RESUMO
MAIN CONCLUSION: The GhEB1C gene of the EB1 protein family functions as microtubule end-binding protein and may be involved in the regulation of microtubule-related pathways to enhance resistance to Verticillium wilt. The expression of GhEB1C is induced by SA, also contributing to Verticillium wilt resistance. Cotton, as a crucial cash and oil crop, faces a significant threat from Verticillium wilt, a soil-borne disease induced by Verticillium dahliae, severely impacting cotton growth and development. Investigating genes associated with resistance to Verticillium wilt is paramount. We identified and performed a phylogenetic analysis on members of the EB1 family associated with Verticillium wilt in this work. GhEB1C was discovered by transcriptome screening and was studied for its function in cotton defense against V. dahliae. The RT-qPCR analysis revealed significant expression of the GhEB1C gene in cotton leaves. Subsequent localization analysis using transient expression demonstrated cytoplasmic localization of GhEB1C. VIGS experiments indicated that silencing of the GhEB1C gene significantly increased susceptibility of cotton to V. dahliae. Comparative RNA-seq analysis showed that GhEB1C silenced plants exhibited altered microtubule-associated protein pathways and flavonogen-associated pathways, suggesting a role for GhEB1C in defense mechanisms. Overexpression of tobacco resulted in enhanced resistance to V. dahliae as compared to wild-type plants. Furthermore, our investigation into the relationship between the GhEB1C gene and plant disease resistance hormones salicylic axid (SA) and jasmonic acid (JA) revealed the involvement of GhEB1C in the regulation of the SA pathway. In conclusion, our findings demonstrate that GhEB1C plays a crucial role in conferring immunity to cotton against Verticillium wilt, providing valuable insights for further research on plant adaptability to pathogen invasion.
Assuntos
Resistência à Doença , Gossypium , Filogenia , Doenças das Plantas , Proteínas de Plantas , Gossypium/genética , Gossypium/microbiologia , Gossypium/imunologia , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Ascomicetos/fisiologia , Ascomicetos/patogenicidade , Ácido Salicílico/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Folhas de Planta/microbiologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Oxilipinas/metabolismo , Verticillium/fisiologia , Ciclopentanos/metabolismoRESUMO
Microtubule-based vesicle trafficking usually relies upon kinesin and dynein motors and few reports describe microtubule polymerisation driving directional vesicle trafficking. Here we show that Arabidopsis END BINDING1b (EB1b), a microtubule plus-end binding protein, directly interacts with SYP121, a SNARE protein that mediates the trafficking of the K+ channel KAT1 and its distribution to the plasma membrane (PM) in Arabidopsis guard cells. Knockout of AtEB1b and its homologous proteins results in a modest but significant change in the distribution of KAT1 and SYP121 in guard cells and consequently delays light-induced stomatal opening. Live-cell imaging reveals that a portion of SYP121-associated endomembrane compartments co-localise with AtEB1b at the growing ends of microtubules, trafficking along with the growth of microtubules for targeting to the PM. Our study reveals a mechanism of vesicle trafficking driven by microtubule growth, which is involved in the redistribution of PM proteins to modulate guard cell movement.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Membrana Celular , Proteínas Associadas aos Microtúbulos , Microtúbulos , Estômatos de Plantas , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Microtúbulos/metabolismo , Estômatos de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Membrana Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Transporte Proteico , Katanina/metabolismo , Katanina/genética , Movimento Celular , Proteínas de Ciclo CelularRESUMO
Family with sequence similarity 161 (Fam161) is an ancient family of microtubule-binding proteins located at the centriole and cilium transition zone (TZ) lumen that exhibit rapid evolution in mice. However, their adaptive role is unclear. Here, we used flies to gain insight into their cell type-specific adaptations. Fam161 is the sole orthologue of FAM161A and FAM161B found in flies. Mutating Fam161 results in reduced male reproduction and abnormal geotaxis behaviour. Fam161 localizes to sensory neuron centrioles and their specialized TZ (the connecting cilium) in a cell type-specific manner, sometimes labelling only the centrioles, sometimes labelling the centrioles and cilium TZ and sometimes labelling the TZ with varying lengths that are longer than other TZ proteins, defining a new ciliary compartment, the extra distal TZ. These findings suggest that Fam161 is an essential centriole and TZ protein with a unique cell type-specific localization in fruit flies that can produce cell type-specific adaptations.
