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Cardiac glycosides (CGs) are natural steroid glycosides, which act as inhibitors of the cellular sodium-potassium ATPase pump. Although traditionally considered toxic to human cells, CGs are widely used as drugs for the treatment of cardiovascular-related medical conditions. More recently, CGs have been explored as potential anti-viral drugs and inhibit replication of a range of RNA and DNA viruses. Previously, a compound screen identified CGs that inhibited vaccinia virus (VACV) infection. However, no further investigation of the inhibitory potential of these compounds was performed, nor was there investigation of the stage(s) of the poxvirus lifecycle they impacted. Here, we investigated the anti-poxvirus activity of a broad panel of CGs. We found that all CGs tested were potent inhibitors of VACV replication. Our virological experiments showed that CGs did not impact virus infectivity, binding, or entry. Rather, experiments using recombinant viruses expressing reporter proteins controlled by VACV promoters and arabinoside release assays demonstrated that CGs inhibited early and late VACV protein expression at different concentrations. Lack of virus assembly in the presence of CGs was confirmed using electron microscopy. Thus, we expand our understanding of compounds with anti-poxvirus activity and highlight a yet unrecognized mechanism by which poxvirus replication can be inhibited.

Urinary tract infection is among the most common infections worldwide, typically studied in animals and cell lines with limited uropathogenic strains. Here, we assessed diverse bacterial species in a human urothelial microtissue model exhibiting full stratification, differentiation, innate epithelial responses, and urine tolerance. Several uropathogens invaded intracellularly, but also commensal Escherichia coli , suggesting that invasion is a shared survival strategy, not solely a virulence hallmark. The E. coli adhesin FimH was required for intracellular bacterial community formation, but not for invasion. Other shared lifestyles included filamentation (Gram-negatives), chaining (Gram-positives), and hijacking of exfoliating cells, while biofilm-like aggregates were formed mainly with Pseudomonas and Proteus . Urothelial cells expelled invasive bacteria in Rab-/LC3-decorated structures, while highly cytotoxic/invasive uropathogens, but not commensals, disrupted host barrier function and strongly induced exfoliation and cytokine production. Overall, this work highlights diverse species-/strain-specific infection strategies and corresponding host responses in a human urothelial microenvironment, providing insights at the microtissue, cell, and molecular level.

Dystonia, a neurological disorder defined by abnormal postures and disorganized movements, is considered to be a neural circuit disorder with dysfunction arising within and between multiple brain regions. Given that spinal neural circuits constitute the final pathway for motor control, we sought to determine their contribution to this movement disorder. Focusing on the most common inherited form of dystonia in humans, DYT1- TOR1A , we generated a conditional knockout of the torsin family 1 member A ( Tor1a ) gene in the mouse spinal cord and dorsal root ganglia (DRG). We found that these mice recapitulated the phenotype of the human condition, developing early-onset generalized torsional dystonia. Motor signs emerged early in the mouse hindlimbs before spreading caudo-rostrally to affect the pelvis, trunk, and forelimbs throughout postnatal maturation. Physiologically, these mice bore the hallmark features of dystonia, including spontaneous contractions at rest and excessive and disorganized contractions, including cocontractions of antagonist muscle groups, during voluntary movements. Spontaneous activity, disorganized motor output, and impaired monosynaptic reflexes, all signs of human dystonia, were recorded from isolated mouse spinal cords from these conditional knockout mice. All components of the monosynaptic reflex arc were affected, including motor neurons. Given that confining the Tor1a conditional knockout to DRG did not lead to early-onset dystonia, we conclude that the pathophysiological substrate of this mouse model of dystonia lies in spinal neural circuits. Together, these data provide new insights into our current understanding of dystonia pathophysiology.

