Dynamin Inhibitor Research Articles

Intersectin-1 enhances calcium-dependent replenishment of the readily releasable pool of synaptic vesicles during development.

Intersectin-1 (Itsn1) is a scaffold protein that plays a key role in coupling exocytosis and endocytosis of synaptic vesicles (SVs). However, it is unclear whether and how Itsn1 regulates these processes to support efficient neurotransmission during development. To address this, we examined the calyx of Held synapse in the auditory brainstem of wild-type and Itsn1 mutant mice before (immature) and after (mature) the onset of hearing. Itsn1 was present in the pre- and postsynaptic compartments at both developmental stages. Loss of function of Itsn1 did not alter presynaptic action potentials, Ca2+ entry via voltage-gated Ca2+ channels (VGCCs), transmitter release or short-term depression (STD) induced by depletion of SVs in the readily releasable pool (RRP) in either age group. Yet, fast Ca2+-dependent recovery from STD was attenuated in mature mutant synapses, while it was unchanged in immature mutant synapses. This deficit at mature synapses was rescued by introducing the DH-PH domains of Itsn1 into the presynaptic terminals. Inhibition of dynamin, which interacts with Itsn1 during endocytosis, had no effect on STD recovery. Interestingly, we found a developmental enrichment of Itsn1 near VGCCs, which may underlie the Itsn1-mediated fast replenishment of the RRP. Consequently, the absence of Itsn1 in mature synapses led to a higher failure rate of postsynaptic spiking during high-frequency synaptic transmission. Taken together, our findings suggest that Itsn1 translocation to the vicinity of VGCCs during development is crucial for accelerating Ca2+-dependent RRP replenishment and sustaining high-fidelity neurotransmission. KEY POINTS: Itsn1 is expressed in the pre- and postsynaptic compartments of the calyx of Held synapse. Developmental upregulation of vesicular glutamate transporter-1 is Itsn1 dependent. Itsn1 does not affect basal synaptic transmission at different developmental stages. Itsn1 is required for Ca2+-dependent recovery from short-term depression in mature synapses. Itsn1 mediates the recovery through its DH-PH domains, independent of its interactive partner dynamin. Itsn1 translocates to the vicinity of presynaptic Ca2+ channels during development. Itsn1 supports high-fidelity neurotransmission by enabling rapid recovery from vesicular depletion during repetitive activity.

Engineering synthetic agonists for targeted activation of Notch signaling.

Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although there are several classes of Notch inhibitors, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins by tethering affinity-matured Notch ligands to antibodies or cytokines that internalize their targets. This bispecific format enables SNAGs to "pull" on mechanosensitive Notch receptors, triggering their activation in the presence of a desired biomarker. We successfully developed SNAGs targeting six independent surface markers, including the tumor antigens PDL1, CD19, and HER2, and the immunostimulatory receptor CD40. SNAGs targeting CD40 increase expansion of central memory γδ T cells from peripheral blood, highlighting their potential to improve the phenotype and yield of low-abundance T cell subsets. These insights have broad implications for the pharmacological activation of mechanoreceptors and will expand our ability to modulate Notch signaling in biotechnology.

Abstract P430: Angiotensin-(1-7) Induces A Decrease In Proinflammatory Genes Expression Through A Mas Receptor-Mediated Translocation To Brainstem Nuclei Of Spontaneously Hypertensive Rats

