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1.
J Immunol Res ; 2022: 9916228, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36093435

RESUMO

Objective: This study explored the colorectal cancer exosome lncRNA prostate cancer associated transcript 1- (PCAT1) mediated circulating tumors and the mechanism of cell colorectal cancer liver metastasis. Methods: Exosomes were extracted from the primary colorectal cancer (CRC) cell lines HCT116 and SW480 and cultured with T84 and human umbilical vein endothelial (HUVE) cells. The expression of PCAT1 and miR-329-3p was detected by real-time quantitative polymerase chain reaction (RT-qPCR), the expression of Netrin-1, CD146, and epithelial mesenchymal transition (EMT) related proteins was detected by Western blot, the proliferation activity of T84 cells was detected by cell counting kit 8 (CCK-8), and cell migration was detected by Transwell. The expression of the F-actin signal was detected by immunofluorescence after coculture of exosomes with human umbilical vein endothelial cells (HUVECs). Changes in subcutaneous tumor and liver nodule size after PCAT1 deletion were observed in a mouse model of liver metastasis from rectal cancer. Results: PCAT1 expression was upregulated in primary cell lines and their exosomes. After exosomes were cocultured with colorectal cancer tumor circulating T84 cells, the expression of Netrin-1 and CD146 was upregulated, the expression of miR-329-3p was downregulated, the proliferation and migration ability of T84 cells were enhanced, and EMT occurred. After knocking down PCAT1, the above phenomenon was reversed. Similarly, after exosomes were cocultured with HUVECs, the expression of the F-actin signal increased, and after PCAT1 was knocked down, the F-actin signal also decreased. PCAT1 regulates miR-329-3p/Netrin-1 and affects the biological behavior of T84 and F-actin signal expression in HUVECs. In a mouse model of colorectal cancer liver metastasis, knocking down PCAT1 significantly reduced the nodules formed by liver metastasis in mice. Conclusions: LncRNA PCAT1 derived from colorectal cancer exosomes regulates the activity of the Netrin-1-CD146 complex in circulating tumor cells (CTCs) to promote the occurrence of colorectal cancer EMT and liver metastasis and provides new molecular targets for the treatment of colorectal cancer liver metastasis.


Assuntos
Neoplasias Colorretais , Neoplasias Hepáticas , MicroRNAs , RNA Longo não Codificante , Actinas/metabolismo , Animais , Antígeno CD146/genética , Antígeno CD146/metabolismo , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Netrina-1/genética , Netrina-1/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
2.
Biomed Res Int ; 2022: 8537959, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36119932

RESUMO

Tendon-derived stem cells (TDSCs) play a vital role in repair of rotator cuff tear injuries by secreting paracrine proteins that regulate resident cell functions. Secreted exosomes may play a role in tendon injury repair by mediating intercellular communication; however, the detailed mechanisms by which TDSC-derived exosomes affect tenocyte development remain unknown. Here, we examined the effects of exosomes isolated from conditioned medium of TDSCs on tenocyte differentiation, migration, and transition to a fibroblastic phenotype in vitro. Successful isolation of exosomes from TDSCs was confirmed by high expression levels of CD81, CD63, CD9, and TSG101. Treatment with TDSC-derived exosomes promoted the growth and migration of cultured rat tenocytes, and increased the levels of the fibrosis markers collagen I, collagen III, scleraxis, tenascin C, and α-smooth muscle actin. Furthermore, vascular endothelial growth factor A (VEGFA) expression was higher in TDSC-derived exosomes than in TDSCs, and genetic knockdown of VEGFA suppressed the stimulatory effect of TDSC-derived exosomes on tenocyte development. Overall, these results demonstrate that VEGFA-enriched exosomes isolated from TDSCs promote differentiation and migration of cultured tenocytes and their transition to a fibroblastic phenotype. These data provide a new potential clinical treatment strategy for tendon injury.


