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Autophagy Reports Aug 2023Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal...
Macroautophagy/autophagy is a degradative pathway that plays an important role in maintaining cellular homeostasis in eukaryotes. During autophagy, cisternal compartments called phagophores are generated to sequester intracellular components; these structures mature into autophagosomes, which deliver the cargo into lysosomes/vacuoles for degradation. Numerous autophagy-related (Atg) proteins are part of the core machinery that mediates autophagosome biogenesis. Atg9, a lipid scramblase and the only multispanning transmembrane protein among the core Atg machinery, traffics between cytoplasmic reservoirs and the phagophore assembly site (PAS) to provide membranes, recruit other Atg proteins and rearrange lipids on the phagophore membrane. However, the factors mediating Atg9 trafficking remain to be fully understood. In our recent study, we found that the yeast dynamin-like GTPase Vps1 (vacuolar protein sorting 1) is involved in autophagy and is important for Atg9 transport to the PAS. Moreover, we showed that Vps1 function in autophagy requires its GTPase and oligomerization activities. Interestingly, specific mutations in DNM2 (dynamin 2), one of the human homologs of Vps1 that have been linked with specific human diseases such as microcytic anemia and Charcot-Marie-Tooth, also impairs Atg9 transport to the PAS, suggesting that a defect in autophagy may underlay the pathophysiology of these severe human pathologies.
PubMed: 38107506
DOI: 10.1080/27694127.2023.2247309 -
Journal of Neurochemistry Feb 2024The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults,...
The aquaporin-4 (AQP4) water channel is abundantly expressed in the glial cells of the central nervous system and facilitates brain swelling following diverse insults, such as traumatic injury or stroke. Lack of specific and therapeutic AQP4 inhibitors highlights the need to explore alternative routes to control the water permeability of glial cell membranes. The cell surface abundance of AQP4 in mammalian cells fluctuates rapidly in response to changes in oxygen levels and tonicity, suggesting a role for vesicular trafficking in its translocation to and from the cell surface. However, the molecular mechanisms of AQP4 trafficking are not fully elucidated. In this work, early and recycling endosomes were investigated as likely candidates of rapid AQP4 translocation together with changes in cytoskeletal dynamics. In transiently transfected HEK293 cells a significant amount of AQP-eGFP colocalised with mCherry-Rab5-positive early endosomes and mCherry-Rab11-positive recycling endosomes. When exposed to hypotonic conditions, AQP4-eGFP rapidly translocated from intracellular vesicles to the cell surface. Co-expression of dominant negative forms of the mCherry-Rab5 and -Rab11 with AQP4-eGFP prevented hypotonicity-induced AQP4-eGFP trafficking and led to concentration at the cell surface or intracellular vesicles respectively. Use of endocytosis inhibiting drugs indicated that AQP4 internalisation was dynamin-dependent. Cytoskeleton dynamics-modifying drugs also affected AQP4 translocation to and from the cell surface. AQP4 trafficking mechanisms were validated in primary human astrocytes, which express high levels of endogenous AQP4. The results highlight the role of early and recycling endosomes and cytoskeletal dynamics in AQP4 translocation in response to hypotonic and hypoxic stress and suggest continuous cycling of AQP4 between intracellular vesicles and the cell surface under physiological conditions.
Topics: Animals; Humans; HEK293 Cells; Protein Transport; Endosomes; Endocytosis; Astrocytes; Aquaporin 4; Mammals
PubMed: 38102893
DOI: 10.1111/jnc.16029 -
Biological Research Dec 2023Growing evidence has suggested that Type I Interferon (I-IFN) plays a potential role in the pathogenesis of Down Syndrome (DS). This work investigates the underlying...
BACKGROUND
Growing evidence has suggested that Type I Interferon (I-IFN) plays a potential role in the pathogenesis of Down Syndrome (DS). This work investigates the underlying function of MX1, an effector gene of I-IFN, in DS-associated transcriptional regulation and phenotypic modulation.
