Tubulin-Na+, K + -ATPase interaction: Involvement in enzymatic regulation and cellular function.

A new function for tubulin was described by our laboratory: acetylated tubulin forms a complex with Na+ ,K + -ATPase (NKA) and inhibits its activity. This process was shown to be a regulatory factor of physiological importance in cultured cells, human erythrocytes, and several rat tissues. Formation of the acetylated tubulin-NKA complex is reversible. We demonstrated that in cultured cells, high concentrations of glucose induce translocation of acetylated tubulin from cytoplasm to plasma membrane with a consequent inhibition of NKA activity. This effect is reversed by adding glutamate, which is coctransported to the cell with Na + . Another posttranslational modification of tubulin, detyrosinated tubulin, is also involved in the regulation of NKA activity: it enhances the NKA inhibition induced by acetylated tubulin. Manipulation of the content of these modifications of tubulin could work as a new strategy to maintain homeostasis of Na + and K + , and to regulate a variety of functions in which NKA is involved, such as osmotic fragility and deformability of human erythrocytes. The results summarized in this review show that the interaction between tubulin and NKA plays an important role in cellular physiology, both in the regulation of Na + /K + homeostasis and in the rheological properties of the cells, which is mechanically different from other roles reported up to now.

Electrochemical regulation of budding yeast polarity.

Cells are naturally surrounded by organized electrical signals in the form of local ion fluxes, membrane potential, and electric fields (EFs) at their surface. Although the contribution of electrochemical elements to cell polarity and migration is beginning to be appreciated, underlying mechanisms are not known. Here we show that an exogenous EF can orient cell polarization in budding yeast (Saccharomyces cerevisiae) cells, directing the growth of mating projections towards sites of hyperpolarized membrane potential, while directing bud emergence in the opposite direction, towards sites of depolarized potential. Using an optogenetic approach, we demonstrate that a local change in membrane potential triggered by light is sufficient to direct cell polarization. Screens for mutants with altered EF responses identify genes involved in transducing electrochemical signals to the polarity machinery. Membrane potential, which is regulated by the potassium transporter Trk1p, is required for polarity orientation during mating and EF response. Membrane potential may regulate membrane charges through negatively charged phosphatidylserines (PSs), which act to position the Cdc42p-based polarity machinery. These studies thus define an electrochemical pathway that directs the orientation of cell polarization.

PMCA activity and membrane tubulin affect deformability of erythrocytes from normal and hypertensive human subjects.

Our previous studies demonstrated formation of a complex between acetylated tubulin and brain plasma membrane Ca(2+)-ATPase (PMCA), and the effect of the lipid environment on structure of this complex and on PMCA activity. Deformability of erythrocytes from hypertensive human subjects was reduced by an increase in membrane tubulin content. In the present study, we examined the regulation of PMCA activity by tubulin in normotensive and hypertensive erythrocytes, and the effect of exogenously added diacylglycerol (DAG) and phosphatidic acid (PA) on erythrocyte deformability. Some of the key findings were that: (i) PMCA was associated with tubulin in normotensive and hypertensive erythrocytes, (ii) PMCA enzyme activity was directly correlated with erythrocyte deformability, and (iii) when tubulin was present in the erythrocyte membrane, treatment with DAG or PA led to increased deformability and associated PMCA activity. Taken together, our findings indicate that PMCA activity is involved in deformability of both normotensive and hypertensive erythrocytes. This rheological property of erythrocytes is affected by acetylated tubulin and its lipid environment because both regulate PMCA activity.

Activation of H(+)-ATPase by glucose in Saccharomyces cerevisiae involves a membrane serine protease.