Assuntos
Exossomos , Traumatismos dos Tendões , Actinas/metabolismo , Animais , Colágeno/metabolismo , Meios de Cultivo Condicionados/farmacologia , Fenótipo , Ratos , Células-Tronco/metabolismo , Tenascina/metabolismo , Traumatismos dos Tendões/terapia , Tendões/metabolismo , Tenócitos , Fator A de Crescimento do Endotélio Vascular/metabolismo
3.
J Cell Biol ; 221(11)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36102863

RESUMO

Mitochondrial damage represents a dramatic change in cellular homeostasis. One rapid response is perimitochondrial actin polymerization, termed acute damage-induced actin (ADA). The consequences of ADA are not understood. In this study, we show evidence suggesting that ADA is linked to rapid glycolytic activation upon mitochondrial damage in multiple cells, including mouse embryonic fibroblasts and effector CD8+ T lymphocytes. ADA-inducing treatments include CCCP, antimycin, rotenone, oligomycin, and hypoxia. The Arp2/3 complex inhibitor CK666 or the mitochondrial sodium-calcium exchanger (NCLX) inhibitor CGP37157 inhibits both ADA and the glycolytic increase within 5 min, supporting ADA's role in glycolytic stimulation. Two situations causing chronic reductions in mitochondrial ATP production, mitochondrial DNA depletion and mutation to the NDUFS4 subunit of complex 1 of the electron transport chain, cause persistent perimitochondrial actin filaments similar to ADA. CK666 treatment causes rapid mitochondrial actin loss and a drop in ATP in NDUFS4 knock-out cells. We propose that ADA is necessary for rapid glycolytic activation upon mitochondrial impairment, to re-establish ATP production.


Assuntos
Actinas , Trifosfato de Adenosina , Mitocôndrias , Actinas/metabolismo , Trifosfato de Adenosina/biossíntese , Animais , Linfócitos T CD8-Positivos , Células Cultivadas , Complexo I de Transporte de Elétrons/metabolismo , Fibroblastos , Glicólise , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Polimerização
4.
Physiol Rep ; 10(18): e15370, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36117313

RESUMO

Proteinuria is a major manifestation of kidney disease, reflecting injuries of glomerular podocytes. Actin cytoskeleton plays a pivotal role in stabilizing the foot processes of podocytes against the hydrostatic pressure of filtration. Calponin is an actin associated protein that regulates mechanical tension-related cytoskeleton functions and its role in podocytes has not been established. Here we studied the kidney phenotypes of calponin isoform 2 knockout (KO) mice. Urine samples were examined to quantify the ratio of albumin and creatinine. Kidney tissue samples were collected for histology and ultrastructural studies. A mouse podocyte cell line (E11) was used to study the expression and cellular localization of calponin 2. In comparison with wild-type (WT) controls, calponin 2 KO mice showed age-progressive high proteinuria and degeneration of renal glomeruli. High levels of calponin 2 are expressed in E11 podocytes and colocalized with actin stress fibers, tropomyosin and myosin IIA. Electron microscopy showed that aging calponin 2 KO mice had effacement of the podocyte foot processes and increased thickness of the glomerular basement membrane as compared to that of WT control. The findings demonstrate that deletion of calponin 2 aggravates age-progressive degeneration of the glomerular structure and function as filtration barrier. The critical role of calponin 2 in podocytes suggests a molecular target for understanding the pathogenesis of proteinuria and therapeutic development.


Assuntos
Actinas , Miosina não Muscular Tipo IIA , Actinas/metabolismo , Albuminas , Animais , Proteínas de Ligação ao Cálcio , Creatinina , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos , Proteinúria/metabolismo , Tropomiosina
5.
Proc Natl Acad Sci U S A ; 119(37): e2208465119, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36067310