METHODS
We performed assay for transposase-accessible chromatin with high-throughout sequencing (ATAC-seq) to explore the difference of chromatin accessibility between DS derived amniocytes (DSACs) and controls. We then combined the annotated differentially expressed genes (DEGs) and enriched transcriptional factors (TFs) targeting the promoter region from ATAC-seq results with the DEGs in RNA-seq, to identify key genes and pathways involved in alterations of biological processes and pathways in DS.
RESULTS
Binding motif analysis showed a significant increase in chromatin accessibility of genes related to neural cell function, among others, in DSACs, which is primarily regulated by members of the activator protein-1 (AP-1) transcriptional factor family. Further studies indicated that MX Dynamin Like GTPase 1 (MX1), defined as one of the key effector genes of I-IFN, is a critical upstream regulator. Its overexpression induced expression of AP-1 TFs and mediated inflammatory response, thus leading to decreased cellular viability of DS cells. Moreover, treatment with specific AP-1 inhibitor T-5224 improved DS-associated phenotypes in DSACs.
CONCLUSIONS
This study demonstrates that MX1-mediated AP-1 activation is partially responsible for cellular dysfunction of DS. T-5224 effectively ameliorated DS-associated phenotypes in DSACs, suggesting it as a potential treatment option for DS patients.
Topics: Humans; Transcription Factor AP-1; Chromatin Immunoprecipitation Sequencing; RNA-Seq; Down Syndrome; Chromatin; Myxovirus Resistance Proteins
PubMed: 38066591
DOI: 10.1186/s40659-023-00474-x -
Neuromuscular Disorders : NMD Feb 2024The Myotubular and Centronuclear Myopathy Registry is an international research database containing key longitudinal data on a diverse and growing cohort of individuals...
The Myotubular and Centronuclear Myopathy Registry is an international research database containing key longitudinal data on a diverse and growing cohort of individuals affected by this group of rare and ultra-rare neuromuscular conditions. It can inform and support all areas of translational research including epidemiological and natural history studies, clinical trial feasibility planning, recruitment for clinical trials or other research studies, stand-alone clinical studies, standards of care development, and provision of real-world evidence data. For ten years, it has also served as a valuable communications tool and provided a link between the scientific and patient communities. With the anticipated advent of disease-modifying therapies for these conditions, the registry is a key resource for the generation of post-authorisation data for regulatory decision-making, real world evidence, and patient-reported outcome measures. In this paper we present some key data from the current 444 registered individuals with the following genotype split: MTM1 n=270, DNM2 n=42, BIN1 n=4, TTN n=4, RYR1 n=12, other n=4, unknown n=108. The data presented are consistent with the current literature and the common understanding of a strong genotype/phenotype correlations in CNM, most notably the data supports the current knowledge that XLMTM is typically the most severe form of CNM. Additionally, we outline the ways in which the registry supports research, and, more generally, the importance of continuous investment and development to maintain the relevance of registries for all stakeholders. Further information on the registry and contact details are available on the registry website at www.mtmcnmregistry.org.
Topics: Humans; Muscle, Skeletal; Translational Research, Biomedical; Dynamin II; Genotype; Myopathies, Structural, Congenital
PubMed: 38061948
DOI: 10.1016/j.nmd.2023.10.014 -
Frontiers in Cellular and Infection... 2023Liver sinusoidal endothelial cells (LSEC) are scavenger cells with a remarkably high capacity for clearance of several blood-borne macromolecules and nanoparticles,...
