Fundamental role of microvilli in the main functions of differentiated cells: Outline of an universal regulating and signaling system at the cell periphery

Until now, the general importance of microvilli present on the surface of almost all differentiated cells has been strongly underestimated and essential functions of these abundant surface organelles remained unrecognized. Commonly, the role of microvilli has been reduced to their putative function of cell‐surface enlargement. In spite of a large body of detailed knowledge about the specific functions of microvilli in sensory receptor cells for sound, light, and odor perception, their functional importance for regulation of basic cell functions remained obscure. Here, a number of microvillar mechanisms involved in fundamental cell functions are discussed. Two structural features enable the extensive functional competence of microvilli: First, the exclusive location of almost all functional important membrane proteins on microvilli of differentiated cells and second, the function of the F‐actin‐based cytoskeletal core of microvilli as a structural diffusion barrier modulating the flow of low molecular substrates and ions into and out of the cell. The specific localization on microvilli of important functional membrane proteins such as glucose transporters, ion channels, ion pumps, and ion exchangers indicate the importance and diversity of microvillar functions. In this review, the microvillar mechanisms of audioreceptor, photoreceptor, and olfactory/taste receptor cells are discussed as highly specialized adaptations of a general type of microvillar mechanisms involved in regulation of important basic cell functions such as glucose transport/energy metabolism, ion channel regulation, generation and modulation of the membrane potential, volume regulation, and Ca signaling. Even the constitutive cellular defence against cytotoxic compounds, also called “multidrug resistance (MDR),” is discussed as a microvillar mechanism. A comprehensive examination of the specific properties of “cable‐like” ion conduction along the microvillar core structure of F‐actin allows the proposal that microvilli are specifically designed for using ionic currents as cellular signals. In view of the multifaceted gating and signaling properties of TRP channels, the possible role of microvilli as a universal gating device for TRP channel regulation is discussed. Combined with the role of the microvillar core bundle of actin filaments as high‐affinity Ca store, microvilli may turn out as highly specialized Ca signaling organelle involved in store‐operated Ca entry (SOCE) and initiation of nonlinear Ca signals such as waves and oscillations. J. Cell. Physiol. 226: 896–927, 2011. © 2010 Wiley‐Liss, Inc.     

Microvillar ion channels: cytoskeletal modulation of ion fluxes

The recently presented theory of microvillar Ca(2+)signaling [Lange, K. (1999) J. Cell. Physiol.180, 19-35], combined with Manning's theory of "condensed counterions" in linear polyelectrolytes [Manning, G. S. (1969). J. Chem. Phys.51, 924-931] and the finding of cable-like ion conductance in actin filaments [Lin, E. C. & Cantiello, H. F. (1993). Biophys. J.65, 1371-1378], allows a systematic interpretation of the role of the actin cytoskeleton in ion channel regulation.Ion conduction through actin filament bundles of microvilli exhibits unique nonlinear transmission properties some of which closely resemble that of electronic semiconductors: (1) bundles of microfilaments display significant resistance to cation conduction and (2) this resistance is decreased by supply of additional energy either as thermal, mechanical or electromagnetic field energy. Other transmission properties, however, are unique for ionic conduction in polyelectrolytes. (1) Current pulses injected into the filaments were transformed into oscillating currents or even into several discrete charge pulses closely resembling that of single-channel recordings. Discontinuous transmission is due to the existence of counterion clouds along the fixed anionic charge centers of the polymer, each acting as an "ionic capacitor". (2) The conductivity of linear polyelectrolytes strongly decreases with the charge number of the counterions; thus, Ca(2+)and Mg(2+)are effective modulator of charge transfer through linear polyelectrolytes. Field-dependent formation of divalent cation plugs on either side of the microvillar conduction line may generate the characteristic gating behavior of cation channels. (3) Mechanical movement of actin filament bundles, e.g. bending of hair cell microvilli, generates charge translocations along the filament structure (mechano-electrical coupling). (4) Energy of external fields, by inducing molecular dipoles within the polyelectrolyte matrix, can be transformed into mechanical movement of the system (electro-mechanical coupling). Because ionic transmission through linear polyelectrolytes is very slow compared with electronic conduction, only low-frequency electromagnetic fields can interact with the condensed counterion systems of linear polyelectrolytes.The delineated characteristics of microvillar ion conduction are strongly supported by the phenomenon of electro-mechanical coupling (reverse transduction) in microvilli of the audioreceptor (hair) cells and the recently reported dynamics of Ca(2+)signaling in microvilli of audio- and photoreceptor cells. Due to the cell-specific expression of different types and combinations of ion channels and transporters in the microvillar tip membrane of differentiated cells, the functional properties of this cell surface organelle are highly variable serving a multitude of different cellular functions including receptor-mediated effects such as Ca(2+)signaling, regulation of glucose and amino acid transport, as well as modulation of membrane potential. Even mechanical channel activation involved in cell volume regulation can be deduced from the systematic properties of the microvillar channel concept. In addition, the specific ion conduction properties of microfilaments combined with their proposed role in Ca(2+)signaling make microvilli the most likely cellular site for the interaction with external electric and magnetic fields.     