BACKGROUND: Glucose induces H(+)-ATPase activation in Saccharomyces cerevisiae. Our previous study showed that (i) S. cerevisiae plasma membrane H(+)-ATPase forms a complex with acetylated tubulin (AcTub), resulting in inhibition of the enzyme activity; (ii) exogenous glucose addition results in the dissociation of the complex and recovery of the enzyme activity. METHODS: We used classic biochemical and molecular biology tools in order to identify the key components in the mechanism that leads to H(+)-ATPase activation after glucose treatment. RESULTS: We demonstrate that glucose-induced dissociation of the complex is due to pH-dependent activation of a protease that hydrolyzes membrane tubulin. Biochemical analysis identified a serine protease with a kDa of 35-40 and an isoelectric point between 8 and 9. Analysis of several knockout yeast strains led to the detection of Lpx1p as the serine protease responsible of tubulin proteolysis. When lpx1Δ cells were treated with glucose, tubulin was not degraded, the AcTub/H(+)-ATPase complex did not undergo dissociation, and H(+)-ATPase activation was significantly delayed. CONCLUSION: Our findings indicate that the mechanism of H(+)-ATPase activation by glucose involves a decrease in the cytosolic pH and consequent activation of a serine protease that hydrolyzes AcTub, accelerating the process of the AcTub/H(+)-ATPase complex dissociation and the activation of the enzyme. GENERAL SIGNIFICANCE: Our data sheds light into the mechanism by which acetylated tubulin dissociates from the yeast H(+)-ATPase, identifying a degradative step that remained unknown. This finding also proposes an indirect way to pharmacologically regulate yeast H(+)-ATPase activity and open the question about mechanistic similarities with other higher eukaryotes.

Tubulin-mediated anatomical and functional changes caused by Ca2+ in human erythrocytes.

In previous research, we observed that tubulin can be found in three fractions within erythrocytes, i.e., attached to the membrane, as a soluble fraction, or as part of a structure that can be sedimented by centrifugation. Given that its differential distribution within these fractions may alter several hemorheological properties, such as erythrocyte deformability, the present work studied how this distribution is in turn affected by Ca2+, another key player in the regulation of erythrocyte cytoskeleton stability. The effect of Ca2+ on some hemorheological parameters was also assessed. The results showed that when Ca2+ concentrations increased in the cell, whether by the addition of ionophore A23187, by specific plasma membrane Ca2 + _ATPase (PMCA) inhibition, or due to arterial hypertension, tubulin translocate to the membrane, erythrocyte deformability decreased, and phosphatidylserine exposure increased. Moreover, increased Ca2+ was associated with an inverse correlation in the distribution of tubulin and spectrin, another important cytoskeleton protein. Based on these findings, we propose the existence of a mechanism of action through which higher Ca2+ concentrations in erythrocytes trigger the migration of tubulin to the membrane, a phenomenon that results in alterations of rheological and molecular aspects of the membrane itself, as well as of the integrity of the cytoskeleton.

Tubulin Regulates Plasma Membrane Ca2+-ATPase Activity in a Lipid Environment-dependent Manner.

Ca2+ plays a crucial role in cell signaling, cytosolic Ca2+ can change up to 10,000-fold in concentration due to the action of Ca2+-ATPases, including PMCA, SERCA and SCR. The regulation and balance of these enzymes are essential to maintain cytosolic Ca2+ homeostasis. Our laboratory has discovered a novel PMCA regulatory system, involving acetylated tubulin alone or in combination with membrane lipids. This regulation controls cytosolic Ca2+ levels and influences cellular properties such as erythrocyte rheology. This review summarizes the findings on the regulatory mechanism of PMCA activity by acetylated tubulin in combination with lipids. The combination of tubulin cytoskeleton and membrane lipids suggests a novel regulatory system for PMCA, which consequently affects cytosolic Ca2+ content, depending on cytoskeletal and plasma membrane dynamics. Understanding the interaction between acetylated tubulin, lipids and PMCA activity provides new insights into Ca2+ signaling and cell function. Further research may shed light on potential therapeutic targets for diseases related to Ca2+ dysregulation. This discovery contributes to a broader understanding of cellular processes and offers opportunities to develop innovative approaches to treat Ca2+-related disorders. By elucidating the complex regulatory mechanisms of Ca2+ homeostasis, we advance our understanding of cell biology and its implications for human health.

Tubulin pools in human erythrocytes: altered distribution in hypertensive patients affects Na+, K+-ATPase activity.

The presence of tubulin in human erythrocytes was demonstrated using five different antibodies. Tubulin was distributed among three operationally distinguishable pools: membrane, sedimentable structure and soluble fraction. It is known that in erythrocytes from hypertensive subjects (HS), the Na(+), K(+)-ATPase (NKA) activity is partially inhibited as compared with erythrocytes from normal subjects (NS). In erythrocytes from HS the membrane tubulin pool is increased by ~150%. NKA was found to be forming a complex with acetylated tubulin that results in inhibition of enzymes. This complex was also increased in erythrocytes from HS. Treatment of erythrocytes from HS with nocodazol caused a decrease of acetylated tubulin in the membrane and stimulation of NKA activity, whereas taxol treatment on erythrocytes from NS had the opposite effect. These results suggest that, in erythrocytes from HS, tubulin was translocated to the membrane, where it associated with NKA with the consequent enzyme inhibition.