RESUMO

Gene expression is tightly regulated by RNA-binding proteins (RBPs) to facilitate cell survival, differentiation, and migration. Previous reports have shown the importance of the Insulin-like Growth Factor II mRNA-Binding Protein (IGF2BP1/IMP1/ZBP1) in regulating RNA fate, including localization, transport, and translation. Here, we generated and characterized a knockout mouse to study RBP regulation. We report that IGF2BP1 is essential for proper brain development and neonatal survival. Specifically, these mice display disorganization in the developing neocortex, and further investigation revealed a loss of cortical marginal cell density at E17.5. We also investigated migratory cell populations in the IGF2BP1[Formula: see text] mice, using BrdU labeling, and detected fewer mitotically active cells in the cortical plate. Since RNA localization is important for cellular migration and directionality, we investigated the regulation of ß-actin messenger RNA (mRNA), a well-characterized target with established roles in cell motility and development. To aid in our understanding of RBP and target mRNA regulation, we generated mice with endogenously labeled ß-actin mRNA (IGF2BP1[Formula: see text]; ß-actin-MS2[Formula: see text]). Using endogenously labeled ß-actin transcripts, we report IGF2BP1[Formula: see text] neurons have increased transcription rates and total ß-actin protein content. In addition, we found decreased transport and anchoring in knockout neurons. Overall, we present an important model for understanding RBP regulation of target mRNA.


Assuntos
Actinas , Encéfalo , Proteínas de Ligação a RNA , Actinas/genética , Actinas/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Movimento Celular/genética , Camundongos , Camundongos Knockout , Neurônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
6.
Front Endocrinol (Lausanne) ; 13: 938596, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36072926

RESUMO

Activation and/or modulation of the membrane-associated receptors plays a critical role in brain development. Thyroid hormone (TH) acts on both nuclear receptors (thyroid hormone receptor, TR) and membrane-associated receptors, particularly integrin αvß3 in neurons and glia. Integrin αvß3-mediated signal transduction mediates various cellular events during development including morphogenesis, migration, synaptogenesis, and intracellular metabolism. However, the involvement of integrin αvß3-mediated TH action during brain development remains poorly understood. Thus, we examined the integrin αvß3-mediated effects of TH (T3, T4, and rT3) in the neurons and astrocytes using primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture of neurons and astrocytes. We found that TH augments dendrite arborization of cerebellar Purkinje cells. This augmentation was suppressed by knockdown of integrin αvß3, as well as TRα and TRß. A selective integrin αvß3 antagonist, LM609, was also found to suppress TH-induced arborization. However, whether this effect was a direct action of TH on Purkinje cells or due to indirect actions of other cells subset such as astrocytes was not clarified. To further study neuron-specific molecular mechanisms, we used Neuro-2A clonal cells and found TH also induces neurite growth. TH-induced neurite growth was reduced by co-exposure with LM609 or knockdown of TRα, but not TRß. Moreover, co-culture of Neuro-2A and astrocytes also increased TH-induced neurite growth, indicating astrocytes may be involved in neuritogenesis. TH increased the localization of synapsin-1 and F-actin in filopodia tips. TH exposure also increased phosphorylation of FAK, Akt, and ERK1/2. Phosphorylation was suppressed by co-exposure with LM609 and TRα knockdown. These results indicate that TRs and integrin αvß3 play essential roles in TH-induced dendritogenesis and neuritogenesis. Furthermore, astrocytes-neuron communication via TR-dependent and TR-independent signaling through membrane receptors and F-actin are required for TH-induced neuritogenesis.


Assuntos
Actinas , Integrina alfaVbeta3 , Actinas/metabolismo , Actinas/farmacologia , Integrina alfaVbeta3/metabolismo , Receptores dos Hormônios Tireóideos/fisiologia , Transdução de Sinais/fisiologia , Receptores beta dos Hormônios Tireóideos , Hormônios Tireóideos/farmacologia , Hormônios Tireóideos/fisiologia
7.
Front Immunol ; 13: 980539, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36059552

