RESUMEN
Septins are GTP-binding proteins that assemble into hetero-oligomers. They can interact with each other end-to-end to form filaments, making them the fourth element of the cytoskeleton. To update the current knowledge on the ever-increasing implications of these fascinating proteins in cellular functions, a hundred expert scientists from across the globe gathered from 12 to 15 October 2021 in Berlin for the first hybrid-format (on site and virtual) EMBO workshop Molecular and Cell Biology of Septins.
Asunto(s)
Proteínas de Unión al GTP , Septinas , Berlin , Citoesqueleto/metabolismo , Proteínas de Unión al GTP/metabolismo , Microtúbulos/metabolismo , Septinas/genética , Septinas/metabolismoRESUMEN
This review extensively reports data from the literature concerning the complex relationships between the stress-induced c-Jun N-terminal kinases (JNKs) and the four main cytoskeleton elements, which are actin filaments, microtubules, intermediate filaments, and septins. To a lesser extent, we also focused on the two membrane-associated cytoskeletons spectrin and ESCRT-III. We gather the mechanisms controlling cytoskeleton-associated JNK activation and the known cytoskeleton-related substrates directly phosphorylated by JNK. We also point out specific locations of the JNK upstream regulators at cytoskeletal components. We finally compile available techniques and tools that could allow a better characterization of the interplay between the different types of cytoskeleton filaments upon JNK-mediated stress and during development. This overview may bring new important information for applied medical research.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animales , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Humanos , Filamentos Intermedios/metabolismo , Microtúbulos/metabolismo , Septinas/metabolismo , Espectrina/metabolismoRESUMEN
Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Acetiltransferasas/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Acetiltransferasas/genética , Animales , Secuencia de Bases , Línea Celular , Humanos , Ratones , Microtúbulos/metabolismo , ARN Interferente PequeñoRESUMEN
The cytoskeleton comprises three polymerizing structures that have been studied for a long time, actin microfilaments, microtubules and intermediate filaments, plus more recently investigated dynamic assemblies like septins or the endocytic-sorting complex required for transport (ESCRT) complex. These filament-forming proteins control several cell functions through crosstalks with each other and with membranes. In this review, we report recent works that address how septins bind to membranes, and influence their shaping, organization, properties and functions, either by binding to them directly or indirectly through other cytoskeleton elements.
RESUMEN
Cell resistance to low doses of paclitaxel (Taxol) involves a modulation of microtubule (MT) dynamics. We applied a proteomic approach based on 2-DE coupled with MS to identify changes in the MT environment of Taxol-resistant breast cancer cells. Having established a proteomic pattern of the microtubular proteins extracted from MDA-MB-231 cells, we verified by Western blotting that in resistant cells, α- and ß-tubulins (more specifically the ßIII and ßIV isotypes) increased. Interestingly, four septins (SEPT2, 8, 9 and 11), which are GTPases involved in cytokinesis and in MT/actin cytoskeleton organization, were overexpressed and enriched in the MT environment of Taxol-resistant cells compared to their sensitive counterpart. Changes in the MT proteome of resistant cells also comprised increased kinesin-1 heavy chain expression and recruitment on MTs while dynein light chain-1 was downregulated. Modulation of motor protein recruitment around MTs might reflect their important role in controlling MT dynamics via the organization of signaling pathways. The identification of proteins previously unknown to be linked to taxane-resistance could also be valuable to identify new biological markers of resistance.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Microtúbulos/metabolismo , Paclitaxel/farmacología , Proteoma/metabolismo , Septinas/metabolismo , Moduladores de Tubulina/farmacología , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Proteómica/métodos , Tubulina (Proteína)/metabolismo , Regulación hacia ArribaRESUMEN
Cell resistance to taxanes involves several complementary mechanisms, among which septin relocalization from actin stress fibers to microtubules plays an early role. By investigating the molecular mechanism underlying this relocalization, we found that acute paclitaxel treatment triggers the release from stress fibers and subsequent proteasome-mediated degradation of binder of Rho GTPases 2 (BORG2)/Cdc42 effector protein 3 (Cdc42EP3) and to a lesser extent of BORG3/Cdc42EP5, two Cdc42 effectors that link septins to actin in interphase cells. BORG2 or BORG3 silencing not only caused septin detachment from stress fibers but also mimicked the effects of paclitaxel by triggering both septin relocalization to microtubules and significant drug resistance. Conversely, BORG2 or BORG3 overexpression retained septins on actin fibers even after paclitaxel treatment, without affecting paclitaxel sensitivity. We found that drug-induced inhibition of Cdc42 resulted in a drop in BORG2 level and in the relocalization of septins to microtubules. Accordingly, although septins relocalized when overexpressing an inactive mutant of Cdc42, the expression of a constitutively active mutant acted locally at actin stress fibers to prevent septin release, even after paclitaxel treatment. These findings reveal the role of Cdc42 upstream of BORG2 and BORG3 in controlling the interplay between septins, actin fibers, and microtubules in basal condition and in response to taxanes.