The blood barriers of the nervous system protect neural environments but can hinder therapeutic accessibility. The blood-brain barrier (BBB) is well characterized, consisting of endothelial cells with specialized tight junctions and low levels of transcytosis, properties conferred by contacting pericytes and astrocytes. In contrast, the blood-nerve barrier (BNB) of the peripheral nervous system is poorly defined. Here, we characterize the structure of the mammalian BNB, identify the processes that confer barrier function, and demonstrate how the barrier can be opened in response to injury. The homeostatic BNB is leakier than the BBB, which we show is due to higher levels of transcytosis. However, the barrier is reinforced by macrophages that specifically engulf leaked materials, identifying a role for resident macrophages as an important component of the BNB. Finally, we demonstrate the exploitation of these processes to effectively deliver RNA-targeting therapeutics to peripheral nerves, indicating new treatment approaches for nervous system pathologies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The techniques collectively known as volume electron microscopy (vEM) each come with their own advantages and challenges, making them more or less suitable for any specific project. SEM array tomography (SEM-AT) is certainly no different in this respect. Requiring microtomy skills, and involving more data alignment post imaging, SEM-AT presents challenges to its users, nevertheless, as perhaps the most flexible, cost effective and potentially accessible vEM approach to regular EM facilities, it benefits those same users with multiple advantages due to its inherently non-destructive nature. The general principles and advantages/disadvantages of SEM-AT are described here, together with a step-by-step guide to the workflow, from block trimming, sectioning and collection on coverslips, to alignment of the high-resolution 3D dataset. With a suitable SEM/backscatter electron detector setup, and equipment readily found in an electron microscopy lab, it should be possible to begin to acquire 3D ultrastructural data. With the addition of appropriate SEM-AT imaging software, this process can be significantly enhanced to automatically image hundreds, potentially thousands, of sections. Hardware and software advances and future improvements will only make this easier, to the extent that SEM-AT could become a routine vEM technique throughout the world, rather than the privilege of a small number of experts in limited specialist facilities., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Although nuclei are the defining features of eukaryotes, we still do not fully understand how the nuclear compartment is duplicated and partitioned during division. This is especially the case for organisms that do not completely disassemble their nuclear envelope upon entry into mitosis. In studying this process in Drosophila neural stem cells, which undergo asymmetric divisions, we find that the nuclear compartment boundary persists during mitosis thanks to the maintenance of a supporting nuclear lamina. This mitotic nuclear envelope is then asymmetrically remodeled and partitioned to give rise to two daughter nuclei that differ in envelope composition and exhibit a >30-fold difference in volume. The striking difference in nuclear size was found to depend on two consecutive processes: asymmetric nuclear envelope resealing at mitotic exit at sites defined by the central spindle, and differential nuclear growth that appears to depend on the available local reservoir of ER/nuclear membranes, which is asymmetrically partitioned between the two daughter cells. Importantly, these asymmetries in size and composition of the daughter nuclei, and the associated asymmetries in chromatin organization, all become apparent long before the cortical release and the nuclear import of cell fates determinants. Thus, asymmetric nuclear remodeling during stem cell divisions may contribute to the generation of cellular diversity by initiating distinct transcriptional programs in sibling nuclei that contribute to later changes in daughter cell identity and fate., Competing Interests: Declarations of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The replication and assembly of vaccinia virus (VACV), the prototypic poxvirus, occurs exclusively in the cytoplasm of host cells. While the role of cellular cytoskeletal components in these processes remains poorly understood, vimentin-a type III intermediate filament-has been shown to associate with viral replication sites and to be incorporated into mature VACV virions. Here, we employed chemical and genetic approaches to further investigate the role of vimentin during the VACV lifecycle. The collapse of vimentin filaments, using acrylamide, was found to inhibit VACV infection at the level of genome replication, intermediate- and late-gene expression. However, we found that CRISPR-mediated knockout of vimentin did not impact VACV replication. Combining these tools, we demonstrate that acrylamide treatment results in the formation of anti-viral granules (AVGs) known to mediate translational inhibition of many viruses. We conclude that vimentin is dispensable for poxvirus replication and assembly and that acrylamide, as a potent inducer of AVGs during VACV infection, serves to bolster cell's anti-viral response to poxvirus infection., (© 2021 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.)

Poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple-membrane particles, termed intracellular enveloped virions (IEVs), are released from infected cells by fusion. Whereas the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to an RNAi screen directed against cellular membrane-trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of MVB-IEVs showed that they account for half of IEV production indicating that MVBs are a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread., (© 2021 Huttunen et al.)

