Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value.

Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell.

Redox regulation of anoikis: reactive oxygen species as essential mediators of cell survival.

Proper attachment to the extracellular matrix (ECM) is essential for cell survival. The loss of integrin-mediated cell-ECM contact results in an apoptotic process termed anoikis. However, mechanisms involved in regulation of cell survival are poorly understood and mediators responsible for anoikis have not been well characterized. Here, we demonstrate that reactive oxygen species (ROS) produced through the involvement of the small GTPase Rac-1 upon integrin engagement exert a mandatory role in transducing a pro-survival signal that ensures that cells escape from anoikis. In particular, we show that ROS are responsible for the redox-mediated activation of Src that trans-phosphorylates epidermal growth factor receptor (EGFR) in a ligand-independent manner. The redox-dependent phosphorylation of EGFR activates both extracellular signal-regulated protein kinase and Akt downstream signalling pathways, culminating in degradation of the pro-apoptotic protein Bim. Hence, our results shed new light on the mechanism granting the adhesion-dependent antiapoptotic effect, highlighting a fundamental role of ROS-mediated Src regulation in ensuring anoikis protection.

Insulin inhibits platelet-derived growth factor-induced cell proliferation.

Cellular behavior can be considered to be the result of a very complex spatial and temporal integration of intracellular and extracellular signals. These signals arise from serum-soluble factors as well as from cell-substrate or cell-cell interactions. The current approach in mitogenesis studies is generally to analyze the effect of a single growth factor on serum-starved cells. In this context, a metabolic hormone such as insulin is found to be a mitogenic agent in many cellular types. In the present study, we have considered the effect of insulin stimulation in platelet-derived growth factor (PDGF)-activated NIH-3T3 and C2C12 cells. Our results show that insulin is able to inhibit strongly both NIH-3T3 and C2C12 cell growth induced by PDGF, one of the most powerful mitotic agents for these cell types. This inhibitory effect of insulin is due primarily to a premature down-regulation of the PDGF receptor. Thus, when NIH-3T3 or C2C12 cells are stimulated with both PDGF and insulin, we observe a decrease in PDGF receptor phosphorylation with respect to cells treated with PDGF alone. In particular, we find that costimulation with insulin leads to a reduced production of H2O2 with respect to cell stimulation with PDGF alone. The relative low concentration of H2O2 in PDGF/insulin-costimulated cell leads to a limited down-regulation of protein tyrosine phosphatases, and, consequently, to a reduced PDGF receptor phosphorylation efficiency. The latter is very likely to be responsible for the insulin-dependent inhibition of PDGF-receptor mitogenic signaling.

Expression of the Stp1 LMW-PTP and inhibition of protein CK2 display a cooperative effect on immunophilin Fpr3 tyrosine phosphorylation and Saccharomyces cerevisiae growth.

Although the yeast genome does not encode bona fide protein tyrosine kinases, tyrosine-phosphorylated proteins are numerous, suggesting that besides dual-specificity kinases, some Ser/Thr kinases are also committed to tyrosine phosphorylation in Saccharomyces cerevisiae. Here we show that blockage of the highly pleiotropic Ser/Thr kinase CK2 with a specific inhibitor synergizes with the overexpression of Stp1 low-molecular-weight protein tyrosine phosphatase (PTP) in inducing a severe growth-defective phenotype, consistent with a prominent role for CK2 in tyrosine phosphorylation in yeast. We also present in vivo evidence that immunophilin Fpr3, the only tyrosine-phosphorylated CK2 substrate recognized so far, interacts with and is dephosphorylated by Spt1. These data disclose a functional correlation between CK2 and LMW-PTPs, and suggest that reversible phosphorylation of Fpr3 plays a role in the regulation of growth rate and budding in S. cerevisiae.

Down-regulation of platelet-derived growth factor receptor signaling during myogenesis.