Assuntos
Centríolos , Cílios , Proteínas de Drosophila , Animais , Centríolos/metabolismo , Cílios/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Masculino , Drosophila melanogaster/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Especificidade de ÓrgãosRESUMO
Subcortical heterotopia is a cortical malformation associated with epilepsy, intellectual disability, and an excessive number of cortical neurons in the white matter. Echinoderm microtubule-associated protein like 1 (EML1) mutations lead to subcortical heterotopia, associated with abnormal radial glia positioning in the cortical wall, prior to malformation onset. This perturbed distribution of proliferative cells is likely to be a critical event for heterotopia formation; however, the underlying mechanisms remain unexplained. This study aimed to decipher the early cellular alterations leading to abnormal radial glia. In a forebrain conditional Eml1 mutant model and human patient cells, primary cilia and centrosomes are altered. Microtubule dynamics and cell cycle kinetics are also abnormal in mouse mutant radial glia. By rescuing microtubule formation in Eml1 mutant embryonic brains, abnormal radial glia delamination and heterotopia volume were significantly reduced. Thus, our new model of subcortical heterotopia reveals the causal link between Eml1's function in microtubule regulation and cell position, both critical for correct cortical development.
Assuntos
Centrossomo , Proteínas Associadas aos Microtúbulos , Microtúbulos , Prosencéfalo , Animais , Centrossomo/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Prosencéfalo/embriologia , Microtúbulos/metabolismo , Camundongos , Cílios/metabolismo , Cílios/patologia , Mutação/genética , Células Ependimogliais/metabolismo , Células Ependimogliais/patologia , Ciclo Celular/genéticaRESUMO
Faithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. Here, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of apolar microbeads establishes their biorientation-like state in mouse cells. NDC80-NUF2 microbeads align at the spindle equator and self-correct alignment errors. The alignment is associated with stable bipolar microtubule attachment and is independent of the outer kinetochore proteins SPC24-SPC25, KNL1, the Mis12 complex, inner kinetochore proteins, and Aurora. Larger microbeads align more rapidly, suggesting a size-dependent biorientation mechanism. This study demonstrates a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.
Assuntos
Proteínas de Ciclo Celular , Segregação de Cromossomos , Cinetocoros , Microesferas , Proteínas Associadas aos Microtúbulos , Microtúbulos , Fuso Acromático , Animais , Camundongos , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fuso Acromático/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is an adult-onset motor neuron disease with a mean survival time of three years. The 97% of the cases have TDP-43 nuclear depletion and cytoplasmic aggregation in motor neurons. TDP-43 prevents non-conserved cryptic exon splicing in certain genes, maintaining transcript stability, including ATG4B, which is crucial for autophagosome maturation and Microtubule-associated proteins 1A/1B light chain 3B (LC3B) homeostasis. In ALS mice (G93A), Atg4b depletion worsens survival rates and autophagy function. For the first time, we observed an elevation of LC3ylation in the CNS of both ALS patients and atg4b-/- mouse spinal cords. Furthermore, LC3ylation modulates the distribution of ATG3 across membrane compartments. Antisense oligonucleotides (ASOs) targeting cryptic exon restore ATG4B mRNA in TARDBP knockdown cells. We further developed multi-target ASOs targeting TDP-43 binding sequences for a broader effect. Importantly, our ASO based in peptide-PMO conjugates show brain distribution post-IV administration, offering a non-invasive ASO-based treatment avenue for neurodegenerative diseases.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas Relacionadas à Autofagia , Cisteína Endopeptidases , Proteínas de Ligação a DNA , Proteínas Associadas aos Microtúbulos , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Masculino , Medula Espinal/metabolismo , Medula Espinal/patologia , Autofagia/fisiologia , Camundongos Knockout , Splicing de RNA/genética , Feminino , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Oligonucleotídeos Antissenso/farmacologiaRESUMO
Enteroviruses are a genus of small RNA viruses that are responsible for approximately one billion global infections annually. These infections range in severity from the common cold and flu-like symptoms to more severe diseases, such as viral myocarditis, pancreatitis, and neurological disorders, that continue to pose a global health challenge with limited therapeutic strategies currently available. In the current study, we sought to understand the interaction between coxsackievirus B3 (CVB3), which is a model enterovirus, and macrophage cells, as there is limited understanding of how this virus interacts with macrophage innate immune cells. Our study demonstrated that CVB3 can robustly activate macrophages without apparent viral replication in these cells. We also showed that myeloid cells lacked the viral entry receptor coxsackievirus and adenovirus receptor (CAR). However, the expression of exogenous CAR in RAW264.7 macrophages was unable to overcome the viral replication deficit. Interestingly, the CAR expression was associated with altered inflammatory responses during prolonged infection. Additionally, we identified the autophagy protein LC3 as a novel stimulus for macrophage activation. These findings provide new insights into the mechanisms of CVB3-induced macrophage activation and its implications for viral pathogenesis.