Asunto(s)
Actinas , Septinas , Actinas/metabolismo , Reguladores de Proteínas de Unión al GTP/metabolismo , Microtúbulos/metabolismo , Septinas/genética , Septinas/metabolismo , Fibras de Estrés/metabolismoRESUMEN
The stress-induced c-Jun N-terminal kinase (JNK) controls microtubule dynamics by enhancing both microtubule growth and rescues. Here, we show that upon cell stress, JNK directly phosphorylates the microtubule rescue factor CLIP-170 in its microtubule-binding domain to increase its rescue-promoting activity. Phosphomimetic versions of CLIP-170 enhance its ability to promote rescue events in vitro and in cells. Furthermore, while phosphomimetic mutations do not alter CLIP-170's capability to form comets at growing microtubule ends, both phosphomimetic mutations and JNK activation increase the occurrence of CLIP-170 remnants on the microtubule lattice at the rear of comets. As the CLIP-170 remnants, which are potential sites of microtubule rescue, display a shorter lifetime when CLIP-170 is phosphorylated, we propose that instead of acting at the time of rescue occurrence, CLIP-170 would rather contribute in preparing the microtubule lattice for future rescues at these predetermined sites.
Asunto(s)
MAP Quinasa Quinasa 4/genética , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Estrés Fisiológico/genética , Animales , Anisomicina/farmacología , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Fibroblastos/ultraestructura , Regulación de la Expresión Génica , Células HeLa , Humanos , MAP Quinasa Quinasa 4/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/efectos de la radiación , Microtúbulos/ultraestructura , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Fosforilación/efectos de la radiación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Cloruro de Sodio/farmacología , Rayos UltravioletaRESUMEN
Cancer cell resistance to taxanes is a complex, multifactorial process, which results from the combination of several molecular and cellular changes. In breast cancer cells adapted to long-term paclitaxel treatment, we previously identified a new adaptive mechanism that contributes to resistance and involves high levels of tubulin tyrosination and long-chain polyglutamylation coupled with high levels of septin expression, especially that of SEPT9_i1. This in turn led to higher CLIP-170 and MCAK recruitment to microtubules to enhance microtubule dynamics and therefore counteract the stabilizing effects of taxanes. Here, we explored to which extent this new mechanism alone could trigger taxane resistance. We show that coupling septins (including SEPT9_i1) overexpression together with long-chain tubulin polyglutamylation induce significant paclitaxel resistance in several naive (taxane-sensitive) cell lines and accordingly stimulate the binding of CLIP-170 and MCAK to microtubules. Strikingly, such resistance was paralleled by a systematic relocalization of septin filaments from actin fibers to microtubules. We further show that this relocalization resulted from the overexpression of septins in a context of enhanced tubulin polyglutamylation and reveal that it could also be promoted by an acute treatment with paclitaxel of sensitve cell displaying a high basal level of SEPT9_i1. These findings point out the functional importance and the complex cellular dynamics of septins in the onset of cell resistance to death caused by microtubule-targeting antimitotic drugs of the taxane family.
