RESUMO
Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.
Assuntos
Actinas , Septinas , MicrotúbulosRESUMO
Long-range membrane traffic is guided by microtubule-associated proteins and posttranslational modifications, which collectively comprise a traffic code. The regulatory principles of this code and how it orchestrates the motility of kinesin and dynein motors are largely unknown. Septins are a large family of GTP-binding proteins, which assemble into complexes that associate with microtubules. Using single-molecule in vitro motility assays, we tested how the microtubule-associated SEPT2/6/7, SEPT2/6/7/9, and SEPT5/7/11 complexes affect the motilities of the constitutively active kinesins KIF5C and KIF1A and the dynein-dynactin-bicaudal D (DDB) motor complex. We found that microtubule-associated SEPT2/6/7 is a potent inhibitor of DDB and KIF5C, preventing mainly their association with microtubules. SEPT2/6/7 also inhibits KIF1A by obstructing stepping along microtubules. On SEPT2/6/7/9-coated microtubules, KIF1A inhibition is dampened by SEPT9, which alone enhances KIF1A, showing that individual septin subunits determine the regulatory properties of septin complexes. Strikingly, SEPT5/7/11 differs from SEPT2/6/7, in permitting the motility of KIF1A and immobilizing DDB to the microtubule lattice. In hippocampal neurons, filamentous SEPT5 colocalizes with somatodendritic microtubules that underlie Golgi membranes and lack SEPT6. Depletion of SEPT5 disrupts Golgi morphology and polarization of Golgi ribbons into the shaft of somato-proximal dendrites, which is consistent with the tethering of DDB to microtubules by SEPT5/7/11. Collectively, these results suggest that microtubule-associated complexes have differential specificities in the regulation of the motility and positioning of microtubule motors. We posit that septins are an integral part of the microtubule-based code that spatially controls membrane traffic.
Assuntos
Dineínas , Cinesinas , Proteínas Associadas aos Microtúbulos , Septinas , Dineínas/metabolismo , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Septinas/metabolismo , Células COS , Células HEK293 , Humanos , Animais , Chlorocebus aethiops , Transporte ProteicoRESUMO
Septins are a family of multimeric GTP-binding proteins, which are abnormally expressed in cancer. Septin 9 (SEPT9) is an essential and ubiquitously expressed septin with multiple isoforms, which have differential expression patterns and effects in breast cancer cells. It is unknown, however, if SEPT9 isoforms associate with different molecular networks and functions. Here, we performed a proteomic screen in MCF-7 breast cancer cells to identify the interactome of GFP-SEPT9 isoforms 1, 4 and 5, which vary significantly in their N-terminal extensions. While all three isoforms associated with SEPT2 and SEPT7, the truncated SEPT9_i4 and SEPT9_i5 interacted with septins of the SEPT6 group more promiscuously than SEPT9_i1, which bound predominately SEPT8. Spatial mapping and functional clustering of non-septin partners showed isoform-specific differences in interactions with proteins of distinct subcellular organelles (e.g., nuclei, centrosomes, cilia) and functions such as cell signalling and ubiquitination. The interactome of the full length SEPT9_i1 was more enriched in cytoskeletal regulators, while the truncated SEPT9_i4 and SEPT9_i5 exhibited preferential and isoform-specific interactions with nuclear, signalling, and ubiquitinating proteins. These data provide evidence for isoform-specific interactions, which arise from truncations in the N-terminal extensions of SEPT9, and point to novel roles in the pathogenesis of breast cancer.
Assuntos
Neoplasias da Mama , Septinas , Feminino , Perfilação da Expressão Gênica , Humanos , Células MCF-7 , Isoformas de Proteínas/genética , Proteômica , Septinas/genética , Septinas/metabolismoRESUMO
There are limited data about the management of patients presenting for elective generator replacements in the setting of previously implanted cardiac resynchronization therapy (CRT) devices that are nearing end-of-life. The individual patient's clinical status and concomitant morbidities may evolve so that considerations may include not only replacement of the pulse generator, but also potentially changing the type of device [e.g. downgrading CRT-defibrillator (CRT-D) to CRT-pacemaker (CRT-P) or ICD or upgrading of CRT-P to CRT-D]. Moreover, the clinical evidence for CRT-D/CRT-P implantation may change over time, with ongoing research and availability of new trial data. In this review we discuss the ethical, clinical and financial implications related to CRT generator replacements and the need for additional clinical trials to better understand which patients should undergo CRT device downgrading or upgrading at the time of battery depletion.
