RESUMEN
An important question in cell biology is how cytoskeletal proteins evolved and drove the development of novel structures and functions. Here we address the origin of SPIRE actin nucleators. Mammalian SPIREs work with RAB GTPases, formin (FMN)-subgroup actin assembly proteins and class-5 myosin (MYO5) motors to transport organelles along actin filaments towards the cell membrane. However, the origin and extent of functional conservation of SPIRE among species is unknown. Our sequence searches show that SPIRE exist throughout holozoans (animals and their closest single-celled relatives), but not other eukaryotes. SPIRE from unicellular holozoans (choanoflagellate), interacts with RAB, FMN and MYO5 proteins, nucleates actin filaments and complements mammalian SPIRE function in organelle transport. Meanwhile SPIRE and MYO5 proteins colocalise to organelles in Salpingoeca rosetta choanoflagellates. Based on these observations we propose that SPIRE originated in unicellular ancestors of animals providing an actin-myosin driven exocytic transport mechanism that may have contributed to the evolution of complex multicellular animals.
Asunto(s)
Actomiosina , Orgánulos , Animales , Orgánulos/metabolismo , Actomiosina/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Actinas/metabolismo , Humanos , Coanoflagelados/metabolismo , Citoesqueleto de Actina/metabolismo , Evolución Biológica , Evolución Molecular , Forminas/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Filogenia , Proteínas NuclearesRESUMEN
BACKGROUND: With recent advances in gene sequencing technology, more than 60 genetic mutations associated with very early onset inflammatory bowel disease (VEO-IBD) have been reported. Most of the genes are associated with immune deficiencies. The Myosin 5B (MYO5B) gene is primarily involved in cell motility and material transport which is associated with congenital intractable diarrhea and cholestasis. No studies have examined the relationship between the MYO5B gene and VEO-IBD. We report a case of a child with a mutation in the MYO5B gene who was diagnosed with VEO-IBD, then we investigated the association between the MYO5B gene and VEO-IBD. CASE PRESENTATION: A 7-month-old baby girl with a chief complaint of "blood in the stool for more than 4 months and vaginal pus and blood discharge for 3 weeks" was diagnosed with VEO-IBD, and her symptoms improved after treatment with mesalazine. The whole-exome sequencing was performed with peripheral blood. Immunohistochemistry was performed on the terminal ileal tissue. Western blotting, quantitative polymerase chain reaction (Q-PCR) and immunofluorescence were performed with cultured organoid tissue from the terminal ileum. Whole-exome sequencing identified heterozygous missense of MYO5B variant of unknown significance (p. [I769N]; [T1546M]). Immunohistochemistry revealed a significant decrease in the expression of MYO5B protein in the terminal ileum of the child with MYO5B mutation; Q-PCR revealed a decrease in the mRNA levels of occludin and ZO-1 and both the mRNA levels and protein levels of MYO5B was downregulated in the patient. Immunofluorescence images showed that MYO5B gene mutation disrupted the apical delivery of transporters SGLT1, NHE3 and AQP7. CONCLUSIONS: MYO5B gene mutation leading to the downregulation of MYO5B protein may promote the occurrence of VEO-IBD by decreasing mRNA and protein levels of intestinal tight junction genes and dislocating the apical transporters.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Mutación , Miosina Tipo V , Humanos , Femenino , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/patología , Miosina Tipo V/genética , Lactante , Edad de Inicio , Secuenciación del Exoma , Cadenas Pesadas de MiosinaRESUMEN
Mucormycosis is an emerging and deadly invasive fungal infection caused by fungi belonging to the Mucorales order. We investigated the myosin superfamily, which encompasses diverse actin-based motor proteins with various cellular functions. Specifically, the role of the Myo5B (ID 179665) protein from the myosin class V family in Mucor lusitanicus was explored by generating silencing phenotypes and null mutants corresponding to the myo5B gene. Silencing fungal transformants exhibited a markedly reduced growth rate and a nearly complete absence of sporulation compared to the wild-type strain. The myo5BΔ null mutant strain displayed atypical characteristics, including abnormally short septa and inflated hyphae. Notably, there were a majority of small yeast-like cells instead of filamentous hyphae in the mutant. These yeast-like cells cannot germinate normally, resulting in a loss of polarity. In vivo virulence assays conducted in the Galleria mellonella invertebrate model revealed that the myo5BΔ mutant strain was avirulent. These findings shed light on the crucial contributions of the Myo5B protein to the dimorphism and pathogenicity of M. lusitanicus. Therefore, the myosin V family is a potential target for future therapeutic interventions aimed at treating mucormycosis.
