RESUMEN
The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and membrane fusion. The AAA+ ATPase motor cytoplasmic dynein regulates ciliary trafficking, mitotic spindle formation and organelle transport, and dissecting its precise functions has been challenging because of its rapid timescale of action and the lack of cell-permeable, chemical modulators. Here we describe the discovery of ciliobrevins, the first specific small-molecule antagonists of cytoplasmic dynein. Ciliobrevins perturb protein trafficking within the primary cilium, leading to their malformation and Hedgehog signalling blockade. Ciliobrevins also prevent spindle pole focusing, kinetochore-microtubule attachment, melanosome aggregation and peroxisome motility in cultured cells. We further demonstrate the ability of ciliobrevins to block dynein-dependent microtubule gliding and ATPase activity in vitro. Ciliobrevins therefore will be useful reagents for studying cellular processes that require this microtubule motor and may guide the development of additional AAA+ ATPase superfamily inhibitors.
Asunto(s)
Citoplasma/enzimología , Dineínas Citoplasmáticas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Quinazolinonas/química , Quinazolinonas/farmacología , Animales , Bovinos , Cilios/efectos de los fármacos , Cilios/metabolismo , Cilios/patología , Dineínas Citoplasmáticas/metabolismo , Proteínas Hedgehog/metabolismo , Cinetocoros/efectos de los fármacos , Cinetocoros/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Melanosomas/efectos de los fármacos , Melanosomas/metabolismo , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Peso Molecular , Movimiento/efectos de los fármacos , Células 3T3 NIH , Peroxisomas/efectos de los fármacos , Peroxisomas/fisiología , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Huso Acromático/patologíaRESUMEN
In several migratory cells, the microtubule-organizing center (MTOC) is repositioned between the leading edge and nucleus, creating a polarized morphology. Although our understanding of polarization has progressed as a result of various scratch-wound and cell migration studies, variations in culture conditions required for such assays have prevented a unified understanding of the intricacies of MTOC and nucleus positioning that result in cell polarization. Here, we employ a new SMRT (for sparse, monolayer, round, triangular) analysis that uses a universal coordinate system based on cell centroid to examine the pathways regulating MTOC and nuclear positions in cells plated in a variety of conditions. We find that MTOC and nucleus positioning are crucially and independently affected by cell shape and confluence; MTOC off-centering correlates with the polarization of single cells; acto-myosin contractility and microtubule dynamics are required for single-cell polarization; and end binding protein 1 and light intermediate chain 1, but not Par3 and light intermediate chain 2, are required for single-cell polarization and directional cell motility. Using various cellular geometries and conditions, we implement a systematic and reproducible approach to identify regulators of MTOC and nucleus positioning that depend on extracellular guidance cues.
Asunto(s)
Núcleo Celular/fisiología , Polaridad Celular , Dineínas Citoplasmáticas/fisiología , Proteínas Asociadas a Microtúbulos/fisiología , Centro Organizador de los Microtúbulos/fisiología , Actinas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Moléculas de Adhesión Celular/fisiología , Proteínas de Ciclo Celular , Movimiento Celular , Núcleo Celular/ultraestructura , Forma de la Célula , Células Cultivadas , Dineínas Citoplasmáticas/antagonistas & inhibidores , Laminas/fisiología , Ratones , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Centro Organizador de los Microtúbulos/ultraestructura , Microtúbulos/fisiología , Miosina Tipo II/fisiologíaRESUMEN
Cytoplasmic dynein-1 (hereafter dynein) is a six-subunit motor complex that transports a variety of cellular components and pathogens along microtubules. Dynein's cellular functions are only partially understood, and potent and specific small-molecule inhibitors and activators of this motor would be valuable for addressing this issue. It has also been hypothesized that an inhibitor of dynein-based transport could be used in antiviral or antimitotic therapy, whereas an activator could alleviate age-related neurodegenerative diseases by enhancing microtubule-based transport in axons. Here, we present the first high-throughput screening (HTS) assay capable of identifying both activators and inhibitors of dynein-based transport. This project is also the first collaborative screening report from the Medical Research Council and AstraZeneca agreement to form the UK Centre for Lead Discovery. A cellular imaging assay was used, involving chemically controlled recruitment of activated dynein complexes to peroxisomes. Such a system has the potential to identify molecules that affect multiple aspects of dynein biology in vivo. Following optimization of key parameters, the assay was developed in a 384-well format with semiautomated liquid handling and image acquisition. Testing of more than 500,000 compounds identified both inhibitors and activators of dynein-based transport in multiple chemical series. Additional analysis indicated that many of the identified compounds do not affect the integrity of the microtubule cytoskeleton and are therefore candidates to directly target the transport machinery.