Microvillar Ca++ signaling: A new view of an old problem

Proceeding from the recent finding that the main components of the Ca⁺⁺ signal pathway are located in small membrane protrusions on the surface of differentiated cells, called microvilli, a novel concept of cellular Ca⁺⁺ signaling was developed. The main features of this concept can be summarized as follows: Microvilli are formed on the cell surface of differentiating or resting cells from exocytic membrane domains, growing out from the cell surface by elongation of an internal bundle of actin filaments. The microvillar tip membranes contain all functional important proteins synthesized such as ion channels and transporters for energy-providing substrates and structural components, which are, in rapidly growing undifferentiated cells, distributed over the whole cell surface by lateral diffusion. The microvillar shaft structure, a bundle of actin filaments, forms a dense cytoskeletal matrix tightly covered by the microvillar lipid membrane and represents an effective diffusion barrier separating the microvillar tip compartment (entrance compartment) from the cytoplasm. This diffusion barrier prevents the passage of low molecular components such as Ca⁺⁺ glucose and other relevant substrates from the entrance compartment into the cytoplasm. The effectiveness of the actin-based diffusion barrier is modulated by various signal pathways and effectors, most importantly, by the actin-depolymerizing/reorganizing activity of the phospholipase C (PLC)-coupled Ca⁺⁺ signaling. Moreover, the microvillar bundle of actin filaments plays a dual role in Ca⁺⁺ signaling. It combines the function of a diffusion barrier, preventing Ca⁺⁺ influx into the resting cell, with that of a high-affinity, ATP-dependent, and IP₃-sensitive Ca⁺⁺ store. Activation of Ca⁺⁺ signaling via PLC-coupled receptors simultaneously empties Ca⁺⁺ stores and activates the influx of external Ca⁺⁺. The presented concept of Ca⁺⁺ signaling is compatible with all established data on Ca⁺⁺ signaling. Properties of Ca⁺⁺ signaling, that could not be reconciled with the basic principles of the current hypothesis, are intrinsic properties of the new concept. Quantal Ca⁺⁺ release, Ca⁺⁺-induced Ca⁺⁺ release (CICR), the coupling phenomen between the filling state of the Ca⁺⁺ store and the activity of the Ca⁺⁺ influx pathway, as well as the various yet unexplained complex kinetics of Ca⁺⁺ uptake and release can be explained on a common mechanistic basis. J. Cell. Physiol. 180:19–34, 1999. © 1999 Wiley-Liss, Inc.     