Cell differentiation is often associated with a block in the cell cycle. Growth factor signaling has been reported to be impaired in differentiated cells, due to the withdrawal of growth factors or to transcriptional down-regulation of their receptors. Our proposal is that the down regulation of growth factor signaling may be achieved through an alternative pathway: the decrease of growth factor receptor activation and the ensuing inhibition of intracellular pathways leading the cell to division. Here we report that platelet-derived growth factor receptor (PDGFr) signaling is down-regulated during muscle differentiation, although its expression level remains unchanged. PDGFr signaling inhibition is achieved through a decrease in the receptor tyrosine phosphorylation level, in particular of Tyr716, Tyr751, Tyr857 and Tyr1021, leading to down-regulation of intracellular signaling pathways. Furthermore, during myogenesis, the expression level of several phosphotyrosine phosphatases (PTPs) increases and most of them shift toward the reduced/activated state. We propose a causal link between the down-regulation of PDGFr tyrosine phosphorylation and the increases in PTP specific activity during myogenesis.

Low molecular weight protein tyrosine phosphatases: small, but smart.

Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of 18-kDa enzymes involved in cell growth regulation. Despite very limited sequence similarity to the PTP superfamily, they display a conserved signature motif in the catalytic site. LMW-PTP associates and dephosphorylate many growth factor receptors, such as platelet-derived growth factor receptor (PDGF-r), insulin receptor and ephrin receptor, thus downregulating many of the tyrosine kinase receptor functions that lead to cell division. In particular, LMW-PTP acts on both growth-factor-induced mitosis, through dephosphorylation of activated PDGF-r, and on cytoskeleton rearrangement, through dephosphorylation of p190RhoGAP and the consequent regulation of the small GTPase Rho. LMW-PTP activity is modulated by tyrosine phosphorylation on two specific residues, each of them with specific characteristics. LMW-PTP activity on specific substrates depends also on its localization. Moreover, LMW-PTP is reversibly oxidized during growth factor signaling, leading to inhibition of its enzymatic activity. Recovery of phosphatase activity depends on the availability of reduced glutathione and involves the formation of an S-S bridge between the two catalytic site cysteines. Furthermore, studies on the redox state of LMW-PTP in contact-inhibited cells and in mature myoblasts suggest that LMW-PTP is a general and versatile modulator of growth inhibition.

Low molecular weight protein-tyrosine phosphatase is involved in growth inhibition during cell differentiation.

Low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme involved in mitogenic signaling and cytoskeletal rearrangement after platelet-derived growth factor (PDGF) stimulation. Recently, we demonstrated that LMW-PTP is regulated by a redox mechanism involving the two cysteine residues of the catalytic site, which turn reversibly from reduced to oxidized state after PDGF stimulation. Since recent findings showed a decrease of intracellular reactive oxygen species in contact inhibited cells and a lower tyrosine phosphorylation level in dense cultures in comparison to sparse ones, we studied if the level of endogenous LMW-PTP is regulated by growth inhibition conditions, such as cell confluence and differentiation. Results show that both cell confluence and cell differentiation up-regulate LMW-PTP expression in C2C12 and PC12 cells. We demonstrate that during myogenesis LMW-PTP is regulated at translational level and that the protein accumulates at the plasma membrane. Furthermore, we showed that both myogenesis and cell-cell contact lead to a dramatic decrease of tyrosine phosphorylation level of PDGF receptor. In addition, we observed an increased association of the receptor with LMW-PTP during myogenesis. Herein, we demonstrate that myogenesis decreases the intracellular level of reactive oxygen species, as observed in dense cultures. As a consequence, LMW-PTP turns from oxidized to reduced form during muscle differentiation, increasing its activity in growth inhibition conditions such as differentiation. These data suggest that LMW-PTP plays a crucial role in physiological processes, which require cell growth arrest such as confluence and differentiation.

Two vicinal cysteines confer a peculiar redox regulation to low molecular weight protein tyrosine phosphatase in response to platelet-derived growth factor receptor stimulation.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H(2)O(2) or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H(2)O(2), evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H(2)O(2) production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.

Expression of the small tyrosine phosphatase (Stp1) in Saccharomyces cerevisiae: a study on protein tyrosine phosphorylation.

Small tyrosine phoshatase 1 (Stp1) is a Schizosaccharomyces pombe low-molecular-mass phosphotyrosine-phosphatase 50% identical to Saccharomyces cerevisiae Ltp1. In order to investigate the role of Stp1 in yeast, a mutant was generated having the characteristic of a dominant negative molecule. Changes in protein tyrosine phosphorylation in S. cerevisiae proteome in response to Stp1 or its dominant negative mutant expression were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The most remarkable result is the modification by phosphorylation on tyrosine of several proteins involved in carbohydrate metabolism. Twelve proteins were identified on the basis of their positions in the anti-phosphotyrosine immunoblot of the 2-D electrophoresis. Ten of these present tyrosyl residues that are within the consensus sequence for protein kinase CK2 (casein kinase-2). These data open the possibility for the identification of Stp1 substrates in yeast and provide hints about the nature of tyrosine phosphorylating agents in yeast and in other organisms where bona fide tyrosine kinases are lacking.

