Littérature scientifique sur le sujet « End-tethering »

The end of the life of a transport vesicle requires a complex series of tethering, docking, and fusion events. Tethering complexes play a crucial role in the recognition of membrane entities and bringing them into close opposition, thereby coordinating and controlling cellular trafficking events. Here we provide a comprehensive RNA interference analysis of the CORVET and HOPS tethering complexes in metazoans. Knockdown of CORVET components promoted RAB-7 recruitment to subapical membranes, whereas in HOPS knockdowns, RAB-5 was found also on membrane structures close to the cell center, indicating the RAB conversion might be impaired in the absence of these tethering complexes. Unlike in yeast, metazoans have two VPS33 homologues, which are Sec1/Munc18 (SM)-family proteins involved in the regulation of membrane fusion. We assume that in wild type, each tethering complex contains a specific SM protein but that they may be able to substitute for each other in case of absence of the other. Of importance, knockdown of both SM proteins allowed bypass of the endosome maturation block in sand-1 mutants. We propose a model in which the SM proteins in tethering complexes are required for coordinated flux of material through the endosomal system.

Abstract Background Due to ongoing left ventricular (LV) remodeling and consecutive geometric displacement of both papillary muscles, end-stage heart failure is frequently associated with relevant functional mitral regurgitation (FMR) Type IIIb. Treatment strategies of FMR and their prognostic impact are still controversial. Case summary We present a case of an 80-year-old patient who suffered from recurrent symptoms of congestive heart failure due to dilated cardiomyopathy and concomitant severe FMR. To specifically address severe tethering of both mitral leaflets heart team decision was to perform minimally invasive mitral valve repair (MVR) including a subannular LV remodeling procedure, instead of an interventional edge-to-edge repair (MitraClip® procedure). In addition to mitral valve ring annuloplasty, standardized relocation of both papillary muscles was performed successfully, leading to a complete resolution of mitral leaflet tethering. There were no procedural complications and the patient was discharged with an excellent functional result without residual mitral regurgitation. Furthermore, after 12 and 24 months, he reported an increase of his functional exercise capacity and a remarkable reverse LV remodeling could be demonstrated. Discussion Novel subannular repair techniques, especially the relocation of both papillary muscles, specifically address severe leaflet tethering in FMR and have an obvious potential to improve long-term competence of MVR. Therefore, they could be considered as a viable therapeutic option even in elderly patients presenting with end-stage cardiomyopathy and severe leaflet tenting.

Cytoplasmic dynein is the major microtubule minus-end–directed cellular motor. Most dynein activities require dynactin, but the mechanisms regulating cargo-dependent dynein–dynactin interaction are poorly understood. In this study, we focus on dynein–dynactin recruitment to cargo by the conserved motor adaptor Bicaudal D2 (BICD2). We show that dynein and dynactin depend on each other for BICD2-mediated targeting to cargo and that BICD2 N-terminus (BICD2-N) strongly promotes stable interaction between dynein and dynactin both in vitro and in vivo. Direct visualization of dynein in live cells indicates that by itself the triple BICD2-N–dynein–dynactin complex is unable to interact with either cargo or microtubules. However, tethering of BICD2-N to different membranes promotes their microtubule minus-end–directed motility. We further show that LIS1 is required for dynein-mediated transport induced by membrane tethering of BICD2-N and that LIS1 contributes to dynein accumulation at microtubule plus ends and BICD2-positive cellular structures. Our results demonstrate that dynein recruitment to cargo requires concerted action of multiple dynein cofactors.

Sec2p is the exchange factor that activates Sec4p, the Rab GTPase controlling the final stage of the yeast exocytic pathway. Sec2p is recruited to secretory vesicles by Ypt32-GTP, a Rab controlling exit from the Golgi. Sec15p, a subunit of the octameric exocyst tethering complex and an effector of Sec4p, binds to Sec2p on secretory vesicles, displacing Ypt32p. Sec2p mutants defective in the region 450–508 amino acids bind to Sec15p more tightly. In these mutants, Sec2p accumulates in the cytosol in a complex with the exocyst and is not recruited to vesicles by Ypt32p. Thus the region 450–508 amino acids negatively regulates the association of Sec2p with the exocyst, allowing it to recycle on to new vesicles. The structures of one nearly full-length exocyst subunit and three partial subunits have been determined and, despite very low sequence identity, all form rod-like structures built of helical bundles stacked end to end. These rods may bind to each other along their sides to form the assembled complex. While Sec15p binds Sec4-GTP on the vesicle, other subunits bind Rho GTPases on the plasma membrane, thus tethering vesicles to exocytic sites. Sec4-GTP also binds Sro7p, a yeast homologue of the Drosophila lgl (lethal giant larvae) tumour suppressor. Sro7 also binds to Sec9p, a SNAP25 (25 kDa synaptosome-associated protein)-like t-SNARE [target-membrane-associated SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)], and can form a Sec4p–Sro7p–Sec9p ternary complex. Overexpression of Sec4p, Sro7p or Sec1p (another SNARE regulator) can bypass deletions of three different exocyst subunits. Thus promoting SNARE function can compensate for tethering defects.