Regulation of cell volume via microvillar ion channels

A novel mechanism of cellular volume regulation is presented, which ensues from the recently introduced concept of transport and ion channel regulation via microvillar structures (Lange K, 1999, J Cell Physiol 180:19-35). According to this notion, the activity of ion channels and transporter proteins located on microvilli of differentiated cells is regulated by changes in the structural organization of the bundle of actin filaments in the microvillar shaft region. Cells with microvillar surfaces represent two-compartment systems consisting of the cytoplasm on the one side and the sum of the microvillar tip (or, entrance) compartments on the other side. The two compartments are separated by the microvillar actin filament bundle acting as diffusion barrier ions and other solutes. The specific organization of ion and water channels on the surface of microvillar cell types enables this two-compartment system to respond to hypo- and hyperosmotic conditions by activation of ionic fluxes along electrochemical gradients. Hypotonic exposure results in swelling of the cytoplasmic compartment accompanied by a corresponding reduction in the length of the microvillar diffusion barrier, allowing osmolyte efflux and regulatory volume decrease (RVD). Hypertonic conditions, which cause shortening of the diffusion barrier via swelling of the entrance compartment, allow osmolyte influx for regulatory volume increase (RVI). Swelling of either the cytoplasmic or the entrance compartment, by using membrane portions of the microvillar shafts for surface enlargement, activates ion fluxes between the cytoplasm and the entrance compartment by shortening of microvilli. The pool of available membrane lipids used for cell swelling, which is proportional to length and number of microvilli per cell, represents the sensor system that directly translates surface enlargements into activation of ion channels. Thus, the use of additional membrane components for osmotic swelling or other types of surface-expanding shape changes (such as the volume-invariant cell spreading or stretching) directly regulates influx and efflux activities of microvillar ion channels. The proposed mechanism of ion flux regulation also applies to the physiological main functions of epithelial cells and the auxiliary action of swelling-induced ATP release. Furthermore, the microvillar entrance compartment, as a finely dispersed ion-accessible peripheral space, represents a cellular sensor for environmental ionic/osmotic conditions able to detect concentration gradients with high lateral resolution. Volume regulation via microvillar surfaces is only one special aspect of the general property of mechanosensitivity of microvillar ionic pathways.     

SR-BI is required for microvillar channel formation and the localization of HDL particles to the surface of adrenocortical cells in vivo

Scavenger receptor class B type I (SR-BI) mediates the selective uptake of HDL cholesteryl ester into liver and steroidogenic tissues. In steroidogenic cells, juxtaposed microvilli, or microvilli snuggled against the plasma membrane create microvillar channels that fill with HDL. Microvillar membranes contain SR-BI and are believed to be the site of HDL cholesteryl ester uptake. A recent study showed that SR-BI expression in insect cells elicits membrane structures that contain SR-BI, bind HDL, and closely resemble the ultrastructure of microvillar channels. In the present study we compared the ultrastructure of adrenal gland microvillar membranes in Srb1+/+ and Srb1-/- mice to test whether SR-BI is required for the formation of microvillar channels. The results show that SR-BI is absolutely required for microvillar channel formation and that the microvillar membranes of Srb1-/- mice are 17% thinner than in Srb1+/+ mice.We conclude that SR-BI has a major influence on plasma membrane ultrastructure and organization in vivo.     

Cellular target of weak magnetic fields: ionic conduction along actin filaments of microvilli

The interaction of weakelectromagnetic fields (EMF) with living cells is a most important butstill unresolved biophysical problem. For this interaction, thermal andother types of noise appear to cause severe restrictions in the actionof weak signals on relevant components of the cell. A recentlypresented general concept of regulation of ion and substrate pathwaysthrough microvilli provides a possible theoretical basis for thecomprehension of physiological effects of even extremely low magneticfields. The actin-based core of microfilaments in microvilli isproposed to represent a cellular interaction site for magnetic fields.Both the central role of F-actin in Ca²⁺ signaling and itspolyelectrolyte nature eliciting specific ion conduction propertiesrender the microvillar actin filament bundle an ideal interaction sitefor magnetic and electric fields. Ion channels at the tip of microvilliare connected with the cytoplasm by a bundle of microfilaments forminga diffusion barrier system. Because of its polyelectrolyte nature, themicrofilament core of microvilli allows Ca²⁺ entry into thecytoplasm via nonlinear cable-like cation conduction through arrays ofcondensed ion clouds. The interaction of ion clouds with periodicallyapplied EMFs and field-induced cation pumping through the cascade ofpotential barriers on the F-actin polyelectrolyte followswell-known physical principles of ion-magnetic field (MF) interactionand signal discrimination as described by the stochastic resonance andBrownian motor hypotheses. The proposed interaction mechanismis in accord with our present knowledge about Ca²⁺signaling as the biological main target of MFs and the postulated extreme sensitivity for coherent excitation by very low field energieswithin specific amplitude and frequency windows. Microvillar F-actinbundles shielded by a lipid membrane appear to function like electronicintegration devices for signal-to-noise enhancement; the influence ofcoherent signals on cation transduction is amplified, whereas that ofrandom noise is reduced.

