RESUMEN
In developing olfactory bulb (OB), mitral cells (MCs) remodel their dendrites to establish the precise olfactory circuit, and these circuits are critical for individuals to sense odors and elicit behaviors for survival. However, how microtubules (MTs) participate in the process of dendritic remodeling remains elusive. Here, we reveal that calmodulin-regulated spectrin-associated proteins (CAMSAPs), a family of proteins that bind to the minus-end of the noncentrosomal MTs, play a crucial part in the development of MC dendrites. We observed that Camsap2 knockout (KO) males are infertile while the reproductive tract is normal. Further study showed that the infertility was due to the severe defects of mating behavior in male mice. Besides, mice with loss-of-function displayed defects in the sense of smell. Furthermore, we found that the deficiency of CAMSAP2 impairs the classical morphology of MCs, and the CAMSAP2-dependent dendritic remodeling process is responsible for this defect. Thus, our findings demonstrate that CAMSAP2 plays a vital role in regulating the development of MCs.
Asunto(s)
Dendritas , Ratones Noqueados , Proteínas Asociadas a Microtúbulos , Bulbo Olfatorio , Olfato , Animales , Femenino , Masculino , Ratones , Dendritas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Morfogénesis/genética , Bulbo Olfatorio/metabolismo , Olfato/fisiologíaRESUMEN
In bovine follicular development, the proliferation of bovine granulosa cells affects follicular selection, atresia, and cystic follicle formation. When cystic follicles appear on the ovaries, granulosa cells stop proliferating, resulting in the reduction of granulosa cells layer. In our previous study, the whole transcriptome sequencing revealed that Bone morphogenetic protein receptor 2 (BMPR2) was differentially expressed between cystic and normal follicular granulosa cells. We speculated that long noncoding RNA may act as competing endogenous RNA targeting microRNAs and then regulating the expression of BMPR2 and the function of granulosa cells, thereby affecting follicular development and cyst formation. In this study, the results elucidated that long noncoding RNA S100PBP (NONBTAT011846.2) directly bound miR-2285bc, which targeted in the BMPR2 3'-UTR. miR-2285bc suppresses granulosa cells proliferation by downregulating BMPR2 expression. Furthermore, long noncoding RNA S100PBP was silenced by small interfering RNA, and long noncoding RNA S100PBP regulated BMPR2 expression by sponging miR-2285bc investigated through cross-verification. When small interfering RNA of long noncoding RNA S100PBP was transfected into granulosa cells, the results revealed similar molecular changes as those transfected with miR-2285bc mimics. Silencing long noncoding RNA S100PBP or overexpressing miR-2285bc altered the expressions of some follicular development-related genes, which could be related to follicular cyst occurrence. In conclusion, our findings support that long noncoding RNA S100PBP regulates the expression of BMPR2 through sponge miR-2285bc, promotes the proliferation of granulosa cells, inhibits their apoptosis, and increases the synthesis and secretion of follicular steroid hormones, thus promoting the development of bovine follicles.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo II , Proliferación Celular , Células de la Granulosa , MicroARNs , ARN Largo no Codificante , Animales , Femenino , Células de la Granulosa/metabolismo , Bovinos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Regulación de la Expresión GénicaRESUMEN
The manchette is a crucial transient structure involved in sperm development, with its composition and regulation still not fully understood. This study focused on investigating the roles of CAMSAP1 and CAMSAP2, microtubule (MT) minus-end binding proteins, in regulating manchette MTs, spermiogenesis, and male fertility. The loss of CAMSAP1, but not CAMSAP2, disrupts the well-orchestrated process of spermiogenesis, leading to abnormal manchette elongation and delayed removal, resulting in deformed sperm nuclei and tails resembling oligoasthenozoospermia symptoms. We investigated the underlying molecular mechanisms by purifying manchette assemblies and comparing them through proteomic analysis, and results showed that the absence of CAMSAP1 disrupted the proper localization of key proteins (CEP170 and KIF2A) at the manchette minus end, compromising its structural integrity and hindering MT depolymerization. These findings highlight the significance of maintaining homeostasis in manchette MT minus-ends for shaping manchette morphology during late spermiogenesis, offering insights into the molecular mechanisms underlying infertility and sperm abnormalities.