Asunto(s)
Dineínas Citoplasmáticas/antagonistas & inhibidores , Ensayos Analíticos de Alto Rendimiento/métodos , Peroxisomas/genética , Bibliotecas de Moléculas Pequeñas/farmacología , Transporte Biológico/efectos de los fármacos , Dineínas Citoplasmáticas/química , Dineínas Citoplasmáticas/genética , Humanos , Transporte Iónico/genética , Microtúbulos/efectos de los fármacosRESUMEN
Aberrant hedgehog (Hh) signaling contributes to the pathogenesis of multiple cancers. Available inhibitors target Smoothened (Smo), which can acquire mutations causing drug resistance. Thus, compounds that inhibit Hh signaling downstream of Smo are urgently needed. We identified dynarrestin, a novel inhibitor of cytoplasmic dyneins 1 and 2. Dynarrestin acts reversibly to inhibit cytoplasmic dynein 1-dependent microtubule binding and motility in vitro without affecting ATP hydrolysis. It rapidly and reversibly inhibits endosome movement in living cells and perturbs mitosis by inducing spindle misorientation and pseudoprometaphase delay. Dynarrestin reversibly inhibits cytoplasmic dynein 2-dependent intraflagellar transport (IFT) of the cargo IFT88 and flux of Smo within cilia without interfering with ciliogenesis and suppresses Hh-dependent proliferation of neuronal precursors and tumor cells. As such, dynarrestin is a valuable tool for probing cytoplasmic dynein-dependent cellular processes and a promising compound for medicinal chemistry programs aimed at development of anti-cancer drugs.
Asunto(s)
Dineínas Citoplasmáticas/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cilios/efectos de los fármacos , Cilios/metabolismo , Dineínas Citoplasmáticas/metabolismo , Proteínas Hedgehog/antagonistas & inhibidores , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Mitosis/efectos de los fármacos , Células 3T3 NIH , Transporte de Proteínas/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.
Asunto(s)
Dineínas Citoplasmáticas/antagonistas & inhibidores , Dineínas Citoplasmáticas/química , Animales , Proteínas Hedgehog/fisiología , Ratones , Estructura Molecular , Células 3T3 NIH , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Quinazolinonas/química , Quinazolinonas/farmacología , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Retroviral capsid (CA) cores undergo uncoating during their retrograde transport (toward the nucleus), and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC) using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable) CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating.
Asunto(s)
Dineínas Citoplasmáticas/metabolismo , VIH-1/fisiología , Interacciones Huésped-Patógeno , Desencapsidación Viral , Transporte Biológico , Cápside/metabolismo , Dineínas Citoplasmáticas/antagonistas & inhibidores , Técnicas de Silenciamiento del Gen , HumanosRESUMEN
Nitric oxide (NO) deficiency contributes to hypertension. We previously showed that neuronal nitric oxide synthase (nNOS) was involved in hypertension and kidney damage in spontaneously hypertensive rats (SHRs). The protein inhibitor of nNOS (PIN) has been reported to inhibit activity of nNOS.Thus, we tested whether increased PIN in the kidney results in hypertension and whether small interfering RNA (siRNA) targeting PIN attenuates hypertension in SHRs. Four-week-old male SHRs were assigned into three groups (n = 6-7/group): SHR; SHR + PIN, SHR that received siRNA targeting PIN; and SHR + NC, SHR treated with random negative control siRNA. Rats were sacrificed at 12 weeks of age. PIN protein expression was inhibited considerably when PIN siRNA was transfected into NRK52E cells (90% siRNA at 1 nM). The increases of BP were attenuated by siRNA targeting PIN in 12-week-old SHRs. Immunostaining of nNOS-α and total nNOS was greater in SHR + PIN group than SHR. Moreover, renal superoxide production and 8-hydroxydeoxyguanosine (8-OHdG) staining were more decreased in the SHR + PIN group than SHRs. We conclude that PIN siRNA reduced PIN expression in vitro and in vivo. PIN siRNA therapy attenuates hypertension in SHRs at 12 weeks of age. Our results suggest that PIN is involved in the development of hypertension.