RESUMO

Strategies based on intracellular expression of artificial binding domains present several advantages over manipulating nucleic acid expression or the use of small molecule inhibitors. Intracellularly-functional nanobodies can be considered as promising macrodrugs to study key signaling pathways by interfering with protein-protein interactions. With the aim of studying the RAS-related small GTPase RHOA family, we previously isolated, from a synthetic phage display library, nanobodies selective towards the GTP-bound conformation of RHOA subfamily proteins that lack selectivity between the highly conserved RHOA-like and RAC subfamilies of GTPases. To identify RHOA/ROCK pathway inhibitory intracellular nanobodies, we implemented a stringent, subtractive phage display selection towards RHOA-GTP followed by a phenotypic screen based on F-actin fiber loss. Intracellular interaction and intracellular selectivity between RHOA and RAC1 proteins was demonstrated by adapting the sensitive intracellular protein-protein interaction reporter based on the tripartite split-GFP method. This strategy led us to identify a functional intracellular nanobody, hereafter named RH28, that does not cross-react with the close RAC subfamily and blocks/disrupts the RHOA/ROCK signaling pathway in several cell lines without further engineering or functionalization. We confirmed these results by showing, using SPR assays, the high specificity of the RH28 nanobody towards the GTP-bound conformation of RHOA subfamily GTPases. In the metastatic melanoma cell line WM266-4, RH28 expression triggered an elongated cellular phenotype associated with a loss of cellular contraction properties, demonstrating the efficient intracellular blocking of RHOA/B/C proteins downstream interactions without the need of manipulating endogenous gene expression. This work paves the way for future therapeutic strategies based on protein-protein interaction disruption with intracellular antibodies.


Assuntos
Anticorpos de Domínio Único , Actinas/metabolismo , Guanosina Trifosfato , Transdução de Sinais , Anticorpos de Domínio Único/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo
8.
Cell Death Dis ; 13(9): 786, 2022 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-36097267

RESUMO

The dynamics of synaptic vesicles (SVs) within presynaptic domains are tightly controlled by synapsin1 phosphorylation; however, the mechanism underlying the anchoring of synapsin1 with F-actin or SVs is not yet fully understood. Here, we found that Syn1 is modified with protein palmitoylation, and examining the roles of Syn1 palmitoylation in neurons led us to uncover that Syn1 palmitoylation is negatively regulated by its phosphorylation; together, they manipulate the clustering and redistribution of SVs. Using the combined approaches of electron microscopy and genetics, we revealed that Syn1 palmitoylation is vital for its binding with F-actin but not SVs. Inhibition of Syn1 palmitoylation causes defects in SVs clustering and a reduced number of total SVs in vivo. We propose a model in which SVs redistribution is triggered by upregulated Syn1 phosphorylation and downregulated Syn1 palmitoylation, and they reversibly promote SVs clustering. The crosstalk of Syn1 palmitoylation and phosphorylation thereby bidirectionally manipulates SVs dynamics in neurons.


Assuntos
Lipoilação , Vesículas Sinápticas , Actinas/metabolismo , Neurônios/metabolismo , Fosforilação , Vesículas Sinápticas/metabolismo
9.
Cytokine ; 159: 156018, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36054965

RESUMO

An increase in the number of mast cells could contribute to inflammatory diseases and pathologic conditions. A receptor activator of NF-κB ligand (RANKL)/RANK system is one of the key signaling pathways accelerating mast cell-mediated allergic inflammatory reactions. However, the biological functions of RANKL in mast cell proliferation remains to be clarified. The aim of the present study is to clarify the role of RANKL in mast cell proliferation. Surprisingly, RANKL remarkably reduced the proliferation of human mast cell line, HMC-1 cells through the inhibition of murine double minute 2 (MDM2) and Ki-67 mRNA expressions in a dose-dependent manner. RANKL significantly reduced cell viability, whereas it increased cellular senescence via increasing levels of p53, phosphorylated(p)-p53, p21, and p16 and decreasing levels of retinoblastoma protein (pRb) and p-pRb in HMC-1 cells. Even in rat peritoneal mast cells, RANKL induced cellular senescence by increasing filamentous-actin polymerization. In addition, RANKL remarkably reduced thymic stromal lymphopoietin (TSLP)-induced mast cell proliferation via the downregulation of MDM2 and Ki-67. RANKL decreased levels of p-signal transducer and activator of transcription 6 in TSLP-stimulated HMC-1 cells. The mast cell growth factor, interleukin-13 was remarkably down-regulated by treatment with RANKL in TSLP-stimulated HMC-1 cells. Furthermore, RANKL increased the number of senescence-associated ß-galactosidase-stained cells and protein levels of p53, p-p53, and p21 in TSLP-stimulated HMC-1 cells. These data suggest that RANKL down-regulates mast cell proliferation by inducing senescence.