Asunto(s)
Proteómica , Semen , Humanos , Masculino , Espermatogénesis/fisiología , Microtúbulos/metabolismo , FertilidadRESUMEN
Soyasaponins are major small molecules that accumulate in soybean (Glycine max) seeds. Among them, type-A soyasaponins, fully acetylated at the terminal sugar of their C22 sugar chain, are responsible for the bitter taste of soybean-derived foods. However, the molecular basis for the acetylation of type-A soyasaponins remains unclear. Here, we identify and characterize GmSSAcT1, encoding a BADH-type soyasaponin acetyltransferase that catalyzes three or four consecutive acetylations on type-A soyasaponins in vitro and in planta. Phylogenetic analysis and biochemical assays suggest that GmSSAcT1 likely evolved from acyltransferases present in leguminous plants involved in isoflavonoid acylation. Loss-of-function mutants of GmSSAcT1 exhibited impaired seed germination, which attribute to the excessive accumulation of null-acetylated type-A soyasaponins. We conclude that GmSSAcT1 not only functions as a detoxification gene for high accumulation of type-A soyasaponins in soybean seeds but is also a promising target for breeding new soybean varieties with lower bitter soyasaponin content.
Asunto(s)
Glycine max , Gusto , Glycine max/genética , Germinación/genética , Acetiltransferasas/genética , Filogenia , Semillas/genética , Fitomejoramiento , AzúcaresRESUMEN
Microtubule networks support many cellular processes and have a highly ordered architecture. However, due to the limited axial resolution of conventional light microscopy, the structural features of these networks cannot be resolved in three-dimensional (3D) space. Here, we use customized ultra-high resolution interferometric single-molecule localization microscopy to characterize the microtubule networks in Caco2 cells. We find that the microtubule minus-ends associated protein CAMSAPs localize at a portion of microtubule intersections. Further investigation shows that depletion of CAMSAP2 and CAMSAP3 leads to the narrowing of the inter-microtubule distance. We find that CAMSAPs recognize microtubule defects, which are often associated with microtubule intersections, and then recruit katanin to remove the damaged microtubules. Therefore, the CAMSAP-katanin complex is a regulating module for the distance between microtubules. Taken together, our results characterize the architecture of the cellular microtubule networks in high resolution and provide molecular insights into how the 3D structure of microtubule networks is controlled.
RESUMEN
Multicolor imaging allows protein colocalizations and organelle interactions to be studied in biological research, which is especially important for single-molecule localization microscopy (SMLM). Here, we propose a multicolor method called excitation-resolved stochastic optical reconstruction microscopy (ExR-STORM). The method, which is based on the excitation spectrum of fluorescent dyes, successfully separated four spectrally very close far-red organic fluorophores utilizing three excitation lasers with cross-talk of less than 3%. Dyes that are only 5 nm apart in the emission spectrum were resolved, resulting in negligible chromatic aberrations. This method was extended to three-dimensional (3D) imaging by combining the astigmatic method, providing a powerful tool for resolving 3D morphologies at the nanoscale.
RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Fu Fang Gang Liu (FFGL) is an effective formula for treating wart proliferation caused by human papillomavirus (HPV) infection and has the potential to treat HPV-related cancers. However, scientific evidence of its anti-tumor activity against cervical cancer, the most common cancer caused by HPV, is lacking. AIM OF THE STUDY: To clarify the anti-tumor effect of an FFGL aqueous extract on human cervical cancer and its possible mechanism of cell cycle arrest in HeLa cells. MATERIALS AND METHODS: The anti-proliferative effect of FFGL on cervical cancer cells was assessed using the cell counting kit-8 assay. The proportion of apoptotic cells, cell cycle distribution, and cell division rate were determined using flow cytometry. Quantitative proteomics was used to identify differentially expressed proteins after FFGL treatment, and bioinformatics analysis was used to identify key nodal proteins affected by FFGL. Immunofluorescence and western blot analyses were used to explore changes in the expression of related proteins in the cell cycle and DNA damage pathways to elucidate the potential mechanism of action of FFGL against HeLa cell proliferation. RESULTS: FFGL inhibited cervical cancer cell proliferation and caused cell cycle arrest. According to quantitative proteomics, CyclinB1 may play an important role in the anti-proliferative effect of FFGL on HeLa cells. Additional experiments showed that FFGL aqueous extract caused ATM-mediated DNA damage, further phosphorylated CHK2, led to the inactivation of Cdc25C, inhibited the activity of the CDK1/CyclinB1 complex, and resulted in cell cycle arrest. CONCLUSIONS: FFGL can inhibit cervical cancer cell proliferation. Furthermore, it can increase CDK1 phosphorylation, block the cell cycle by causing DNA damage, and inhibit HeLa cell proliferation.