Cell fate transitions are key to development and homeostasis. It is thus essential to understand the cellular mechanisms controlling fate transitions. Cell division has been implicated in fate decisions in many stem cell types, including neuronal and epithelial progenitors. In other stem cells, such as embryonic stem (ES) cells, the role of division remains unclear. Here, we show that exit from naive pluripotency in mouse ES cells generally occurs after a division. We further show that exit timing is strongly correlated between sister cells, which remain connected by cytoplasmic bridges long after division, and that bridge abscission progressively accelerates as cells exit naive pluripotency. Finally, interfering with abscission impairs naive pluripotency exit, and artificially inducing abscission accelerates it. Altogether, our data indicate that a switch in the division machinery leading to faster abscission regulates pluripotency exit. Our study identifies abscission as a key cellular process coupling cell division to fate transitions., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Adaptor protein (AP) complexes mediate key sorting decisions in the cell through selective incorporation of transmembrane proteins into vesicles. Little is known of the roles of AP-4, despite its loss of function leading to a severe early onset neurological disorder, AP-4 deficiency syndrome. Here we demonstrate an AP-4 epsilon subunit knockout mouse model that recapitulates characteristic neuroanatomical phenotypes of AP-4 deficiency patients. We show that ATG9A, critical for autophagosome biogenesis, is an AP-4 cargo, which is retained within the trans -Golgi network (TGN) in vivo and in culture when AP-4 function is lost. TGN retention results in depletion of axonal ATG9A, leading to defective autophagosome generation and aberrant expansions of the distal axon. The reduction in the capacity to generate axonal autophagosomes leads to defective axonal extension and de novo generation of distal axonal swellings containing accumulated ER, underlying the impaired axonal integrity in AP-4 deficiency syndrome. Abbreviations : AP: adaptor protein; AP4B1: adaptor-related protein complex AP-4, beta 1; AP4E1: adaptor-related protein complex AP-4, epsilon 1; ATG: autophagy-related; EBSS: Earle's balanced salt solution; ER: endoplasmic reticulum; GFAP: glial fibrillary acidic protein; GOLGA1/Golgin-97/GOLG97: golgi autoantigen, golgin subfamily a, 1; GOLGA2/GM130: golgi autoantigen, golgin subfamily a, 2; HSP: hereditary spastic paraplegia; LC3/MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MAP2: microtubule-associated protein 2; MAPK8IP1/JIP1: mitogen-acitvated protein kinase 8 interacting protein 1; NEFH/NF200: neurofilament, heavy polypeptide; RBFOX3/NeuN (RNA binding protein, fox-1 homolog [C. elegans] 3); SQSTM1/p62: sequestosome 1; TGN: trans-Golgi network; WIPI2: WD repeat domain, phosphoinositide interacting protein 2.

Junctional complexes between endothelial cells form a dynamic barrier that hinders passive diffusion of blood constituents into interstitial tissues. Remodelling of junctions is an essential process during leukocyte trafficking, vascular permeability, and angiogenesis. However, for many junctional proteins, the mechanisms of junctional remodelling have yet to be determined. Here, we used receptor mutagenesis, horseradish peroxidase (HRP), and ascorbate peroxidase 2 (APEX-2) proximity labelling, alongside light and electron microscopy (EM), to map the intracellular trafficking routes of junctional adhesion molecule-C (JAM-C). We found that JAM-C cotraffics with receptors associated with changes in permeability such as vascular endothelial cadherin (VE-Cadherin) and neuropilin (NRP)-1 and 2, but not with junctional proteins associated with the transmigration of leukocytes. Dynamic JAM-C trafficking and degradation are necessary for junctional remodelling during cell migration and angiogenesis. By identifying new potential trafficking machinery, we show that a key point of regulation is the ubiquitylation of JAM-C by the E3 ligase Casitas B-lineage lymphoma (CBL), which controls the rate of trafficking versus lysosomal degradation., Competing Interests: The authors have declared that no competing interests exist.

Lymph nodes (LNs) act as filters, constantly sampling peripheral cues. This is facilitated by the conduit network, a tubular structure of aligned extracellular matrix (ECM) fibrils ensheathed by fibroblastic reticular cells (FRCs). LNs undergo rapid 3- to 5-fold expansion during adaptive immune responses, but these ECM-rich structures are not permanently damaged. Whether conduit flow or filtering function is affected during LN expansion is unknown. Here, we show that conduits are partially disrupted during acute LN expansion, but FRC-FRC contacts remain connected. We reveal that polarized FRCs deposit ECM basolaterally using LL5-β and that ECM production is regulated at transcriptional and secretory levels by the C-type lectin CLEC-2, expressed by dendritic cells. Inflamed LNs maintain conduit size exclusion, and flow is disrupted but persists, indicating the robustness of this structure despite rapid tissue expansion. We show how dynamic communication between peripheral tissues and LNs provides a mechanism to prevent inflammation-induced fibrosis in lymphoid tissue., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

