the theory behind this method and we show the 3D reconstruction of nanometer microvilli structures on the apical membrane of OK cells.Work supported in part by NIH-P41 P41-RRO3155 and P50-GM076516

Possible role of class I myosins in molecule segregation at B cell microvilli structures (78.37)

Abstract Cell cytoskeleton plasticity is now recognized as an important element of cell to cell and cell to antigen interactions. It has been shown that T cells and dendritic cells use shape changes in order to establish effective contacts between them. B lymphocytes also show cytoskeleton rearrangements during antigen recognition. The B cell surface change upon activation with LPS or anti-CD40 plus IL-4; these stimuli induce the generation of long thin microvilli, longer than those observed in resting conditions. These structures are formed by a central actin filaments bundle with bridging associated proteins that links them to plasma membrane. One of these proteins are class I myosins, which are molecular motors that interacts both with actin fibers and plasma membrane phosphoinositides; Myo1c and Myo1g are importantly expressed in B cells and appears to increase upon activation. By using live-cell microscopy we determined that they are present across microvilli structures and seems to be involved in their formation. Finally, our data suggest a role for microvilli by segregating molecules such as MHC-II, LFA-1, CD40, etc. possibly mediated by class I myosins. Supported by CONACyT, project 56836