Low molecular weight protein-tyrosine phosphatase controls the rate and the strength of NIH-3T3 cells adhesion through its phosphorylation on tyrosine 131 or 132.

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement. Our previous results demonstrated that LMW-PTP is able to bind and dephosphorylate activated PDGF receptor, thus inhibiting cell proliferation. Recently we have shown that LMW-PTP is specifically phosphorylated by c-Src in a cytoskeleton-associated fraction in response to PDGF, and this phosphorylation increases LMW-PTP activity about 20-fold. LMW-PTP strongly influences cell adhesion, spreading, and chemotaxis induced by PDGF stimulation, by regulating the phosphorylation level of p190Rho-GAP, a protein that is able to regulate Rho activity and hence cytoskeleton rearrangement. In the present study we investigate the physiological role of the two LMW-PTP tyrosine phosphorylation sites, using LMW-PTP mutants on tyrosine 131 or 132. We demonstrate that each tyrosine residue is involved in specific LMW-PTP functions. Both of them are phosphorylated during PDGF signaling. Phosphorylation on tyrosine 131 influences mitogenesis, dephosphorylating activated PDGF-R and cytoskeleton rearrangement, acting on p190RhoGAP. Phosphorylation on tyrosine 132 leads to an increase in the strength of cell substrate adhesion, down-regulating matrix metalloproteases expression, through the inhibition of Grb2/MAPK pathway. In conclusion, LMW-PTP tyrosine phosphorylation on both Tyr(131) or Tyr(132) cooperate to determine a faster and stronger adhesion to extracellular matrix, although these two events may diverge in timing and relative amount.

Acylphosphatase is a strong apoptosis inducer in HeLa cell line.

Acylphosphatase (AcP) is a low-molecular-weight protein widely distributed in many vertebrate tissues with a yet unknown physiologic function. To study the in vivo behavior of AcP, HeLa cells were transiently transfected with a vector expressing the AcP/EGFP fusion protein. Analysis of the transfected cells showed a high level of cellular death in cells expressing the AcP/EGFP fusion protein with respect to control cells expressing EGFP alone. Flow cytometry and time lapse analysis of AcP/EGFP transfected cells evidenced a typical pattern of apoptosis. Surprisingly, cells transfected with a mutated form of AcP, with negligible in vitro acylphosphatase activity, undergo apoptosis as well as cells transfected with wild-type protein, suggesting that the physiologic role of AcP could be not related to this catalytic activity.

The inhibitory effect of the 5' untranslated region of muscle acylphosphatase mRNA on protein expression is relieved during cell differentiation.

Previous experiments suggested that the upstream AUG triplet present in the 5' untranslated region (UTR) of muscle acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5'UTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5'UTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5'UTR of muscle acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5'UTR of the muscle acylphosphatase mRNA.

The low M(r) protein-tyrosine phosphatase is involved in Rho-mediated cytoskeleton rearrangement after integrin and platelet-derived growth factor stimulation.

The low molecular weight protein-tyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. In fact, LMW-PTP is able to specifically bind and dephosphorylate activated PDGF receptor, thus modulating PDGF-induced mitogenesis. In particular, LMW-PTP is involved in pathways that regulate the transcription of the immediately early genes myc and fos in response to growth factor stimulation. Recently, we have found that LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its phosphorylation, LMW-PTP increases its catalytic activity about 20-fold. In this study, our interest was to investigate the role of LMW-PTP phosphorylation in cellular response to PDGF stimulation. To address this issue, we have transfected in NIH-3T3 cells a mutant form of LMW-PTP in which the c-Src phosphorylation sites (Tyr(131) and Tyr(132)) were mutated to alanine. We have established that LMW-PTP phosphorylation by c-Src after PDGF treatment strongly influences both cell adhesion and migration. In addition, we have discovered a new LMW-PTP substrate localized in the cytoskeleton that becomes tyrosine-phosphorylated after PDGF treatment: p190Rho-GAP. Hence, LMW-PTP plays multiple roles in PDGF receptor-mediated mitogenesis, since it can bind and dephosphorylate PDGF receptor, and, at the same time, the cytoskeleton-associated LMW-PTP, through the regulation of the p190Rho-GAP phosphorylation state, controls the cytoskeleton rearrangement in response to PDGF stimulation.