To achieve efficient binding and subsequent fusion, most enveloped viruses encode between one and five proteins 1 . For many viruses, the clustering of fusion proteins-and their distribution on virus particles-is crucial for fusion activity 2,3 . Poxviruses, the most complex mammalian viruses, dedicate 15 proteins to binding and membrane fusion 4 . However, the spatial organization of these proteins and how this influences fusion activity is unknown. Here, we show that the membrane of vaccinia virus is organized into distinct functional domains that are critical for the efficiency of membrane fusion. Using super-resolution microscopy and single-particle analysis, we found that the fusion machinery of vaccinia virus resides exclusively in clusters at virion tips. Repression of individual components of the fusion complex disrupts fusion-machinery polarization, consistent with the reported loss of fusion activity 5 . Furthermore, we show that displacement of functional fusion complexes from virion tips disrupts the formation of fusion pores and infection kinetics. Our results demonstrate how the protein architecture of poxviruses directly contributes to the efficiency of membrane fusion, and suggest that nanoscale organization may be an intrinsic property of these viruses to assure successful infection.

Peripheral nerves are highly regenerative, in contrast to the poor regenerative capabilities of the central nervous system (CNS). Here, we show that adult peripheral nerve is a more quiescent tissue than the CNS, yet all cell types within a peripheral nerve proliferate efficiently following injury. Moreover, whereas oligodendrocytes are produced throughout life from a precursor pool, we find that the corresponding cell of the peripheral nervous system, the myelinating Schwann cell (mSC), does not turn over in the adult. However, following injury, all mSCs can dedifferentiate to the proliferating progenitor-like Schwann cells (SCs) that orchestrate the regenerative response. Lineage analysis shows that these newly migratory, progenitor-like cells redifferentiate to form new tissue at the injury site and maintain their lineage, but can switch to become a non-myelinating SC. In contrast, increased plasticity is observed during tumourigenesis. These findings show that peripheral nerves have a distinct mechanism for maintaining homeostasis and can regenerate without the need for an additional stem cell population.This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The formation of myelinating Schwann cells (mSCs) involves the remarkable biogenic process, which rapidly generates the myelin sheath. Once formed, the mSC transitions to a stable homeostatic state, with loss of this stability associated with neuropathies. The histone deacetylases histone deacetylase 1 (HDAC1) and HDAC2 are required for the myelination transcriptional program. Here, we show a distinct role for HDAC3, in that, while dispensable for the formation of mSCs, it is essential for the stability of the myelin sheath once formed-with loss resulting in progressive severe neuropathy in adulthood. This is associated with the prior failure to downregulate the biogenic program upon entering the homeostatic state leading to hypertrophy and hypermyelination of the mSCs, progressing to the development of severe myelination defects. Our results highlight distinct roles of HDAC1/2 and HDAC3 in controlling the differentiation and homeostatic states of a cell with broad implications for the understanding of this important cell-state transition., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo., Summary: Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

