Klaus Lange

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.

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.

Calcium storage and release properties of F-actin: evidence for the involvement of F-actin in cellular calcium signaling

Preceding studies have shown that the bulk of the ATP‐dependent, inositol 1,4,5‐trisphosphate (IP3)‐sensitive Ca2+ store of hamster insulinoma (HIT) cells is located in microvilli on the cell surface. Similar results were obtained with isolated rat hepatocytes. Moreover, in vesicles of microvillar origin, passive fluxes of Ca2+, ATP, and IP3 occur through cation and anion channels, respectively, suggesting that Ca2+ storage is due to ATP‐dependent Ca2+ binding to an intravesicular component. Here we demonstrate that F‐actin may be a possible candidate for this function. ATP‐actin monomers bind Ca2+ with high affinity (K d = 2−8 nM) to their divalent cation binding sites. Polymerization of actin monomers decreases the rate constant for divalent cation exchange at this binding site by more than 3 orders of magnitude rendering bound cations nearly unavailable. F‐actin‐bound Ca2+ can be released by depolymerization and dissociation from Ca2+‐ADP‐actin monomers (K d = 375 nM). We now provide additional evidence for the possible involvement of actin in Ca2+ storage. (1) Preincubation of surface‐derived Ca2+‐storing vesicles from HIT cells with the F‐actin stabilizer, phalloidin, strongly inhibited ATP‐dependent Ca2+ uptake, reducing the IP3‐sensitive Ca2+ pool by 70%. Phalloidin, when added after the loading process, affected neither the amount of stored Ca2+ nor IP3 action on the store. (2) F‐actin polymerized in the presence of Mg2+ in nominally Ca2+‐free buffer still contained about half of the high affinity sites occupied with Ca2+ (Mg/Ca‐F‐actin). (3) Using the fura‐2 technique, we found that in the presence of ATP, Mg/Ca‐F‐actin incorporated free Ca2+ at a relatively low rate. Short pulses of ultrasound (3–10s) strongly accelerated Ca2+ uptake, decreasing free Ca2+ from 500 nM to below 100 nM. (4) In the presence of physiological levels of Mg2+ (0.5 mM), sonication liberated large amounts of Ca2+ from Mg/Ca‐F‐actin. (5) Ca‐F‐actin released bound Ca2+ at a very slow rate. Short ultrasonic pulses rapidly elevated free Ca2+ from about 50 nM up to 500 nM. (6) Small amounts of profilin, an actin‐binding protein, released Ca2+ both from Ca‐and Mg/Ca‐F‐actin and also inhibited uptake of Ca2+ into Mg/Ca‐F‐actin. (7) Phalloidin completely inhibited Ca‐uptake into Mg/Ca‐F‐actin even during ultrasonic treatment. These findings suggest that Ca2+ storage may occur by addition of Ca‐ATP‐actin monomers to reactive ends of the polymer and emptying of this store by profilin‐stimulated release of Ca‐ADP‐actin. Thus, receptor‐operated Ca2+ signaling, initiated by phospholipase C activation, may proceed via the well‐known phosphatidylinositol phosphate‐regulated profilin/gelsolin pathway of actin reorganization/depolemerization. The importance of the proposed microvillar Ca2+ signaling system for living cells remains to be established.

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. J. Cell. Physiol. 185:21–35, 2000.

F-actin-based Ca signaling-a critical comparison with the current concept of Ca signaling.

A short comparative survey on the current idea of Ca signaling and the alternative concept of F‐actin‐based Ca signaling is given. The two hypotheses differ in one central aspect, the mechanism of Ca storage. The current theory rests on the assumption of Ca‐accumulating endoplasmic/sarcoplasmic reticulum‐derived vesicles equipped with an ATP‐dependent Ca pump and IP3‐ or ryanodine‐sensitive channel‐receptors for Ca‐release. The alternative hypothesis proceeds from the idea of Ca storage at the high‐affinity binding sites of actin filaments. Cellular sites of F‐actin‐based Ca storage are microvilli and the submembrane cytoskeleton. Several specific features of Ca signaling such as store‐channel coupling, quantal Ca release, spiking and oscillations, biphasic and “phasic” uptake kinetics, and Ca‐induced Ca release (CICR), which are not adequately described by the current concept, are inherent properties of the F‐actin system and its dynamic state of treadmilling. J. Cell. Physiol. 209: 270–287, 2006.

Insulin-responsive glucose transporters are concentrated in a cell surfacederived 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 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.