     

Two microvillar organs, new to Crustacea, in the Mystacocarida

The mystacocarid crustacean Derocheilocaris typica has two microvillar organs, one new, the other previously unappreciated in crustacean literature. The first is situated on the head-shield and consists of three pairs of cells: one with microvilli and a ballooned nucleus; one smaller and without special features; the third large and investing the other two and extending down to the foregut. We call this new organ the "cephalic microvillar organ" and discuss the value of the concept "dorsal organ", to which it might have been included. The second organ consists of about 21 cells that cover the proximal part of the dorsal surface of the labrum. The cells are alike, being characterized by an apical field of microvilli and a large residual body. This organ is here called the "labral microvillar organ". Both organs are neither sensory nor secretory and do not qualify for membership in any of the other recognized organ systems. We are unable to deduce their Dero-cheilocaris functions.     

Microvillar elongation following parthenogenetic activation of sea urchin eggs

Parthenogenetic activation of unfertilized sea urchin eggs with ammonium chloride at pH 8.0 resulted in a slow, but dramatic, reorganization of surface microvilli in four species of sea urchin eggs. Following NH4Cl treatment, elongation of microvilli on the egg surface was observed concomitant with the formation of microfilament bundles within the microvillar cores. A minimum of 2 h of treatment was required for elongation and microfilament bundle formation to occur. The maintenance of elongated microvilli was pH-sensitive; removal of the activating agent resulted in the retraction of extended microvilli while readdition of NH4Cl caused microvilli to elongate again. Accompanying microvillar elongation in activated eggs, there was an increased calcium uptake as measured by 45Ca uptake. Blocking calcium uptake by incubation in lanthanum chloride or zero-calcium seawater containing 2 mM EGTA prevented microvillar elongation. These results suggested that elongation of microvilli following parthenogenetic activation by NH4Cl is pH- and calcium-dependent and is similar to that observed during normal fertilization.     

Calcium-independent inhibition of glucose transport in PC-12 and L6 cells by calcium channel antagonists

The goal of these studies was todetermine whether different calcium channel antagonists affect glucosetransport in a neuronal cell line. Rat pheochromocytoma (PC-12) cellswere treated with L-, T-, and N-type calcium channel antagonists beforemeasurement of accumulation of 2-[³H]deoxyglucose(2-[³H]DG). The L-type channel antagonistsnimodipine, nifedipine, verapamil, and diltiazem all inhibited glucosetransport in a dose-dependent manner (2-150 µM) withnimodipine being the most potent and diltiazem only moderatelyinhibiting transport. T- and N-type channel antagonists had no effecton transport. The L-type channel agonist l-BAY K 8644 alsoinhibited uptake of 2-[³H]DG. The ability of these drugsto inhibit glucose transport was significantly diminished by thepresence of unlabeled 2-DG in the uptake medium. Some experiments wereperformed in the presence of EDTA (4 mM) or in uptake buffer withoutcalcium. The absence of calcium in the uptake medium had no effect oninhibition of glucose transport by nimodipine or verapamil. To examinethe effects of these drugs on a cell model of a peripheral tissue, westudied rat L6 muscle cells. The drugs inhibited glucose transport in L6 myoblasts in a dose-dependent manner that was independent of calciumin the uptake medium. These studies suggest that the calcium channelantagonists inhibit glucose transport in cells through mechanisms otherthan the antagonism of calcium channels, perhaps by acting directly onglucose transporters.

