RESUMO
MOTIVATION: High-content imaging screens provide a cost-effective and scalable way to assess cell states across diverse experimental conditions. The analysis of the acquired microscopy images involves assembling and curating raw cellular measurements into morphological profiles suitable for testing biological hypotheses. Despite being a critical step, general-purpose and adaptable tools for morphological profiling are lacking and no solution is available for the high-performance Julia programming language. RESULTS: Here, we introduce BioProfiling.jl, an efficient end-to-end solution for compiling and filtering informative morphological profiles in Julia. The package contains all the necessary data structures to curate morphological measurements and helper functions to transform, normalize and visualize profiles. Robust statistical distances and permutation tests enable quantification of the significance of the observed changes despite the high fraction of outliers inherent to high-content screens. This package also simplifies visual artifact diagnostics, thus streamlining a bottleneck of morphological analyses. We showcase the features of the package by analyzing a chemical imaging screen, in which the morphological profiles prove to be informative about the compounds' mechanisms of action and can be conveniently integrated with the network localization of molecular targets. AVAILABILITY AND IMPLEMENTATION: The Julia package is available on GitHub: https://github.com/menchelab/BioProfiling.jl. We also provide Jupyter notebooks reproducing our analyses: https://github.com/menchelab/BioProfilingNotebooks. The data underlying this article are available from FigShare, at https://doi.org/10.6084/m9.figshare.14784678.v2. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Linguagens de Programação , Software , MicroscopiaRESUMO
CD4+ T cell trafficking is a fundamental property of adaptive immunity. In this study, we uncover a novel role for histone deacetylase 1 (HDAC1) in controlling effector CD4+ T cell migration, thereby providing mechanistic insight into why a T cell-specific deletion of HDAC1 protects against experimental autoimmune encephalomyelitis (EAE). HDAC1-deficient CD4+ T cells downregulated genes associated with leukocyte extravasation. In vitro, HDAC1-deficient CD4+ T cells displayed aberrant morphology and migration on surfaces coated with integrin LFA-1 ligand ICAM-1 and showed an impaired ability to arrest on and to migrate across a monolayer of primary mouse brain microvascular endothelial cells under physiological flow. Moreover, HDAC1 deficiency reduced homing of CD4+ T cells into the intestinal epithelium and lamina propria preventing weight-loss, crypt damage and intestinal inflammation in adoptive CD4+ T cell transfer colitis. This correlated with reduced expression levels of LFA-1 integrin chains CD11a and CD18 as well as of selectin ligands CD43, CD44 and CD162 on transferred circulating HDAC1-deficient CD4+ T cells. Our data reveal that HDAC1 controls T cell-mediated autoimmunity via the regulation of CD4+ T cell trafficking into the CNS and intestinal tissues.
Assuntos
Autoimunidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Quimiotaxia de Leucócito/imunologia , Histona Desacetilase 1/metabolismo , Inflamação/etiologia , Inflamação/metabolismo , Animais , Biomarcadores , Adesão Celular , Quimiotaxia de Leucócito/genética , Modelos Animais de Doenças , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/diagnóstico , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Células Endoteliais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Histona Desacetilase 1/genética , Imuno-Histoquímica , Inflamação/diagnóstico , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos KnockoutRESUMO
Cytokinesis in many eukaryotes requires a contractile actomyosin ring that is placed at the division site. In fission yeast, which is an attractive organism for the study of cytokinesis, actomyosin ring assembly and contraction requires the myosin II heavy chain Myo2p. Although myo2-E1, a temperature-sensitive mutant defective in the upper 50â kDa domain of Myo2p, has been studied extensively, the molecular basis of the cytokinesis defect is not understood. Here, we isolate myo2-E1-Sup2, an intragenic suppressor that contains the original mutation in myo2-E1 (G345R) and a second mutation in the upper 50â kDa domain (Y297C). Unlike myo2-E1-Sup1, a previously characterized myo2-E1 suppressor, myo2-E1-Sup2 reverses actomyosin ring contraction defects in vitro and in vivo Structural analysis of available myosin motor domain conformations suggests that a steric clash in myo2-E1, which is caused by the replacement of a glycine with a bulky arginine, is relieved in myo2-E1-Sup2 by mutation of a tyrosine to a smaller cysteine. Our work provides insight into the function of the upper 50â kDa domain of Myo2p, informs a molecular basis for the cytokinesis defect in myo2-E1, and may be relevant to the understanding of certain cardiomyopathies.