Mas receptor (MasR) displays differential trafficking in brainstem neurons from spontaneously hypertensive rats (SHRs): the fraction of dynamin-mediated internalized MasRs into early endosomes was larger and the fraction of MasRs recycled back to the plasma membrane was smaller in SHR than in Wistar neurons. Surprisingly, in SHR neurons but not in Wistar neurons, Ang-(1-7) induced MasR translocation to the nucleus. Our aim was to investigate the biological response elicited by MasR in the nucleus and whether MasR translocation was due to interaction with other receptors. We evaluated: 1) nitric oxide (NO) generation by DAF (diaminofluorescein) fluorescence; 2) interleukin (IL) 6, IL-1β, cyclooxygenase 2 (COX2) and NFkB expression by real time PCR; and 3) MasR interaction with AT1R and the dopamine type 2 receptor (D2R) by proximity ligation assay in brainstem neuronal primary cultures from Wistar and SHR rats incubated in the presence or absence of Ang-(1-7) or Ang-(1-7) + dynasore (DYN) (dynamin inhibitor) or Ang-(1-7) + A779 (MasR antagonist) during 30 min. Ang-(1-7) (100 nM) induced a decrease in the pro-inflammatory factors IL-6, IL-1β, COX2 and NFkB expression in brainstem neurons from Wistar and SHR rats which was blocked by A779, suggesting a MasR-mediated response. The effect of Ang-(1-7) was prevented when MasR internalization was prevented by DYN only in SHRs neurons but not in Wistar neurons, suggesting that the effect of Ang-(1-7) in SHR neurons is mediated by MasR translocation to the nucleus. Treatment of neurons with Ang-(1-7) induced an increase in NO in the nucleus of SHR but not of Wistar neurons and this effect was prevented by DYN and A779, suggesting that the Ang-(1-7)-induced NO production in the nucleus was due to MasR translocation to this compartment. MasR stimulation with Ang-(1-7) induced an increase in MasR-D2R heteromerization in SHR but not in Wistar neurons, and no change in MasR-AT1R heteromerization. Ou study showed that MasR translocation to the nucleus resulted in a reduction in pro-inflammatory genes expression and an increase in NO generation in SHR neurons. A fraction of MasR was translocated through the interaction with D2Rs, suggesting that this heteomer may be involved in the anti-inflammatory responses elicited by Mas translocation to the nucleus.

A Drosophila model for mechanistic investigation of tau protein spread.

Brain protein aggregates are a hallmark of neurodegenerative disease. Previous work indicates that specific protein components of these aggregates are toxic, including tau (encoded by MAPT) in Alzheimer's disease and related tauopathies. Increasing evidence also indicates that these toxic proteins traffic between cells in a prion-like fashion, thereby spreading pathology from one brain region to another. However, the mechanisms involved in trafficking are poorly understood. We therefore developed a transgenic Drosophila model to facilitate rapid evaluation of candidate tau trafficking modifiers. Our model uses the bipartite Q system to drive co-expression of tau and GFP in the fly eye. We found age-dependent spread of tau into the brain, represented by detection of tau, but not of GFP. We also found that tau trafficking was attenuated upon inhibition of the endocytic factor dynamin (encoded by shi) or knockdown of glycogen synthase kinase-3β (GSK-3β, encoded by sgg). Further work revealed that dynamin promoted tau uptake in recipient tissues, whereas GSK-3β appeared to promote tau spread via direct phosphorylation of tau. Our robust and flexible system will promote the identification of tau-trafficking components involved in the pathogenesis of neurodegenerative diseases.

Three-Dimensional Lymphatics-on-a-Chip Reveals Distinct, Size-Dependent Nanoparticle Transport Mechanisms in Lymphatic Drug Delivery.

Although nanoparticle-based lymphatic drug delivery systems promise better treatment of cancer, infectious disease, and immune disease, their clinical translations are limited by low delivery efficiencies and unclear transport mechanisms. Here, we employed a three-dimensional (3D) lymphatics-on-a-chip featuring an engineered lymphatic vessel (LV) capable of draining interstitial fluids including nanoparticles. We tested lymphatic drainage of different sizes (30, 50, and 70 nm) of PLGA-b-PEG nanoparticles (NPs) using the lymphatics-on-a-chip device. In this study, we discovered that smaller NPs (30 and 50 nm) transported faster than larger NPs (70 nm) through the interstitial space, as expected, but the smaller NPs were captured by lymphatic endothelial cells (LECs) and accumulated within their cytosol, delaying NP transport into the lymphatic lumen, which was not observed in larger NPs. To examine the mechanisms of size-dependent NP transports, we employed four inhibitors, dynasore, nystatin, amiloride, and adrenomedullin, to selectively block dynamin-, caveolin-, macropinocytosis-mediated endocytosis-, and cell junction-mediated paracellular transport. Inhibiting dynamin using dynasore enhanced the transport of smaller NPs (30 and 50 nm) into the lymphatic lumen, minimizing cytosolic accumulation, but showed no effect on larger NP transport. Interestingly, the inhibition of caveolin by nystatin decreased the lymphatic transport of larger NPs without affecting the smaller NP transport, indicating distinct endocytosis mechanisms used by different sizes of NPs. Macropinocytosis inhibition by amiloride did not change the drainage of all sizes of NPs; however, paracellular transport inhibition by adrenomedullin blocked the lymphatic transport of NPs of all sizes. We further revealed that smaller NPs were captured in the Rab7-positive late-stage lymphatic endosomes to delay their lymphatic drainage, which was reversed by dynamin inhibition, suggesting that Rab7 is a potential target to enhance the lymphatic delivery of smaller NPs. Together, our 3D lymphatics-on-a-chip model unveils size-dependent NP transport mechanisms in lymphatic drug delivery.