The effects of mitofusin 2 (MFN2) deficiency, on mitochondrial morphology and the mitochondria-junctional sarcoplasmic reticulum (jSR) complex in the adult heart, have been previously investigated using 2D electron microscopy, an approach which is unable to provide a 3D spatial assessment of these imaging parameters. Here, we use 3D electron tomography to show that MFN2-deficient mitochondria are larger in volume, more elongated, and less rounded; have fewer mitochondria-jSR contacts, and an increase in the distance between mitochondria and jSR, when compared to WT mitochondria. In comparison to 2D electron microscopy, 3D electron tomography can provide further insights into mitochondrial morphology and the mitochondria-jSR complex in the adult heart., (© 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Weibel-Palade bodies (WPBs), the storage organelles of endothelial cells, are essential to normal haemostatic and inflammatory responses. Their major constituent protein is von Willebrand factor (VWF) which, following stimulation with secretagogues, is released into the blood vessel lumen as large platelet-catching strings. This exocytosis changes the protein composition of the cell surface and also results in a net increase in the amount of plasma membrane. Compensatory endocytosis is thought to limit changes in cell size and retrieve fusion machinery and other misplaced integral membrane proteins following exocytosis; however, little is known about the extent, timing, mechanism and precise function of compensatory endocytosis in endothelial cells. Using biochemical assays, live-cell imaging and correlative spinning-disk microscopy and transmission electron microscopy assays we provide the first in-depth high-resolution characterisation of this process. We provide a model of compensatory endocytosis based on rapid clathrin- and dynamin-mediated retrieval. Inhibition of this process results in a change of exocytic mode: WPBs then fuse with previously fused WPBs rather than the plasma membrane, leading, in turn, to the formation of structurally impaired tangled VWF strings.This article has an associated First Person interview with the first authors of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Interferon inducible transmembrane proteins (IFITMs) are broad-spectrum antiviral factors. In cell culture the entry of many enveloped viruses, including orthomyxo-, flavi-, and filoviruses, is inhibited by IFITMs, though the mechanism(s) involved remain unclear and may vary between viruses. We demonstrate that Sindbis and Semliki Forest virus (SFV), which both use endocytosis and acid-induced membrane fusion in early endosomes to infect cells, are restricted by the early endosomal IFITM3. The late endosomal IFITM2 is less restrictive and the plasma membrane IFITM1 does not inhibit normal infection by either virus. IFITM3 inhibits release of the SFV capsid into the cytosol, without inhibiting binding, internalization, trafficking to endosomes or low pH-induced conformational changes in the envelope glycoprotein. Infection by SFV fusion at the cell surface was inhibited by IFITM1, but was equally inhibited by IFITM3. Furthermore, an IFITM3 mutant (Y20A) that is localized to the plasma membrane inhibited infection by cell surface fusion more potently than IFITM1. Together, these results indicate that IFITMs, in particular IFITM3, can restrict alphavirus infection by inhibiting viral fusion with cellular membranes. That IFITM3 can restrict SFV infection by fusion at the cell surface equivalently to IFITM1 suggests that IFITM3 has greater antiviral potency against SFV., (© 2016 The Authors. Traffic published by John Wiley & Sons Ltd.)

Weibel-Palade bodies (WPBs) are endothelial storage organelles that mediate the release of molecules involved in thrombosis, inflammation and angiogenesis, including the pro-thrombotic glycoprotein von Willebrand factor (VWF). Although many protein components required for WPB formation and function have been identified, the role of lipids is almost unknown. We examined two key phosphatidylinositol kinases that control phosphatidylinositol 4-phosphate levels at the trans-Golgi network, the site of WPB biogenesis. RNA interference of the type II phosphatidylinositol 4-kinases PI4KIIα and PI4KIIβ in primary human endothelial cells leads to formation of an increased proportion of short WPB with perturbed packing of VWF, as exemplified by increased exposure of antibody-binding sites. When stimulated with histamine, these cells release normal levels of VWF yet, under flow, form very few platelet-catching VWF strings. In PI4KIIα-deficient mice, immuno-microscopy revealed that VWF packaging is also perturbed and these mice exhibit increased blood loss after tail cut compared to controls. This is the first demonstration that lipid kinases can control the biosynthesis of VWF and the formation of WPBs that are capable of full haemostatic function., (© 2016. Published by The Company of Biologists Ltd.)

Arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome is caused by deficiencies in the trafficking proteins VPS33B or VIPAR, and is associated with a bleeding diathesis and a marked reduction in platelet α-granules. We generated a tamoxifen-inducible mouse model of VPS33B deficiency, Vps33b(fl/fl)-ER(T2), and studied the platelet phenotype and α-granule biogenesis. Ultrastructural analysis of Vps33b(fl/fl)-ER(T2) platelets identified a marked reduction in α-granule count and the presence of small granule-like structures in agreement with the platelet phenotype observed in ARC patients. A reduction of ∼65% to 75% was observed in the α-granule proteins von Willebrand factor and P-selectin. Although platelet aggregation responses were not affected, a defect in δ-granule secretion was observed. Under arteriolar shear conditions, Vps33b(fl/fl)-ER(T2) platelets were unable to form stable aggregates, and tail-bleeding measurement revealed a bleeding diathesis. Analysis of bone marrow-derived megakaryocytes (MKs) by conventional and immuno-electron microscopy from Vps33b(fl/fl)-ER(T2) mice revealed a reduction in mature type-II multivesicular bodies (MVB II) and an accumulation of large vacuoles. Proteins that are normally stored in α-granules were underrepresented in MVB II and proplatelet extensions. These results demonstrate that abnormal protein trafficking and impairment in MVB maturation in MKs underlie the α-granule deficiency in Vps33b(fl/fl)-ER(T2) mouse and ARC patients., (© 2015 by The American Society of Hematology.)