Assuntos
Actomiosina/metabolismo , Citocinese/genética , Cadeias Pesadas de Miosina/fisiologia , Miosina Tipo II/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Sequência de Aminoácidos , Divisão Celular , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo II/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin ß4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin ß4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the ß- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.
Assuntos
Actinas/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Humanos , PichiaRESUMO
In fission yeast, the lengths of interphase microtubule (iMT) arrays are adapted to cell length to maintain cell polarity and to help centre the nucleus and cell division ring. Here, we show that length regulation of iMTs is dictated by spatially regulated competition between MT-stabilising Tea2/Tip1/Mal3 (Kinesin-7) and MT-destabilising Klp5/Klp6/Mcp1 (Kinesin-8) complexes at iMT plus ends. During MT growth, the Tea2/Tip1/Mal3 complex remains bound to the plus ends of iMT bundles, thereby restricting access to the plus ends by Klp5/Klp6/Mcp1, which accumulate behind it. At cell ends, Klp5/Klp6/Mcp1 invades the space occupied by the Tea2/Tip1/Tea1 kinesin complex triggering its displacement from iMT plus ends and MT catastrophe. These data show that in vivo, whilst an iMT length-dependent model for catastrophe factor accumulation has validity, length control of iMTs is an emergent property reflecting spatially regulated competition between distinct kinesin complexes at the MT plus tip.
Assuntos
Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citologia , Polaridade Celular , Interfase/fisiologia , Cinesinas/genética , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Actin cytoskeleton remodeling sustains the ability of cytotoxic T cells to search for target cells and eliminate them. We here investigated the relationship between energetic status, actin remodeling, and functional fitness in human CD8+ effector T cells. Cell spreading during migration or immunological synapse assembly mirrored cytotoxic activity. Morphological and functional fitness were boosted by interleukin-2 (IL-2), which also stimulated the transcription of glycolytic enzymes, actin isoforms, and actin-related protein (ARP)2/3 complex subunits. This molecular program scaled with F-actin content and cell spreading. Inhibiting glycolysis impaired F-actin remodeling at the lamellipodium, chemokine-driven motility, and adhesion, while mitochondrial oxidative phosphorylation blockade impacted cell elongation during confined migration. The severe morphological and functional defects of ARPC1B-deficient T cells were only partially corrected by IL-2, emphasizing ARP2/3-mediated actin polymerization as a crucial energy state integrator. The study therefore underscores the tight coordination between metabolic and actin remodeling programs to sustain the cytotoxic activity of CD8+ T cells.
Assuntos
Actinas , Linfócitos T CD8-Positivos , Humanos , Actinas/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Interleucina-2/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismoRESUMO
Motility is a crucial activity of immune cells allowing them to patrol tissues as they differentiate, sample or exchange information, and execute their effector functions. Although all immune cells are highly migratory, each subset is endowed with very distinct motility patterns in accordance with functional specification. Furthermore individual immune cell subsets adapt their motility behaviour to the surrounding tissue environment. This review focuses on how the generation and adaptation of diversified motility patterns in immune cells is sustained by actin cytoskeleton dynamics. In particular, we review the knowledge gained through the study of inborn errors of immunity (IEI) related to actin defects. Such pathologies are unique models that help us to uncover the contribution of individual actin regulators to the migration of immune cells in the context of their development and function.