Asunto(s)
Proteínas Fúngicas , Hifa , Mucor , Hifa/crecimiento & desarrollo , Hifa/genética , Mucor/genética , Mucor/patogenicidad , Mucor/crecimiento & desarrollo , Virulencia , Animales , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Mucormicosis/microbiología , Mariposas Nocturnas/microbiología , Humanos , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/genéticaRESUMEN
Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.
Asunto(s)
Caveolina 1 , Movimiento Celular , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Humanos , Caveolina 1/metabolismo , Caveolina 1/genética , Macrófagos/metabolismo , Fosforilación , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/genética , Transporte Biológico , Cicatrización de Heridas/fisiología , Orgánulos/metabolismoRESUMEN
Transport and localization of melanosome at the periphery region of melanocyte are depended on myosin-5a (Myo5a), which associates with melanosome by interacting with its adaptor protein melanophilin (Mlph). Mlph contains four functional regions, including Rab27a-binding domain, Myo5a GTD-binding motif (GTBM), Myo5a exon F-binding domain (EFBD), and actin-binding domain (ABD). The association of Myo5a with Mlph is known to be mediated by two specific interactions: the interaction between the exon-F-encoded region of Myo5a and Mlph-EFBD and that between Myo5a-GTD and Mlph-GTBM. Here, we identify a third interaction between Myo5a and Mlph, that is, the interaction between the exon-G-encoded region of Myo5a and Mlph-ABD. The exon-G/ABD interaction is independent from the exon-F/EFBD interaction and is required for the association of Myo5a with melanosome. Moreover, we demonstrate that Mlph-ABD interacts with either the exon-G or actin filament, but cannot interact with both of them simultaneously. Based on above findings, we propose a new model for the Mlph-mediated Myo5a transportation of melanosomes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Melanosomas , Miosina Tipo V , Unión Proteica , Melanosomas/metabolismo , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Animales , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Humanos , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/genética , Melanocitos/metabolismoRESUMEN
Migrasomes, enriched with signaling molecules such as chemokines, cytokines and angiogenic factors, play a pivotal role in the spatially defined delivery of these molecules, influencing critical physiological processes including organ morphogenesis and angiogenesis. The mechanism governing the accumulation of signaling molecules in migrasomes has been elusive. In this study, we show that secretory proteins, including signaling proteins, are transported into migrasomes by secretory carriers via both the constitutive and regulated secretion pathways. During cell migration, a substantial portion of these carriers is redirected to the rear of the cell and actively transported into migrasomes, driven by the actin-dependent motor protein Myosin-5a. Once at the migrasomes, these carriers fuse with the migrasome membrane through SNARE-mediated mechanisms. Inhibiting migrasome formation significantly reduces secretion, suggesting migrasomes as a principal secretion route in migrating cells. Our findings reveal a specialized, highly localized secretion paradigm in migrating cells, conceptually paralleling the targeted neurotransmitter release observed in neuronal systems.
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Movimiento Celular , Humanos , Animales , Transducción de Señal , Transporte de Proteínas , Miosina Tipo V/metabolismo , Proteínas SNARE/metabolismo , RatonesRESUMEN
Myosin Va (Myo Va) is one of three protein complexes involved in melanosome transport. In this study, we identified BMP-2 as an up-regulator of Myo Va expression using 2-methyl-naphtho[1,2,3-de]quinolin-8-one (MNQO). Our results showed that MNQO reduced the mRNA and protein expression of Myo Va and BMP-2 in melanocytes. Knockdown of BMP-2 by siRNA also affected Myo Va mRNA and protein expression, confirming that MNQO regulates Myo Va through BMP-2. Furthermore, phosphorylation of Smad1/5/8 by BMP2 treatment confirmed that the BMP-2/Smad signaling pathway regulates Myo Va expression in Melan-a melanocytes. Smad-binding elements were found in the Myo Va promoter and phosphorylated Smad1/5/8 bind directly to the Myo Va promoter to activate Myo Va transcription and BMP-2 enhances this binding. These findings provide insight into a new role for BMP-2 in Melan-a melanocytes and a mechanism of regulation of Myo Va expression that may be beneficial in the treatment of albinism or hyperpigmentation disorders.