Assuntos
Interleucina-13 , Proteínas Proto-Oncogênicas c-mdm2 , Actinas/metabolismo , Animais , Proliferação de Células , Citocinas/metabolismo , Humanos , Interleucina-13/metabolismo , Antígeno Ki-67/metabolismo , Ligantes , Mastócitos/metabolismo , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Proteína do Retinoblastoma , Fator de Transcrição STAT6/metabolismo , Fator de Células-Tronco , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismo
10.
Int J Mol Sci ; 23(17)2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36077249

RESUMO

Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations-after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.


Assuntos
Actinas , Cadeias Leves de Miosina , Actinas/metabolismo , Animais , Células Endoteliais/metabolismo , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Cadeias Leves de Miosina/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Quinases Associadas a rho/metabolismo
11.
Cells ; 11(17)2022 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-36078161

RESUMO

Ageing is accompanied by dramatic changes in chromatin structure organization and genome function. Two essential components of chromatin, the linker histone Hho1p and actin-related protein 4 (Arp4p), have been shown to physically interact in Saccharomyces cerevisiae cells, thus maintaining chromatin dynamics and function, as well as genome stability and cellular morphology. Disrupting this interaction has been proven to influence the stability of the yeast genome and the way cells respond to stress during chronological ageing. It has also been proven that the abrogated interaction between these two chromatin proteins elicited premature ageing phenotypes. Alterations in chromatin compaction have also been associated with replicative ageing, though the main players are not well recognized. Based on this knowledge, here, we examine how the interaction between Hho1p and Arp4p impacts the ageing of mitotically active yeast cells. For this purpose, two sets of strains were used-haploids (WT(n), arp4, hho1Δ and arp4 hho1Δ) and their heterozygous diploid counterparts (WT(2n), ARP4/arp4, HHO1/hho1Δ and ARP4 HHO1/arp4 hho1Δ)-for the performance of extensive morphological and physiological analyses during replicative ageing. These analyses included a comparative examination of the yeast cells' chromatin structure, proliferative and reproductive potential, and resilience to stress, as well as polysome profiles and chemical composition. The results demonstrated that the haploid chromatin mutants arp4 and arp4 hho1Δ demonstrated a significant reduction in replicative and total lifespan. These findings lead to the conclusion that the importance of a healthy interaction between Arp4p and Hho1p in replicative ageing is significant. This is proof of the concomitant importance of Hho1p and Arp4p in chronological and replicative ageing.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Actinas/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo
12.
J Gen Physiol ; 154(10)2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36053243

RESUMO

The ß-myosin heavy chain expressed in ventricular myocardium and the myosin heavy chain (MyHC) in slow-twitch skeletal Musculus soleus (M. soleus) type-I fibers are both encoded by MYH7. Thus, these myosin molecules are deemed equivalent. However, some reports suggested variations in the light chain composition between M. soleus and ventricular myosin, which could influence functional parameters, such as maximum velocity of shortening. To test for functional differences of the actin gliding velocity on immobilized myosin molecules, we made use of in vitro motility assays. We found that ventricular myosin moved actin filaments with ∼0.9 µm/s significantly faster than M. soleus myosin (0.3 µm/s). Filaments prepared from isolated actin are not the native interaction partner of myosin and are believed to slow down movement. Yet, using native thin filaments purified from M. soleus or ventricular tissue, the gliding velocity of M. soleus and ventricular myosin remained significantly different. When comparing the light chain composition of ventricular and M. soleus ß-myosin, a difference became evident. M. soleus myosin contains not only the "ventricular" essential light chain (ELC) MLC1sb/v, but also an additional longer and more positively charged MLC1sa. Moreover, we revealed that on a single muscle fiber level, a higher relative content of MLC1sa was associated with significantly slower actin gliding. We conclude that the ELC MLC1sa decelerates gliding velocity presumably by a decreased dissociation rate from actin associated with a higher actin affinity compared to MLC1sb/v. Such ELC/actin interactions might also be relevant in vivo as differences between M. soleus and ventricular myosin persisted when native thin filaments were used.