InterFeron Inducible TransMembrane proteins 1-3 (IFITM1, IFITM2 and IFITM3) are a family of proteins capable of inhibiting the cellular entry of numerous human and animal viruses. IFITM1-3 are unique amongst the currently described viral restriction factors in their apparent ability to block viral entry. This restrictive property is dependant on the localisation of the proteins to plasma and endosomal membranes, which constitute the main portals of viral entry into cells. The topology of the IFITM proteins within cell membranes is an unresolved aspect of their biology. Here we present data from immunofluorescence microscopy, protease cleavage, biotin-labelling and immuno-electron microscopy assays, showing that human IFITM1 has a membrane topology in which the N-terminal domain resides in the cytoplasm, and the C-terminal domain is extracellular. Furthermore, we provide evidence that this topology is conserved for all of the human interferon-induced IFITM proteins. This model is consistent with that recently proposed for murine IFITM3, but differs from that proposed for murine IFITM1.

Schwann cells are highly plastic cells that dedifferentiate to a progenitor-like state following injury. However, deregulation of this plasticity, may be involved in the formation of neurofibromas, mixed-cell tumors of Schwann cell (SC) origin that arise upon loss of NF1. Here, we show that adult myelinating SCs (mSCs) are refractory to Nf1 loss. However, in the context of injury, Nf1-deficient cells display opposing behaviors along the wounded nerve; distal to the injury, Nf1(-/-) mSCs redifferentiate normally, whereas at the wound site Nf1(-/-) mSCs give rise to neurofibromas in both Nf1(+/+) and Nf1(+/-) backgrounds. Tracing experiments showed that distinct cell types within the tumor derive from Nf1-deficient SCs. This model of neurofibroma formation demonstrates that neurofibromas can originate from adult SCs and that the nerve environment can switch from tumor suppressive to tumor promoting at a site of injury. These findings have implications for both the characterization and treatment of neurofibromas., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Following damage to peripheral nerves, a remarkable process of clearance and regeneration takes place. Axons downstream of the injury degenerate, while the nerve is remodeled to direct axonal regrowth. Schwann cells are important for this regenerative process. "Sensing" damaged axons, they dedifferentiate to a progenitor-like state, in which they aid nerve regeneration. Here, we demonstrate that activation of an inducible Raf-kinase transgene in myelinated Schwann cells is sufficient to control this plasticity by inducing severe demyelination in the absence of axonal damage, with the period of demyelination/ataxia determined by the duration of Raf activation. Remarkably, activation of Raf-kinase also induces much of the inflammatory response important for nerve repair, including breakdown of the blood-nerve barrier and the influx of inflammatory cells. This reversible in vivo model identifies a central role for ERK signaling in Schwann cells in orchestrating nerve repair and is a powerful system for studying peripheral neuropathies and cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

N-cadherin is a homophilic adhesion protein that remains expressed at mature excitatory synapses beyond its developmental role in synapse formation. We investigated the trans-synaptic activity of N-cadherin in regulating synapse function in rodent cultured hippocampal neurons using optical methods and electrophysiology. Interfering with N-cadherin in postsynaptic neurons reduced basal release probability (p(r)) at inputs to the neuron, and this trans-synaptic impairment of release accompanied impaired vesicle endocytosis. Moreover, loss of the GluA2 AMPA-type glutamate receptor subunit, which decreased p(r) by itself, occluded the interference with postsynaptic N-cadherin. The loss of postsynaptic N-cadherin activity, however, did not affect the compensatory upregulation of p(r) induced by chronic activity silencing, whereas postsynaptic β-catenin deletion blocked this presynaptic homeostatic adaptation. Our findings suggest that postsynaptic N-cadherin helps link basal pre- and postsynaptic strengths to control the p(r) offset, whereas the p(r) gain adjustment requires a distinct trans-synaptic pathway involving β-catenin.