Assuntos
Dispositivos de Terapia de Ressincronização Cardíaca/estatística & dados numéricos , Terapia de Ressincronização Cardíaca/estatística & dados numéricos , Tomada de Decisão Clínica/métodos , Remoção de Dispositivo/estatística & dados numéricos , Análise de Falha de Equipamento/métodos , Insuficiência Cardíaca/prevenção & controle , Idoso , Análise de Falha de Equipamento/estatística & dados numéricos , Medicina Baseada em Evidências , Feminino , Humanos , Masculino , Resultado do TratamentoRESUMO
OBJECTIVE: Inflammatory attacks of familial Mediterranean fever (FMF) are characterised by circulation and influx of high number of polymorphonuclear neutrophils (PMN) in the affected sites and profound therapeutic effect of IL-1ß inhibitors. We investigated the role of neutrophil extracellular traps (NET) in the pathogenesis of FMF, and their involvement in IL-1ß production. METHODS: Blood samples were obtained from six FMF patients during remissions and from three patients during attacks. NET formation and NET components were studied by fluorescence techniques, immunobloting and MPO-DNA complex ELISA. RESULTS: PMNs from patients released NETs decorated with IL-1ß during disease attacks. On the other hand, PMNs from patients during remission were resistant to inflammatory stimuli that induce NET release in PMNs from control subjects. Lower basal autophagy levels were identified in PMNs during remission, while induction of autophagy facilitated NET release, suggesting that autophagy is involved in the regulation of NET release. During the resolution of attacks, inhibition of NET formation by negative feedback mechanism was also observed. The anti-inflammatory agents, colchicine and DNAse I, inhibited IL-1ß production in PMNs and IL-1ß activity in NETs, respectively. CONCLUSIONS: We suggest two additive events for triggering the FMF attack; the production of IL-1ß by PMNs and its release through NETs. At the same time NETs, homeostatically, downregulate further NETosis, facilitating the resolution of attack. Compensatorly, lower basal autophagy of PMNs may protect from crises by attenuating the release of pro-inflammatory NETs.
Assuntos
Armadilhas Extracelulares/imunologia , Febre Familiar do Mediterrâneo/imunologia , Interleucina-1beta/imunologia , Neutrófilos/imunologia , Adulto , Anti-Inflamatórios/farmacologia , Autofagia/imunologia , Estudos de Casos e Controles , Colchicina/farmacologia , Desoxirribonuclease I/farmacologia , Armadilhas Extracelulares/efeitos dos fármacos , Retroalimentação Fisiológica , Feminino , Humanos , Interleucina-1beta/biossíntese , Masculino , Pessoa de Meia-Idade , Neutrófilos/efeitos dos fármacos , Indução de Remissão , Adulto JovemRESUMO
At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are together required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.
Assuntos
Anáfase , Cinesinas , Microtúbulos , Fuso Acromático , Humanos , Fuso Acromático/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Dineínas/metabolismo , Dineínas/genética , Torque , Segregação de Cromossomos , Citoesqueleto de Actina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genéticaRESUMO
At each cell division, nanometer-scale motors and microtubules give rise to the micron-scale spindle. Many mitotic motors step helically around microtubules in vitro, and most are predicted to twist the spindle in a left-handed direction. However, the human spindle exhibits only slight global twist, raising the question of how these molecular torques are balanced. Here, using lattice light sheet microscopy, we find that anaphase spindles in the epithelial cell line MCF10A have a high baseline twist, and we identify factors that both increase and decrease this twist. The midzone motors KIF4A and MKLP1 are redundantly required for left-handed twist at anaphase, and we show that KIF4A generates left-handed torque in vitro. The actin cytoskeleton also contributes to left-handed twist, but dynein and its cortical recruitment factor LGN counteract it. Together, our work demonstrates that force generators regulate twist in opposite directions from both within and outside the spindle, preventing strong spindle twist during chromosome segregation.