The host GTPase Dynamin 2 modulates apical junction structure to control cell-to-cell spread of Listeria monocytogenes.

The food-borne pathogen Listeria monocytogenes uses actin-based motility to generate plasma membrane protrusions that mediate the spread of bacteria between host cells. In polarized epithelial cells, efficient protrusion formation by L. monocytogenes requires the secreted bacterial protein InlC, which binds to a carboxyl-terminal Src homology 3 (SH3) domain in the human scaffolding protein Tuba. This interaction antagonizes Tuba, thereby diminishing cortical tension at the apical junctional complex and enhancing L. monocytogenes protrusion formation and spread. Tuba contains five SH3 domains apart from the domain that interacts with InlC. Here, we show that human GTPase Dynamin 2 associates with two SH3 domains in the amino-terminus of Tuba and acts together with this scaffolding protein to control the spread of L. monocytogenes. Genetic or pharmacological inhibition of Dynamin 2 or knockdown of Tuba each restored normal protrusion formation and spread to a bacterial strain deleted for the inlC gene (∆inlC). Dynamin 2 localized to apical junctions in uninfected human cells and protrusions in cells infected with L. monocytogenes. Localization of Dynamin 2 to junctions and protrusions depended on Tuba. Knockdown of Dynamin 2 or Tuba diminished junctional linearity, indicating a role for these proteins in controlling cortical tension. Infection with L. monocytogenes induced InlC-dependent displacement of Dynamin 2 from junctions, suggesting a possible mechanism of antagonism of this GTPase. Collectively, our results show that Dynamin 2 cooperates with Tuba to promote intercellular tension that restricts the spread of ∆inlC Listeria. By expressing InlC, wild-type L. monocytogenes overcomes this restriction.

Dynamin 2-mediated endocytosis of BLT1 is required for IL-8 production in HMC-1 cells induced by Trichomonas vaginalis-derived secretory products.

We previously reported that leukotriene B4 (LTB4) contained in Trichomonas vaginalis-derived secretory products (TvSP) play an essential role in interleukin-8 (IL-8) production in human mast cell line (HMC-1 cells) via LTB4 receptor (BLT)-mediated Nuclear Factor-kappa B (NF-кB) activation. Dynamin, a GTPase, has been known to be involved in endocytosis of receptors for signaling of production of cytokine or chemokines. In the present study, we investigated the role of dynamin-mediated BLT1 endocytosis in TvSP-induced IL-8 production. When HMC-1 cells were transfected with BLT1 or BLT2 siRNA, TvSP-induced IL-8 production was significantly inhibited compared with that in cells transfected with control siRNA. In addition, pretreatment of HMC-1 cells with a dynamin inhibitor (Dynasore) reduced IL-8 production induced by TvSP or LTB4. TvSP- or LTB4- induced phosphorylation of NF-кB was also attenuated by pretreatment with Dynasore. After exposing HMC-1 cells to TvSP or LTB4, BLT1 was translocated from the intracellular compartments to the plasma membrane within 30 min. At 60 min after stimulation with TvSP or LTB4, BLT1 remigrated from the cell surface to intracellular areas. Pretreatment of HMC-1 cells with dynamin-2 siRNA blocked internalization of BLT1 induced by TvSP or LTB4. Co-immunoprecipitation experiments revealed that dynamin-2 strongly interacted with BLT1 60 min after stimulation with TvSP or LTB4. These results suggest that T. vaginalis-secreted LTB4 induces IL-8 production in HMC-1 cells via dynamin 2-mediated endocytosis of BLT1 and phosphorylation of NF-кB.