Assuntos
Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Enterovirus Humano B , Ativação de Macrófagos , Macrófagos , Internalização do Vírus , Animais , Humanos , Camundongos , Autofagia , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/metabolismo , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Infecções por Coxsackievirus/virologia , Infecções por Coxsackievirus/imunologia , Enterovirus Humano B/fisiologia , Macrófagos/virologia , Macrófagos/imunologia , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Células RAW 264.7 , Replicação ViralRESUMO
Autophagy of glycogen (glycophagy) is crucial for the maintenance of cellular glucose homeostasis and physiology in mammals. STBD1 can serve as an autophagy receptor to mediate glycophagy by specifically recognizing glycogen and relevant key autophagic factors, but with poorly understood mechanisms. Here, we systematically characterize the interactions of STBD1 with glycogen and related saccharides, and determine the crystal structure of the STBD1 CBM20 domain with maltotetraose, uncovering a unique binding mode involving two different oligosaccharide-binding sites adopted by STBD1 CBM20 for recognizing glycogen. In addition, we demonstrate that the LC3-interacting region (LIR) motif of STBD1 can selectively bind to six mammalian ATG8 family members. We elucidate the detailed molecular mechanism underlying the selective interactions of STBD1 with ATG8 family proteins by solving the STBD1 LIR/GABARAPL1 complex structure. Importantly, our cell-based assays reveal that both the STBD1 LIR/GABARAPL1 interaction and the intact two oligosaccharide binding sites of STBD1 CBM20 are essential for the effective association of STBD1, GABARAPL1, and glycogen in cells. Finally, through mass spectrometry, biochemical, and structural modeling analyses, we unveil that STBD1 can directly bind to the Claw domain of RB1CC1 through its LIR, thereby recruiting the key autophagy initiation factor RB1CC1. In all, our findings provide mechanistic insights into the recognitions of glycogen, ATG8 family proteins, and RB1CC1 by STBD1 and shed light on the potential working mechanism of STBD1-mediated glycophagy.
Assuntos
Família da Proteína 8 Relacionada à Autofagia , Autofagia , Glicogênio , Animais , Humanos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/química , Sítios de Ligação , Cristalografia por Raios X , Glicogênio/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Ligação ProteicaRESUMO
Microtubule (MT) consists of α-tubulin and ß-tubulin. The dynamic instability regulated by various microtubule associated proteins (MAPs) is essential for MT functions. To analyze the interaction between tubulin/MT and MAP in vivo, we usually need tubulin and MAP co-expressed. Here, we constructed a dual-transgene vector expressing rice (Oryza sativa) α-tubulin and MAP simultaneously. To construct this vector, plant expression vector pCambia1301 was used as the plasmid backbone and Gibson assembly cloning technology was used. We first fused and cloned the GFP fragment, α-tubulin open reading frame (ORF), and NOS terminator into the vector pCambia1301 to construct the p35S::GFP-α-tubulin vector that expressed GFP-α-tubulin fusion protein. Subsequently, we fused and cloned the CaMV 35S promoter, mCherry fragment, and NOS terminator into the p35S::GFP-α-tubulin vector to generate the universal dual-transgene expression vector (p35S::GFP-α-tubulin-p35S::mCherry vector). With the p35S::GFP-α-tubulin-p35S::mCherry vector, MAP ORF can be cloned into the site of 5' or 3' terminus of mCherry to co-express GFP-α-tubulin and MAP-mCherry/mCherry-MAP. To validate the availability and universality of the dual-transgene expression vector, a series of putative rice MAP genes including GL7, OsKCBP, OsCLASP, and OsMOR1 were cloned into the vector respectively, transformed into Agrobacterium tumefaciens strain, and expressed in Nicotiana benthamiana leaves. The results indicated that all of the MAPs were co-expressed with α-tubulin and localized to MTs, validating the availability and universality of the vector and that GL7, OsKCBP, OsCLASP, and OsMOR1 might be MAPs. The application of the co-expression vector constructed by us would facilitate studies on the interaction between tubulin/MT and MAP in tobacco transient expression systems or transgenic rice.