Assuntos
Actinas , Cadeias Leves de Miosina , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosinas Ventriculares
13.
Kidney360 ; 3(5): 843-858, 2022 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-36128477

RESUMO

Background: Patients with chronic kidney disease (CKD) frequently have compromised physical performance, which increases their mortality; however, their skeletal muscle dysfunction has not been characterized at the single-fiber and molecular levels. Notably, interventions to mitigate CKD myopathy are scarce. Methods: The effect of CKD in the absence and presence of iron supplementation on the contractile function of individual skeletal muscle fibers from the soleus and extensor digitorum longus muscles was evaluated in 16-week-old mice. CKD was induced by the adenine diet, and iron supplementation was by weekly iron dextran injections. Results: Maximally activated and fatigued fiber force production was decreased 24%-52% in untreated CKD, independent of size, by reducing strongly bound myosin/actin cross-bridges and/or decreasing myofilament stiffness in myosin heavy chain (MHC) I, IIA, and IIB fibers. Additionally, myosin/actin interactions in untreated CKD were slower for MHC I and IIA fibers and unchanged or faster in MHC IIB fibers. Iron supplementation improved anemia and did not change overall muscle mass in CKD mice. Iron supplementation ameliorated CKD-induced myopathy by increasing strongly bound cross-bridges, leading to improved specific tension, and/or returning the rate of myosin/actin interactions toward or equivalent to control values in MHC IIA and IIB fibers. Conclusions: Skeletal muscle force production was significantly reduced in untreated CKD, independent of fiber size, indicating that compromised physical function in patients is not solely due to muscle mass loss. Iron supplementation improved multiple aspects of CKD-induced myopathy, suggesting that timely correction of iron imbalance may aid in ameliorating contractile deficits in CKD patients.


Assuntos
Cadeias Pesadas de Miosina , Insuficiência Renal Crônica , Actinas/metabolismo , Adenina/metabolismo , Animais , Dextranos/metabolismo , Suplementos Nutricionais , Ferro/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosinas/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico
14.
Cell Rep ; 40(12): 111372, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36130510

RESUMO

Golgi outposts (GOPs) in dendrites are known for their role in promoting branch extension, but whether GOPs have other functions is unclear. We found that terminal branches of Drosophila class IV dendritic arborization (C4da) neurons actively grow during the early third-instar (E3) larval stage but retract in the late third (L3) stage. Interestingly, the Fringe (Fng) glycosyltransferase localizes increasingly at GOPs in distal dendritic regions through the E3 to the L3 stage. Expression of the endopeptidase Furin 2 (Fur2), which proteolyzes and inactivates Fng, decreases from E3 to L3 in C4da neurons, thereby increasing Fng-positive GOPs in dendrites. The epidermal Delta ligand and neuronal Notch receptor, the substrate for Fng-mediated O-glycosylation, also negatively regulate dendrite growth. Fng inhibits actin dynamics in dendrites, linking dendritic branch retraction to suppression of the C4da-mediated thermal nociception response in late larval stages. Thus, Fng-positive GOPs function in dendrite retraction, which would add another function to the repertoire of GOPs in dendrite arborization.


Assuntos
Dendritos , Proteínas de Drosophila , Actinas/metabolismo , Animais , Dendritos/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Furina/metabolismo , Glicosiltransferases/metabolismo , Larva/metabolismo , Ligantes , Receptores Notch/metabolismo , Células Receptoras Sensoriais/metabolismo
15.
Nat Commun ; 13(1): 5444, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114192

RESUMO

The Rho-family GTPase Rac1 activates the WAVE regulatory complex (WRC) to drive Arp2/3 complex-mediated actin polymerization in many essential processes. Rac1 binds to WRC at two distinct sites-the A and D sites. Precisely how Rac1 binds and how the binding triggers WRC activation remain unknown. Here we report WRC structures by itself, and when bound to single or double Rac1 molecules, at ~3 Å resolutions by cryogenic-electron microscopy. The structures reveal that Rac1 binds to the two sites by distinct mechanisms, and binding to the A site, but not the D site, drives WRC activation. Activation involves a series of unique conformational changes leading to the release of sequestered WCA (WH2-central-acidic) polypeptide, which stimulates the Arp2/3 complex to polymerize actin. Together with biochemical and cellular analyses, the structures provide a novel mechanistic understanding of how the Rac1-WRC-Arp2/3-actin signaling axis is regulated in diverse biological processes and diseases.