Asunto(s)
Infecciones por Papillomavirus , Neoplasias del Cuello Uterino , Femenino , Humanos , Células HeLa , Neoplasias del Cuello Uterino/patología , Proliferación Celular , ADN , ApoptosisRESUMEN
Glutamatergic and GABAergic synaptic transmission controls excitation and inhibition of postsynaptic neurons, whereas activity of ion channels modulates neuronal intrinsic excitability. However, it is unclear how excessive neuronal excitation affects intrinsic inhibition to regain homeostatic stability under physiological or pathophysiological conditions. Here, we report that a seizure-like sustained depolarization can induce short-term inhibition of hippocampal CA3 neurons via a mechanism of membrane shunting. This depolarization-induced shunting inhibition (DShI) mediates a non-synaptic, but neuronal intrinsic, short-term plasticity that is able to suppress action potential generation and postsynaptic responses by activated ionotropic receptors. We demonstrate that the TRESK channel significantly contributes to DShI. Disruption of DShI by genetic knockout of TRESK exacerbates the sensitivity and severity of epileptic seizures of mice, whereas overexpression of TRESK attenuates seizures. In summary, these results uncover a type of homeostatic intrinsic plasticity and its underlying mechanism. TRESK might represent a therapeutic target for antiepileptic drugs.
Asunto(s)
Potenciales de Acción/fisiología , Canales de Potasio/metabolismo , Convulsiones/fisiopatología , Potenciales de Acción/efectos de los fármacos , Animales , Calcio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Canales Iónicos/metabolismo , Ligandos , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/deficiencia , Canales de Potasio/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Convulsiones/genética , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Neurogenesis in the developing neocortex relies on extensive mitosis of radial glial cells (RGCs) in the apical surface. The nuclear migration of epithelial-like RGCs is fundamentally important for proper mitosis, but how the apical processes of RGCs are anchored to ensure the nucleokinetic behavior of RGCs remains unclear. Here we find that Talpid3, related to Joubert syndrome, is localized to the mother centriole of RGCs and is required for their apical mitosis. Genetic silencing of Talpid3 causes abnormal RGC delamination and thereby impairs their interkinetic nuclear migration in both cell-autonomous and non-autonomous manners. Further analyses reveal that Talpid3 associates with Ninein to regulate microtubule organization and maintain the integrity of adherens junctions to anchor RGCs. Moreover, genetic ablation of Talpid3 results in synchronized, ectopic mitosis of neural progenitors and dysregulated neurogenesis. Our study provides an intriguing perspective for the non-ciliogenic role of centriolar proteins in mediating cortical neurogenesis.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centrosoma/metabolismo , Neurogénesis/inmunología , Uniones Adherentes/metabolismo , Animales , Humanos , RatonesRESUMEN
The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.