The study of actin in regulated exocytosis has a long history with many different results in numerous systems. A major limitation on identifying precise mechanisms has been the paucity of experimental systems in which actin function has been directly assessed alongside granule content release at distinct steps of exocytosis of a single secretory organelle with sufficient spatiotemporal resolution. Using dual-color confocal microscopy and correlative electron microscopy in human endothelial cells, we visually distinguished two sequential steps of secretagogue-stimulated exocytosis: fusion of individual secretory granules (Weibel-Palade bodies [WPBs]) and subsequent expulsion of von Willebrand factor (VWF) content. Based on our observations, we conclude that for fusion, WPBs are released from cellular sites of actin anchorage. However, once fused, a dynamic ring of actin filaments and myosin II forms around the granule, and actomyosin II contractility squeezes VWF content out into the extracellular environment. This study therefore demonstrates how discrete actin cytoskeleton functions within a single cellular system explain actin filament-based prevention and promotion of specific exocytic steps during regulated secretion.

The δ-opioid receptor (DOR) undergoes ligand-induced downregulation by endosomal sorting complex required for transport (ESCRT)-dependent endocytic trafficking to lysosomes. In contrast to a number of other signaling receptors, the DOR can downregulate effectively when its ubiquitination is prevented. We explored the membrane trafficking basis of this behavior. First, we show that internalized DORs traverse the canonical multivesicular body (MVB) pathway and localize to intralumenal vesicles (ILVs). Second, we show that DOR ubiquitination stimulates, but is not essential for, receptor transfer to ILVs and proteolysis of the receptor endodomain. Third, we show that receptor ubiquitination plays no detectable role in the early sorting of internalized DORs out of the recycling pathway. Finally, we show that DORs undergo extensive proteolytic fragmentation in the ectodomain, even when receptor ubiquitination is prevented or ILV formation itself is blocked. Together, these results are sufficient to explain why DORs downregulate effectively in the absence of ubiquitination, and they place a discrete molecular sorting operation in the MVB pathway effectively upstream of the ESCRT. More generally, these findings support the hypothesis that mammalian cells can control the cytoplasmic accessibility of internalized signaling receptors independently from their ultimate trafficking fate., (© 2010 John Wiley & Sons A/S.)

The neuronal ceroid lipofuscinoses (NCLs) constitute a group of autosomal recessive neurodegenerative diseases affecting children. To date, the disease pathogenesis remains unknown, although the role of lysosomal impairment is widely recognized across the different diseases. Recently, the creation of simple models of juvenile NCL (Batten disease) has provided additional insights into the disease mechanism at the molecular level. We report defects in metabolism identified in the Schizosacchromyces pombe yeast model, where btn1, the orthologue of CLN3, has been deleted, using a metabolomics approach based on high resolution 1H and 13C NMR spectroscopy. Such changes represent the first documented metabolic changes associated with deletion of btn1. A decrease in extracellular glucose and increases in the concentration of extracellular ethanol and alanine labelling demonstrate increased glycolytic flux that may arise from vacuolar impairment, whilst amino acid changes were detected which were also in accordance with defective vacuolar functionality. That these changes were detected using a metabolomic based approach advocates its use to further analyse other yeast models of human disease to better understand the function of orthologue genes.

The epidermal growth factor receptor (EGFR) is a critical determinator of cell fate. Signalling from this receptor tyrosine kinase is spatially regulated by progression through the endocytic pathway, governing receptor half-life and accessibility to signalling proteins and phosphatases. Endocytosis of EGFR is required for interaction with the protein tyrosine phosphatase PTP1B (ref. 1), which localizes to the cytoplasmic face of the endoplasmic reticulum (ER), raising the question of how PTP1B comes into contact with endosomal EGFR. We show that EGFR-PTP1B interaction occurs by means of direct membrane contacts between the perimeter membrane of multivesicular bodies (MVBs) and the ER. The population of EGFR interacting with PTP1B is the same population that undergo ESCRT-mediated (endosomal sorting complex required for transport) sorting within MVBs, and PTP1B activity promotes the sequestration of EGFR on to MVB internal vesicles. Membrane contacts between endosomes and the ER form in both the presence and absence of stimulation by EGF. Thus membrane contacts between endosomes and the ER may represent a global mechanism for direct interaction between proteins on these two organelles.