Dissecting the roles of dynamin and clathrin in platelet pinocytosis

Platelets endocytose many molecules from their environment. However, this process of pinocytosis in platelets is poorly understood. Key endocytic regulators such as dynamin, clathrin, CDC42 and Arf6 are expressed in platelets but their roles in pinocytosis is not known. Stimulated platelets form two subpopulations of pro-aggregatory and procoagulant platelets. The effect of stimulation on pinocytosis is also poorly understood. In this study, washed human platelets were treated with a range of endocytosis inhibitors and stimulated using different activators. The rate of pinocytosis was assessed using pHrodo green, a pH-sensitive 10 kDa dextran. In unstimulated platelets, pHrodo fluorescence increased over time and accumulated as intracellular puncta indicating constituently active pinocytosis. Stimulated platelets (both pro-aggregatory and procoagulant) had an elevated pinocytosis rate compared to unstimulated platelets. Dynamin inhibition blocked pinocytosis in unstimulated, pro-aggregatory and procoagulant platelets indicating that most platelet pinocytosis is dynamin dependent. Although pinocytosis was clathrin-independent in unstimulated and procoagulant populations, clathrin partially contributed to pinocytosis in pro-aggregatory platelets.

Velogenic Newcastle disease virus invades chicken brain by infecting brain microvascular endothelial cells to increase blood-brain barrier permeability1

The blood-brain barrier (BBB) keeps poisons and infections out of the brain. Some viruses can pass through this barrier and replicate in the central nervous system (CNS). Velogenic Newcastle disease virus (VNDV) is a neurotropic virus that causes avian nonsuppurative encephalitis. VNDV often develops into a chronic infection that seriously affects poultry health in partially immune birds. The routes by which the virus enters the chicken brain are poorly understood. In this study, we discovered that VNDV increased BBB permeability in vivo and in vitro by breaking the tight junction protein zona occludens-1 (ZO-1) continuity of chicken brain microvascular endothelial cells (chBMECs). By investigating the susceptibility of chBMECs to NDV infection, we found that VNDV but not lentogenic NDV was detected in the basolateral compartment in transwell assays after apical infection, suggesting efficient replication and transcellular transport of the virus across the BBB in vitro. Furthermore, viral replication and BBB permeability were reduced during the early stage of infection by using the dynamin inhibitor dynasore. Our data demonstrate that VNDV invades the chicken brain by infecting and damaging the tight junction of chBMECs directly to increase BBB permeability. VNDV could infect chBMECs via endocytosis. As a result, our findings provide compelling evidence for VNDV entrance into the brain via the BBB, paving the way for the development of medications for NDV prevention and therapy.

Neonatal Human Enteroids Take Up Human Milk Extracellular Vesicles That Survive Neonatal Human Digestion