Inhibition of flippase-like activity by tubulin regulates phosphatidylserine exposure in erythrocytes from hypertensive and diabetic patients.

Plasma membrane tubulin is an endogenous regulator of P-ATPases and the unusual accumulation of tubulin in the erythrocyte membrane results in a partial inhibition of some their activities, causing hemorheological disorders like reduced cell deformability and osmotic resistance. These disorders are of particular interest in hypertension and diabetes, where the abnormal increase in membrane tubulin may be related to the disease development. Phosphatidylserine (PS) is more exposed on the membrane of diabetic erythrocytes than in healthy cells. In most cells, PS is transported from the exoplasmic to the cytoplasmic leaflet of the membrane by lipid flippases. Here, we report that PS is more exposed in erythrocytes from both hypertensive and diabetic patients than in healthy erythrocytes, which could be attributed to the inhibition of flippase activity by tubulin. This is supported by: (i) the translocation rate of a fluorescent PS analog in hypertensive and diabetic erythrocytes was slower than in healthy cells, (ii) the pharmacological variation of membrane tubulin in erythrocytes and K562 cells was linked to changes in PS translocation and (iii) the P-ATPase-dependent PS translocation in inside-out vesicles (IOVs) from human erythrocytes was inhibited by tubulin. These results suggest that tubulin regulates flippase activity and hence, the membrane phospholipid asymmetry.

Erythrocyte plasma membrane potential: past and current methods for its measurement.

The plasma membrane functions both as a natural insulator and a diffusion barrier to the movement of ions. A wide variety of proteins transport and pump ions to generate concentration gradients that result in voltage differences, while ion channels allow ions to move across the membrane down those gradients. Plasma membrane potential is the difference in voltage between the inside and the outside of a biological cell, and it ranges from ~- 3 to ~- 90 mV. Most of the most significant discoveries in this field have been made in excitable cells, such as nerve and muscle cells. Nevertheless, special attention has been paid to some events controlled by changes in membrane potential in non-excitable cells. The origins of several blood disorders, for instance, are related to disturbances at the level of plasma membrane in erythrocytes, the structurally simplest red blood cells. The high simplicity of erythrocytes, in particular, made them perfect candidates for the electrophysiological studies that laid the foundations for understanding the generation, maintenance, and roles of membrane potential. This article summarizes the methodologies that have been used during the past decades to determine Δψ in red blood cells, from seminal microelectrodes, through the use of nuclear magnetic resonance or lipophilic radioactive ions to quantify intra and extracellular ions, to continuously renewed fluorescent potentiometric dyes. We have attempted to highlight the advantages and disadvantages of each methodology, as well as to provide a description of the technical aspects involved.

Activation of PMCA by calmodulin or ethanol in plasma membrane vesicles from rat brain involves dissociation of the acetylated tubulin/PMCA complex.

We have recently shown that acetylated tubulin interacts with plasma membrane Na(+),K(+)-ATPase and inhibits its enzyme activity in several types of cells. H(+)-ATPase of Saccharomyces cerevisiae is similarly inhibited by interaction with acetylated tubulin. The activities of both these ATPases are restored upon dissociation of the acetylated tubulin/ATPase complex. Here, we report that in plasma membrane vesicles isolated from brain synaptosomes, another P-type ATPase, plasma membrane Ca(2+)-ATPase (PMCA), undergoes enzyme activity regulation by its association/dissociation with acetylated tubulin. The presence of acetylated tubulin/PMCA complex in membrane vesicles was demonstrated by analyzing the behavior of acetylated tubulin in a detergent partition, and by immunoprecipitation experiments. PMCA is known to be stimulated by ethanol and calmodulin at physiological concentrations. We found that treatment of plasma membrane vesicles with these reagents induced dissociation of the complex, with a concomitant restoration of enzyme activity. Conversely, incubation of vesicles with exogenous tubulin induced the association of acetylated tubulin with PMCA, and the inhibition of enzyme activity. These findings indicate that activation of synaptosomal PMCA by ethanol and calmodulin involves dissociation of the acetylated tubulin/PMCA complex. This regulatory mechanism was shown to also operate in living cells.