Asunto(s)
Polaridad Celular/fisiología , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/fisiología , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Inmunoprecipitación , Ratones , Proteínas Asociadas a Microtúbulos/genética , Neuronas , Paclitaxel , Unión ProteicaRESUMEN
Rationale: Mitochondrial disorders preferentially affect tissues with high energy requirements, such as the retina and corneal endothelium, in human eyes. Mesenchymal stem cell (MSC)-based treatment has been demonstrated to be beneficial for ocular degeneration. However, aside from neuroprotective paracrine actions, the mechanisms underlying the beneficial effect of MSCs on retinal and corneal tissues are largely unknown. In this study, we investigated the fate and associated characteristics of mitochondria subjected to intercellular transfer from MSCs to ocular cells. Methods: MSCs were cocultured with corneal endothelial cells (CECs), 661W cells (a photoreceptor cell line) and ARPE-19 cells (a retinal pigment epithelium cell line). Immunofluorescence, fluorescence activated cell sorting and confocal microscopy imaging were employed to investigate the traits of intercellular mitochondrial transfer and the fate of transferred mitochondria. The oxygen consumption rate of recipient cells was measured to investigate the effect of intercellular mitochondrial transfer. Transcriptome analysis was performed to investigate the expression of metabolic genes in recipient cells with donated mitochondria. Results: Mitochondrial transport is a ubiquitous intercellular mechanism between MSCs and various ocular cells, including the corneal endothelium, retinal pigmented epithelium, and photoreceptors. Additionally, our results indicate that the donation process depends on F-actin-based tunneling nanotubes. Rotenone-pretreated cells that received mitochondria from MSCs displayed increased aerobic capacity and upregulation of mitochondrial genes. Furthermore, living imaging determined the ultimate fate of transferred mitochondria through either degradation by lysosomes or exocytosis as extracellular vesicles. Conclusions: For the first time, we determined the characteristics and fate of mitochondria undergoing intercellular transfer from MSCs to various ocular cells through F-actin-based tunneling nanotubes, helping to characterize MSC-based treatment for ocular tissue regeneration.
Asunto(s)
Comunicación Celular , Metabolismo Energético , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , Actinas/metabolismo , Animales , Línea Celular , Movimiento Celular , Técnicas de Cocultivo , Córnea/citología , Córnea/metabolismo , Córnea/patología , ADN Mitocondrial/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Distrofia Endotelial de Fuchs/genética , Distrofia Endotelial de Fuchs/patología , Distrofia Endotelial de Fuchs/terapia , Humanos , Inyecciones Intraoculares , Células Madre Mesenquimatosas/citología , Ratones , Mitocondrias/genética , Modelos Animales , Atrofia Óptica Autosómica Dominante/genética , Atrofia Óptica Autosómica Dominante/patología , Atrofia Óptica Autosómica Dominante/terapia , Atrofia Óptica Hereditaria de Leber/genética , Atrofia Óptica Hereditaria de Leber/patología , Atrofia Óptica Hereditaria de Leber/terapia , Células Fotorreceptoras de Vertebrados/citología , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patologíaRESUMEN
In the present work, a tubular nano-copper sulfide was successfully synthesized by hydrothermal method. The physical and chemical properties of the prepared materials were characterized by XRD, SEM, TEM, and BET. The synthesized copper sulfide was used as an adsorbent for removing 17α-ethynyl estradiol (EE2) and exhibited excellent adsorption properties. At 25 °C, 15 mg of adsorbent was applied for 50 mL of 5 mg/L EE2 solution, adsorption equilibrium was reached after 180 min, and the adsorption rate reached nearly 90%. In addition, the kinetics, isothermal adsorption, and thermodynamics of the adsorption process were discussed on the basis of theoretical calculations and experimental results. The theoretical maximum adsorption capacity of copper sulfide was calculated to be 147.06 mg/g. The results of this study indicated that copper sulfide was a stable and efficient adsorbent with promising practical applications.