Asunto(s)
Proteína Morfogenética Ósea 2 , Melanocitos , Cadenas Pesadas de Miosina , Miosina Tipo V , Transducción de Señal , Miosina Tipo V/metabolismo , Miosina Tipo V/genética , Melanocitos/metabolismo , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Cadenas Pesadas de Miosina/metabolismo , Cadenas Pesadas de Miosina/genética , Humanos , Proteínas Smad/metabolismo , Regiones Promotoras Genéticas/genética , Fosforilación , Ratones , Animales , Regulación de la Expresión GénicaRESUMEN
Molecular motors employ chemical energy to generate unidirectional mechanical output against a track while navigating a chaotic cellular environment, potential disorder on the track, and against Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions.
Asunto(s)
Actinas , Miosina Tipo V , Actinas/química , Miosinas/química , Citoesqueleto de Actina/química , Movimiento (Física) , Miosina Tipo V/químicaRESUMEN
OBJECTIVE: To explore the clinical and genetic characteristics of a neonate with Microvillus inclusion disease (MVID). METHODS: A neonate with MVID admitted to the First Affiliated Hospital of Zhengzhou University in May 2019 was selected as the study subject. Clinical data were collected. Whole exome sequencing (WES) was carried out, and candidate variants were verified by Sanger sequencing and multiple ligation-dependent probe amplification (MLPA). A literature was also carried out to summarize the clinical and genetic characteristics of MVID. RESULTS: The prematurely born neonate had presented with unexplained refractory diarrhea and metabolic acidosis. Active symptomatic treatment was ineffective, and the child had died at 2 months old. WES revealed that he had harbored compound heterozygous variants of the MYO5B gene, namely c.1591C>T (p.R531W) and deletion of exon 9. Sanger sequencing showed that the R531W variant was inherited form his father, and MLPA confirmed that the exon 9 deletion was inherited from his mother. Seven children with MVID were reported in China, of which one was lost during follow-up and six had deceased. One hundred eighty eight patients were reported worldwide and only one was cured. The clinical features of MVID had included refractory diarrhea, metabolic acidosis and poor prognosis. CONCLUSION: The child was diagnosed with MVID due to the compound heterozygous variants of the MYO5B gene, which has provided a basis for genetic counseling and prenatal diagnosis.
Asunto(s)
Acidosis , Síndromes de Malabsorción , Microvellosidades , Mucolipidosis , Miosina Tipo V , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Embarazo , Diarrea/genética , Síndromes de Malabsorción/genética , Microvellosidades/patología , Mucolipidosis/genética , Cadenas Pesadas de Miosina , Miosina Tipo V/genéticaRESUMEN
Robust linkage between adherens junctions and the actomyosin cytoskeleton allows cells to change shape and move during morphogenesis without tearing tissues apart. The Drosophila multidomain protein Canoe and its mammalian homolog afadin are crucial for this, as in their absence many events of morphogenesis fail. To define the mechanism of action for Canoe, we are taking it apart. Canoe has five folded protein domains and a long intrinsically disordered region. The largest is the Dilute domain, which is shared by Canoe and myosin V. To define the roles of this domain in Canoe, we combined biochemical, genetic and cell biological assays. AlphaFold was used to predict its structure, providing similarities and contrasts with Myosin V. Biochemical data suggested one potential shared function - the ability to dimerize. We generated Canoe mutants with the Dilute domain deleted (CnoΔDIL). Surprisingly, they were viable and fertile. CnoΔDIL localized to adherens junctions and was enriched at junctions under tension. However, when its dose was reduced, CnoΔDIL did not provide fully wild-type function. Furthermore, canoeΔDIL mutants had defects in the orchestrated cell rearrangements of eye development. This reveals the robustness of junction-cytoskeletal connections during morphogenesis and highlights the power of natural selection to maintain protein structure.