Assuntos
Vetores Genéticos , Proteínas Associadas aos Microtúbulos , Oryza , Tubulina (Proteína) , Oryza/genética , Oryza/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Vetores Genéticos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Microtúbulos/metabolismo , Microtúbulos/genéticaRESUMO
Lysosomes, essential for intracellular degradation and recycling, employ damage-control strategies such as lysophagy and membrane repair mechanisms to maintain functionality and cellular homeostasis. Our study unveils migratory autolysosome disposal (MAD), a response to lysosomal damage where cells expel LAMP1-LC3 positive structures via autolysosome exocytosis, requiring autophagy machinery, SNARE proteins, and cell migration. This mechanism, crucial for mitigating lysosomal damage, underscores the role of cell migration in lysosome damage control and facilitates the release of small extracellular vesicles, highlighting the intricate relationship between cell migration, organelle quality control, and extracellular vesicle release.
Assuntos
Autofagia , Movimento Celular , Lisossomos , Lisossomos/metabolismo , Humanos , Exocitose , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Proteínas de Membrana Lisossomal/metabolismo , Proteínas de Membrana Lisossomal/genética , Animais , Vesículas Extracelulares/metabolismo , Células HeLa , Proteína 1 de Membrana Associada ao LisossomoRESUMO
White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. Genome-wide association studies identified TRIM47 at the 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found highly expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we predicted a highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing Trim47 significantly increased autophagy with ULK1 phosphorylation induction, transcription, and vacuole formation. Together, we demonstrate that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation.
Assuntos
Autofagia , Encéfalo , Células Endoteliais , Animais , Camundongos , Humanos , Células Endoteliais/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Substância Branca/metabolismo , Substância Branca/patologia , Fatores de Risco , Masculino , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Camundongos Endogâmicos C57BL , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Bronchial asthma is a chronic condition characterized by airway inflammation and remodeling, which pose complex pathophysiological challenges. Autophagy has been identified as a practical strategy to regulate inflammation and remodeling processes in chronic inflammatory diseases with pathological characteristics, such as asthma. PF (Paeoniflorin) is a potential new autophagy regulatory compound. Previous studies have reported that PF can inhibit airway inflammation to alleviate allergic asthma, but whether this is mediated through the regulation of autophagy and the molecular mechanism of action remains unclear. PURPOSE: The aim of this study was to evaluate the inhibitory effect of natural small molecule PF on asthma by regulating epithelial autophagy. METHODS: The rat asthma model was established through intraperitoneal injection of OVA and aluminum hydroxide suspension, followed by atomized inhalation of OVA for a period of two weeks. Following treatment with PF, histopathology was observed using Masson and H&E staining, while airway Max Rrs was evaluated using a pulmonary function apparatus. Levels of inflammatory cells in BALF were detected using a blood cell analyzer, and levels of inflammatory factors in BALF were detected through Elisa. Expressions of p-PRAS40 and p-Raptor were observed through immunohistochemistry, and levels of Beclin1 and LC3B were observed through immunofluorescence. The structure and quantity of autophagosomes and autophagolysosomal were observed through TEM. An autophagy model of 16HBE cells was established after treatment with 10ng/mL IL13 for 30 minutes. PRAS40 (AKT1S1) overexpression and mutation of PF and Raptor binding site (K207M& L302I& Q417H) were introduced in 16HBE cells. Autophagy in cells was measured by mFRP-GFP-LC3 ADV fluorescent tracer. The binding sites of PF and Raptor were analyzed using the Autodock Tool. The p-mTOR, p-Raptor, p-PRAS40, LC3II/LC3I were detected through Western blot, and interaction between PRAS40-Raptor and Raptor-mTOR was detected through Co-IP. RESULTS: The results showed that PF effectively reduced airway inflammation, improved airway pathological changes and remodeling, and maintained lung function. Additionally, PF was found to reverse excessive autophagy in airway epithelial cells. Interestingly, PF activated the mTORC1 subunit PRAS40 and Raptor in airway epithelial cells by regulating their phosphorylation. PRAS40 is an endogenous mTOR inhibitor that promotes autophagy. PF competitively binds Raptor to PRAS40, promoting Raptor-mTOR interactions to activate mTORC1, an outcome that can be reversed by PRAS40 overexpression and site-specific amino acid codon mutations in Raptor. CONCLUSION: These findings suggest that PF intervention and inhibition of PRAS40-Raptor interaction are effective treatments for bronchial asthma. By activating mTORC1, PF effectively reverses excessive autophagy in airway epithelial cells, leading to improved airway function and reduced inflammation.