RESUMEN
WDR62 is the second most common genetic alteration associated with microcephaly. It has been shown that Wdr62 is required for germ cell meiosis initiation in mice, and the majority of male germ cells are lost in the meiotic defect of first wave spermatogenesis in Wdr62 mutants. Strikingly, in this study, we found that the initiation of meiosis following spermatogenesis was not affected and the germ cells were gradually repopulated at later developmental stages. However, most germ cells were arrested at metaphase of meiosis I and no mature sperm were detected in epididymides. Further, this study demonstrated that metaphase I arrest of Wdr62-deficient spermatocytes was caused by asymmetric distribution of the centrosome and aberrant spindle assembly. Also, mechanistic studies demonstrated that WDR62 interacts with centrosome-associated protein CEP170, and deletion of Wdr62 causes downregulation of the CEP170 protein, which in turn leads to the aberrant spindle assembly. In summary, this study indicates that the meiosis of first wave spermatogenesis and the following spermatogenesis started from spermatogonium is probably regulated by different mechanisms. We also demonstrated a new function of WDR62 in germ cell meiosis, through its interaction with CEP170.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Huso Acromático/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Centrosoma/metabolismo , Masculino , Meiosis/genética , Meiosis/fisiología , Metafase/genética , Metafase/fisiología , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas del Tejido Nervioso/genética , Unión Proteica , Espermatocitos/citología , Espermatocitos/metabolismo , Espermatogénesis/genética , Espermatogénesis/fisiologíaRESUMEN
The molecular mechanisms that guide each neuron to become polarized, forming a single axon and multiple dendrites, remain unknown. Here we show that CAMSAP3 (calmodulin-regulated spectrin-associated protein 3), a protein that regulates the minus-end dynamics of microtubules, plays a key role in maintaining neuronal polarity. In mouse hippocampal neurons, CAMSAP3 was enriched in axons. Although axonal microtubules were generally acetylated, CAMSAP3 was preferentially localized along a less-acetylated fraction of the microtubules. CAMSAP3-mutated neurons often exhibited supernumerary axons, along with an increased number of neurites having nocodazole-resistant/acetylated microtubules compared with wild-type neurons. Analysis using cell lines showed that CAMSAP3 depletion promoted tubulin acetylation, and conversely, mild overexpression of CAMSAP3 inhibited it, suggesting that CAMSAP3 works to retain nonacetylated microtubules. In contrast, CAMSAP2, a protein related to CAMSAP3, was detected along all neurites, and its loss did not affect neuronal polarity, nor did it cause increased tubulin acetylation. Depletion of α-tubulin acetyltransferase-1 (αTAT1), the key enzyme for tubulin acetylation, abolished CAMSAP3 loss-dependent multiple-axon formation. These observations suggest that CAMSAP3 sustains a nonacetylated pool of microtubules in axons, interfering with the action of αTAT1, and this process is important to maintain neuronal polarity.
Asunto(s)
Polaridad Celular , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/metabolismo , Neuronas/metabolismo , Acetilación , Animales , Hipocampo/citología , Ratones , Ratones Noqueados , Tubulina (Proteína)/metabolismoRESUMEN
Proper differentiation of trophoblast cells in the human placenta is a prerequisite for a successful pregnancy, and dysregulation of this process may lead to malignant pregnancy outcomes, such as preeclampsia. Finding specific markers for different types of trophoblast cells is essential for understanding trophoblast differentiation. Here, we report that placenta-specific protein 8 (PLAC8) is specifically expressed in the interstitial extravillous trophoblast cells (iEVTs) on the fetomaternal interface. Using model systems, including placental villi-decidua co-culture, iEVTs induction by using primary trophoblast cells or explants, etc., we found that PLAC8 promotes invasion and migration of iEVTs. Mechanistically, time-lapse imaging, GTPase activity assay, co-immunoprecipitation and RNA-seq studies show that PLAC8 increases the Cdc42 and Rac1 activities, and further induces the formation of filopodia at the leading edge of the migratory trophoblast cells. More interestingly, PLAC8 is significantly upregulated under hypoxia and expression of PLAC8 is higher in iEVTs from preeclamptic placentas when compared with those from the normal control placentas. Together, PLAC8 is a new marker for iEVTs and plays an important role in promoting trophoblast invasion and migration.
Asunto(s)
Placenta/citología , Placenta/fisiología , Proteínas/fisiología , Trofoblastos/fisiología , Biomarcadores/metabolismo , Estudios de Casos y Controles , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Movimiento Celular/genética , Movimiento Celular/fisiología , Vellosidades Coriónicas/anatomía & histología , Técnicas de Cocultivo , Decidua/citología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de Unión al GTP Monoméricas/metabolismo , Placenta/irrigación sanguínea , Preeclampsia/genética , Preeclampsia/patología , Preeclampsia/fisiopatología , Embarazo , Proteínas/antagonistas & inhibidores , Proteínas/genética , ARN Interferente Pequeño/genética , Regulación hacia Arriba , Proteína de Unión al GTP cdc42/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The behavior of visceral endoderm cells was examined as the anterior visceral endoderm (AVE) formed from the distal visceral endoderm (DVE) using the mouse lines R26-H2B-EGFP and R26-PHA7-EGFP to visualize cell nuclei and adherens junction, respectively. The analysis using R26-H2B-EGFP demonstrated global cell rearrangement that was not specific to the DVE cells in the monolayer embryonic visceral endoderm sheet; each population of the endoderm cells moved collectively in a swirling movement as a whole. Most of the AVE cells at E6.5 were not E5.5 DVE cells but were E5.5 cells that were located caudally behind them, as previously reported (Hoshino et al., 2015; Takaoka et al., 2011). In the rearrangement, the posterior embryonic visceral endoderm cells did not move, as extraembryonic visceral endoderm cells did not, and they constituted a distinct population during the process of anterior-posterior axis formation. The analysis using R26-PHA7-EGFP suggested that constriction of the apical surfaces of the cells in prospective anterior portion of the DVE initiated the global cellular movement of the embryonic visceral endoderm to drive AVE formation.