Assuntos
Actinas/fisiologia , Leucócitos/fisiologia , Actinas/ultraestrutura , Animais , Movimento Celular , Humanos , Leucócitos/ultraestruturaRESUMO
Cytokinesis in many eukaryotes is dependent on a contractile actomyosin ring (AMR), composed of F-actin, myosin II, and other actin and myosin II regulators. Through fluorescence recovery after photobleaching experiments, many components of the AMR have been shown to be mobile and to undergo constant exchange with the cytosolic pools. However, how the mobility of its components changes at distinct stages of mitosis and cytokinesis has not been addressed. Here, we describe the mobility of eight Schizosaccharomyces pombe AMR proteins at different stages of mitosis and cytokinesis using an approach we have developed. We identified three classes of proteins, which showed 1) high (Ain1, Myo2, Myo51), 2) low (Rng2, Mid1, Myp2, Cdc12), and 3) cell cycle-dependent (Cdc15) mobile fractions. We observed that the F-BAR protein Cdc15 undergoes a 20-30% reduction in its mobile fraction after spindle breakdown and initiation of AMR contraction. Moreover, our data indicate that this change in Cdc15 mobility is dependent on the septation initiation network (SIN). Our work offers a novel strategy for estimating cell cycle-dependent mobile protein fractions in cellular structures and provides a valuable dataset, that is of interest to researchers working on cytokinesis.
Assuntos
Actomiosina/metabolismo , Proteínas Contráteis/metabolismo , Citocinese/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/fisiologia , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Contráteis/fisiologia , Citocinese/genética , Proteínas do Citoesqueleto/metabolismo , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Ligação ao GTP/metabolismo , Mitose/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo II/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ T cells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.
Assuntos
Forma Celular , Imageamento Tridimensional , Células Matadoras Naturais/citologia , Linfócitos T/citologia , Complexo 2-3 de Proteínas Relacionadas à Actina/deficiência , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Adolescente , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linhagem Celular , Forma Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Exocitose/efeitos dos fármacos , Humanos , Sinapses Imunológicas/efeitos dos fármacos , Sinapses Imunológicas/metabolismo , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Masculino , Compostos Organosselênicos/farmacologia , Compostos de Organossilício/farmacologia , Análise de Célula Única , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tionas/farmacologia , Uracila/análogos & derivados , Uracila/farmacologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Eukaryotic cells assemble actomyosin rings during cytokinesis to function as force-generating machines to drive membrane invagination and to counteract the intracellular pressure and the cell surface tension. How the extracellular matrix affects actomyosin ring contraction has not been fully explored. While studying the Schizosaccharomyces pombe 1,3-ß-glucan-synthase mutant cps1-191, which is defective in division septum synthesis and arrests with a stable actomyosin ring, we found that weakening of the extracellular glycan matrix caused the generated spheroplasts to divide under the nonpermissive condition. This nonmedial slow division was dependent on a functional actomyosin ring and vesicular trafficking, but independent of normal septum synthesis. Interestingly, the high intracellular turgor pressure appears to play a minimal role in inhibiting ring contraction in the absence of cell wall remodeling in cps1-191 mutants, as decreasing the turgor pressure alone did not enable spheroplast division. We propose that during cytokinesis, the extracellular glycan matrix restricts actomyosin ring contraction and membrane ingression, and remodeling of the extracellular components through division septum synthesis relieves the inhibition and facilitates actomyosin ring contraction.
Assuntos
Actomiosina/metabolismo , Membrana Celular , Citocinese , Schizosaccharomyces/metabolismo , Actomiosina/fisiologia , Parede Celular , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Glucosiltransferases/genética , Mutação , Schizosaccharomyces/fisiologia , Proteínas de Schizosaccharomyces pombeRESUMO
Tropomyosin is a coiled-coil actin binding protein key to the stability of actin filaments. In muscle cells, tropomyosin is subject to calcium regulation, but its regulation in nonmuscle cells is not understood. Here, we provide evidence that the fission yeast tropomyosin, Cdc8, is regulated by phosphorylation of a serine residue. Failure of phosphorylation leads to an increased number and stability of actin cables and causes misplacement of the division site in certain genetic backgrounds. Phosphorylation of Cdc8 weakens its interaction with actin filaments. Furthermore, we show through in vitro reconstitution that phosphorylation-mediated release of Cdc8 from actin filaments facilitates access of the actin-severing protein Adf1 and subsequent filament disassembly. These studies establish that phosphorylation may be a key mode of regulation of nonmuscle tropomyosins, which in fission yeast controls actin filament stability and division site placement.