     

Activation of Calcium Signaling in Isolated Rat Hepatocytes Is Accompanied by Shape Changes of Microvilli

Preceding studies using the hamster insulinoma cell line, HIT, and isolated rat hepatocytes have shown that two essential components of the Ca²⁺signaling pathway, the ATP-dependent Ca²⁺store and the store-coupled Ca²⁺influx pathway, are both located in microvilli covering the surface of these cells. Microvilli-derived vesicles from both cell types exhibited anion and cation pathways which could be inhibited by anion and cation channel-specific inhibitors. These findings suggested that the microvillar tip compartment forms a space which is freely accessible for external Ca²⁺, ATP, and IP₃. The entry of Ca²⁺into the cytoplasm, however, is largely restricted by the microvillar core structure, the dense bundle of actin microfilaments acting as a diffusion barrier between the microvillar tip compartment and the cell body. Moreover, evidence has been presented that F-actin may function as ATP-dependent and IP₃-sensitive Ca²⁺store that can be emptied by profilin-induced depolymerization or reorganization [K. Lange and U. Brandt (1996)FEBS Lett.395, 137–142]. Here we demonstrate the tight connection between microvillar shape changes and the activation of the Ca²⁺signaling system in isolated rat hepatocytes. Using a combination of scanning electron microscopy (SEM) and fura-2 fluorescence technique, we confirmed a consequence of the “diffusion barrier” concept of Ca²⁺signaling: Irrespective of the type of the applied stimulus, activation of the Ca²⁺influx pathway is accompanied by changes in the structural organization of microvilli indicative of the loss of their diffusion barrier function. We further show that the cell surfaces of unstimulated hepatocytes isolated by either the collagenase or the EDTA perfusion technique are densely covered with microvilli predominantly of a short and slender type. Beside this rather uniformly shaped type of microvilli, a number of dilated surface protrusions were observed. Under these conditions the cells displayed the well known rather high basal [Ca²⁺]_iof 200–250 nMas repeatedly demonstrated for freshly isolated hepatocytes. However, addition of the serine protease inhibitor, phenylmethanesulfonyl fluoride (PMSF), to the cell suspension immediately after its preparation reduced the basal cytoplasmic Ca²⁺level to about 100 nM.Concomitantly, dilated surface protrusions disappeared, and cell surfaces exclusively displayed short, slender microvilli. Activation of the Ca²⁺signaling pathway by vasopressin, as well as by the IP₃-independent acting Ca²⁺store inhibitor, thapsigargin, was accompanied by a conspicuous shortening and dilation of microvilli following the same time courses as the respective increases of [Ca²⁺]_iinduced by the effectors. Furthermore, the abundance of the large form of surface protrusions on isolated hepatocytes positively correlated with the size of a cellular Ca²⁺/Fura-2 compartment which is rapidly depleted from Ca²⁺by extracellular EGTA. These findings support the postulated localization of the store-coupled Ca²⁺influx pathway in microvilli of HIT cells also for hepatocytes and are in accord with the notion of a cytoskeletal diffusion barrier regulating the flux of external Ca²⁺via the microvillar tip region in the cytoplasm.     

Mitogen-induced proliferation increases biotin uptake into human peripheral blood mononuclear cells

