Properties of ATP-ase of the microvillus membrane isolated from epithelial cells of rabbit small intestine

For the purpose to investigate the physiological functions of microvillus ATPase, general properties of the enzyme were studied on the microvillus membranes isolated from rabbit intestinal epithelial cells. 1) ATPase of the microvillus membranes was activated with Mg2+. Mg.ATP complex was thought to be a subStrate of the enzyme. The Michaelis constant for ATP of the ATPase was a value of 0.8 to I .0 mM. 2) The microvillus ATPase was also activated with Ca2+, but the affinity was lower than a half of that of Mg2+. 3) The optimum pH of the ATPase was about 7.8. 4) Activity of the microvillus ATPase was markedly inhibited by treating with deoxycholate (DOC), and the activity inhibited was partially restored by washing the microvillus membrane with distilled water. The structure of the membranes destroyed by treating with DOC was also partially restored by the same procedure. 5) Ultrasonic treatment also markedly destroyed the microvillus membrane and inhibited ATPase activity. Damaged ultrastructure and ATPase activity both were partially restored by treating with phospholipid, EPL. 6) Simultaneous presence of Na+ and K + stimulated scarcely the ATPase of purified microvillus membranes. 7) The microvillus ATPase was slightly activated in the presence of n-glucose. Phloridin gave little effect on the activity of the microvillus ATPase.</p

Biophysical modelling of a drosophila photoreceptor

It remains unclear how visual information is co-processed by different layers of neurons in the retina. In particular, relatively little is known how retina translates vast environmental light changes into neural responses of limited range. We began examining this question in a bottom-up way in a relatively simple °y eye. To gain understanding of how complex bio-molecular interactions govern the conversion of light input into voltage output (phototransduction), we are building a biophysical model of the Drosophila R1-R6 photoreceptor. Our model, which relates molecular dynamics of the underlying biochemical reactions to external light input, attempts to capture the molecular dynamics of phototransduction gain control in a quantitative way

Molecular Model of the Microvillar Cytoskeleton and Organization of the Brush Border

BACKGROUND. Brush border microvilli are ~1-µm long finger-like projections emanating from the apical surfaces of certain, specialized absorptive epithelial cells. A highly symmetric hexagonal array of thousands of these uniformly sized structures form the brush border, which in addition to aiding in nutrient absorption also defends the large surface area against pathogens. Here, we present a molecular model of the protein cytoskeleton responsible for this dramatic cellular morphology. METHODOLOGY/PRINCIPAL FINDINGS. The model is constructed from published crystallographic and microscopic structures reported by several groups over the last 30+ years. Our efforts resulted in a single, unique, self-consistent arrangement of actin, fimbrin, villin, brush border myosin (Myo1A), calmodulin, and brush border spectrin. The central actin core bundle that supports the microvillus is nearly saturated with fimbrin and villin cross-linkers and has a density similar to that found in protein crystals. The proposed model accounts for all major proteinaceous components, reproduces the experimentally determined stoichiometry, and is consistent with the size and morphology of the biological brush border membrane. CONCLUSIONS/SIGNIFICANCE. The model presented here will serve as a structural framework to explain many of the dynamic cellular processes occurring over several time scales, such as protein diffusion, association, and turnover, lipid raft sorting, membrane deformation, cytoskeletal-membrane interactions, and even effacement of the brush border by invading pathogens. In addition, this model provides a structural basis for evaluating the equilibrium processes that result in the uniform size and structure of the highly dynamic microvilli.Boston University (Graduate Student Research Fellowship); National Institutes of Health (GM62886

Random Photon Absorption Model Elucidates How Early Gain Control in Fly Photoreceptors Arises from Quantal Sampling

Many diurnal photoreceptors encode vast real-world light changes effectively, but how this performance originates from photon sampling is unclear. A 4-module biophysically-realistic fly photoreceptor model, in which information capture is limited by the number of its sampling units (microvilli) and their photon-hit recovery time (refractoriness), can accurately simulate real recordings and their information content. However, sublinear summation in quantum bump production (quantum-gain-nonlinearity) may also cause adaptation by reducing the bump/photon gain when multiple photons hit the same microvillus simultaneously. Here, we use a Random Photon Absorption Model (RandPAM), which is the 1st module of the 4-module fly photoreceptor model, to quantify the contribution of quantum-gain-nonlinearity in light adaptation. We show how quantum-gain-nonlinearity already results from photon sampling alone. In the extreme case, when two or more simultaneous photon-hits reduce to a single sublinear value, quantum-gain-nonlinearity is preset before the phototransduction reactions adapt the quantum bump waveform. However, the contribution of quantum-gain-nonlinearity in light adaptation depends upon the likelihood of multi-photon-hits, which is strictly determined by the number of microvilli and light intensity. Specifically, its contribution to light-adaptation is marginal (≤1%) in fly photoreceptors with many thousands of microvilli, because the probability of simultaneous multi-photon-hits on any one microvillus is low even during daylight conditions. However, in cells with fewer sampling units, the impact of quantum-gain-nonlinearity increases with brightening light

Postprandial morphological response of the intestinal epithelium of the Burmese python (Python molurus)