Tubulin is retained throughout the human hematopoietic/erythroid cell differentiation process and plays a structural role in sedimentable fraction of mature erythrocytes.

We investigated the properties of tubulin present in the sedimentable fraction ("Sed-tub") of human erythrocytes, and tracked the location and organization of tubulin in various types of cells during the process of hematopoietic/erythroid differentiation. Sed-tub was sensitive to taxol/nocodazole (drugs that modify microtubule assembly/disassembly), but was organized as part of a protein network rather than in typical microtubule form. This network had a non-uniform "connected-ring" structure, with tubulin localized in the connection areas and associated with other proteins. When tubulin was eliminated from Sed-tub fraction, this connected-ring structure disappeared. Spectrin, a major protein component in Sed-tub fraction, formed a complex with tubulin. During hematopoietic differentiation, tubulin shifts from typical microtubule structure (in pro-erythroblasts) to a disorganized structure (in later stages), and is retained in reticulocytes following enucleation. Thus, tubulin is not completely lost when erythrocytes mature; it continues to play a structural role in the Sed-tub fraction.

Alterations of hemorheological parameters and tubulin content in erythrocytes from diabetic subjects.

Treatment of human erythrocytes with high glucose concentrations altered the content and distributions of three tubulin isotypes, with consequent reduction of erythrocyte deformability and osmotic resistance. In erythrocytes from diabetic subjects (D erythrocytes), (i) tubulin in the membrane-associated fraction (Mem-Tub) was increased and tubulin in the sedimentable fraction (Sed-Tub) was decreased, (ii) deformability was lower than in erythrocytes from normal subjects (N erythrocytes), and (iii) detyrosinated/acetylated tubulin content was higher in the Mem-Tub fraction and tyrosinated/acetylated tubulin content was higher in the Sed-Tub fraction, in comparison with N erythrocytes. Similar properties were observed for human N erythrocytes treated with high glucose concentrations, and for erythrocytes from rats with streptozotocin-induced diabetes. In N erythrocytes, high-glucose treatment caused translocation of tubulin from the Sed-Tub to Mem-Tub fraction, thereby reducing deformability and inducing acetylation/tyrosination in the Sed-Tub fraction. The increased tubulin acetylation in these cells resulted from inhibition of deacetylase enzymes. Increased tubulin acetylation and translocation of this acetylated tubulin to the Mem-Tub fraction were both correlated with reduced osmotic resistance. Our findings suggest that (i) high glucose concentrations promote tubulin acetylation and translocation of this tubulin to the membrane, and (ii) this tubulin is involved in regulation of erythrocyte deformability and osmotic fragility.

Activation of the plasma membrane H-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H(+)-ATPase-acetylated tubulin complex.

In the yeast Saccharomyces cerevisiae, plasma membrane H(+)-ATPase is activated by d-glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usually displays hydrophilic properties, but behaves as a hydrophobic compound when complexed with H(+)-ATPase, and therefore partitions into a detergent phase. When cells were treated with glucose, the H(+)-ATPase-tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H(+)-ATPase activity increased as a function of glucose concentration, as measured by both ATP-hydrolyzing capacity and H(+)-pumping activity. The addition of 2-deoxy-d-glucose inhibited the above glucose-induced phenomena, suggesting the involvement of glucose transporters. Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentrated near the plasma membrane. Results from immunoprecipitation experiments using anti-(acetylated tubulin) and anti-(H(+)-ATPase) immunoglobulins indicated a physical interaction between H(+)-ATPase and acetylated tubulin in the membranes of glucose-starved cells. When cells were pretreated with 1 mm glucose, this interaction was disrupted. Double immunofluorescence, observed by confocal microscopy, indicated that H(+)-ATPase and acetylated tubulin partially colocalize at the periphery of glucose-starved cells, with predominance at the outer and inner sides of the membrane, respectively. Colocalization was not observed when cells were pretreated with 1 mm glucose, reinforcing the idea that glucose treatment produces dissociation of the H(+)-ATPase-tubulin complex. Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H(+)-ATPase activity by acetylated tubulin and concomitant increase of the H(+)-ATP ase-tubulin complex.