Asunto(s)
Proteínas de Drosophila , Miosina Tipo V , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Miosina Tipo V/metabolismo , Citoesqueleto/metabolismo , Uniones Intercelulares/metabolismo , Uniones Adherentes/metabolismo , Morfogénesis , Cadherinas/metabolismo , Mamíferos/metabolismoRESUMEN
Depending on the chemical energy from ATP hydrolysis, myosin V can drive the multistep and continuous coupled cycling process to transport cellular cargo to targeted regions. However, it is still obscure how the molecular memory induced by the multistep coupled transported process could regulate the dynamic behavior of the motor state of myosin V. Here, we propose a novel non-Markovian polymorphic mechanochemical model to investigate the effect of the molecular memory on the mechanic of noise attenuation of myosin V system. We first define an effective transition rate for a multistep coupled reaction process which is the function of memory and system states to transform equivalently the non-Markovian process into the classical Markov process. By noise decomposition technology, it is observed that both the intrinsic and extrinsic noises of the ADP-myosin V bound state (AM â ADP) exhibit a monotonically decreasing trend with lengthening the molecular memory. Molecular memory as a regulation factor can amplify the contribution of intrinsic noise to the overall noise while reducing the influence of extrinsic noise on the AM â ADP. Moreover, the modulation of molecular memory could induce stochastic focusing. These results indicate that the role of molecular memory in the myosin V state transition can not only offer a handle to maintain the robustness of the motion system but also serve as a paradigm for studying more complex molecular motors.
Asunto(s)
Miosina Tipo V , Miosina Tipo V/química , Miosina Tipo V/metabolismo , Comunicación Celular , Adenosina Trifosfato/metabolismo , Actinas/químicaRESUMEN
Cellular vesicle long-distance transport along the cytoplasmic actin network has recently been uncovered in several cell systems. In metaphase mouse oocytes, the motor protein myosin-5b (Myo5b) and the actin nucleation factor Spire are recruited to the Rab11a-positive vesicle membrane, forming a ternary complex of Myo5b/Spire/Rab11a that drives the vesicle long-distance transport to the oocyte cortex. However, the mechanism underlying the intermolecular regulation of the Myo5b/Spire/Rab11a complex remains unknown. In this study, we expressed and purified Myo5b, Spire2, and Rab11a proteins, and performed ATPase activity measurements, pulldown and single-molecule motility assays. Our results demonstrate that both Spire2 and Rab11a are required to activate Myo5b motor activity under physiological ionic conditions. The GTBM fragment of Spire2 stimulates the ATPase activity of Myo5b, while Rab11a enhances this activation. This activation occurs by disrupting the head-tail interaction of Myo5b. Furthermore, at the single-molecule level, we observed that the GTBM fragment of Spire2 and Rab11a coordinate to stimulate the Myo5b motility activity. Based on our results, we propose that upon association with the vesicle membrane, Myo5b, Spire2 and Rab11a form a ternary complex, and the inhibited Myo5b is synergistically activated by Spire2 and Rab11a, thereby triggering the long-distance transport of vesicles.
Asunto(s)
Actinas , Miosina Tipo V , Ratones , Animales , Actinas/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Microvillus inclusion disease (MVID) is a rare condition that is present from birth and affects the digestive system. People with MVID experience severe diarrhea that is difficult to control, cannot absorb dietary nutrients, and struggle to grow and thrive. In addition, diverse clinical manifestations, some of which are life-threatening, have been reported in cases of MVID. MVID can be caused by variants in the MYO5B, STX3, STXBP2, or UNC45A gene. These genes produce proteins that have been functionally linked to each other in intestinal epithelial cells. MVID associated with STXBP2 variants presents in a subset of patients diagnosed with familial hemophagocytic lymphohistiocytosis type 5. MVID associated with UNC45A variants presents in most patients diagnosed with osteo-oto-hepato-enteric syndrome. Furthermore, variants in MYO5B or STX3 can also cause other diseases that are characterized by phenotypes that can co-occur in subsets of patients diagnosed with MVID. Recent studies involving clinical data and experiments with cells and animals revealed connections between specific phenotypes occurring outside of the digestive system and the type of gene variants that cause MVID. Here, we have reviewed these patterns and correlations, which are expected to be valuable for healthcare professionals in managing the disease and providing personalized care for patients and their families.