The postprandial morphological changes of the intestinal epithelium of Burmese pythons were examined using fasting pythons and at eight time points after feeding. In fasting pythons, tightly packed enterocytes possess very short microvilli and are arranged in a pseudostratified fashion. Enterocyte width increases by 23% within 24 h postfeeding, inducing significant increases in villus length and intestinal mass. By 6 days postfeeding, enterocyte volume had peaked, following as much as an 80% increase. Contributing to enterocyte hypertrophy is the cellular accumulation of lipid droplets at the tips and edges of the villi of the proximal and middle small intestine, but which were absent in the distal small intestine. At 3 days postfeeding, conventional and environmental scanning electron microscopy revealed cracks and lipid extrusion along the narrow edges of the villi and at the villus tips. Transmission electron microscopy demonstrated the rapid postprandial lengthening of enterocyte microvilli, increasing 4.8-fold in length within 24 h, and the maintaining of that length through digestion. Beginning at 24 h postfeeding, spherical particles were found embedded apically within enterocytes of the proximal and middle small intestine. These particles possessed an annular-like construction and were stained with the calcium-stain Alizarine red S suggesting that they were bone in origin. Following the completion of digestion, many of the postprandial responses were reversed, as observed by the atrophy of enterocytes, the shortening of villi, and the retraction of the microvilli. Further exploration of the python intestine will reveal the underlying mechanisms of these trophic responses and the origin and fate of the engulfed particles

Rab8a and Rab8b are essential for several apical transport pathways but insufficient for ciliogenesis

The small GTP-binding protein Rab8 is known to play an essential role in intracellular transport and cilia formation. We have previously demonstrated that Rab8a is required for localising apical markers in various organisms. Rab8a has a closely related isoform, Rab8b. To determine whether Rab8b can compensate for Rab8a, we generated Rab8b-knockout mice. Although the Rab8b-knockout mice did not display an overt phenotype, Rab8a and Rab8b double-knockout mice exhibited mislocalisation of apical markers and died earlier than Rab8a-knockout mice. The apical markers accumulated in three intracellular patterns in the double-knockout mice. However, the localisation of basolateral and/or dendritic markers of the double-knockout mice seemed normal. The morphology and the length of various primary and/or motile cilia, and the frequency of ciliated cells appeared to be identical in control and double-knockout mice. However, an additional knockdown of Rab10 in double-knockout cells greatly reduced the percentage of ciliated cells. Our results highlight the compensatory effect of Rab8a and Rab8b in apical transport, and the complexity of the apical transport process. In addition, neither Rab8a nor Rab8b are required for basolateral and/or dendritic transport. However, simultaneous loss of Rab8a and Rab8b has little effect on ciliogenesis, whereas additional loss of Rab10 greatly affects ciliogenesis

L-selectin mediated leukocyte tethering in shear flow is controlled by multiple contacts and cytoskeletal anchorage facilitating fast rebinding events

L-selectin mediated tethers result in leukocyte rolling only above a threshold in shear. Here we present biophysical modeling based on recently published data from flow chamber experiments (Dwir et al., J. Cell Biol. 163: 649-659, 2003) which supports the interpretation that L-selectin mediated tethers below the shear threshold correspond to single L-selectin carbohydrate bonds dissociating on the time scale of milliseconds, whereas L-selectin mediated tethers above the shear threshold are stabilized by multiple bonds and fast rebinding of broken bonds, resulting in tether lifetimes on the timescale of $10^{-1}$ seconds. Our calculations for cluster dissociation suggest that the single molecule rebinding rate is of the order of $10^4$ Hz. A similar estimate results if increased tether dissociation for tail-truncated L-selectin mutants above the shear threshold is modeled as diffusive escape of single receptors from the rebinding region due to increased mobility. Using computer simulations, we show that our model yields first order dissociation kinetics and exponential dependence of tether dissociation rates on shear stress. Our results suggest that multiple contacts, cytoskeletal anchorage of L-selectin and local rebinding of ligand play important roles in L-selectin tether stabilization and progression of tethers into persistent rolling on endothelial surfaces.Comment: 9 pages, Revtex, 4 Postscript figures include

The nuclear receptor hepatocyte nuclear factor 4α acts as a morphogen to induce the formation of microvilli

Microvilli are actin-based organelles found on apical plasma membranes that are involved in nutrient uptake and signal transduction. Numerous components, including ezrin/radixin/moesin (ERM) proteins, have been identified that link filamentous actins to transmembrane proteins, but the signals driving microvillus biogenesis are not known. In this study, we show that the conditional and/or ectopic expression of a nuclear receptor, hepatocyte nuclear factor 4α (HNF4α), triggers microvillus morphogenesis. We also demonstrate that HNF4α expression induces ERM-binding phosphoprotein 50 (EBP50) expression and that attenuation of EBP50 using RNA interference inhibits microvillus development. We conclude that HNF4α acts as a morphogen to trigger microvillus formation

Complex polarity: building multicellular tissues through apical membrane traffic

The formation of distinct subdomains of the cell surface is crucial for multicellular organism development. The most striking example of this is apical-basal polarization. What is much less appreciated is that underpinning an asymmetric cell surface is an equally dramatic intracellular endosome rearrangement. Here, we review the interplay between classical cell polarity proteins and membrane trafficking pathways, and discuss how this marriage gives rise to cell polarization. We focus on those mechanisms that regulate apical polarization, as this is providing a number of insights into how membrane traffic and polarity are regulated at the tissue level

WASp is required for the correct temporal morphogenesis of rhabdomere microvilli

Microvilli are actin-based fingerlike membrane projections that form the basis of the brush border of enterocytes and the Drosophila melanogaster photoreceptor rhabdomere. Although many microvillar cytoskeletal components have been identified, the molecular basis of microvillus formation is largely undefined. Here, we report that the Wiskott-Aldrich syndrome protein (WASp) is necessary for rhabdomere microvillus morphogenesis. We show that WASp accumulates on the photoreceptor apical surface before microvillus formation, and at the time of microvillus initiation WASp colocalizes with amphiphysin and moesin. The loss of WASp delays the enrichment of F-actin on the apical photoreceptor surface, delays the appearance of the primordial microvillar projections, and subsequently leads to malformed rhabdomeres