Asunto(s)
Tipificación del Cuerpo , Embrión de Mamíferos/citología , Endodermo/citología , Vísceras/embriología , Animales , Ciclo Celular , Núcleo Celular/metabolismo , Rastreo Celular , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Imagen de Lapso de TiempoRESUMEN
Microtubules (MTs) play essential roles in many steps of autophagy, an important degradation pathway in the maintenance of cellular homoeostasis. In many cells, MT networks are comprised of centrosomal MTs and noncentrosomal MTs. However, it is unknown whether noncentrosomal MTs and its binding proteins are involved in autophagy. Here, we show in HeLa cells that calmodulin-regulated spectrin-associated protein 2 (CAMSAP2), a noncentrosomal MT minus-end stabilizing protein, regulates retrograde transport of autophagosomes through MT dynamics. CAMSAP2 cooperates with EB1 to regulate end-binding protein 1 (EB1) behaviour at MT plus ends, MT growth directions and autophagosome transport. An association between CAMSAP2 and EB1 in the cytosol may modulate EB1 binding to MT plus ends. Collectively, our data indicate that noncentrosomal MTs regulate autophagy through a cross-talk between CAMSAP2 and EB1.
Asunto(s)
Autofagosomas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Autofagia , Transporte Biológico , Citosol/metabolismo , Células HeLa , Humanos , Unión ProteicaRESUMEN
The epithelium has an apico-basal axis polarity that plays an important role in absorption, excretion and other physiological functions. In epithelial cells, a substantial number of non-centrosomal microtubules (MTs) are scattered in the cytoplasm with an apico-basal polarity and reorientate as epithelial cells perform different functions. Several previous studies have found that non-centrosomal MTs are nucleated at the centrosome, and then released and translocated elsewhere. However, the detailed process and molecular mechanism remain largely unknown. In this study, we found that Nezha, also called calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), a non-centrosomal MT minus-end protein, accumulates in the pericentrosomal area and accompanies the release of MTs from the centrosome; whereas depletion of CAMSAP3 prevented MT release and instead caused focusing of MTs at centrosomes. Further studies demonstrated that CAMSAP3 precisely coordinates with dynein and katanin to regulate the MT detachment process. In conclusion, our results indicate that CAMSAP3 is a key molecule for generation of non-centrosomal MTs.
Asunto(s)
Centrosoma/metabolismo , Katanina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Animales , Línea Celular Tumoral , Dineínas/metabolismo , Células Epiteliales/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The Golgi assembly pattern varies among cell types. In fibroblast cells, the Golgi apparatus concentrates around the centrosome that radiates microtubules; whereas in epithelial cells, whose microtubules are mainly noncentrosomal, the Golgi apparatus accumulates around the nucleus independently of centrosome. Little is known about the mechanisms behind such cell type-specific Golgi and microtubule organization. Here, we show that the microtubule minus-end binding protein Nezha/CAMSAP3 (calmodulin-regulated spectrin-associated protein 3) plays a role in translocation of Golgi vesicles in epithelial cells. This function of CAMSAP3 is supported by CG-NAP (centrosome and Golgi localized PKN-associated protein) through their binding. Depletion of either one of these proteins similarly induces fragmentation of Golgi membranes. Furthermore, we find that stathmin-dependent microtubule dynamics is graded along the radial axis of cells with highest activity at the perinuclear region, and inhibition of this gradient disrupts perinuclear distribution of the Golgi apparatus. We propose that the assembly of the Golgi apparatus in epithelial cells is induced by a multi-step process, which includes CAMSAP3-dependent Golgi vesicle clustering and graded microtubule dynamics.