Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of the BBSome, a cargo adaptor essential for export of transmembrane receptors from cilia. Although actin-dependent ectocytosis has been proposed to compensate defective cargo retrieval, its molecular basis remains unclear, especially in relation to BBS pathology. In this study, we investigated how actin polymerization and ectocytosis are regulated within the cilium. Our findings reveal that ciliary CDC42, a RHO-family GTPase triggers in situ actin polymerization, ciliary ectocytosis, and cilia shortening in BBSome-deficient cells. Activation of the Sonic Hedgehog pathway further enhances CDC42 activity specifically in BBSome-deficient cilia. Inhibition of CDC42 in BBSome-deficient cells decreases the frequency and duration of ciliary actin polymerization events, causing buildup of G protein coupled receptor 161 (GPR161) in bulges along the axoneme during Sonic Hedgehog signaling. Overall, our study identifies CDC42 as a key trigger of ciliary ectocytosis. Hyperactive ciliary CDC42 and ectocytosis and the resulting loss of ciliary material might contribute to BBS disease severity.
- MeSH
- aktiny * metabolismus MeSH
- Bardetův-Biedlův syndrom metabolismus genetika patologie MeSH
- cdc42 protein vázající GTP * metabolismus genetika MeSH
- cilie * metabolismus MeSH
- lidé MeSH
- myši MeSH
- proteiny hedgehog * metabolismus MeSH
- receptory spřažené s G-proteiny metabolismus genetika MeSH
- signální transdukce * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
MICAL proteins play a crucial role in cellular dynamics by binding and disassembling actin filaments, impacting processes like axon guidance, cytokinesis, and cell morphology. Their cellular activity is tightly controlled, as dysregulation can lead to detrimental effects on cellular morphology. Although previous studies have suggested that MICALs are autoinhibited, and require Rab proteins to become active, the detailed molecular mechanisms remained unclear. Here, we report the cryo-EM structure of human MICAL1 at a nominal resolution of 3.1 Å. Structural analyses, alongside biochemical and functional studies, show that MICAL1 autoinhibition is mediated by an intramolecular interaction between its N-terminal catalytic and C-terminal coiled-coil domains, blocking F-actin interaction. Moreover, we demonstrate that allosteric changes in the coiled-coil domain and the binding of the tripartite assembly of CH-L2α1-LIM domains to the coiled-coil domain are crucial for MICAL activation and autoinhibition. These mechanisms appear to be evolutionarily conserved, suggesting a potential universality across the MICAL family.
- MeSH
- aktiny metabolismus chemie MeSH
- alosterická regulace MeSH
- calponiny MeSH
- elektronová kryomikroskopie * MeSH
- lidé MeSH
- mikrofilamenta metabolismus ultrastruktura MeSH
- mikrofilamentové proteiny metabolismus chemie ultrastruktura MeSH
- molekulární modely MeSH
- oxygenasy se smíšenou funkcí MeSH
- proteinové domény MeSH
- proteiny s doménou LIM metabolismus chemie genetika MeSH
- vazba proteinů * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Various studies have correlated the mechanical properties of the aortic wall with its biochemical parameters and inner structure. Very few studies have addressed correlations with the cohesive properties, which are crucial for understanding fracture phenomena such as aortic dissection, i.e. a life-threatening process. Aimed at filling this gap, we conducted a comprehensive biochemical and histological analysis of human aortas (the ascending and descending thoracic and infrarenal abdominal aorta) from 34 cadavers obtained post-mortem during regular autopsies. The pentosidine, hydroxyproline and calcium contents, calcium/phosphorus molar ratio, degree of atherosclerosis, area fraction of elastin, collagen type I and III, alpha smooth muscle actin, vasa vasorum, vasa vasorum density, aortic wall thickness, thicknesses of the adventitia, media and intima were determined and correlated with the delamination forces in the longitudinal and circumferential directions of the vessel as determined from identical cadavers. The majority of the parameters determined did not indicate significant correlation with age, except for the calcium content and collagen maturation (enzymatic crosslinking). The main results concern differences between enzymatic and non-enzymatic crosslinking and those caused by the presence of atherosclerosis. The enzymatic crosslinking of collagen increased with age and was accompanied by a decrease in the delamination strength, while non-enzymatic crosslinking tended to decrease with age and was accompanied by an increase in the delamination strength. As the rate of calcification increased, the presence of atherosclerosis led to the formation of calcium phosphate plaques with higher solubility than the tissue without or with only mild signs of atherosclerosis. STATEMENT OF SIGNIFICANCE: This study presents a detailed biochemical and histological analysis of human aortic samples (ascending thoracic aorta, descending thoracic aorta and infrarenal abdominal aorta) taken from 34 cadavers. The contribution of this scientific study lies in the detailed biochemical comparison of the enzymatic and non-enzymatic glycosylation-derived crosslinks of vascular tissues and their influence on the delamination strength of the human aorta since, to the best of our knowledge, no such comprehensive studies exist in the literature. A further benefit concerns the notification of the limitations of the various analytical methods applied; an important factor that must be taken into account in such studies.