Asunto(s)
Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Neoplasias/tratamiento farmacológico , Paclitaxel/farmacología , Septinas/biosíntesis , Tubulina (Proteína)/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Células CHO , Línea Celular Tumoral , Cricetulus , Resistencia a Antineoplásicos , Células HeLa , Humanos , Cinesinas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Septinas/metabolismoRESUMEN
Recently, the role of the peroxisome proliferator-activated receptor alpha (PPARalpha) in the hepatic inflammatory response has been associated to the decrease of acute phase protein transcription, although the molecular mechanisms are still to be elucidated. Here, we were interested in the regulation by Wy-14643 (PPARalpha agonist) of alpha1-acid glycoprotein (AGP), a positive acute phase protein, after stimulation by Dexamethasone (Dex), a major modulator of the inflammatory response. In cultured rat hepatocytes, we demonstrate that PPARalpha inhibits at the transcriptional level the Dex-induced AGP gene expression. PPARalpha exerts this inhibitory effect by antagonizing the CCAAT/enhancer binding protein (C/EBPbeta) transcription factor that is involved in Dex-dependent up-regulation of AGP gene expression. Overexpression of C/EBPbeta alleviates the repressive effect of PPARalpha, thus restoring the Dex-stimulated AGP promoter activity. Furthermore, glutathione-S-transferase GST pull-down and coimmunoprecipitation experiments evidenced, for the first time, a physical interaction between PPARalpha and the C-terminal DNA binding region of C/EBPbeta, thus preventing it from binding to specific sequence elements of the AGP promoter. Altogether, these results provide an additional molecular mechanism of negative regulation of acute phase protein gene expression by sequestration of the C/EBPbeta transcription factor by PPARalpha and reveal the high potency of the latter in controlling inflammation.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica/fisiología , Orosomucoide/genética , PPAR alfa/metabolismo , Animales , Dexametasona/farmacología , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Coactivador 2 del Receptor Nuclear , Orosomucoide/biosíntesis , Proliferadores de Peroxisomas/farmacología , Regiones Promotoras Genéticas , Pirimidinas/farmacología , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
Since the initial discovery of septin family GTPases, the understanding of their molecular organization and cellular roles keeps being refined. Septins have been involved in many physiological processes and the misregulation of specific septin gene expression has been implicated in diverse human pathologies, including neurological disorders and cancer. In this minireview, we focus on the importance of the subunit composition and subcellular localization of septins relevant to tumor initiation, progression, and metastasis. We especially underline the importance of septin polymer composition and of their association with the plasma membrane, actin, or microtubules in cell functions involved in cancer and in resistance to cancer therapies. Through their scaffolding role, their function in membrane compartmentalization or through their protective function against protein degradation, septins also emerge as critical organizers of membrane-associated proteins and of signaling pathways implicated in cancer-associated angiogenesis, apoptosis, polarity, migration, proliferation, and in metastasis. Also, the question as to which of the free monomers, hetero-oligomers, or filaments is the functional form of mammalian septins is raised and the control over their spatial and temporal localization is discussed. The increasing amount of crosstalks identified between septins and cellular signaling mediators reinforces the exciting possibility that septins could be new targets in anti-cancer therapies or in therapeutic strategies to limit drug resistance.
RESUMEN
In the present study, we analyzed the influence of retinoic acids on the expression of alpha-1 acid glycoprotein (AGP). We show that in rat primary hepatocytes, 9-cis retinoic acid and all-trans retinoic acid increase AGP gene expression at the transcriptional level. Transient transfections of rat primary hepatocytes with a reporter construct driven by the rat AGP gene promoter indicated that retinoids regulate AGP gene expression via the -763/-138 region of the AGP promoter. Furthermore, cotransfection experiments with retinoic acid receptor alpha (RARalpha) and retinoid X receptor alpha (RXRalpha) expression vectors in NIH3T3 cells demonstrated that both RXRalpha/RXRalpha homodimer and RXRalpha/RARalpha heterodimer are competent for ligand-induced transactivation of the AGP promoter. Unilateral deletion and site-directed mutagenesis identified two retinoic-acid responsive elements (RARE), RARE-I and RARE-II, which interestingly correspond to a direct repeat of two TGACCT-related hexanucleotides separated by a single bp only (DR1-type response element). Cotransfection assays showed that RXRalpha and RARalpha activate AGP gene transcription through these two elements either as a homodimer (RXRalpha/RXRalpha) or as a heterodimer (RXRalpha/RARalpha). The RXRalpha/RXRalpha homodimer acts most efficiently through the RARE-I response element to promote AGP transactivation, whereas the RXRalpha/RARalpha heterodimer mediates transactivation better via the RARE-II responsive element.