Asunto(s)
Dineínas Citoplasmáticas/antagonistas & inhibidores , Terapia Genética/métodos , Hipertensión/terapia , Riñón/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Animales , Western Blotting , Técnicas de Cultivo de Célula , Línea Celular , Dineínas Citoplasmáticas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Hipertensión/genética , Hipertensión/metabolismo , Inmunohistoquímica , Masculino , Estrés Oxidativo , Ratas Endogámicas SHR , Ratas Wistar , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
We have previously described a novel modulator of the actin cytoskeleton that also regulates Ras and mitogen-activated protein kinase activities in TGFß-sensitive epithelial cells. Here we examined the functional role of this signaling regulatory protein (km23-1) in mediating the migration, invasion, and tumor growth of human colorectal carcinoma (CRC) cells. We show that small interfering RNA (siRNA) depletion of km23-1 in human CRC cells inhibited constitutive extracellular signal-regulated kinase (ERK) activation, as well as pro-invasive ERK effector functions that include phosphorylation of Elk-1, constitutive regulation of c-Fos-DNA binding, TGFß1 promoter transactivation, and TGFß1 secretion. In addition, knockdown of km23-1 reduced the paracrine effects of CRC cell-secreted factors in conditioned medium and in fibroblast co-cultures. Moreover, km23-1 depletion in human CRC cells reduced cell migration and invasion, as well as expression of the ERK-regulated, metastasis-associated scaffold protein Ezrin. Finally, km23-1 inhibition significantly suppressed tumor formation in vivo. Thus, our results implicate km23-1 as a novel anti-metastasis target for human colon carcinoma cells, capable of decreasing tumor growth and invasion via a mechanism involving suppression of various pro-migratory features of CRC. These include a reduction in ERK signaling, diminished TGFß1 production, decreased expression of the plasma membrane-cytoskeletal linker Ezrin, as well as attenuation of the paracrine effects of colon carcinoma-secreted factors on fibroblast migration and mitogenesis. As such, km23-1 inhibitors may represent a viable therapeutic strategy for interfering with colon cancer progression and invasion.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Dineínas Citoplasmáticas/genética , Regulación Neoplásica de la Expresión Génica , ARN Interferente Pequeño/genética , Animales , Línea Celular Tumoral , Movimiento Celular , Colágeno/química , Neoplasias Colorrectales/metabolismo , Dineínas Citoplasmáticas/antagonistas & inhibidores , Dineínas Citoplasmáticas/metabolismo , Proteínas del Citoesqueleto/antagonistas & inhibidores , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Cámaras de Difusión de Cultivos , Combinación de Medicamentos , Quinasas MAP Reguladas por Señal Extracelular/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Vectores Genéticos , Humanos , Laminina/química , Lentivirus/genética , Ratones , Ratones Desnudos , Invasividad Neoplásica , Trasplante de Neoplasias , Proteoglicanos/química , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Carga Tumoral/genética , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
Rapid ligand-induced trafficking of glucocorticoid nuclear hormone receptor (GR) from the cytoplasm to the nucleus is an extensively studied model for intracellular retrograde cargo transport employed in constructive morphogenesis and many other cellular functions. Unfortunately, potent and selective small-molecule disruptors of this process are lacking, which has restricted pharmacological investigations. We describe here the development and validation of a 384-well high-content screening (HCS) assay to identify inhibitors of the rapid ligand-induced retrograde translocation of cytoplasmic glucocorticoid nuclear hormone receptor green fluorescent fusion protein (GR-GFP) into the nuclei of 3617.4 mouse mammary adenocarcinoma cells. We selected 3617.4 cells, because they express GR-GFP under the control of a tetracycline (Tet)-repressible promoter and are exceptionally amenable to image acquisition and analysis procedures. Initially, we investigated the time-dependent expression of GR-GFP in 3617.4 cells under Tet-on and Tet-off control to determine the optimal conditions to measure dexamethasone (Dex)-induced GR-GFP nuclear translocation on the ArrayScan-VTI automated imaging