Assuntos
Asma , Autofagia , Células Epiteliais , Glucosídeos , Alvo Mecanístico do Complexo 1 de Rapamicina , Monoterpenos , Animais , Humanos , Masculino , Ratos , Asma/tratamento farmacológico , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Glucosídeos/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Monoterpenos/farmacologia , Ovalbumina , Ratos Sprague-Dawley , Proteína Regulatória Associada a mTOR/metabolismoRESUMO
Microtubules are dynamic cytoskeletal polymers essential for cell division, motility, and intracellular transport. Microtubule dynamics are characterized by dynamic instability-the ability of individual microtubules to switch between phases of growth and shrinkage. Dynamic instability can be explained by the GTP-cap model, suggesting that a "cap" of GTP-tubulin subunits at the growing microtubule end has a stabilizing effect, protecting against microtubule catastrophe-the switch from growth to shrinkage. Although the GTP-cap is thought to protect the growing microtubule end, whether the GTP-cap size affects microtubule stability in cells is not known. Notably, microtubule end-binding proteins, EBs, recognize the nucleotide state of tubulin and display comet-like localization at growing microtubule ends, which can be used as a proxy for the GTP-cap. Here, we employ high spatiotemporal resolution imaging to compare the relationship between EB comet size and microtubule dynamics in interphase LLC-PK1 cells to that measured in vitro. Our data reveal that the GTP-cap size in cells scales with the microtubule growth rate in the same way as in vitro. However, we find that microtubule ends in cells can withstand transition to catastrophe even after the EB comet is lost. Thus, our findings suggest that the presence of the GTP-cap is not the determinant of microtubule end stability in cells.
Assuntos
Guanosina Trifosfato , Proteínas Associadas aos Microtúbulos , Microtúbulos , Tubulina (Proteína) , Microtúbulos/metabolismo , Guanosina Trifosfato/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Proteínas Associadas aos Microtúbulos/metabolismo , Suínos , Células LLC-PK1 , Interfase/fisiologiaRESUMO
Autophagy is the primary intracellular degradation system, and it plays an important role in many biological and pathological processes. Studies of autophagy involvement in developmental processes are important for understanding various processes. Among them are fibrosis, degenerative diseases, cancer development, and metastasis formation. Diabetic kidney disease is one of the main causes of chronic kidney disease and end-stage renal failure. The aim of this study was to investigate the immunohistochemical expression patterns of LC3B, LAMP2A, and GRP78 during different developmental stages of early-developing human kidneys and in samples from patients with type II diabetes mellitus. During the 7/8th DW, moderate expression of LC3B and LAMP2A and strong expression of GRP78 were found in the mesonephric glomeruli and tubules. In the 9/10th DW, the expression of LC3B and LAMP2A was even more pronounced in the mesonephric tubules. LC3B, LAMP2A, and GRP78 immunoreactivity was also found in the paramesonephric and mesonephric ducts and was stronger in the 9/10th DW compared with the 7/8th DW. In addition, the expression of LC3B, LAMP2A, and GRP78 also appeared in the mesenchyme surrounding the paramesonephric duct in the 9/10th DW. In the 15/16th DW, the expression of LC3B in the glomeruli was weak, that of LAMP2A was moderate, and that of GRP78 was strong. In the tubuli, the expression of LC3B was moderate, while the expression of LAMP2A and GRP78 was strong. The strongest expression of LC3B, LAMP2A, and GRP78 was observed in the renal medullary structures, including developing blood vessels. In postnatal human kidneys, the most extensive LC3B, LAMP2A, and GRP78 expression in the cortex was found in the epithelium of the proximal convoluted tubules, with weak to moderate expression in the glomeruli. The medullary expression of LC3B was weak, but the expression of LAMP2A and GRP78 was the strongest in the medullary tubular structures. Significantly lower expression of LC3B was found in the glomeruli of the diabetic patients in comparison with the nondiabetic patients, but there was no difference in the expression of LC3B in the tubule-interstitial compartment. The expression of LAMP2A was significantly higher in the tubule-interstitial compartments of the diabetic patients in comparison with the nondiabetic patients, while its expression did not differ in the glomeruli. Extensive expression of GRP78 was found in the glomeruli and the tubule-interstitial compartments, but there was no difference in the expression between the two groups of patients. These data give us new information about the expression of LC3B, LAMP2A, and GRP78 during embryonic, fetal, and early postnatal development. The spatiotemporal expression of LC3B, LAMP2A, and GRP78 indicates the important role of autophagy during the early stages of renal development. In addition, our data suggest a disturbance in autophagy processes in the glomeruli and tubuli of diabetic kidneys as an important factor in the pathogenesis of diabetic kidney disease.