Asunto(s)
Orosomucoide/biosíntesis , Retinoides/metabolismo , Alitretinoína , Animales , Northern Blotting , Células Cultivadas , Dimerización , Eliminación de Gen , Genes Reporteros , Vectores Genéticos , Hepatocitos/metabolismo , Ligandos , Luciferasas/metabolismo , Ratones , Células 3T3 NIH , Plásmidos/metabolismo , Regiones Promotoras Genéticas , Ratas , Ratas Sprague-Dawley , Receptores de Ácido Retinoico/biosíntesis , Elementos de Respuesta , Receptor alfa de Ácido Retinoico , Receptores X Retinoide , Factores de Transcripción/biosíntesis , Transcripción Genética , Activación Transcripcional , Transfección , Tretinoina/farmacologíaRESUMEN
The mechanisms of cancer cell adaptation to the anti-microtubule agents of the taxane family are multifaceted and still poorly understood. Here, in a model of breast cancer cells which display amplified microtubule dynamics to resist Taxol®, we provide evidence that septin filaments containing high levels of SEPT9_i1 bind to microtubules in a way that requires tubulin long chain polyglutamylation. Reciprocally, septin filaments provide a scaffold for elongating and trimming polyglutamylation enzymes to finely tune the glutamate side-chain length on microtubules to an optimal level. We also demonstrate that tubulin retyrosination and/or a high level of tyrosinated tubulin is crucial to allow the interplay between septins and polyglutamylation on microtubules and that together, these modifications result in an enhanced CLIP-170 and MCAK recruitment to microtubules. Finally, the inhibition of tubulin retyrosination, septins, tubulin long chain polyglutamylation or of both CLIP-170 and MCAK allows the restoration of cell sensitivity to taxanes, providing evidence for a new integrated mechanism of resistance.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Paclitaxel/farmacología , Ácido Poliglutámico/metabolismo , Septinas/metabolismo , Tubulina (Proteína)/metabolismo , Línea Celular Tumoral , Resistencia a Antineoplásicos , Humanos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismoRESUMEN
Microtubules are highly dynamic polymers of α/ß tubulin heterodimers that play key roles in cell division and in organizing cell cytoplasm. Although they have been discovered more than two decades ago, tubulin post-translational modifications recently gained a new interest as their role was increasingly highlighted in neuron differentiation and neurodegenerative disorders. Here, we specifically focus on tubulin acetylation from its discovery to recent studies that provide new insights into how it is regulated in health and disease and how it impacts microtubule functions. Even though new mechanisms involving tubulin acetylation are regularly being uncovered, the molecular links between its location inside the microtubule lumen and its regulators and effectors is still poorly understood. This review highlights the emerging roles of tubulin acetylation in multiple cellular functions, ranging from cell motility, cell cycle progression or cell differentiation to intracellular trafficking and signalling. It also points out that tubulin acetylation should no longer be seen as a passive marker of microtubule stability, but as a broad regulator of microtubule functions.
Asunto(s)
Tubulina (Proteína)/metabolismo , Acetilación , Humanos , Enfermedades Neurodegenerativas/metabolismo , Neurogénesis , Procesamiento Proteico-Postraduccional , Tubulina (Proteína)/fisiologíaRESUMEN
Involved in a wide range of cellular processes such as signal transduction, microtubules are highly dynamic polymers that accumulate various post-translational modifications including polyglutamylation, polyglycylation, carboxyterminal cleavage and acetylation, the functions of which just begin to be uncovered. The molecular chaperone Hsp90, which is essential for the folding and activity of numerous client proteins involved in cell proliferation and apoptosis, associates with the microtubule network but the effects of tubulin post-translational modifications on its microtubule binding has not yet been investigated. Herein, we show that both the constitutive (beta) and the inducible (alpha) Hsp90 isoforms bind to microtubules in a way that depends on the level of tubulin acetylation. Tubulin acetylation also stimulates the binding and the signaling function of at least two of its client proteins, the kinase Akt/PKB and the transcription factor p53. This study highlights the role of tubulin acetylation in modulating microtubule-based transport of Hsp90-chaperoned proteins and thus in regulating signaling dynamics in the cytoplasm.