- MeSH
- aktiny metabolismus MeSH
- aorta * metabolismus MeSH
- arginin analogy a deriváty MeSH
- ateroskleróza metabolismus patologie MeSH
- dospělí MeSH
- elastin metabolismus MeSH
- hydroxyprolin metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- lysin analogy a deriváty metabolismus MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- stárnutí * fyziologie MeSH
- vápník metabolismus MeSH
- vasa vasorum metabolismus MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Cíl: Srovnání cervikálního stromatu u pacientek s pokročilými stadii karcinomu děložního hrdla s kontrolní skupinou: v období před léčbou srovnat hematologické parametry u pacientek se stejnými parametry v kontrolní skupině; ověřit souvislost stromálních markerů s s prognostickými faktory karcinomu děložního hrdla. Materiál a metody: Prospektivně bylo hodnoceno 16 pacientek s diagnózou pokročilého invazivního karcinomu děložního hrdla. Kontrolní skupinu tvořilo 22 pacientek (s děložním myomem). Bylo provedeno imunohistochemické vyšetření k detekci hladkosvalového aktinu alfa (SMA – alpha-smooth muscle actin) a fibroblasty aktivujícího proteinu alfa (FAP – fibroblast activation protein alpha). Výsledky imunohistochemického vyšetření a hematologické parametry byly vyhodnoceny pomocí Fisherova exaktního testu a Mann-Whitneyho testu. Výsledky: Silné imunobarvení FAP bylo častější u pacientek s karcinomem děložního hrdla v porovnání s pacientkami s děložním myomem (p = 0,0002). Co se týká SMA, silné imunobarvení bylo také ve větší míře zjištěno ve skupině pacientek s karcinomem oproti kontrolní skupině (p < 0,00001). Poměr neutrofily/lymfocyty (NLR) byl vyšší u pacientek s neoplazií děložního hrdla ve srovnání s kontrolní skupinou (p = 0,0019). Souvislost mezi zkoumanými parametry a prognostickými faktory nebyla prokázána. Závěr: Silné imunohistochemické barvení FAP a SMA je ve srovnání s kontrolní skupinou častější u pacientek s karcinomem děložního hrdla. Poměr NLR u nich byl rovněž vyšší.
Objective: To compare cervical stroma in advanced cervical cancer with the control group; to compare, in the pre-treatment period, hemogram parameters in patients with advanced cervical cancer with the same parameters as the control group; and to verify if there is an association of stromal markers with prognostic factors in cervical cancer. Materials and methods: We prospectively evaluated 16 patients diagnosed with advanced invasive cervical cancer. A control group of 22 patients was used (uterine leiomyoma). Immunohistochemistry was performed to verify the stromal immunostaining of alpha-smooth muscle actin (SMA) and fibroblast activation protein alpha (FAP). Immunostainings and hemogram parameters were compared using Fisher's exact and Mann-Whitney Test, respectively. Results: Strong FAP immunostaining was more frequent in patients with cervical cancer when compared with patients with leiomyoma (P = 0.0002). Regarding SMA, strong immunostaining was also found more in the group of cancer patients compared to the control group (P < 0.00001). The neutrophil-lymphocyte ratio (NLR) values were higher in the cancer patient group compared to the control group (P = 0.0019). There was no association of the parameters studied with prognostic factors. Conclusions: Strong FAP and SMA immunostaining was found more in patients with cervical cancer when compared to the control group. NLR values were also higher in cervical cancer.