Asunto(s)
Síndromes de Malabsorción , Microvellosidades , Mucolipidosis , Fenotipo , Humanos , Mucolipidosis/genética , Mucolipidosis/patología , Microvellosidades/patología , Microvellosidades/genética , Síndromes de Malabsorción/genética , Síndromes de Malabsorción/patología , Animales , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Mutación , Predisposición Genética a la EnfermedadRESUMEN
N-methyl-D-aspartate receptor (NMDAR) trafficking is a key process in the regulation of synaptic efficacy and brain function. However, the molecular mechanism underlying the surface transport of NMDARs is largely unknown. Here we identified myosin Va (MyoVa) as the specific motor protein that traffics NMDARs in hippocampal neurons. We found that MyoVa associates with NMDARs through its cargo binding domain. This association was increased during NMDAR surface transport. Knockdown of MyoVa suppressed NMDAR transport. We further demonstrated that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates NMDAR transport through its direct interaction with MyoVa. Furthermore, MyoVa employed Rab11 family-interacting protein 3 (Rab11/FIP3) as the adaptor proteins to couple themselves with NMDARs during their transport. Accordingly, the knockdown of FIP3 impairs hippocampal memory. Together, we conclude that in hippocampal neurons, MyoVa conducts active transport of NMDARs in a CaMKII-dependent manner.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Hipocampo , Miosina Tipo V , Neuronas , Receptores de N-Metil-D-Aspartato , Animales , Hipocampo/metabolismo , Neuronas/metabolismo , Miosina Tipo V/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Transporte de Proteínas/fisiología , Cadenas Pesadas de Miosina/metabolismo , Ratas , Células Cultivadas , Humanos , Ratas Sprague-DawleyRESUMEN
Head and neck squamous carcinoma (HNSC) poses a significant public health challenge due to its substantial morbidity. Nevertheless, despite advances in current treatments, the prognosis for HNSC remains unsatisfactory. To address this, single-cell RNA sequencing (RNA-seq) and bulk RNA-seq data combined with in vitro studies were conducted to examine the role of MYO5A (Myosin VA) in HNSC. Our investigation revealed an overexpression of MYO5A in HNSC that promotes HNSC migration in vitro. Remarkably, knockdown of MYO5A suppressed vimentin expression. Furthermore, analyzing the TCGA database evidenced that MYO5A is a risk factor for human papillomavirus positive (HPV+) HNSC (HR = 0.81, P < 0.001). In high MYO5A expression HNSC, there was a low count of tumor infiltrating lymphocytes (TIL), including activated CD4+ T cells, CD8+ T cells, and B cells. Of note, CD4+ T cells and B cells were positively associated with improved HPV+ HNSC outcomes. Correlation analysis demonstrated a decreased level of immunostimulators in high MYO5A-expressing HNSC. Collectively, these findings suggest that MYO5A may promote HNSC migration through vimentin and involve itself in the process of immune infiltration in HNSC, advancing the understanding of the mechanisms and treatment of HNSC.