Liver sinusoidal endothelial cells (LSEC) are scavenger cells with a remarkably high capacity for clearance of several blood-borne macromolecules and nanoparticles, including some viruses. Endocytosis in LSEC is mainly via the clathrin-coated pit mediated route, which is dynamin-dependent. LSEC can also be a site of infection and latency of betaherpesvirus, but mode of virus entry into these cells has not yet been described. In this study we have investigated the role of dynamin in the early stage of muromegalovirus muridbeta1 (MuHV-1, murid betaherpesvirus 1, murine cytomegalovirus) infection in mouse LSECs. LSEC cultures were freshly prepared from C57Bl/6JRj mouse liver. We first examined dose- and time-dependent effects of two dynamin-inhibitors, dynasore and MitMAB, on cell viability, morphology, and endocytosis of model ligands via different LSEC scavenger receptors to establish a protocol for dynamin-inhibition studies in these primary cells. LSECs were challenged with MuHV-1 (MOI 0.2) ± dynamin inhibitors for 1h, then without inhibitors and virus for 11h, and nuclear expression of MuHV-1 immediate early antigen (IE1) measured by immune fluorescence. MuHV-1 efficiently infected LSECs . Infection was significantly and independently inhibited by dynasore and MitMAB, which block dynamin function via different mechanisms, suggesting that initial steps of MuHV-1 infection is dynamin-dependent in LSECs. Infection was also reduced in the presence of monensin which inhibits acidification of endosomes. Furthermore, competitive binding studies with a neuropilin-1 antibody blocked LSEC infection. This suggests that MuHV-1 infection in mouse LSECs involves virus binding to neuropilin-1 and occurs via endocytosis.
Topics: Mice; Animals; Muromegalovirus; Endothelial Cells; Neuropilin-1; Liver; Dynamins
PubMed: 38029264
DOI: 10.3389/fcimb.2023.1249894 -
Molecular Biology of the Cell Feb 2024Drp1 is a dynamin family GTPase required for mitochondrial and peroxisomal division. Oligomerization increases Drp1 GTPase activity through interactions between...
Drp1 is a dynamin family GTPase required for mitochondrial and peroxisomal division. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600. Commonly, phosphorylation of S579 is considered activating, while S600 phosphorylation is considered inhibiting. However, direct effects of phosphorylation on Drp1 GTPase activity have not been investigated in detail. Here, we compare effects of S579 and S600 phosphorylation on purified Drp1, using phosphomimetic mutants and in vitro phosphorylation. Both phosphomimetic mutants are shifted toward smaller oligomers. Both phosphomimetic mutations maintain basal GTPase activity, but eliminate GTPase stimulation by actin and decrease GTPase stimulation by cardiolipin, Mff, and MiD49. Phosphorylation of S579 by Erk2 produces similar effects. When mixed with wildtype Drp1, both S579D and S600D phosphomimetic mutants reduce the actin-stimulated GTPase activity of Drp1-WT. Conversely, a Drp1 mutant (K38A) lacking GTPase activity stimulates Drp1-WT GTPase activity under both basal and actin-stimulated conditions. These results suggest that the effect of S579 phosphorylation is not to activate Drp1 directly. In addition, our results suggest that nearest neighbor interactions within the Drp1 oligomer affect catalytic activity.
Topics: Actins; Cardiolipins; Phosphorylation; Dynamins; GTP Phosphohydrolases; Mitochondrial Proteins; Mitochondrial Dynamics
PubMed: 38019609
DOI: 10.1091/mbc.E23-11-0427 -
MBio Nov 2023Mitochondria constitute major sources of HO and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in...
Mitochondria constitute major sources of HO and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in cell biology and relies on highly regulated mechano-GTPases that are oligomerization dependent and belong to the dynamin-related protein family, like DnmA. Our previous work demonstrated that HO induces mitochondrial constriction, division, and remodeling of the outer membrane. Here, we show that HO also induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. The study of this response uncovered that HO induces the depolymerization and reorganization of actin as well as the critical role that cysteines 450 and 776 play in DnmA function. Our results provide new insights into the mechanisms of reactive oxygen species cell signaling and how they can regulate the dynamics of the actin cytoskeleton and the division of mitochondria and peroxisomes.
PubMed: 38014993
DOI: 10.1128/mbio.02822-23 -
Acta Biochimica Et Biophysica Sinica Jan 2024Epithelial-mesenchymal transformation (EMT) plays an important role in the progression of diabetic nephropathy. Dexmedetomidine (DEX) has shown renoprotective effects...