Assuntos
Autofagia , Nefropatias Diabéticas , Chaperona BiP do Retículo Endoplasmático , Rim , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas Associadas aos Microtúbulos , Humanos , Chaperona BiP do Retículo Endoplasmático/metabolismo , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Rim/metabolismo , Rim/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Biomarcadores/metabolismo , Feminino , Masculino , Proteínas de Choque Térmico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologiaRESUMO
Cytopathology induced by methamphetamine (METH) is reminiscent of degenerative disorders such as Parkinson's disease, and it is characterized by membrane organelles arranged in tubulo-vesicular structures. These areas, appearing as clusters of vesicles, have never been defined concerning the presence of specific organelles. Therefore, the present study aimed to identify the relative and absolute area of specific membrane-bound organelles following a moderate dose (100 µM) of METH administered to catecholamine-containing PC12 cells. Organelles and antigens were detected by immunofluorescence, and they were further quantified by plain electron microscopy and in situ stoichiometry. This analysis indicated an increase in autophagosomes and damaged mitochondria along with a decrease in lysosomes and healthy mitochondria. Following METH, a severe dissipation of hallmark proteins from their own vesicles was measured. In fact, the amounts of LC3 and p62 were reduced within autophagy vacuoles compared with the whole cytosol. Similarly, LAMP1 and Cathepsin-D within lysosomes were reduced. These findings suggest a loss of compartmentalization and confirm a decrease in the competence of cell clearing organelles during catecholamine degeneration. Such cell entropy is consistent with a loss of energy stores, which routinely govern appropriate subcellular compartmentalization.
Assuntos
Autofagossomos , Lisossomos , Metanfetamina , Metanfetamina/farmacologia , Animais , Células PC12 , Ratos , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Catepsina D/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Although centrosomes help organize spindles in most cell types, oocytes of most species lack these structures. During acentrosomal spindle assembly in C. elegans oocytes, microtubule minus ends are sorted outwards away from the chromosomes where they form poles, but then these outward forces must be balanced to form a stable bipolar structure. Simultaneously, microtubule dynamics must be precisely controlled to maintain spindle length and organization. How forces and dynamics are tuned to create a stable bipolar structure is poorly understood. Here, we have gained insight into this question through studies of ZYG-8, a conserved doublecortin-family kinase; the mammalian homolog of this microtubule-associated protein is upregulated in many cancers and has been implicated in cell division, but the mechanisms by which it functions are poorly understood. We found that ZYG-8 depletion from oocytes resulted in overelongated spindles with pole and midspindle defects. Importantly, experiments with monopolar spindles revealed that ZYG-8 depletion led to excess outward forces within the spindle and suggested a potential role for this protein in regulating the force-generating motor BMK-1/kinesin-5. Further, we found that ZYG-8 is also required for proper microtubule dynamics within the oocyte spindle and that kinase activity is required for its function during both meiosis and mitosis. Altogether, our findings reveal new roles for ZYG-8 in oocytes and provide insights into how acentrosomal spindles are stabilized to promote faithful meiosis.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Microtúbulos , Oócitos , Fuso Acromático , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Microtúbulos/metabolismo , Microtúbulos/genética , Fuso Acromático/metabolismo , Fuso Acromático/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Oócitos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Centrossomo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Diabetes-related bone loss represents a significant complication that persistently jeopardizes the bone health of individuals with diabetes. Primary cilia proteins have been reported to play a vital role in regulating osteoblast differentiation in diabetes-related bone loss. However, the specific contribution of KIAA0753, a primary cilia protein, in bone loss induced by diabetes remains unclear. In this investigation, we elucidated the pivotal role of KIAA0753 as a promoter of osteoblast differentiation in diabetes. RNA sequencing demonstrated a marked downregulation of KIAA0753 expression in pro-bone MC3T3 cells exposed to a high glucose environment. Diabetes mouse models further validated the downregulation of KIAA0753 protein in the femur. Diabetes was observed to inhibit osteoblast differentiation in vitro, evidenced by downregulating the protein expression of OCN, OPN and ALP, decreasing primary cilia biosynthesis, and suppressing the Hedgehog signalling pathway. Knocking down KIAA0753 using shRNA methods was found to shorten primary cilia. Conversely, overexpression KIAA0753 rescued these changes. Additional insights indicated that KIAA0753 effectively restored osteoblast differentiation by directly interacting with SHH, OCN and Gli2, thereby activating the Hedgehog signalling pathway and mitigating the ubiquitination of Gli2 in diabetes. In summary, we report a negative regulatory relationship between KIAA0753 and diabetes-related bone loss. The clarification of KIAA0753's role offers valuable insights into the intricate mechanisms underlying diabetic bone complications.