We sought todetermine whether the proliferation of immune cells affects thecellular uptake of the vitamin biotin. Peripheral blood mononuclearcells (PBMC) were isolated from healthy adults. The proliferationof PBMC was induced by either pokeweed lectin, concanavalin A, orphytohemagglutinin. When the medium contained a physiologicalconcentration of[³H]biotin,nonproliferating PBMC accumulated 406 ± 201 amol[³H]biotin · 10⁶cells¹ · 30 min¹. For proliferatingPBMC, [³H]biotinuptake increased to between 330 and 722% of nonproliferating values.Maximal transport rates of[³H]biotin inproliferating PBMC were also about four times greater than those innonproliferating PBMC, suggesting that proliferation was associatedwith an increase in the number of biotin transporters on the PBMCmembrane. The biotin affinities and specificities of the transporterfor proliferating and nonproliferating PBMC were similar, providingevidence that the same transporter mediates biotin uptake in bothstates. [¹⁴C]ureauptake values for proliferating and nonproliferating PBMC were similar,suggesting that the increased[³H]biotin uptake wasnot caused by a global upregulation of transporters duringproliferation. We conclude that PBMC proliferation increases thecellular accumulation of biotin.     

Relative contribution of chloride channels and transporters to regulatory volume decrease in human glioma cells

Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl^– channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V_Post-RVD < V_baseline). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl^– channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd²⁺ (V_Post-RVD > 1.4*V_baseline). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA), a known K⁺-Cl^– cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd²⁺ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl^– channels vs. transporters to this process is 60–70% and 30–40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl^– channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a. voltage-gated chloride channel family; potassium-chloride cotransporters; peritumoral edema     

Insulin-responsive glucose transporters are concentrated in a cell surface-derived membrane fraction of 3T3-L1 adipocytes

The recently proposed mechanistic concept of a receptor-regulated entrance compartment for hexose transport formed by microvilli on 3T3-L1 adipocytes predicted a preferential localization of glucose transporters in these structures. The cytochalasin B-binding technique was used to determine in basal and insulin-stimulated cells the distribution of glucose transporters between plasma membranes, low density microsomes (LDM) and two cell surface-derived membrane fractions prepared by a hydrodynamic shearing technique. The shearing procedure applied prior to homogenization yielded a low density surface-derived vesicle (LDSV) fraction which contained nearly 60% of the cellular glucose transporters and the total insulin-sensitive transporter pool. The rest of the glucose transporter population was localized within the plasma membrane (5%) and the LDM fraction (37%). Pretreatment of the cells with insulin (20 mU/ml for 10 min) reduced the transporter content of the LDSV fraction by 40% and increased that of the plasma membrane fraction 4-fold. The transporter containing LDSV fraction was clearly differentiated from the LDM fraction by its low specific galactosyltransferase activity and its insulin-sensitivity. Scanning electron microscopy revealed that the LDSV fraction contained a rather uniform population of spherical vesicles of 100-200 nm in diameter.     

Regulation of Turgor Pressure by Suspension-Cultured Tobacco Cells

Turgor regulation and effects of high NaCl and water deficiton growth and internal solutes were studied after transferringtobacco cells from control culture medium (osmotic pressure= 0.13–0.15 MPa at time of transfer) to culture mediumcontaining either 82 mol m^–3 NaCl or 150 mol m^–3melibiose (osmotic pressure of media = 0.62 MPa). Followingtransfer to media with higher osmotic pressure, expansion rateand turgor pressure were reduced. Within 24 h of imposing thewater deficit, expansion rate had returned to that of cellsin control culture medium. However, by 24 h, turgor pressurehad only risen from 0.2 MPa to 0.65 MPa in the NaCl treatmentand to 0.53 MPa in the melibiose treatment, while it was 0.73MPa in the control treatment. Furthermore, turgor pressure remainedwithin 0.05 MPa of these respective values for the rest of the(75 h) experiment. These results suggest differences in bothcell wall properties (extensibility and/or threshold turgor)and the level at which turgor is maintained for cells in thevarious treatments. Solutes contributing nearly all (82–97%) of the osmoticpressure in cells were identified. The initial (up to 24 h)increases in turgor pressure were mainly due to increases insolute concentrations caused by relatively slow expansion rates.However, increased Na⁺ and Cl ^– uptake contributed toincreased turgor pressure in the NaCl treatment and caused turgorpressure of cells in this treatment to increase faster thanin the melibiose treatment. Likewise, expansion rate rose morequickly in the NaCl than in the melibiose treatment. After 24h, maximum expansion rate was reached and concentrations ofmost internal solutes began to decrease. Nevertheless, turgorpressure remained relatively constant. The constancy of turgorpressure was due to increased glucose uptake rates relativeto controls, with consequent increases in concentrations ofsucrose, glucose and fructose and, in cells in the melibiosetreatment, of organic acids. Glucose uptake was slower in theNaCl than in the melibiose treatment but higher turgor pressurewas maintained in the NaCl treatment due to high uptake of Na⁺and Cl^–. Glucose uptake appears to respond to a systemof turgor regulation, but further experiments are required toconfirm this and to determine whether Na⁺ and Cl^– uptakealso respond to a system of turgor regulation. Key words: Salinity, water deficit, growth     