Background: Human milk (HM) is the ideal infant nutrition and reduces infant death and disease. HM contains thousands of bioactive components, including extracellular vesicles (HMEVs). HMEVs are lipid bilayer-encased nanoparticles released from mammary epithelial cells that carry biological cargo to the infant. Pre-clinical models suggest that HMEVs may enhance intestinal function and limit inflammation; however, it is unknown if HMEVs or their cargo survive neonatal human digestion. This limits the ability to leverage HMEV cargo as additives to infant nutrition or as therapeutics. This study aims to develop a neonatal enteroid model and EV isolation pipeline to study HMEV-intestinal epithelial cell (IEC) interactions with the ultimate goal of therapy development. Hypothesis: HMEVs survive in vivo neonatal digestion to be taken up by infant IECs. Methods: All studies were conducted under approved IRB protocols. Neonatal intestinal contents (digesta) were collected after gastric feeding from post-pyloric tubes. HMEVs were isolated from raw and pasteurized human milk and digesta (n=3 each) by density-gradient ultracentrifugation following two-step skimming, acid precipitation of caseins, and multi-step filtration. EVs were validated by electron microscopy, nanoparticle tracking analysis (NTA), and western blotting. HMEV sub-populations were examined by super-resolution microscopy. HMEV uptake was tested using apical-out neonatal human enteroids isolated from a 2-month-old patient undergoing surgery for ileal atresia and validated by qPCR for proliferation and differentiation markers (mean ± SEM). Enteroid uptake was assessed with CMTPX dye-labeled-HMEVs, dynamin inhibitor, and confocal microscopy. Results: Our HMEV isolation pipeline reliably isolates HMEVs exhibiting classic EV morphology and enriched in extracellular vesicle markers CD81 and CD9, but depleted of b-casein and lactalbumin from HM and digesta. NTA data indicates that only 17.8 ± 2.9% (p>0.0001) of input HMEVs survive to reach the neonatal intestine. Human mammary gland-derived protein BTN1A1 is present in digesta EVs. Neonatal apical-out human enteroids represent a less proliferative and more differentiated epithelium, e.g. down-regulated KI67 (0.05 ± 0.03-fold relative to basal-out, p<0.0001, n=4-6) and elevated CHGA (14.22 ± 9.30-fold relative to basal-out, p=0.0072, n=4-6) as measured by qRT-PCR and normalized to housekeeping gene RPLP0. Neonatal enteroids take up digested HMEVs largely via dynamin-mediated endocytosis, as measured by CMTPX dye intensity normalized to enteroid area using Fiji (2.05 x 105 ± 2.95 x 104 vs 5.38 x 104 ± 6.08 x 103, p<0.0001, n=62-115 enteroids, HMEV-treatment alone and HMEVs + dynamin inhibitor, respectively).Conclusion: Our data suggest that HMEVs survive to the neonatal intestine and can be absorbed by neonatal enteroids largely via dynamin-mediated endocytosis. These data are an important first step to leveraging HMEVs and their cargo to treat neonatal intestinal inflammation. Funding: NIH K01DK129401, NIH R01HD097367, USDA NIFA, Collins Medical Trust, Medical Research Foundation, OHSU Exploratory Research Seed Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mechanisms of ingested polystyrene micro-nanoplastics (MNPs) uptake and translocation in an in vitro tri-culture small intestinal epithelium

Micro and nanoplastics (MNPs) are now ubiquitous contaminants of food and water. Many cellular and animal studies have shown that ingested MNPs can breach the intestinal barrier to reach the circulation. To date however, the cellular mechanisms involved in intestinal absorption of MNPs have not been investigated with physiologically relevant models, and thus remain unknown. We employed in vitro simulated digestion, a tri-culture small intestinal epithelium model, and a panel of inhibitors to assess the contributions of the possible mechanisms to absorption of 26 nm carboxylated polystyrene (PS26C) MNPs. Inhibition of ATP synthesis reduced translocation by only 35 %, suggesting uptake by both active endocytic pathways and passive diffusion. Translocation was also decreased by inhibition of dynamin and clathrin, suggesting involvement of clathrin mediated endocytosis (CME) and fast endophilin-mediated endocytosis (FEME). Inhibition of actin polymerization also significantly reduced translocation, suggesting involvement of macropinocytosis or phagocytosis. However, inhibition of the Na+-H+ exchanger had no effect on translocation, thus ruling out macropinocytosis. Together these results suggest uptake by passive diffusion as well as by active phagocytosis, CME, and FEME pathways. Further studies are needed to assess uptake mechanisms for other environmentally relevant MNPs as a function of polymer, surface chemistry, and size.

Live Imaging and Analysis of Meiotic Cytokinesis in Drosophila Testes.

All living organisms require the division of a cell into daughter cells for their growth and maintenance. During cell division, both genetic and cytoplasmic contents are equally distributed between the two daughter cells. At the end of cell division, cytoplasmic contents and the plasma membrane are physically separated between the two daughter cells via a process known as cytokinesis. Hundreds of proteins and lipids involved in the cytokinetic process have been identified; however, much less is known about the mechanisms by which these molecules regulate cytokinesis, being therefore an intense area of current research. Male meiotic cytokinesis in Drosophila melanogaster testes has been shown to be an excellent model to study cytokinesis in vivo. Currently, several excellent protocols are available to study cytokinesis in Drosophila testes. However, improved methods are required to study cytokinesis under in vitro and ex vivo conditions. Here, we demonstrate a simple method to perform live imaging on individual spermatocyte cysts isolated from adult testes. We evaluate amenability of this in vitro method for treatment with pharmacological agents. We show that cytokinesis is strongly inhibited upon treatment with Dynasore, a dynamin inhibitor known to block clathrin-mediated endocytosis. In addition, we also demonstrate an ex vivo method to perform live imaging on whole mount adult testes on gas permeable membrane chambers. We believe the protocols described here are valuable tools to study cytokinetic mechanisms under various genetic and treatment conditions. Key features • In vitro method to study male meiotic cytokinesis in dissected spermatocyte cysts. • In vitro method allows acute treatment with various pharmacological agents to study cytokinesis. • Ex vivo method to image male meiosis cytokinesis in intact adult testes. • Requires 15-60 min to set up and could be imaged up to 6-12 h.