When airway smooth muscle is contracted in vitro, the airway lumen continues to narrow with increasing concentrations of agonist until complete airway closure occurs. Although there remains some controversy regarding whether airways can close in vivo, recent work has clearly demonstrated that, if the airway is sufficiently stimulated with contractile agonists, complete closure of even large cartilaginous conducting airways can readily occur with the lung at functional residual capacity (Brown RH and Mitzner W. J Appl Physiol 85: 2012–2017, 1998). This result suggests that the tethering of airways in situ by parenchymal attachments is small at functional residual capacity. However, at lung volumes above functional residual capacity, the outward tethering of airways should increase, because both the parenchymal shear modulus and tethering forces increase in proportion to the transpulmonary pressure. In the present study, we tested whether we could prevent airway closure in vivo by increasing lung volume with positive end-expiratory pressure (PEEP). Airway smooth muscle was stimulated with increasing methacholine doses delivered directly to airway smooth muscle at three levels of PEEP (0, 6, and 10 cmH2O). Our results show that increased lung volume shifted the airway methacholine dose-response curve to the right, but, in many airways in most animals, airway closure still occurred even at the highest levels of PEEP.

Polymeric maleimido–quaternary ammonium (QA) compounds have been shown to function as molecular tape measures when covalently tethered to external cysteine residues of a Shaker K+ channel (Blaustein R.O., P.A. Cole, C. Williams, and C. Miller. 2000. Nat. Struct. Biol. 7:309–311). For sufficiently long compounds, the cysteine–maleimide tethering reaction creates a high concentration, at the channel's pore, of a TEA-like moiety that irreversibly blocks current. This paper investigates a striking feature of the maleimide–cysteine tethering kinetics. Strong blockers—those that induce substantial levels (>80%) of irreversible inhibition of current—react with channel cysteines much more rapidly than weak blockers and, when delivered to channels with four cysteine targets, react with multiexponential kinetics. This behavior is shown to arise from the ability of a strong blocker to concentrate its maleimide end near a channel's cysteine target by exploiting the reversible pore-blocking affinity of its QA headgroup.