Effects of detyrosinated tubulin on Na+,K+-ATPase activity and erythrocyte function in hypertensive subjects.

Formation of tubulin/Na(+),K(+)-ATPase (NKA) complex in erythrocytes of hypertensive subjects results in a 50% reduction in NKA activity. We demonstrate here that detyrosinated tubulin, which is increased in hypertensive erythrocytes membranes, enhances the inhibitory effect of acetylated tubulin on NKA activity. Moreover, we report a reduced content and activity of the enzyme tubulin tyrosine ligase in erythrocytes of hypertensive subjects. Such alterations are related to changes in erythrocyte deformability. Our findings indicate that the detyrosination/tyrosination cycle of tubulin is important in regulation of NKA activity, and that abnormalities in this cycle are involved in hypertension development.

Tubulin-mediated anatomical and functional changes caused by Ca2+ in human erythrocytes

In previous research, we observed that tubulin can be found in three fractions within erythrocytes, i.e., attached to the membrane, as a soluble fraction, or as part of a structure that can be sedimented by centrifugation. Given that its differential distribution within these fractions may alter several hemorheological properties, such as erythrocyte deformability, the present work studied how this distribution is in turn affected by Ca2+, another key player in the regulation of erythrocyte cytoskeleton stability. The effect of Ca2+ on some hemorheological parameters was also assessed. The results showed that when Ca2+ concentrations increased in the cell, whether by the addition of ionophore A23187, by specific plasma membrane Ca2 + _ATPase (PMCA) inhibition, or due to arterial hypertension, tubulin translocate to the membrane, erythrocyte deformability decreased, and phosphatidylserine exposure increased. Moreover, increased Ca2+ was associated with an inverse correlation in the distribution of tubulin and spectrin, another important cytoskeleton protein. Based on these findings, we propose the existence of a mechanism of action through which higher Ca2+ concentrations in erythrocytes trigger the migration of tubulin to the membrane, a phenomenon that results in alterations of rheological and molecular aspects of the membrane itself, as well as of the integrity of the cytoskeleton. (AU)

Electrochemical regulation of cell polarity and the cytoskeleton.

Cell polarity plays a key role in regulating cell-cell communication, tissue architecture, and development. Both internal and external cues participate in directing polarity and feedback onto each other for robust polarization. One poorly appreciated layer of polarity regulation comes from electrochemical signals spatially organized at the level of the cell or the tissue. These signals which include ion fluxes, membrane potential gradients, or even steady electric fields, emerge from the polarized activation of specific ion transporters, and may guide polarity in wound-healing, development or regeneration. How a given electrochemical cue may influence cytoskeletal elements and cell polarity remains unclear. Here, we review recent progress highlighting the role of electrochemical signals in cell and tissue spatial organization, and elucidating the mechanisms for how such signals may regulate cytoskeletal assembly for cell polarity.

Involvement of membrane tubulin in erythrocyte deformability and blood pressure.

OBJECTIVE: To test the hypothesis that erythrocyte deformability is influenced by changes in the content of membrane tubulin (Mem-tub). METHODS AND RESULTS: Human erythrocytes contain tubulin distributed in three pools (membrane, sedimentable, soluble). Erythrocytes from hypertensive humans have a higher proportion of Mem-tub. Increased Mem-tub content in hypertensive patients was correlated with decreased erythrocyte deformability. Treatment of erythrocytes from normotensive individuals with taxol increased Mem-tub content and reduced deformability, whereas treatment of hypertensive patients erythrocytes with nocodazole had the opposite effect. In-vivo experiments with rats were performed to examine the possible relationship between Mem-tub content, erythrocyte deformability, and blood pressure. Spontaneously hypertensive rats (SHRs) showed lower erythrocyte deformability than normotensive Wistar rats. During the development of hypertension in SHR, tubulin in erythrocytes is translocated to the membrane, and this process is correlated with decreased deformability. In-vivo treatment (intraperitoneal injection) of SHR with nocodazole decreased Mem-tub content, increased erythrocyte deformability, and decreased blood pressure, whereas treatment of Wistar rats with taxol had the opposite effects. CONCLUSION: These findings indicate that increased Mem-tub content contributes to reduced erythrocyte deformability in hypertensive animals.