The dynamin inhibitor, dynasore, prevents zoledronate-induced viability loss in human gingival fibroblasts by partially blocking zoledronate uptake and inhibiting endosomal acidification.

For treatment of medication-related osteonecrosis of the jaw, one proposed approach is the use of a topical agent to block entry of these medications in oral soft tissues. We tested the ability of phosphonoformic acid (PFA), an inhibitor of bisphosphonate entry through certain sodium-dependent phosphate contransporters (SLC20A1, 20A2, 34A1-3) as well as Dynasore, a macropinocytosis inhibitor, for their abilities to prevent zoledronate-induced (ZOL) death in human gingival fibroblasts (HGFs). MTT assay dose-response curves were performed to determine non-cytotoxic levels of both PFA and Dynasore. In the presence of 50 μM ZOL, optimized PFA and Dynasore doses were tested for their ability to restore HGF viability. To determine SLC expression in HGFs, total HGF RNA was subjected to quantitative real-time RT-PCR. Confocal fluorescence microscopy was employed to see if Dynasore inhibited macropinocytotic HGF entry of AF647-ZOL. Endosomal acidification in the presence of Dynasore was measured by live cell imaging utilizing LysoSensor Green DND-189. As a further test of Dynasore's ability to interfere with ZOL-containing endosomal maturation, perinuclear localization of mature endosomes containing AF647-ZOL or TRITC-dextran as a control were assessed via confocal fluorescence microscopy with CellProfiler™ software analysis of the resulting photomicrographs. 0.5 mM PFA did not rescue HGFs from ZOL-induced viability loss at 72 hours while 10 and 30 μM geranylgeraniol did partially rescue. HGFs did not express the SLC transporters as compared to the expression in positive control tissues. 10 μM Dynasore completely prevented ZOL-induced viability loss. In the presence of Dynasore, AF647-ZOL and FITC-dextran co-localized in endosomes. Endosomal acidification was inhibited by Dynasore and perinuclear localization of both TRITC-dextran- and AF647-ZOL-containing endosomes was inhibited by 30 μM Dynasore. Dynasore prevents ZOL-induced viability loss in HGFs by partially interfering with macropinocytosis and by inhibiting the endosomal maturation pathway thought to be needed for ZOL delivery to the cytoplasm.

Dyngo-4a Induces Neuroblastoma Cell Differentiation Through The AKT and ERK1/2 Pathway.