Assuntos
Diferenciação Celular , Proteínas Associadas aos Microtúbulos , Osteoblastos , Transdução de Sinais , Animais , Humanos , Masculino , Camundongos , Linhagem Celular , Cílios/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/genética , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Osteogênese/genética , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
Mitophagy selectively eliminates damaged or dysfunctional mitochondria, playing a crucial role in maintaining mitochondrial quality control. However, it remains unclear whether mitophagy can be fully activated and how it evolves after SCI. Our RNA-seq analysis of animal samples from sham and 1, 3, 5, and 7 days post-SCI indicated that mitophagy was indeed inhibited during the acute and subacute early stages. In vitro experiments showed that this inhibition was closely related to excessive production of reactive oxygen species (ROS) and the downregulation of BNIP3. Excessive ROS led to the blockage of mitophagy flux, accompanied by further mitochondrial dysfunction and increased neuronal apoptosis. Fortunately, ligustilide (LIG) was found to have the ability to reverse the oxidative stress-induced downregulation of BNIP3 and enhance mitophagy through BNIP3-LC3 interaction, alleviating mitochondrial dysfunction and ultimately reducing neuronal apoptosis. Further animal experiments demonstrated that LIG alleviated oxidative stress and mitophagy inhibition, rescued neuronal apoptosis, and promoted tissue repair, ultimately leading to improved motor function. In summary, this study elucidated the state of mitophagy inhibition following SCI and its potential mechanisms, and confirmed the effects of LIG-enhanced mitophagy through BNIP3-LC3, providing new therapeutic targets and strategies for repairing SCI.
Assuntos
4-Butirolactona , Apoptose , Proteínas de Membrana , Mitofagia , Neurônios , Estresse Oxidativo , Ratos Sprague-Dawley , Traumatismos da Medula Espinal , Animais , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Neurônios/metabolismo , Traumatismos da Medula Espinal/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Masculino , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Associadas aos MicrotúbulosRESUMO
Background: This study investigated the genetic characteristics of five Chinese families with keratoconus (KC). Methods: In the five families affected by KC, medical records, clinical observations, and blood samples were collected from all individuals. All KC family members (n = 20) underwent both whole exome sequencing of genomic DNA and Sanger sequencing to confirm the variants. Online software was utilized to analyze all variants, and the online server I-TASSER was employed for in silico predictions of the three-dimensional protein structures of the variants. The newly discovered variants and single nucleotide polymorphisms were further examined in 322 sporadic KC patients. Results: The Pentacam tomographic composite index in those affected first-degree family members of the probands showed a pathological change. Five new variants were detected in the five probands and other affected members in their families: a heterozygous missense variant g.19043832C>T (p.Ser145Asn) in the homer scaffolding protein 3 (HOMER3) gene; a heterozygous missense variant g.99452113G>A (p.Gly483Arg) in the insulin-like growth factor 1 receptor (IGF1R) gene; a heterozygous missense variant g.55118280G>T (p.Trp843Leu) in the echinoderm microtubule-associated protein like 6 (EML6) gene; a heterozygous frameshift variant c. 1226_1227del (p.Gln410Glufs*17) in the DOP1 leucine zipper-like protein B (DOP1B) gene; and a heterozygous splice-site variant c.7776+2T>A in the neurobeachin-like protein 2 (NBEAL2) gene. These variations were predicted to be potentially pathogenic and associated with KC. Conclusion: Five novel variants in HOMER3, IGF1R, EML6, DOP1B, and NBEAL2 genes were identified in this study and may be associated with the pathogenesis of KC. This study provides new information about the gene variants and their protein changes in KC patients. The findings should be explored further and could potentially be applied to the early diagnosis of KC before clinical onset.