The IP3-sensitive calcium store of HIT cells is located in a surface-derived vesicle fraction

Electron microscopic and biochemical techniques were used to study the cellular localization of the ATP‐dependent, IP3‐sensitive, Ca2+ store in the glucose‐ and phosphatidylinositol(PI) agonist‐sensitive hamster insulinoma cell line HIT‐T15. Scanning electron microscopy revealed conspicuous shape changes of the microvilli following stimulation of these cells with bombesin or thapsigargin. These changes closely resemble those previously shown to accompany stimulation of hexose transport in adipocytes with insulin [J. Cell. Physiol. 142 (1990) 1‐14]. Using a hydrodynamic shearing technique for the isolation of microvilli, two cell surface‐derived vesicle fractions were prepared containing 80% of the total cellular Ca2+‐storing activity. In contrast, subcellular fractionation using normal homogenization with a glass/teflon homogenizer yielded the well‐known distribution of the Ca2+‐storing activity which is then predominantly recovered within the microsomal fraction. The surface‐derived vesicle fraction was clearly distinguished from the microsomal fraction by its high content of Na+/K+‐ATPase and an immunoreactive fragment of the GluT‐1 glucose transporter isoform which both are not detectable in the microsomal fraction isolated from homogenates from sheared cells. The Ca2+ uptake properties of the cell surface‐derived vesicle fractions including the vanadate, A23187, and thapsigargin sensitivity were found to be identical with those described for the microsomal Ca2+ stores of various cell types. Inositol 1,4,5‐trisphosphate (IP3) at 1 μM induced a maximal release of 35–40% of the stored Ca2+ from these vesicles.

Restricted localization of the adipocyte/muscle glucose transporter species to a cell surface-derived vesicle fraction of 3T3-L1 adipocytes: Inhibited lateral mobility of integral plasma membrane proteins in newly inserted membrane areas of differentiated 3T3-L1 cells

The recent demonstration of a large cell surface‐derived pool of insulin‐sensitive glucose transporters, presumably concentrated in the microvilli of 3T3‐L1 adipocytes, induced the assumption that in differentiated adipocytes, newly inserted plasma membrane areas may display restricted lateral mobility, thereby preventing diffusion of integral membrane proteins out of these areas into the adjoining plasma membrane. In order to test this assumption, the cell surface distributions of the two glucose transporter species expressed by 3T3‐L1 cells were determined using specific antisera against the HepG2/erythrocyte transporter, GluT1, which is synthesized in both fibroblasts and adipocytes, and the adipocyte/muscle‐specific transporter, GluT4, expressed for the first time 3–4 days after induction of adipose conversion. GluT1 was shown to be localized in the plasma membrane of both 3T3‐L1 preadipocytes and adipocytes, whereas GluT4 was almost entirely restricted to the low density surface‐derived vesicle (LDSV) fraction of 3T3‐L1 adipocytes most likely consisting of microvilli‐derived vesicles. In contrast to the minor portion of GluT4 found in the adipocyte plasma membrane fraction, equal amounts of the GluT1 protein were detected in both the plasma membrane and the LDSV fractions of adipocytes. Both transporter species were present in the microsomal and the LDSV fractions of adipocytes. The observed distribution of the two transporter species is in accordance with the postulated restriction of the lateral mobility in plasma membrane areas formed by newly inserted transgolgi vesicles of differentiated adipocytes.

A new concept for risk assessment of the hazards of non-genotoxic chemicals — electronmicroscopic studies of the cell surface. Evidence for the action of lipophilic chemicals on the Ca2+ signaling system

Recently, we presented evidence for the localization of components of the cellular Ca2+ signaling pathway in microvilli. On stimulation of this pathway, microvilli undergo characteristic morphological changes which can be detected by scanning electron microscopy (SEM) of the cell surface. Here we show that both receptor-mediated (vasopressin) and unspecific stimulation of the Ca2+ signaling system by the lipophilic tumor promoters thapsigargin (TG) and phorbolmyristateacetate (PMA) are accompanied by the same type of morphological changes of the cell surface. Since stimulated cell proliferation accelerates tumor development and sustained elevation of the intracellular Ca2+ concentrations is a precondition for stimulated cell proliferation, activated Ca2+ signaling is one possible mechanism of non-genomic tumor promotion. Using isolated rat hepatocytes we show that all tested lipophilic chemicals with known tumor promoter action, caused characteristic microvillar shape changes. On the other hand, lipophilic solvents that were used as differentiating agents in cell cultures such as dimethylsulfoxide (DMSO) and dimethylformamide also, failed to change the microvillar shapes. Instead DMSO stabilized the original appearance of microvilli. The used technique provides a convenient method for the evaluation of non-genomic carcinogenicity of chemicals prior to their industrial application.