Impaired trafficking of choline transporter-like protein-1 at plasma membrane and inhibition of choline transport in THP-1 monocyte-derived macrophages

The present study investigates choline transport processes and regulation of choline transporter-like protein-1 (CTL1) in human THP-1 monocytic cells and phorbol myristate 13-acetate (PMA)-differentiated macrophages. Choline uptake is saturable and therefore protein-mediated in both cell types, but its transport characteristics change soon after treatments with PMA. The maximal rate of choline uptake intrinsic to monocytic cells is greatly diminished in differentiated macrophages as demonstrated by alterations in V_max values from 1,973 ± 118 to 380 ± 18 nmol·mg^–1·min^–1, when the binding affinity did not change significantly (K_m values 56 ± 8 and 53 ± 6 µM, respectively). Treatments with hemicholinim-3 effectively inhibit most of the choline uptake, establishing that a choline-specific transport protein rather than a general transporter is responsible for the observed kinetic parameters. mRNA screening for the expression of various transporters reveals that CTL1 is the most plausible candidate that possesses the described kinetic and inhibitory properties. Fluorescence-activated cell sorting analyses at various times after PMA treatments further demonstrate that the disappearance of CTL1 protein from the cell surface follows the same trend as the reduction in choline uptake. Importantly, the loss of functional CTL1 from the cell surface occurs without significant changes in total CTL1 protein or its mRNA level indicating that an impaired CTL1 trafficking is the key contributing factor to the reduced choline uptake, subsequent to the PMA-induced THP-1 differentiation to macrophages. protein trafficking     

Prominin‐1 (CD133) modulates the architecture and dynamics of microvilli

Prominin‐1 is a cell surface biomarker that allows the identification of stem and cancer stem cells from different organs. It is also expressed in several differentiated epithelial and non‐epithelial cells. Irrespective of the cell type, prominin‐1 is associated with plasma membrane protrusions. Here, we investigate its impact on the architecture of membrane protrusions using microvilli of Madin‐Darby canine kidney cells as the main model. Our high‐resolution analysis revealed that upon the overexpression of prominin‐1 the number of microvilli and clusters of them increased. Microvilli with branched and/or knob‐like morphologies were observed and stimulated by mutations in the ganglioside‐binding site of prominin‐1. The altered phenotypes were caused by the interaction of prominin‐1 with phosphoinositide 3‐kinase and Arp2/3 complex. Mutation of tyrosine 828 of prominin‐1 impaired its phosphorylation and thereby inhibited the aforementioned interactions abolishing altered microvilli. This suggests that the interplay of prominin‐1‐ganglioside membrane complexes, phosphoinositide 3‐kinase and cytoskeleton components regulates microvillar architecture. Lastly, the expression of prominin‐1 and its mutants modified the structure of filopodia emerging from fibroblast‐like cells and silencing human prominin‐1 in primary hematopoietic stem cells resulted in the loss of uropod‐associated microvilli. Altogether, these findings strengthen the role of prominin‐1 as an organizer of cellular protrusions.      