Assuntos
Ceratocone , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , China , População do Leste Asiático/genética , Sequenciamento do Exoma , Predisposição Genética para Doença/genética , Proteínas de Homeodomínio/genética , Ceratocone/genética , Proteínas Associadas aos Microtúbulos/genética , Mutação de Sentido Incorreto , Linhagem , Polimorfismo de Nucleotídeo Único , Receptor IGF Tipo 1/genética , CriançaRESUMO
OBJECTIVE: To study the biological role and related mechanism of autophagy in acute lung injury (ALI) of hemorrhagic shock mice. METHODS: According to random number table method, wild-type male C57BL/6 mice were divided into control group, ALI group, rapamycin group and 3-methyladenine (3-MA) group, with 8 mice in each group. Light chain 3 (LC3) gene knockout mice with C57BL/6 background were divided into LC3 knockout group and LC3 knockout+ALI group, with 8 mice in each group. Control group, ALI group, LC3 knockout group, LC3 knockout+ALI group were intraperitoneally injected with 2 mL/kg normal saline, rapamycin group was intraperitoneally injected with 3 mg/kg autophagy activator rapamycin, 3-MA group was intraperitoneally injected with 15 mg/kg autophagy inhibitor 3-MA, all of which were given for 3 consecutive days. 2 hours after the last administration, the hemorrhagic shock induced ALI model was established. 24 hours after modeling, the lung index was calculated. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissue and lung injury score was performed. The expressions of autophagy genes LC3- II/LC3- I and Beclin-1 in lung tissue were detected by Western blotting. The contents of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and malondialdehyde (MDA) in lung tissue were detected according to the steps of the kit. RESULTS: Compared with the control group, the lung tissue structure was destroyed and exudation increased, lung index, lung injury score, the expressions of LC3- II/LC3- I, Beclin-1, and the contents of TNF-α, IL-6 and MDA in lung tissue significantly increased in the ALI group. Compared with the ALI group, the structural damage and exudation of lung tissue were reduced in the rapamycin group, lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue decreased, while the expressions of LC3- II/LC3- I and Beclin-1 in lung tissue increased [lung index: (7.56±0.39)% vs. (9.12±0.59)%, lung injury score: 3.04±0.58 vs. 9.32±2.14, TNF-α (ng/mg): 1.85±0.32 vs. 3.51±0.62, IL-6 (ng/mg): 1.61±0.32 vs. 2.52±0.44, MDA (nmol/mg): 1.03±0.16 vs. 1.88±0.24, LC3- II/LC3- I: 1.21±0.12 vs. 0.39±0.05, Beclin-1/ß-actin: 1.10±0.12 vs. 0.58±0.06, all P < 0.05], while lung tissue structure damage was aggravated and exudation was further increased in the 3-MA group, lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue increased, the expressions of LC3- II/LC3- I and Beclin-1 in lung tissue decreased [lung index: (10.44±0.62)% vs. (9.12±0.59)%, lung injury score: 11.59±2.28 vs. 9.32±2.14, TNF-α (ng/mg): 4.77±0.71 vs. 3.51±0.62, IL-6 (ng/mg): 3.44±0.52 vs. 2.52±0.44, MDA (nmol/mg): 2.71±0.42 vs. 1.88±0.24, LC3- II/LC3- I: 0.25±0.04 vs. 0.39±0.05, Beclin-1/ß-actin: 0.21±0.03 vs. 0.58±0.06, all P < 0.05]. Lung index, lung injury score and the contents of TNF-α, IL-6 and MDA in lung tissue of LC3 knockout ALI mice were higher than those of wild-type ALI mice [lung index: (10.44±0.75)% vs. (9.12±0.59)%, lung injury score: 12.41±2.86 vs. 9.32±2.14, TNF-α (ng/mg): 4.85±0.72 vs. 3.51±0.62, IL-6 (ng/mg): 3.28±0.51 vs. 2.52±0.44, MDA (nmol/mg): 2.75±0.41 vs. 1.88±0.24, all P < 0.05]. CONCLUSIONS: Autophagy plays a protective role in ALI of hemorrhagic shock mice, and the related molecular mechanism is the inhibition of inflammatory response and oxidative stress response.