Asunto(s)
Neoplasias de Cabeza y Cuello , Miosina Tipo V , Infecciones por Papillomavirus , Humanos , Vimentina/genética , Neoplasias de Cabeza y Cuello/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Procesos Neoplásicos , Pronóstico , Linfocitos Infiltrantes de Tumor , Cadenas Pesadas de Miosina/genética , Miosina Tipo V/genéticaRESUMEN
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is a prevalent malignant tumor worldwide. Circular RNA (circRNA) is of great value in tumorigenesis progression. However, the mechanism of circFNDC3B in ESCC remains to be clarified. METHODS: Firstly, the circular characteristics of circFNDC3B were evaluated by Actinomycin D and RNase R measurements. The functions of circFNDC3B in ESCC cells were examined by CCK-8, EdU and flow cytometry. Subsequently, the molecular mechanism of circFNDC3B was explained using luciferase reporter gene detection. Finally, we constructed xenograft model to prove the role of circFNDC3B in vivo. RESULTS: Our study revealed that circFNDC3B was more stable than its linear RNA and prominently upregulated in ESCC. Functional findings suggested that silencing of circFNDC3B reduced the proliferation and enhanced apoptosis of ESCC cells in vitro. Meanwhile, knockdown of circFNDC3B attenuated tumor progression in vivo. Next, miR-370-3p/miR-136-5p was discovered to bind circFNDC3B. miR-370-3p/miR-136-5p reversed the promotive effect on cell proliferation and the inhibitory effect on cell apoptosis of circFNDC3B. MYO5A was a downstream target of miR-370-3p/miR-136-5p. CircFNDC3B served as a sponge for miR-370-3p/miR-136-5p and alleviated the prohibitory effect of miR-370-3p/miR-136-5p on MYO5A, which accelerated ESCC progression. CONCLUSION: circFNDC3B positively adjusted the MYO5A expression via spongy miR-370-3p/miR-136-5p, hence achieving the cancer-promoting effect on ESCC. circFNDC3B was a prospective diagnosis marker for ESCC.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , MicroARNs , Miosina Tipo V , ARN Circular , Humanos , Neoplasias Esofágicas/genética , Carcinoma de Células Escamosas de Esófago/genética , Genes Reporteros , MicroARNs/genética , Cadenas Pesadas de Miosina , Estudios Prospectivos , ARN Circular/genéticaRESUMEN
Microvillus inclusion disease (MVID), caused by loss-of-function mutations in the motor protein myosin Vb (MYO5B), is a severe infantile disease characterized by diarrhea, malabsorption, and acid/base instability, requiring intensive parenteral support for nutritional and fluid management. Human patient-derived enteroids represent a model for investigation of monogenic epithelial disorders but are a rare resource from MVID patients. We developed human enteroids with different loss-of function MYO5B variants and showed that they recapitulated the structural changes found in native MVID enterocytes. Multiplex immunofluorescence imaging of patient duodenal tissues revealed patient-specific changes in localization of brush border transporters. Functional analysis of electrolyte transport revealed profound loss of Na+/H+ exchange (NHE) activity in MVID patient enteroids with near-normal chloride secretion. The chloride channel-blocking antidiarrheal drug crofelemer dose-dependently inhibited agonist-mediated fluid secretion. MVID enteroids exhibited altered differentiation and maturation versus healthy enteroids. γ-Secretase inhibition with DAPT recovered apical brush border structure and functional Na+/H+ exchange activity in MVID enteroids. Transcriptomic analysis revealed potential pathways involved in the rescue of MVID cells including serum/glucocorticoid-regulated kinase 2 (SGK2) and NHE regulatory factor 3 (NHERF3). These results demonstrate the utility of patient-derived enteroids for developing therapeutic approaches to MVID.
Asunto(s)
Síndromes de Malabsorción , Mucolipidosis , Miosina Tipo V , Humanos , Microvellosidades/genética , Cadenas Pesadas de Miosina/genética , Miosina Tipo V/genética , Enterocitos/metabolismo , Síndromes de Malabsorción/genética , Síndromes de Malabsorción/terapia , Síndromes de Malabsorción/metabolismo , Mucolipidosis/genética , Mucolipidosis/terapia , Mucolipidosis/metabolismoRESUMEN
Myosin X forms an antiparallel dimer and moves processively on actin bundles. How the antiparallel dimer affects the stepping mechanism of myosin X remains elusive. Here, we generated several chimeras using domains of myosin V and X and performed single-molecule motility assays. We found that the chimera containing the motor domain from myosin V and the lever arm and antiparallel coiled-coil domain from myosin X has multiple forward step sizes and moves processively, similar to full-length myosin X. The chimera containing the motor domain and lever arm from myosin X and the parallel coiled-coil from myosin V takes steps of â¼40 nm at lower ATP concentrations but was nonprocessive at higher ATP concentrations. Furthermore, mutant myosin X with four mutations in the antiparallel coiled-coil domain failed to dimerize and was nonprocessive. These results imply that the antiparallel coiled-coil domain is necessary for multiple forward step sizes of myosin X.