Assuntos
Actinas , Família de Proteínas da Síndrome de Wiskott-Aldrich , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Citoplasma/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
16.
Bull Exp Biol Med ; 173(4): 481-485, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36058963

RESUMO

Western blot analysis is used for evaluation of the level of proteins production in organs and tissues, and housekeeping proteins GAPDH, actin, and tubulin are usually used as the reference proteins. The signal of the target protein is normalized to the corresponding signal of the reference protein. The data on the intensity of actin, tubulin, and GAPDH synthesis are fragmentary: their expression differs in different organs and can vary depending on age, which is often not taken into account in experimental studies. We studied the features of the production of reference proteins in the liver, heart, brain, and lungs of newborn, prepubertal, and adult male Wistar rats. Age-related differences in the expression of ß-actin, ß-tubulin, and GAPDH in the myocardium and dorsal prefrontal cortex were revealed. GAPDH expression in the dorsal prefrontal cortex in adult rats was significantly higher than in prepubertal rats; GAPDH expression in the myocardium of adult rats was significantly higher than in newborns. The level of actin in the dorsal prefrontal cortex in newborn rats was significantly higher than in prepubertal and adult rats. In the liver and lungs, the expression of actin, tubulin, and GAPDH did not differ in newborn, prepubertal, and adult rats. When choosing the reference protein for Western blotting, animals age and the studied organ should be taken into account.


Assuntos
Actinas , Tubulina (Proteína) , Actinas/genética , Actinas/metabolismo , Fatores Etários , Animais , Western Blotting , Masculino , Ratos , Ratos Wistar , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
17.
J Am Heart Assoc ; 11(18): e026292, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36073642

RESUMO

Background Human cardiac biopsies are widely used in clinical and fundamental research to decipher molecular events that characterize cardiac physiological and pathophysiological states. One of the main approaches relies on the analysis of semiquantitative immunoblots that reveals alterations in protein expression levels occurring in diseased hearts. To maintain semiquantitative results, expression level of target proteins must be standardized. The expression of HKP (housekeeping proteins) is commonly used to this purpose. Methods and Results We evaluated the stability of HKP expression (actin, ß-tubulin, GAPDH, vinculin, and calsequestrin) and total protein staining within control (coefficient of variation) and comparatively with ischemic human heart biopsies (P value). All HKP exhibited a high level of intragroup (ie, actin, ß-tubulin, and GAPDH) and/or intergroup variability (ie, GAPDH, vinculin, and calsequestrin). Among all, we found total protein staining to exhibit the highest degree of stability within and between groups, which makes this reference the best to study protein expression level in human biopsies from ischemic hearts and age-matched controls. In addition, we illustrated that using an inappropriate reference protein marker misleads interpretation on SERCA2 (sarco/endoplasmic reticulum Ca2+ ATPase) and cMyBPC (cardiac myosin binding protein-C) expression level after myocardial infarction. Conclusions These reemphasize the need to standardize the level of protein expression with total protein staining in comparative immunoblot studies on human samples from control and diseased hearts.


Assuntos
Actinas , Calsequestrina , Actinas/metabolismo , Biópsia , Miosinas Cardíacas/metabolismo , Grupos Controle , Trabalho Doméstico , Humanos , Isquemia , Tubulina (Proteína)/metabolismo , Vinculina/metabolismo
18.
J Am Heart Assoc ; 11(18): e026365, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36102251