Assuntos
Actinas/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Tropomiosina/metabolismo , FosforilaçãoRESUMO
The position of the division site dictates the size and fate of daughter cells in many organisms. In animal cells, division-site placement involves overlapping mechanisms, including signaling from the central spindle microtubules, astral microtubules, and spindle poles and through polar contractions [1-3]. In fission yeast, division-site positioning requires overlapping mechanisms involving the anillin-related protein Mid1 and the tip complex (comprising the Kelch-repeat protein Tea1, the Dyrk-kinase Pom1, and the SH3-domain protein Tea4) [4-11]. In addition to these factors, cell shape has also been shown to participate in the maintenance of the position of the actomyosin ring [12-14]. The first principles guiding actomyosin ring placement, however, have not been elucidated in any organism. Because actomyosin ring positioning, ring assembly, and cell morphogenesis are genetically separable in fission yeast, we have used it to derive actomyosin ring placement mechanisms from first principles. We report that, during ring assembly in the absence of cytokinetic cues (anillin-related Mid1 and tip-complex proteins), actin bundles follow the path of least curvature and assemble actomyosin rings in an equatorial position in spherical protoplasts and along the long axis in cylindrical cells and compressed protoplasts. The equatorial position of rings is abolished upon treatment of protoplasts with an actin-severing compound or by slowing down actin polymerization. We propose that the physical properties of actin filaments/bundles play key roles in actomyosin ring assembly and positioning, and that key cytokinetic molecules may modulate the length of actin filaments to promote ring assembly along the short axis.
Assuntos
Actomiosina/metabolismo , Citocinese/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Divisão Celular/fisiologia , Citoesqueleto/metabolismo , Toxinas Marinhas , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Protoplastos/fisiologia , Schizosaccharomyces/metabolismo , Esferoplastos/fisiologiaRESUMO
Cytokinesis in many eukaryotes requires an actomyosin-based contractile ring [1]. In fission yeast, cytokinesis involves the type II myosins Myo2p and Myp2p and the type V myosin Myo51p [2]. A recent study by Laplante et al.[3], using deletion mutants of myp2 and myo51 and the mis-sense mutant myo2-E1 [4], concluded that each myosin has distinct functions and proposed that Myp2p plays the dominant role in actomyosin ring contraction. Here we present evidence that Myo2p, not Myp2p, is likely to be the major motor driving actomyosin ring contractility. Since the previous work [3] was performed at 25°C, the permissive temperature for myo2-E1, we compared cytokinesis timings in myo2-E1 and myo2Δ at 25°C and found that myo2-E1 is only partially compromised at 25°C. Furthermore, we find that myp2Δ and myp2Δ myo51Δ double mutants contract actomyosin rings at â¼90% of the rate of wild-type cells at 30°C and 36°C, suggesting that Myp2p plays a minimal role in ring contraction at these temperatures. Finally, ring contraction in our myo2-E1 strain took longer at 25°C than previously reported [3]. Although faster-acting alleles of myo2 will be required to evaluate its contribution at 25°C, our work establishes that Myo2p is the major motor involved in ring contraction, under most, if not all, conditions.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Actomiosina , Constrição , Citocinese , Cadeias Pesadas de Miosina , Miosina Tipo II , MiosinasRESUMO
Cytokinesis in many eukaryotes involves a tension-generating actomyosin-based contractile ring. Many components of actomyosin rings turn over during contraction, although the significance of this turnover has remained enigmatic. Here, using Schizosaccharomyces japonicus, we investigate the role of turnover of actin and myosin II in its contraction. Actomyosin ring components self-organize into â¼1-µm-spaced clusters instead of undergoing full-ring contraction in the absence of continuous actin polymerization. This effect is reversed when actin filaments are stabilized. We tested the idea that the function of turnover is to ensure actin filament homeostasis in a synthetic system, in which we abolished turnover by fixing rings in cell ghosts with formaldehyde. We found that these rings contracted fully upon exogenous addition of a vertebrate myosin. We conclude that actin turnover is required to maintain actin filament homeostasis during ring contraction and that the requirement for turnover can be bypassed if homeostasis is achieved artificially.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Citocinese , Schizosaccharomyces/metabolismo , Fixadores/química , Formaldeído/química , Homeostase , Microscopia Confocal , Microscopia de Vídeo , Schizosaccharomyces/genética , Fatores de Tempo , Imagem com Lapso de TempoRESUMO