Asunto(s)
Miosina Tipo V , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Dominios Proteicos , Dimerización , Adenosina TrifosfatoRESUMEN
Circular RNAs (circRNAs) are a class of key regulators in cancers via regulating gene levels by acting as sponges of miRNAs. This study was devoted to explore the functional mechanism of circRNA fibronectin type III domain-containing protein 3B (circ-FNDC3B) in esophageal squamous cell carcinoma (ESCC). RNA levels were examined via reverse transcription-quantitative polymerase chain reaction assay. Cell viability detection was performed using Cell Counting Kit-8 assay. The proliferation ability was determined through colony formation assay and EDU assay. Flow cytometry was applied for analysis of apoptosis. Invasion ability was assessed via transwell assay. Target binding was analyzed by dual-luciferase reporter assay. The protein expression was measured using western blot. In vivo research was conducted via xenograft model in mice. Circ-FNDC3B exhibited significant upregulation in ESCC tissues and cells. Downregulation of circ-FNDC3B inhibited ESCC cell proliferation and invasion but accelerated cell apoptosis. Circ-FNDC3B interacted with miR-136-5p or miR-370-3p. The function of circ-FNDC3B was achieved by sponging miR-136-5p or miR-370-3p. Myosin VA (MYO5A) acted as a downstream target of miR-136-5p or miR-370-3p. MYO5A reversed miR-136-5p/miR-370-3p-induced tumor inhibition in ESCC cells. Circ-FNDC3B targeted miR-136-5p or miR-370-3p to affect MYO5A expression. Circ-FNDC3B knockdown reduced tumor growth in vivo by inhibiting miR-136-5p or miR-370-3p-mediated MYO5A expression. These findings demonstrated that circ-FNDC3B contributed to malignant progression of ESCC cells via miR-136-5p/MYO5A or miR-370-3p/MYO5A axis.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , MicroARNs , Miosina Tipo V , Humanos , Animales , Ratones , Carcinoma de Células Escamosas de Esófago/genética , Neoplasias Esofágicas/genética , MicroARNs/genética , Apoptosis , Supervivencia Celular , Proliferación Celular , Línea Celular Tumoral , Cadenas Pesadas de Miosina/genética , FibronectinasRESUMEN
Observing the structural dynamics of biomolecules is vital to deepening our understanding of biomolecular functions. High-speed (HS) atomic force microscopy (AFM) is a powerful method to measure biomolecular behavior at near physiological conditions. In the AFM, measured image profiles on a molecular surface are distorted by the tip shape through the interactions between the tip and molecule. Once the tip shape is known, AFM images can be approximately deconvolved to reconstruct the surface geometry of the sample molecule. Thus, knowing the correct tip shape is an important issue in the AFM image analysis. The blind tip reconstruction (BTR) method developed by Villarrubia (J Res Natl Inst Stand Technol 102:425, 1997) is an algorithm that estimates tip shape only from AFM images using mathematical morphology operators. While the BTR works perfectly for noise-free AFM images, the algorithm is susceptible to noise. To overcome this issue, we here propose an alternative BTR method, called end-to-end differentiable BTR, based on a modern machine learning approach. In the method, we introduce a loss function including a regularization term to prevent overfitting to noise, and the tip shape is optimized with automatic differentiation and backpropagations developed in deep learning frameworks. Using noisy pseudo-AFM images of myosin V motor domain as test cases, we show that our end-to-end differentiable BTR is robust against noise in AFM images. The method can also detect a double-tip shape and deconvolve doubled molecular images. Finally, application to real HS-AFM data of myosin V walking on an actin filament shows that the method can reconstruct the accurate surface geometry of actomyosin consistent with the structural model. Our method serves as a general post-processing for reconstructing hidden molecular surfaces from any AFM images. Codes are available at https://github.com/matsunagalab/differentiable_BTR .