LMW-PTP exerts a differential regulation on PDGF- and insulin-mediated signaling.

Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is able to specifically bind and dephosphorylate activated PDGF and insulin receptors, modulating the onset of mitogenic process. LMW-PTP is present in two distinct intracellular locations. While the cytosolic LMW-PTP pool interacts directly with activated insulin or PDGF receptors, the cytoskeleton-associated LMW-PTP is tyrosine phosphorylated upon PDGF stimulation and is involved in cytoskeleton rearrangement acting on p190Rho-GAP. We investigated the differential role of LMW-PTP in PDGF- or insulin-induced mitogenesis and cytoskeleton rearrangement. Dominant negative LMW-PTP influences both PDGF- and insulin-induced mitogenesis with a different extent and it induces a decrease in cellular adhesion and chemotaxis after PDGF but not insulin treatment. PDGF but not insulin stimulation leads to tyrosine phosphorylation of LMW-PTP. We propose that the differential effect of LMW-PTP on PDGF and insulin signaling is mainly due to the fact that during insulin signaling LMW-PTP does not become phosphorylated and thus does not act on its cytoskeleton-associated substrate/s.

Acylphosphatase expression during macrophage differentiation and activation of U-937 cell line.

The two acylphosphatase isoenzymes (muscle type and common type) are differently involved in cell differentiation processes. In this paper we investigate the expression of the two isoenzymes during macrophage differentiation and activation. The U-937 human promonocytic cell line is a model for cell differentiation induced by the tumor promoter phorbol myristic acetate (PMA). Here we show that only the expression of the muscle type acylphosphatase increases during U-937 differentiation and macrophage activation, confirming that the two isoenzymes are differently regulated. Moreover, we determined, in the same conditions, the level of specific mRNA. Results show that after an initial two-fold decrease during PMA stimulation, the muscle type acylphosphatase mRNA levels remain constant also after the treatment with lipopolysaccharide and gamma-interferon, treatments that lead to macrophage activation. It is possible that post-transcription regulation is responsible for the regulation of muscle type acylphosphatase in the cell during differentiation and macrophage activation.

Inhibitory effect of full-length human endostatin on in vitro angiogenesis.

Endostatin, a C-terminal product of collagen XVIII, is a very powerful angiogenesis inhibitor. In vivo experiments in mice indicate that endostatin dramatically reduces tumor mass without causing the onset of any resistance to the treatment. Recently, a 12-aa shorter human endostatin has been purified from plasma, but is ineffective in in vitro angiogenesis assays. Here we report that the full-length human recombinant endostatin has a potent inhibitory activity in in vitro angiogenesis assays. Two powerful angiogenic factors were used to stimulate endothelial cells: FGF-2 and VEGF-165. Endostatin prevented cell growth both in the basal condition and after stimulation with FGF-2 or VEGF-165. Migration of microvascular endothelial cells toward FGF-2 or VEGF-165 was impaired, both when cells were pretreated with the inhibitor and when endostatin was added together with the growth factors. Furthermore, experiments of inhibition of proliferation performed on nonmicroendothelial cells showed that endostatin was ineffective. This study indicates that human endostatin is a potent angiogenesis inhibitor and suggests its use in human anticancer therapy.

The low Mr phosphotyrosine protein phosphatase behaves differently when phosphorylated at Tyr131 or Tyr132 by Src kinase.

The low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) is phosphorylated by Src and Src-related kinases both in vitro and in vivo; in Jurkat cells, and in NIH-3T3 cells, it becomes tyrosine-phosphorylated upon stimulation by PDGF. In this study we show that pp60Src phosphorylates in vitro the enzyme at two tyrosine residues, Tyr131 and Tyr132, previously indicated as the main phosphorylation sites of the enzyme, whereas phosphorylation by the PDGF-R kinase is much less effective and not specific. The effects of LMW-PTP phosphorylation at each tyrosine residue were investigated by using Tyr131 and Tyr132 mutants. We found that the phosphorylation at either residue has differing effects on the enzyme behaviour: Tyr131 phosphorylation is followed by a strong (about 25-fold) increase of the enzyme specific activity, whereas phosphorylation at Tyr132 leads to Grb2 recruitment. These differing effects are discussed on the light of the enzyme structure.