L'instabilità del genoma è una delle caratteristiche delle cellule tumorali e può essere causata da difetti nella riparazione del DNA. In particolare, le rotture del doppio filamento del DNA (DSBs) sono lesioni altamente citotossiche che possono formarsi accidentalmente durante la replicazione del DNA o in seguito all'esposizione ad agenti genotossici, e devono essere correttamente riparate al fine di garantire la stabilità genomica. Per far fronte a queste lesioni del DNA, le cellule eucariotiche attivano la risposta al danno del DNA (DDR) e utilizzano due meccanismi principali per la riparazione dei DSBs: l’unione terminale non omologa (NHEJ) e la ricombinazione omologa (HR). La risposta cellulare ai DSBs ha inizio con il reclutamento dei complessi Ku e MRX/MRN alle due estremità rotte di un DSB. Inoltre, il complesso MRX recluta al DSB anche Tel1/ATM, una chinasi coinvolta nel checkpoint da danno al DNA. Tel1, a sua volta, consente di promuovere e stabilizzare l'associazione del complesso MRX sia ai DSBs che ai telomeri in un ciclo a feedback positivo. Ku, MRX/MRN e Tel1/ATM sono anche necessari per mantenere la lunghezza dei telomeri, strutture nucleoproteiche specializzate situate alle estremità dei cromosomi eucariotici. Il DNA telomerico deve inoltre essere distinto dalle estremità dei DSBs intracromosomici attraverso diversi complessi proteici, i quali vengono reclutati ai telomeri al fine di prevenire l'attivazione della DDR. Nel lievito S. cerevisiae, Rif2 e Rap1 costituiscono due delle principali proteine che compongono tali complessi. Sia Rif2 che Rap1 contrastano l'attivazione di Tel1, la degradazione nucleolitica e l’unione terminale non omologa ai telomeri. Rif2 sembra esercitare tutte queste funzioni inibendo l'associazione del complesso MRX al DNA telomerico; tuttavia, restava ancora da determinare come Rap1 controllasse negativamente l'attività di MRX alle estremità del DNA. Nella prima parte del mio dottorato di ricerca, ho contribuito a dimostrare che Rif2 contrasta l'associazione del complesso MRX sia ai DSBs che ai telomeri in modo dipendente da Rap1. Rap1, a sua volta, può inibire le funzioni di MRX in modo sia dipendente sia indipendente da Rif2, e le funzioni di Rap1 alle estremità del DNA sono influenzate dalle modalità con cui questa proteina lega il DNA. In merito al NHEJ, una questione importante è rappresentata dal mantenimento delle estremità di un DSB in stretta prossimità fra loro, necessario per consentire una corretta rilegatura. Questa funzione è chiamata end-tethering e sebbene alcuni dati in E.coli abbiano suggerito un coinvolgimento del complesso Ku in questo meccanismo di controllo, restava ancora da chiarire quale fosse il suo esatto ruolo nell’end-tethering. Nella seconda parte del mio dottorato, ho quindi studiato questa problematica tramite la generazione di una variante mutante della proteina Ku70 in grado di aumentare la persistenza del complesso Ku ai DSBs. La caratterizzazione dell'allele ku70-C85Y ha consentito di dimostrare che il complesso Ku promuove l’end-tethering del DNA e la mutazione C85Y migliora tale funzione aumentando la ritenzione di Ku in stretta prossimità alle estremità di un DSB. Inoltre, la funzione svolta da Ku nel DSB end-tethering è regolata da Tel1/ATM, che antagonizza tale funzione del complesso Ku limitandone la persistenza alle estremità dei DSBs. Poiché la presenza del complesso Ku alle estremità dei DSBs impedisce l'accesso delle nucleasi di resezione, la regolazione dell'associazione di Ku alle estremità rotte del DNA mediata da Tel1 fornisce un importante livello di controllo nella scelta tra il meccanismo di NHEJ e di HR, suggerendo una nuova funzione di Tel1 nella risposta al danno al DNA. Tutti questi risultati hanno contribuito a chiarire i meccanismi molecolari che modulano la riparazione del DNA in risposta ai DSBs, con un focus specifico sulle funzioni e sulla regolazione dei complessi MRX e Ku.
Genome instability is one of the hallmarks of cancer cells and it can be caused by DNA repair defects. Among several types of DNA damage, DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can form accidentally during DNA replication or upon exposure to genotoxic agents. DSBs must be repaired to avoid loss of genetic information and to ensure genomic stability. Eukaryotic cells repair DSBs by activating the DNA damage response (DDR) and by using two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). The cellular response to DSBs is initiated by the recruitment of Ku (Ku70-Ku80) and MRX/N (Mre11-Rad50-Xrs2/Nbs1) complexes at the two DSB broken ends. MRX in turn recruits Tel1/ATM, a kinase involved in the DNA damage checkpoint, a surveillance mechanism that couples DSB repair and cell-cycle progression. Tel1 allows to promote and stabilize MRX association at both DSBs and telomeres in a positive feedback loop. Ku, MRX/MRN, and Tel1/ATM are also required to maintain the length of telomeres, specialized nucleoprotein complexes at the ends of eukaryotic chromosomes. Furthermore, telomeric DNA must be distinguished from intrachromosomal DSBs ends through different protein complexes, which are recruited to telomeres in order to prevent DDR activation. In S. cerevisiae, Rif2 and Rap1 are two of the main proteins that compose these complexes. Both Rif2 and Rap1 counteract Tel1 activation, nucleolytic degradation, and NHEJ at telomeres. Rif2 appears to exert all these functions by inhibiting MRX association with telomeric DNA, however how Rap1 negatively controls MRX activity at DNA ends remained to be determined. In the first part of my PhD, I contributed to show that Rif2 counteracts MRX association at both DSBs and telomeres in a Rap1-dependent manner. Rap1 in turn can inhibit MRX functions in a Rif2-dependent and -independent manner, and Rap1 functions at DNA ends are influenced by its DNA binding mode. An important issue in NHEJ is the maintenance of the DSB ends in close proximity to allow their correct re-ligation. This function is called end-tethering and some data in E.coli suggested an involvement of the Ku complex in this control mechanism. However, a Ku role in end-tethering remained to be determined. In the second part of my PhD, I investigated this issue by generating a Ku70 mutant variant that increases Ku persistence at DSBs. The characterization of the ku70-C85Y allele has allowed to show that the Ku complex promotes DSB end-tethering and the C85Y mutation enhances this bridging function by increasing Ku retention very close to the DSB ends. The function of Ku in DSB end-tethering is also regulated by Tel1/ATM, which antagonizes this Ku function by limiting Ku persistence at the DSB ends. As the presence of Ku at the DSB ends prevents the access of resection nucleases, the Tel1-mediated regulation of Ku association with the DSB ends provides an important layer of control in the choice between NHEJ and HR mechanism, suggesting a new function of Tel1 in the DNA damage response. All these findings contributed to elucidate the molecular mechanisms that modulate DNA repair and maintain genome stability in response to DSBs, with a specific focus on the functions and regulation of MRX and Ku complexes.