platform. We then miniaturized the assay into a 384-well format and validated the performance of the GR-GFP nuclear translocation HCS assay in our 3-day assay signal window and dimethylsulfoxide validation tests. The molecular chaperone heat shock protein 90 (Hsp90) plays an essential role in the regulation of GR steroid binding affinity and ligand-induced retrograde trafficking to the nucleus. We verified that the GR-GFP HCS assay captured the concentration-dependent inhibition of GR-GFP nuclear translocation by 17-AAG, a benzoquinone ansamycin that selectively blocks the binding and hydrolysis of ATP by Hsp90. We screened the 1280 compound library of pharmacologically active compounds set in the Dex-induced GR-GFP nuclear translocation assay and used the multi-parameter HCS data to eliminate cytotoxic compounds and fluorescent outliers. We identified five qualified hits that inhibited the rapid retrograde trafficking of GR-GFP in a concentration-dependent manner: Bay 11-7085, 4-phenyl-3-furoxancarbonitrile, parthenolide, apomorphine, and 6-nitroso-1,2-benzopyrone. The data presented here demonstrate that the GR-GFP HCS assay provides an effective phenotypic screen and support the proposition that screening a larger library of diversity compounds will yield novel small-molecule probes that will enable the further exploration of intracellular retrograde transport of cargo along microtubules, a process which is essential to the morphogenesis and function of all cells.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Dineínas Citoplasmáticas/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Receptores de Glucocorticoides/metabolismo , Animales , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Dineínas Citoplasmáticas/fisiología , Dexametasona/química , Dexametasona/metabolismo , Dexametasona/farmacología , Ratones , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiologíaRESUMEN
BACKGROUND: Photoreceptors of the retina are highly compartmentalized cells that function as the primary sensory neurons for receiving and initiating transmission of visual information. Proper morphogenesis of photoreceptor neurons is essential for their normal function and survival. We have characterized a zebrafish mutation, cannonball, that completely disrupts photoreceptor morphogenesis. RESULTS: Analysis revealed a non-sense mutation in cytoplasmic dynein heavy chain 1 (dync1h1), a critical subunit in Dynein1, to underlie the cannonball phenotypes. Dynein1 is a large minus-end directed, microtubule motor protein complex that has been implicated in multiple, essential cellular processes. In photoreceptors, Dynein1 is thought to mediate post-Golgi vesicle trafficking, while Dynein2 is thought to be responsible for outer segment maintenance. Surprisingly, cannonball embryos survive until larval stages, owing to wild-type maternal protein stores. Retinal photoreceptor neurons, however, are significantly affected by loss of Dync1h1, as transmission electron microscopy and marker analyses demonstrated defects in organelle positioning and outer segment morphogenesis and suggested defects in post-Golgi vesicle trafficking. Furthermore, dosage-dependent antisense oligonucleotide knock-down of dync1h1 revealed outer segment abnormalities in the absence of overt inner segment polarity and trafficking defects. Consistent with a specific function of Dync1h1 within the outer segment, immunolocalization showed that this protein and other subunits of Dynein1 and Dynactin localized to the ciliary axoneme of the outer segment, in addition to their predicted inner segment localization. However, knock-down of Dynactin subunits suggested that this protein complex, which is known to augment many Dynein1 activities, is only essential for inner segment processes as outer segment morphogenesis was normal. CONCLUSIONS: Our results indicate that Dynein1 is required for multiple cellular processes in photoreceptor neurons, including organelle positioning, proper outer segment morphogenesis, and potentially post-Golgi vesicle trafficking. Titrated knock-down of dync1h1 indicated that outer segment morphogenesis was affected in photoreceptors that showed normal inner segments. These observations, combined with protein localization studies, suggest that Dynein1 may have direct and essential functions in photoreceptor outer segments, in addition to inner segment functions.