K+Nutrition and Na+Toxicity: The Basis of Cellular K+/Na+Ratios

The capacity of plants to maintain a high cytosolic K⁺/Na⁺ratiois likely to be one of the key determinants of plant salt tolerance.Important progress has been made in recent years regarding theidentification and characterization of genes and transportersthat contribute to the cytosolic K⁺/Na⁺ratio. For K⁺uptake,K⁺efflux and K⁺translocation to the shoot, genes have been isolatedthat encode K⁺uptake and K⁺release ion channels and K⁺carriersthat are coupled to either a H⁺or Na⁺gradient. Although thepicture is less clear for the movement of Na⁺, one pathway,in the form of non-selective ion channels, is likely to playa role in Na⁺uptake, whereas Na⁺efflux and compartmentationare likely to be mediated by H⁺-coupled antiport. In addition,several proteins have been characterized that play prominentroles in the regulation of K⁺and/or Na⁺fluxes. In this BotanicalBriefing we will discuss the functions and interactions of thesegenes and transporters in the broader context of K⁺nutritionand Na⁺toxicity. Copyright 1999 Annals of Botany Company Salinty, K⁺/N⁺ratio, transporter, membrane.     

Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

Lipid rafts in epithelial brush borders: atypical membrane microdomains with specialized functions

Epithelial cells that fulfil high-throughput digestive/absorptive functions, such as small intestinal enterocytes and kidney proximal tubule cells, are endowed with a dense apical brush border. It has long been recognized that the microvillar surface of the brush border is organized in cholesterol/sphingolipid-enriched membrane microdomains commonly known as lipid rafts. More recent studies indicate that microvillar rafts, in particular those of enterocytes, have some unusual properties in comparison with rafts present on the surface of other cell types. Thus, microvillar rafts are stable rather than transient/dynamic, and their core components include glycolipids and the divalent lectin galectin-4, which together can be isolated as "superrafts", i.e., membrane microdomains resisting solubilization with Triton X-100 at physiological temperature. These glycolipid/lectin-based rafts serve as platforms for recruitment of GPI-linked and transmembrane digestive enzymes, most likely as an economizing effort to secure and prolong their digestive capability at the microvillar surface. However, in addition to microvilli, the brush border surface also consists of membrane invaginations between adjacent microvilli, which are the only part of the apical surface sterically accessible for membrane fusion/budding events. Many of these invaginations appear as pleiomorphic, deep apical tubules that extend up to 0.5-1 microm into the underlying terminal web region. Their sensitivity to methyl-beta-cyclodextrin suggests them to contain cholesterol-dependent lipid rafts of a different type from the glycolipid-based rafts at the microvillar surface. The brush border is thus an example of a complex membrane system that harbours at least two different types of lipid raft microdomains, each suited to fulfil specialized functions. This conclusion is in line with an emerging, more varied view of lipid rafts being pluripotent microdomains capable of adapting in size, shape, and content to specific cellular functions.     

Functional Characterization of Two Ripening-related Sucrose Transporters from Grape Berries

The ripening of grape (Vitis vinifera L.) berries is associatedwith a large accumulation of glucose and fructose in the vacuolesof the fruit cells. These hexoses are derived from sucrose,which is released from the phloem and may be taken up by parenchymacells prior to hydrolysis. We have expressed two putative ripening-relatedsucrose transporters from grape berries, VvSUC11 (synonymouswith VvSUT1) and VvSUC12, in an invertase deficient yeast strainto characterize their transport activities. Sucrose was takenup by yeast transformed with either transporter at an optimumpH of <4.5 and with a Michaelis constant (K_m) of 0.9–1.4m M. The uptake of sucrose through VvSUC11 and VvSUC12 was inhibitedby protonophores and by vanadate. This is consistent with anactive uptake mechanism involving proton cotransport, typicalof sucrose/H⁺symporters. The transporters from grape berrieswere functionally similar to Scr1, a sucrose transporter fromRicinus cotyledons. It is likely that in grape berries VvSUC11and VvSUC12 facilitate the loading of sucrose from the apoplastinto the parenchyma cells. Copyright 2001 Annals of Botany Company Fruit, grape berries, plasma membrane, sugars, sucrose transporters, Vitis vinifera