RESUMO

Background Clinically, myocardial fibrosis is one of the most common complications caused by chronic kidney disease (CKD). However, the potential mechanisms of CKD-induced myocardial fibrosis have not been clarified. Methods and Results In our in vivo study, a rat model of CKD with 5/6 nephrectomy was established. The CKD model was treated with the glioma 1 (Gli-1) inhibitor GANT-61, and myocardial fibrosis and serum intact fibroblast growth factor 23 levels were assessed 16 weeks after nephrectomy. Finally, we found that Gli-1 and Smoothened in the Sonic Hedgehog (Shh) signaling pathway were activated and that collagen-1 and collagen-3, which constitute the fibrotic index, were expressed in CKD myocardial tissue. After administering the Gli-1 inhibitor GANT-61, the degree of myocardial fibrosis was reduced, and Gli-1 expression was also inhibited. We also measured blood pressure, cardiac biomarkers, and other indicators in rats and performed hematoxylin-eosin staining of myocardial tissue. Furthermore, in vitro studies showed that intact fibroblast growth factor 23 promoted cardiac fibroblast proliferation and transdifferentiation into myofibroblasts by activating the Shh signaling pathway, thereby promoting cardiac fibrosis, as manifested by increased expression of the Shh, Patch 1, and Gli-1 mRNAs and Shh, Smoothened, and Gli-1 proteins in the Shh signaling pathway. The protein and mRNA levels of other fibrosis indicators, such as α-smooth muscle actin, which are also markers of transdifferentiation, collagen-1, and collagen-3, were increased. Conclusions On the basis of these results, intact fibroblast growth factor 23 promotes CKD-induced myocardial fibrosis by activating the Shh signaling pathway.


Assuntos
Proteínas Hedgehog , Insuficiência Renal Crônica , Actinas/metabolismo , Animais , Colágeno Tipo I , Amarelo de Eosina-(YS) , Fator de Crescimento de Fibroblastos 23 , Fibrose , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Hematoxilina , RNA Mensageiro , Ratos , Transdução de Sinais/fisiologia
19.
J Vis Exp ; (186)2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36121261

RESUMO

Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely "tunneling nanotubes" (TNTs). TNTs are defined as open-ended, lipid bilayer-encircled membrane extensions that mediate continuity between neighboring cells of diameters ranging between 50 nm and 1 µm. TNTs were demonstrated initially in neuronal cells, but successive studies have revealed the existence of TNTs in several cell types and diseases, such as neurodegenerative diseases, viral infections, and cancer. Several studies have referred to close-ended, electrically coupled membrane nanostructures between neighboring cells as TNTs or TNT-like structures. The elucidation of ultrastructure in terms of membrane continuity at the endpoint is technically challenging. In addition, studies on cell-cell communication are challenging in terms of the characterization of TNTs using conventional methods due to the lack of specific markers. TNTs are primarily defined as F-actin-based, open-ended membrane protrusions. However, one major limitation is that F-actin is present in all types of protrusions, making it challenging to differentiate TNTs from other protrusions. One of the notable characteristics of F-actin-based TNTs is that these structures hover between two cells without touching the substratum. Therefore, distinct F-actin-stained TNTs can conveniently be distinguished from other protrusions such as filopodia and neurites based on their hovering between cells. We have recently shown that the internalization of oligomeric amyloid-ß1-42 (oAß) via actin-dependent endocytosis stimulates activated p21-activated kinase-1 (PAK1), which mediates the formation of F-actin-containing TNTs coexpressed with phospho-PAK1 between SH-SY5Y neuronal cells. This protocol outlines a 3D volume analysis method to identify and characterize TNTs from the captured z-stack images of F-actin- and phospho-PAK1-immunostained membrane protrusions in oAß-treated neuronal cells. Further, TNTs are distinguished from developing neurites and neuronal outgrowths based on F-actin- and ß-III tubulin-immunostained membrane conduits.


Assuntos
Nanotubos , Neuroblastoma , Actinas/metabolismo , Humanos , Bicamadas Lipídicas , Nanotubos/química , Tubulina (Proteína) , Quinases Ativadas por p21
20.
J Cell Biol ; 221(11)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36121394

RESUMO

Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKß/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKß/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKß is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKß signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKß is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKß signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.


Assuntos
Actomiosina , Miotonina Proteína Quinase , Fagocitose , Actinas/metabolismo , Actomiosina/metabolismo , Miosina Tipo II/metabolismo , Miotonina Proteína Quinase/metabolismo , Fagocitose/fisiologia , Proteínas Tirosina Quinases , Receptores Fc , c-Mer Tirosina Quinase/metabolismo
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