The aim of the study is to check whether dyngo-4a can inhibit neuroblastoma (NB) proliferation and induce NB cell differentiation Background: Dynamin plays a role in regulating neurotransmission, signaling pathways, nutrient uptake, and pathogen infection, enhancing cell proliferation, tumor invasion, and metastasis. Studies have reported that dyngo-4a, a dynamin inhibitor, can be used to identify potential biomarkers and promising novel therapeutic targets for cancer treatment. To our knowledge, no published reports are showing that dynamin inhibitors can reduce NB cell proliferation and induce differentiation. In this study, we report that dyngo-4a can inhibit NB proliferation and induce NB cell differentiation. In this study, mouse neuroblastoma (Neuro-2a) cells were cultured in the presence or absence of dyngo-4a or retinoic acid (RA), or in the presence of both dyngo-4a and RA, or in the presence of sequential administration of dyngo-4a and RA to compare the effects on the inhibition of cell proliferation and effects on neuroblastoma cell differentiation induction. The neural cell markers, Nestin and Tuj 1 (Neuron-specific class III beta-tubulin), were used to demonstrate that the differentiated cells have neuronal cell features. The phosphorylation of Protein Kinase B (AKT), extracellular signalregulated kinases1/2 (ERK1/2), and epidermal growth factor receptor (EGFR) were determined to examine the potential mechanisms of induced differentiation. Dyngo-4a or RA or dyngo-4a with subsequent RA administration induced Neuro-2a cell differentiation. However, RA with subsequent dyngo-4a administration results in almost total death of the Neuro-2a cells. The differentiation rate induced by dyngo-4a was significantly higher than the rate by RA treatment (72.5 ± 1.4% vs. 52.9 ± 3.1% with neuron features, P<0.05; 39.0 ± 0.8% vs. 29.9 ± 1.8% for axons under light microscopy, p<0.05). The differentiation rate of cells treated with dyngo-4a first, followed by RA, was greater than when they were added together (74.8 ± 3.8% vs. 10.6 ± 3.6%; 45.5 ± 1.6% vs. 12.4 ± 0.6%, p<0.01). Co-administration of dyngo-4a and RA at the same time diminished differentiation efficacy significantly. Dyngo-4a induced Neuro-2a cell differentiation and increased Tuj-1 positive staining by the 6th day post- treatment. Dyngo-4a also inhibited Neuro-2a cell proliferation in a dose-dependent manner. Regarding the mechanism, dyngo-4a treatment showed a significant increase in p-AKT and p-ERK1/2 but not in p-EGFR. At a level comparable to RA, dynamin inhibition with dyngo-4a lowers proliferation and causes differentiation of Neuro-2a mouse NB cells in vitro. The AKT pathway is activated by dynago- 4a, which results in differentiation. The combination of RA with dynago-4a reduces the efficiency of differentiation. The application of dynago-4a followed by RA, on the other hand, enhances the differentiating effect, implying alternative mechanistic roles in the process.

Infection of liver sinusoidal endothelial cells with Muromegalovirus muridbeta1 involves binding to neuropilin-1 and is dynamin-dependent.

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 in vitro. 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.

Biogenesis, inheritance, and 3D ultrastructure of the microsporidian mitosome.

During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar genome, and most canonical functions. Here, we combine electron tomography, stereology, immunofluorescence microscopy, and bioinformatics to characterise mechanisms of growth, division, and inheritance of this minimal mitochondrion in two microsporidia species (grown within a mammalian RK13 culture-cell host). Mitosomes of Encephalitozoon cuniculi (2-12/cell) and Trachipleistophora hominis (14-18/nucleus) displayed incremental/non-phasic growth and division and were closely associated with an organelle identified as equivalent to the fungal microtubule-organising centre (microsporidian spindle pole body; mSPB). The mitosome-mSPB association was resistant to treatment with microtubule-depolymerising drugs nocodazole and albendazole. Dynamin inhibitors (dynasore and Mdivi-1) arrested mitosome division but not growth, whereas bioinformatics revealed putative dynamins Drp-1 and Vps-1, of which, Vps-1 rescued mitochondrial constriction in dynamin-deficient yeast (Schizosaccharomyces pombe). Thus, microsporidian mitosomes undergo incremental growth and dynamin-mediated division and are maintained through ordered inheritance, likely mediated via binding to the microsporidian centrosome (mSPB).

ORFV entry into host cells via clathrin-mediated endocytosis and macropinocytosis

Orf virus (ORFV), also known as infectious pustular virus, leads to an acute contagious zoonotic infectious disease. ORFV can directly contact and infect epithelial cells of skin and mucosa, causing damage to tissue cells. So far, the pathway of ORFV entry into cells is unclear. Therefore, finding the internalization pathway of ORFV will help to elucidate the cellular and molecular mechanisms of ORFV infection and invasion, which in turn will provide a certain reference for the prevention and treatment of ORFV. In the present study, chemical inhibitors were used to analyze the mechanism of ORFV entry into target cells. The results showed that the inhibitor of clathrin-mediated endocytosis could inhibit ORFV entry into cells. However, the inhibitor of caveolae-mediated endocytosis cannot inhibit ORFV entry into cells. In addition, inhibition of macropinocytosis pathway also significantly reduced ORFV internalization. Furthermore, the inhibitors of acidification and dynamin also prevented ORFV entry. However, results demonstrated that inhibitors inhibited ORFV entry but did not inhibit ORFV binding. Notably, extracellular trypsin promoted ORFV entry into cells directly, even when the endocytic pathway was inhibited. In conclusion, ORFV enters into its target cells by clathrin-mediated endocytosis and macropinocytosis, while caveolae-dependent endocytosis has little effects on this process. In addition, the entry into target cells by ORFV required an acid environment and the effect of dynamin. Meanwhile, we emphasize that broad-spectrum antiviral inhibitors and extracellular enzyme inhibitors are likely to be effective strategies for the prevention and treatment of ORFV infection.