Cytokinesis depends on a contractile actomyosin ring in many eukaryotes [1-3]. Myosin II is a key component of the actomyosin ring, although whether it functions as a motor or as an actin cross-linker to exert its essential role is disputed [1, 4, 5]. In Schizosaccharomyces pombe, the myo2-E1 mutation affects the upper 50 kDa sub-domain of the myosin II heavy chain, and cells carrying this lethal mutation are defective in actomyosin ring assembly at the non-permissive temperature [6, 7]. myo2-E1 also affects actomyosin ring contraction when rings isolated from permissive temperature-grown cells are incubated with ATP [8]. Here we report isolation of a compensatory suppressor mutation in the lower 50 kDa sub-domain (myo2-E1-Sup1) that reverses the inability of myo2-E1 to form colonies at the restrictive temperature. myo2-E1-Sup1 is capable of assembling normal actomyosin rings, although rings isolated from myo2-E1-Sup1 are defective in ATP-dependent contraction in vitro. Furthermore, the product of myo2-E1-Sup1 does not translocate actin filaments in motility assays in vitro. Superimposition of myo2-E1 and myo2-E1-Sup1 on available rigor and blebbistatin-bound myosin II structures suggests that myo2-E1-Sup1 may represent a novel actin translocation-defective allele. Actomyosin ring contraction and viability of myo2-E1-Sup1 cells depend on the late cytokinetic S. pombe myosin II isoform, Myp2p, a non-essential protein that is normally dispensable for actomyosin ring assembly and contraction. Our work reveals that Myo2p may function in two different and essential modes during cytokinesis: a motor activity-independent form that can promote actomyosin ring assembly and a motor activity-dependent form that supports ring contraction.
Assuntos
Miosina Tipo II/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Citoesqueleto de Actina/metabolismo , Actomiosina/fisiologia , CitocineseRESUMO
Many eukaryotes assemble a ring-shaped actomyosin network that contracts to drive cytokinesis. Unlike actomyosin in sarcomeres, which cycles through contraction and relaxation, the cytokinetic ring disassembles during contraction through an unknown mechanism. Here we find in Schizosaccharomyces japonicus and Schizosaccharomyces pombe that, during actomyosin ring contraction, actin filaments associated with actomyosin rings are expelled as micron-scale bundles containing multiple actomyosin ring proteins. Using functional isolated actomyosin rings we show that expulsion of actin bundles does not require continuous presence of cytoplasm. Strikingly, mechanical compression of actomyosin rings results in expulsion of bundles predominantly at regions of high curvature. Our work unprecedentedly reveals that the increased curvature of the ring itself promotes its disassembly. It is likely that such a curvature-induced mechanism may operate in disassembly of other contractile networks.
Assuntos
Citoesqueleto de Actina/genética , Actomiosina/metabolismo , Citocinese/genética , Contração Muscular/genética , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actomiosina/química , Citoplasma/genética , Citoplasma/metabolismo , Contração Muscular/fisiologia , Sarcômeros/genética , Sarcômeros/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/fisiologiaRESUMO
Protein S-nitrosylation, the covalent attachment of a nitroso moiety to thiol groups of specific cysteine residues, is one of the major pathways of nitric oxide signaling. Hundreds of proteins are subject to this transient post-translational modification and for some the functional consequences have been identified. Biochemical assays for the analysis of protein S-nitrosylation have been established and can be used to study if and under what conditions a given protein is S-nitrosylated. In contrast, the equally desirable subcellular localization of specific S-nitrosylated protein isoforms has not been achieved to date. In the current study we attempted to specifically localize S-nitrosylated α- and ß-tubulin isoforms in primary neurons after fixation. The approach was based on in situ replacement of the labile cysteine nitroso modification with a stable tag and the subsequent use of antibodies which recognize the tag in the context of the tubulin polypeptide sequence flanking the cysteine residue of interest. We established a procedure for tagging S-nitrosylated proteins in cultured primary neurons and obtained polyclonal anti-tag antibodies capable of specifically detecting tagged proteins on immunoblots and in fixed cells. However, the antibodies were not specific for tubulin isoforms. We suggest that different tagging strategies or alternative methods such as fluorescence resonance energy transfer techniques might be more successful.