RESUMO
Endothelial cell migration is critical for vascular angiogenesis and is compromised to facilitate tumor metastasis. The migratory process requires the coordinated assembly and disassembly of focal adhesions (FA), actin, and microtubules (MT). MT dynamics at FAs deliver vesicular cargoes and enhance actomyosin contractility to promote FA turnover and facilitate cell advance. Noncentrosomal (NC) MTs regulate FA dynamics and are sufficient to drive cell polarity, but how NC MTs target FAs to control FA turnover is not understood. Here, we show that Rac1 induces the assembly of FA-proximal septin filaments that promote NC MT growth into FAs and inhibit mitotic centromere-associated kinesin (MCAK)-associated MT disassembly, thereby maintaining intact MT plus ends proximal to FAs. Septin-associated MT rescue is coupled with accumulation of Aurora-A kinase and cytoplasmic linker-associated protein (CLASP) localization to the MT between septin and FAs. In this way, NC MTs are strategically positioned to undergo MCAK- and CLASP-regulated bouts of assembly and disassembly into FAs, thereby regulating FA turnover and cell migration.
Assuntos
Adesões Focais , Septinas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Movimento Celular/fisiologia , Adesões Focais/metabolismo , Microtúbulos/metabolismo , Septinas/metabolismoRESUMO
Septins are an integral component of the cytoskeleton, assembling into higher-order oligomers and filamentous polymers that associate with actin filaments, microtubules and membranes. Here, we review septin interactions with actin and microtubules, and septin-mediated regulation of the organization and dynamics of these cytoskeletal networks, which is critical for cellular morphogenesis. We discuss how actomyosin-associated septins function in cytokinesis, cell migration and host defense against pathogens. We highlight newly emerged roles of septins at the interface of microtubules and membranes with molecular motors, which point to a 'septin code' for the regulation of membrane traffic. Additionally, we revisit the functions of microtubule-associated septins in mitosis and meiosis. In sum, septins comprise a unique module of cytoskeletal regulators that are spatially and functionally specialized and have properties of bona fide actin-binding and microtubule-associated proteins. With many questions still outstanding, the study of septins will continue to provide new insights into fundamental problems of cytoskeletal organization and function.
Assuntos
Actinas , Microtúbulos , Septinas , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Humanos , Microtúbulos/metabolismo , Septinas/metabolismoRESUMO
Septins are GTP-binding proteins that associate with the microtubule (MT) and actin cytoskeleton. Septins affect MT organization and posttranslational modifications, but their role in MT dynamics is less understood. Here, we reconstituted MT dynamics in the presence of the MT-binding septin (SEPT9) using an in vitro cell-free assay, which images the polymerization of tubulin from guanosine-5'-[(α,ß)-methyleno]triphosphate (GMPCPP)-stabilized MT seeds. We found that submicromolar concentrations of SEPT9 suppress MT catastrophe and enhance the growth of MT plus ends to great lengths, while low micromolar concentrations of SEPT9 stabilize MTs by inhibiting dynamic instability. We show that SEPT9 associates preferentially with the lattice of GMPCPP-stabilized MT seeds and surprisingly recruits soluble tubulin to the MT lattice. Notably, the effects of SEPT9 on MT dynamics are dependent on its G-G dimerization interface, which is formed by the pockets of the GTP-binding domains. A mutation (H530D) that disrupts G-G dimerization abrogates the effects of SEPT9 on MT dynamics and diminishes its ability to recruit tubulin to the MT lattice. Taken together, these results suggest that SEPT9 promotes the formation and maintenance of long stable MTs through a mechanism that may involve recruitment of unpolymerized tubulin to the MT lattice.