Evidence for a clathrin-independent endocytic pathway for APP internalization in the neuronal somatodendritic compartment

Amyloid precursor protein (APP) internalization via clathrin-/dynamin-mediated endocytosis (CME) mediated by its YENPTY motif into endosomes containing β-secretase is proposed to be critical for amyloid-beta (Aβ) production. Here, we show that somatodendritic APP internalization in primary rodent neurons is not blocked by inhibiting dynamin or mutating the YENPTY motif, in contrast to non-neuronal cell lines. These phenomena, confirmed in induced human neurons under dynamin inhibition, occur during basal conditions and chemical long-term-depression stimulus, pointing to a clathrin-independent internalization pathway for somatodendritic APP. Mutating the YENPTY motif does not alter APP recycling, degradation, or endolysosomal colocalization. However, both dynamin inhibition and the YENPTY mutant significantly decrease secreted Aβ in neurons, suggesting that internalized somatodendritic APP may not constitute a major source of Aβ. Interestingly, like APP, somatodendritic low-density lipoprotein receptor (LDLR) internalization does not require its CME motif. These results highlight intriguing differences in neuronal internalization pathways and refine our understanding of Aβ production and secretion.

ATP Consumption Is Coupled with Endocytosis in Exudated Neutrophils.

Neutrophil energy metabolism during phagocytosis has been previously reported, and adenosine triphosphate (ATP) plays a crucial role in endocytosis. Neutrophils are prepared by intraperitoneal injection of thioglycolate for 4 h. We previously reported a system established for measuring particulate matter endocytosis by neutrophils using flow cytometry. In this study, we utilized this system to investigate the relationship between endocytosis and energy consumption in neutrophils. A dynamin inhibitor suppressed ATP consumption triggered by neutrophil endocytosis. In the presence of exogenous ATP, neutrophils behave differently during endocytosis depending on ATP concentration. The inhibition of ATP synthase and nicotinamide adenine dinucleotide phosphate oxidase but not phosphatidylinositol-3 kinase suppresses neutrophil endocytosis. The nuclear factor kappa B was activated during endocytosis and inhibited by I kappa B kinase (IKK) inhibitors. Notably, IKK inhibitors restored endocytosis-triggered ATP consumption. Furthermore, data from the NLR family pyrin domain containing three knockout mice suggest that inflammasome activation is not involved in neutrophil endocytosis or concomitant ATP consumption. To summarize, these molecular events occur via endocytosis, which is closely related to ATP-centered energy metabolism.

Dynamin-related protein 2 interacts with the membrane-associated methyltransferase domain of plantago asiatica mosaic virus replicase and promotes viral replication

Positive-strand RNA viruses replicate their RNA in the viral replication complex, a spherical structure formed by remodeling of host intracellular membranes. This process also requires the interaction between viral membrane-associated replication proteins and host factors. We previously identified the membrane-associated determinant of the replicase of plantago asiatica mosaic virus (PlAMV), a positive-strand RNA virus of the genus Potexvirus, in its methyltransferase (MET) domain, and suggested that its interaction with host factors is required to establish viral replication. Here we identified Nicotiana benthamiana dynamin-related protein 2 (NbDRP2) as an interactor of the MET domain of the PlAMV replicase by co-immunoprecipitation (Co-IP) and mass spectrometry analysis. NbDRP2 is closely related to the DRP2 subfamily proteins in Arabidopsis thaliana, AtDRP2A and AtDRP2B. Confocal microscopy observation and Co-IP confirmed the interaction between the MET domain and NbDRP2. Also, the expression of NbDRP2 was induced by PlAMV infection. PlAMV accumulation was reduced when the expression of NbDRP2 gene was suppressed by virus-induced gene silencing. In addition, PlAMV accumulation was reduced in protoplasts treated with dynamin inhibitor. These results indicate a proviral role of the interaction of NbDRP2 with the MET domain in PlAMV replication.