- MeSH
- aktiny analýza metabolismus MeSH
- endopeptidasy MeSH
- klinická studie jako téma MeSH
- leiomyom patologie MeSH
- lidé MeSH
- membránové proteiny analýza metabolismus MeSH
- nádory děložního čípku * patologie MeSH
- serinové endopeptidasy analýza metabolismus MeSH
- želatinasy analýza metabolismus MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
ANTXR1 is one of two cell surface receptors mediating the uptake of the anthrax toxin into cells. Despite substantial research on its role in anthrax poisoning and a proposed function as a collagen receptor, ANTXR1's physiological functions remain largely undefined. Pathogenic variants in ANTXR1 lead to the rare GAPO syndrome, named for its four primary features: Growth retardation, Alopecia, Pseudoanodontia, and Optic atrophy. The disease is also associated with a complex range of other phenotypes impacting the cardiovascular, skeletal, pulmonary and nervous systems. Aberrant accumulation of extracellular matrix components and fibrosis are considered to be crucial components in the pathogenesis of GAPO syndrome, contributing to the shortened life expectancy of affected individuals. Nonetheless, the specific mechanisms connecting ANTXR1 deficiency to the clinical manifestations of GAPO syndrome are largely unexplored. In this study, we present evidence that ANTXR1 deficiency initiates a senescent phenotype in human fibroblasts, correlating with defects in nuclear architecture and actin dynamics. We provide novel insights into ANTXR1's physiological functions and propose GAPO syndrome to be reconsidered as a progeroid disorder highlighting an unexpected role for an integrin-like extracellular matrix receptor in human aging.
- MeSH
- aktiny metabolismus MeSH
- alopecie * metabolismus patologie genetika MeSH
- anodoncie * MeSH
- dědičné atrofie optického nervu genetika metabolismus MeSH
- fibroblasty * metabolismus MeSH
- lidé MeSH
- mikrofilamentové proteiny * MeSH
- poruchy růstu * MeSH
- progerie genetika patologie metabolismus MeSH
- receptory buněčného povrchu metabolismus genetika nedostatek MeSH
- stárnutí buněk * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: To investigate the structural bases of human oocytes' cytoplasmic abnormalities and the causative mechanism of their emergence. Knowledge of an abnormal oocyte's intracellular organization is vital to establishing reliable criteria for clinical evaluation of oocyte morphology. DESIGN: Laboratory-based study on experimental material provided by a private assisted reproduction clinic. SETTING: University laboratory and imaging center. PATIENTS: A total of 105 women undergoing hormonal stimulation for in vitro fertilization (IVF) donated their spare oocytes for this study. INTERVENTIONS: Transmission electron microscopy (TEM) was used to analyze the fine morphology of 22 dysmorphic IVF oocytes exhibiting different types of cytoplasmic irregularities, namely, refractile bodies; centrally located cytoplasmic granularity (CLCG); smooth endoplasmic reticulum (SER) disc; and vacuoles. A total of 133 immature oocytes were exposed to cytoskeleton-targeting compounds or matured in control conditions, and their morphology was examined using fluorescent and electron microscopy. MAIN OUTCOME MEASURES: The ultrastructural morphology of dysmorphic oocytes was analyzed. Drug-treated oocytes had their maturation efficiency, chromosome-microtubule configurations, and fine intracellular morphology examined. RESULTS: TEM revealed ultrastructural characteristics of common oocyte aberrations and indicated that excessive organelle clustering was the underlying cause of 2 of the studied morphotypes. Inhibition experiments showed that disruption of actin, not microtubules, allows for inordinate aggregation of subcellular structures, resembling the ultrastructural pattern seen in morphologically abnormal oocytes retrieved in IVF cycles. These results imply that actin serves as a regulator of organelle distribution during human oocyte maturation. CONCLUSION: The ultrastructural analogy between dysmorphic oocytes and oocytes, in which actin network integrity was perturbed, suggests that dysfunction of the actin cytoskeleton might be implicated in generating common cytoplasmic aberrations. Knowledge of human oocytes' inner workings and the origin of morphological abnormalities is a step forward to a more objective oocyte quality assessment in IVF practice.
- MeSH
- aktiny * MeSH
- cytoplazma MeSH
- cytoskelet MeSH
- lidé MeSH
- mikrotubuly MeSH
- oocyty * ultrastruktura MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2 ) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator.