Assuntos
Microtúbulos/metabolismo , Septinas/metabolismo , Microscopia , Tubulina (Proteína)/metabolismoRESUMO
Septins (SEPTs) are filamentous guanosine-5'-triphosphate (GTP)-binding proteins, which affect microtubule (MT)-dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus-end growth and elongation, while higher and intermediate concentrations inhibit and pause plus-end growth, respectively. We show that SEPT2/6/7 has a modest preference for GTP- over guanosine diphosphate (GDP)-bound MT lattice and competes with end-binding protein 1 (EB1) for binding to guanosine 5'-O-[γ-thio]triphosphate (GTPγS)-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus-end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus-end growth and the tracking of plus end-binding proteins and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Septinas/metabolismo , Animais , Humanos , Modelos Biológicos , Ligação Proteica , Ratos Sprague-DawleyRESUMO
Abscission is the terminal step of mitosis that physically separates two daughter cells [1, 2]. Abscission requires the endocytic sorting complex required for transport (ESCRT), a molecular machinery of multiple subcomplexes (ESCRT-I/II/III) that promotes membrane remodeling and scission [3-5]. Recruitment of ESCRT-I/II complexes to the midbody of telophase cells initiates ESCRT-III assembly into two rings, which subsequently expand into helices and spirals that narrow down to the incipient site of abscission [6-8]. ESCRT-III assembly is highly dynamic and spatiotemporally ordered, but the underlying mechanisms are poorly understood. Here, we report that, after cleavage furrow closure, septins form a membrane-bound double ring that controls the organization and function of ESCRT-III. The septin double ring demarcates the sites of ESCRT-III assembly into rings and disassembles before ESCRT-III rings expand into helices and spirals. We show that septin 9 (SEPT9) depletion, which abrogates abscission, impairs recruitment of VPS25 (ESCRT-II) and CHMP6 (ESCRT-III). Strikingly, ESCRT-III subunits (CHMP4B and CHMP2A/B) accumulate to the midbody, but they are highly disorganized, failing to form symmetric rings and to expand laterally into the cone-shaped helices and spirals of abscission. We found that SEPT9 interacts directly with the ubiquitin E2 variant (UEV) domain of ESCRT-I protein TSG101 through two N-terminal PTAP motifs, which are required for the recruitment of VPS25 and CHMP6, and the spatial organization of ESCRT-III (CHMP4B and CHMP2B) into functional rings. These results reveal that septins function in the ESCRT-I-ESCRT-II-CHMP6 pathway of ESCRT-III assembly and provide a framework for the spatiotemporal control of the ESCRT machinery of cytokinetic abscission.
Assuntos
Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Mitose , Septinas/metabolismo , Animais , Citoesqueleto/metabolismo , Cães , Células Madin Darby de Rim CaninoRESUMO
BACKGROUND: Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly. RESULTS: We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. CONCLUSIONS: Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/metabolismo , Fuso Acromático/metabolismo , Proteínas de Xenopus/fisiologia , Adenosina Trifosfatases/análise , Adenosina Trifosfatases/metabolismo , Animais , Células Cultivadas , DNA Helicases/análise , DNA Helicases/metabolismo , DNA Helicases/fisiologia , Drosophila/genética , Drosophila/metabolismo , Drosophila/ultraestrutura , Células HeLa , Humanos , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/análise , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Fuso Acromático/ultraestrutura , Xenopus , Proteínas de Xenopus/análise , Proteínas de Xenopus/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Proteína ran de Ligação ao GTP/fisiologiaRESUMO
Mitotic regulators exhibiting gain of function in tumor cells are considered useful cancer therapeutic targets for the development of small-molecule inhibitors. The human Aurora kinases are a family of such targets. In this study, from a panel of 105 potential small-molecule inhibitors, two compounds Tripolin A and Tripolin B, inhibited Aurora A kinase activity in vitro. In human cells however, only Tripolin A acted as an Aurora A inhibitor. We combined in vitro, in vivo single cell and in silico studies to demonstrate the biological action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the localization of pAurora A on spindle microtubules (MTs), affected centrosome integrity, spindle formation and length, as well as MT dynamics in interphase, consistent with Aurora A inhibition by RNAi or other specific inhibitors, such as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind Aurora A similarly but not identical to MLN8054, therefore it could be used to dissect pathways orchestrated by Aurora kinases as well as a scaffold for further inhibitor development.