Preferential accumulation of muscle type acylphosphatase in the nucleus during differentiation.

In a previous paper we observed a direct involvement of acylphosphatase in differentiation, associated with enhanced levels of the enzyme in the cell. We have here investigated the subcellular localization of the two known acylphosphatase isoforms during this process. We show that in C2C12 myoblast cells, muscle type acylphosphatase accumulates in the nucleus during differentiation. The same pattern of accumulation is observed also in K562 erythroleukemia cells, although at a lower extent: this fact indicates that this phenomenon is not restricted to muscular cells but rather it could be of general importance in the differentiative process. The common type acylphosphatase, showing an 8-fold increase in the cytoplasm during differentiation, does not accumulate in the nucleus, suggesting distinct roles of the two isoenzymes in this process.

The contribution of acidic residues to the conformational stability of common-type acylphosphatase.

Common-type acylphosphatase is a small cytosolic enzyme whose catalytic properties and three-dimensional structure are known in detail. All the acidic residues of the enzyme have been replaced by noncharged residues in order to assess their contributions to the conformational stability of acylphosphatase. The enzymatic activity parameters and the conformational free energy of each mutant were determined by enzymatic activity assays and chemically induced unfolding, respectively. Some mutants exhibit very similar conformational stability, DeltaG(H2O), and specific activity values as compared to the wild-type enzyme. By contrast, six mutants show a significant reduction of conformational stability and two mutants are more stable than the wild-type protein. Although none of the mutated acidic residues is directly involved in the catalytic mechanism of the enzyme, our results indicate that mutations of residues located on the surface of the protein are responsible for a structural distortion which propagate up to the active site. We found a good correlation between the free energy of unfolding and the enzymatic activity of acylphosphatase. This suggests that enzymatic activity measurements can provide valuable indications on the conformational stability of acylphosphatase mutants, provided the mutated residue lies far apart from the active site. Moreover, our results indicate that the distortion of hydrogen bonds rather than the loss of electrostatic interactions, contributes to the decrease of the conformational stability of the protein.

Low molecular weight protein-tyrosine phosphatase tyrosine phosphorylation by c-Src during platelet-derived growth factor-induced mitogenesis correlates with its subcellular targeting.

The low molecular weight phosphotyrosine phosphatase (LMW-PTP) is an enzyme that is involved in the early events of platelet-derived growth factor (PDGF) receptor signal transduction. Our previous results have shown that LMW-PTP is able to specifically bind and dephosphorylate activated PDGF receptor, thus modulating PDGF-induced mitogenesis. In particular LMW-PTP is involved in pathways that regulate the transcription of the immediately early genes myc and fos in response to growth factor stimulation. In this study we have established that, in nontransformed NIH3T3 cells, LMW-PTP exists constitutively in cytosolic and cytoskeleton-associated localization and that, after PDGF stimulation, c-Src is able to bind and to phosphorylate LMW-PTP only in the cytoskeleton-associated fraction. As a consequence of its tyrosine phosphorylation, LMW-PTP significantly increases its catalytic activity. After PDGF stimulation these two LMW-PTP pools act on distinct substrates, contributing in different manners to the PDGF receptor signaling. The cytoplasmic LMW-PTP fraction exerts its well known action on activated PDGF receptor. On the other hand we have now demonstrated that the cytoskeleton-associated LMW-PTP acts specifically on a few not yet identified proteins that become tyrosine-phosphorylated in response to the PDGF receptor activation. Finally, these two LMW-PTP pools markedly differ in the timing of the processes in which they are involved. The cytoplasmic LMW-PTP pool exerts its action within a few minutes from PDGF receptor activation (short term action), while tyrosine phosphorylation of cytoskeleton-associated LMW-PTP lasts for more than 40 min (long term action). In conclusion LMW-PTP is a striking example of an enzyme that exerts different functions and undergoes different regulation in consequence of its subcellular localization.