Central ghrelin signaling seems to play important role in addiction as well as memory processing. Antagonism of the growth hormone secretagogue receptor (GHS-R1A) has been recently proposed as a promising tool for the unsatisfactory drug addiction therapy. However, molecular aspects of GHS-R1A involvement in specific brain regions remain unclear. The present study demonstrated for the first time that acute as well as subchronic (4 days) administration of the experimental GHS-R1A antagonist JMV2959 in usual intraperitoneal doses including 3 mg/kg, had no influence on memory functions tested in the Morris Water Maze in rats as well as no significant effects on the molecular markers linked with memory processing in selected brain areas in rats, specifically on the β-actin, c-Fos, two forms of the calcium/calmodulin-dependent protein kinase II (CaMKII, p-CaMKII) and the cAMP-response element binding protein (CREB, p-CREB), within the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), dorsal striatum, and hippocampus (HIPP). Furthermore, following the methamphetamine intravenous self-administration in rats, the 3 mg/kg JMV2959 pretreatment significantly reduced or prevented the methamphetamine-induced significant decrease of hippocampal β-actin and c-Fos as well as it prevented the significant decrease of CREB in the NAC and mPFC. These results imply, that the GHS-R1A antagonist/JMV2959 might reduce/prevent some of the memory-linked molecular changes elicited by methamphetamine addiction within brain structures associated with memory (HIPP), reward (NAc), and motivation (mPFC), which may contribute to the previously observed significant JMV2959-induced reduction of the methamphetamine self-administration and drug-seeking behavior in the same animals. Further research is necessary to corroborate these results.
- MeSH
- aktiny MeSH
- ghrelin farmakologie MeSH
- krysa rodu rattus MeSH
- methamfetamin * farmakologie MeSH
- proteinkinasa závislá na vápníku a kalmodulinu typ 2 MeSH
- receptory ghrelinu * MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Recombinant adeno-associated viral vectors (AAVs) are an effective system for gene transfer. AAV serotype 2 (AAV2) is commonly used to deliver transgenes to retinal ganglion cells (RGCs) via intravitreal injection. The AAV serotype however is not the only factor contributing to the effectiveness of gene therapies. Promoters influence the strength and cell-selectivity of transgene expression. This study compares five promoters designed to maximise AAV2 cargo space for gene delivery: chicken β-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken β-actin/short β-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN). The promoters driving enhanced green fluorescent protein (eGFP) were examined in adult C57BL/6J mice eyes and tissues of the visual system. eGFP expression was strongest in the retina, optic nerves and brain when driven by the sCAG and SYN promoters. CBA, CMV, and PGK had moderate expression by comparison. The SYN promoter had almost exclusive transgene expression in RGCs. The PGK promoter had predominant expression in both RGCs and AII amacrine cells. The ubiquitous CBA, CMV, and sCAG promoters expressed eGFP in a variety of cell types across multiple retinal layers including Müller glia and astrocytes. We also found that these promoters could transduce human retina ex vivo, although expression was predominantly in glial cells due to low RGC viability. Taken together, this promoter comparison study contributes to optimising AAV-mediated transduction in the retina, and could be valuable for research in ocular disorders, particularly those with large or complex genetic cargos.
- MeSH
- aktiny genetika metabolismus MeSH
- cytomegalovirové infekce * genetika metabolismus MeSH
- Dependovirus genetika metabolismus MeSH
- genetické vektory genetika MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- Parvovirinae * genetika MeSH
- retinální gangliové buňky metabolismus MeSH
- transdukce genetická MeSH
- transgeny MeSH
- zelené fluorescenční proteiny genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Axonal swellings (AS) are one of the neuropathological hallmark of axonal injury in several disorders from trauma to neurodegeneration. Current evidence proposes a role of perturbed Ca2+ homeostasis in AS formation, involving impaired axonal transport and focal distension of the axons. Mechanisms of AS formation, in particular moments following injury, however, remain unknown. Here we show that AS form independently from intra-axonal Ca2+ changes, which are required primarily for the persistence of AS in time. We further show that the majority of axonal proteins undergoing de/phosphorylation immediately following injury belong to the cytoskeleton. This correlates with an increase in the distance of the actin/spectrin periodic rings and with microtubule tracks remodeling within AS. Observed cytoskeletal rearrangements support axonal transport without major interruptions. Our results demonstrate that the earliest axonal response to injury consists in physiological adaptations of axonal structure to preserve function rather than in immediate pathological events signaling axonal destruction.
- MeSH
- aktiny metabolismus MeSH
- axonální transport fyziologie MeSH
- axony patologie MeSH
- lidé MeSH
- spektrin * metabolismus MeSH
- traumatické poranění mozku * patologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