Epithelial-mesenchymal transformation (EMT) plays an important role in the progression of diabetic nephropathy. Dexmedetomidine (DEX) has shown renoprotective effects against ischemic reperfusion injury; however, whether and how DEX prevents high glucose-induced EMT in renal tubular epithelial cells is incompletely known. Here, we conduct experiments using HK-2 cells, a human tubular epithelial cell line. Our results demonstrate that high glucose increases the expressions of EMT-related proteins, including Vimentin, Slug, Snail and Twist, while decreasing the expression of E-cadherin and increasing Cdk5 expression in HK-2 cells. Both knockdown and inhibition by roscovitine increase the expressions of E-cadherin while decreasing the expressions of other EMT-related markers. DEX inhibits Cdk5 expression without affecting cell viability and changes the expressions of EMT-related markers, similar to effects of Cdk5 inhibition. Furthermore, Cdk5 is found to interact with Drp1 at the protein level and mediate the phosphorylation of Drp1. In addition, Drp1 inhibition with mdivi-1 could also restrain the high glucose-induced EMT process in HK-2 cells. Immunofluorescence results show that roscovitine, Mdivi-1 and DEX inhibit high glucose-induced intracellular ROS accumulation, while the oxidant H O eliminates the protective effect of DEX on the EMT process. These results indicate that DEX mitigates high glucose-induced EMT progression in HK-2 cells via inhibition of the Cdk5/Drp1/ROS pathway.
Topics: Humans; Cadherins; Dexmedetomidine; Epithelial Cells; Epithelial-Mesenchymal Transition; Glucose; Reactive Oxygen Species; Roscovitine; Signal Transduction; Cyclin-Dependent Kinase 5; Dynamins
PubMed: 38013469
DOI: 10.3724/abbs.2023220 -
The Journal of Cell Biology Dec 2023Live super-resolution microscopy has allowed for new insights into recently identified mitochondria-lysosome contact sites, which mediate crosstalk between mitochondria...
Live super-resolution microscopy has allowed for new insights into recently identified mitochondria-lysosome contact sites, which mediate crosstalk between mitochondria and lysosomes, including co-regulation of Rab7 GTP hydrolysis and Drp1 GTP hydrolysis. Here, we highlight recent findings and future perspectives on this dynamic pathway and its roles in health and disease.
Topics: Guanosine Triphosphate; Lysosomes; Microscopy; Mitochondria; Mitochondrial Membranes; rab7 GTP-Binding Proteins; Dynamins
PubMed: 37917024
DOI: 10.1083/jcb.202305032 -
Advanced Science (Weinheim,... Dec 2023Excessive mitochondrial fission following ischemia and hypoxia relies on the formation of contacts between the endoplasmic reticulum and mitochondria (ER-Mito); however,...
Excessive mitochondrial fission following ischemia and hypoxia relies on the formation of contacts between the endoplasmic reticulum and mitochondria (ER-Mito); however, the specific mechanisms behind this process remain unclear. Confocal microscopy and time course recording are used to investigate how ischemia and hypoxia affect the activation of dynamin-related protein 1 (Drp1), a protein central to mitochondrial dynamics, ER-Mito interactions, and the consequences of modifying the expression of Drp1, shroom (Shrm) 4, and inverted formin (INF) 2 on ER-Mito contact establishment. Both Drp1 activation and ER-Mito contact initiation cause excessive mitochondrial fission and dysfunction under ischemic-hypoxic conditions. The activated form of Drp1 aids in ER-Mito contact initiation by recruiting Shrm4 and promoting actin bundling between the ER and mitochondria. This process relies on the structural interplay between INF2 and scattered F-actin on the ER. This study uncovers new roles of cytoplasmic Drp1, providing valuable insights for devising strategies to manage mitochondrial imbalances in the context of ischemic-hypoxic injury.
Topics: Humans; Actins; Dynamins; Mitochondria; Endoplasmic Reticulum; Ischemia; Hypoxia
PubMed: 37909346
DOI: 10.1002/advs.202304885