Activated EGFR (epidermal growth factor receptor) undergoes ESCRT (endosomal sorting complex required for transport)-mediated sorting on to the intraluminal vesicles of MVBs (multivesicular bodies) before degradation in the lysosome. Sorting of endocytosed EGFR on to the intraluminal vesicles of MVBs removes the catalytic domain of the EGFR from the cytoplasm, resulting in termination of receptor signalling. The formation of intraluminal vesicles that contain EGFR is promoted by EGF stimulation in a mechanism that depends on the EGFR substrate, annexin 1. Signalling from endocytosed EGFR is also subject to down-regulation through receptor dephosphorylation by PTPs (protein tyrosine phosphatases), such as PTP1B, an enzyme thought to reside on the ER (endoplasmic reticulum). In the present paper, we review how the phosphorylation state of components of the MVB sorting machinery, as well as the EGFR, may play a critical role in regulating EGFR sorting and signalling.

Annexins are calcium- and phospholipid-binding proteins that have been proposed to have multiple roles in membrane traffic. Historically, this has been based on the in vitro properties of annexins and their localization to specific membrane compartments. However, recent functional evidence supports a role for annexins in specific membrane traffic steps, although the requirement for annexins may be highly dependent on the cellular context. Here we review the roles of annexins in traffic within the endocytic pathway, focusing on clathrin-dependent internalization from the plasma membrane, multivesicular endosome/body (MVB) biogenesis and MVB-lysosome fusion.

Rab11-FIP3 is an endosomal recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11-FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11-FIP3 and expression of a mutant of Rab11-FIP3 that is Rab11-binding deficient cause loss of all ERC-marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore-labelled transferrin cannot access the pericentrosomal region of cells in which Rab11-FIP3 function has been perturbed. We find that this Rab11-FIP3 function appears to be specific because expression of the equivalent Rab11-binding deficient mutant of Rab-coupling protein does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11-FIP3 function. Finally, we demonstrate the presence of an extensive coiled-coil region between residues 463 and 692 of Rab11-FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11-FIP3 is necessary for the structural integrity of the pericentrosomal ERC.

Here we show that EGF and EGF receptor (EGFR) are trafficked through a subpopulation of multivesicular endosomes/bodies (MVBs) that are distinct from morphologically identical vacuoles that label for the late endosomal marker lyso-bisphosphatidic acid (LBPA). EGF stimulation increases both MVB biogenesis and inward vesiculation within EGFR-containing MVBs. Deletion of annexin 1, a substrate of EGFR tyrosine kinase, abolishes the effect of EGF stimulation on inward vesiculation. This phenotype is reversible by transfection with wild-type but not Y21F phosphorylation mutant annexin 1. Deletion of annexin 1 has no effect on EGF-stimulated MVB biogenesis, suggesting that MVB biogenesis and inward vesiculation within MVB are mediated by separate mechanisms. Loss or depletion of annexin 1 has no effect on EGF degradation and causes only a small delay in EGFR degradation, indicating that annexin 1 operates downstream of Hrs- and ESCRT-mediated sorting and is required solely for EGF-stimulated inward vesiculation. Annexin 1 accumulates on internal vesicles of MVB after EGF-stimulated inward vesiculation, suggesting that it may be required for a late stage in inward vesiculation.

We present the first observation of substantial persistent flow in superfluid 3He-A in thick simply connected slabs in a zero magnetic field, but only in l textures with domain walls. The flow is induced in a rotating cryostat using a torsional oscillator as a probe. The hysteretic dependences of the trapped vorticity on the maximal angular velocity of rotation are fairly universal for different densities of domain walls and slab thicknesses. A model of a critical state set by either the critical velocity for vortex nucleation or pinning strength explains all observations.

The accessory sigma factor sigmaB controls a general stress response that is thought to be important for Staphylococcus aureus survival and may contribute to virulence. The strain of choice for genetic studies, 8325-4, carries a small deletion in rsbU, which encodes a positive regulator of sigmaB activity. Consequently, to enable the role of sigmaB in virulence to be addressed, we constructed an rsbU(+) derivative, SH1000, using a method that does not leave behind an antibiotic resistance marker. The phenotypic properties of SH1000 (8325-4 rsbU(+)) were characterized and compared to those of 8325-4, the rsbU mutant, parent strain. A recognition site for sigmaB was located in the promoter region of katA, the gene encoding the sole catalase of S. aureus, by primer extension analysis. However, catalase expression and activity were similar in SH1000 (8325-4 rsbU(+)), suggesting that this promoter may have a minor role in catalase expression under normal conditions. Restoration of sigmaB activity in SH1000 (8325-4 rsbU(+)) resulted in a marked decrease in the levels of the exoproteins SspA and Hla, and this is likely to be mediated by reduced expression of agr in this strain. By using Western blotting and a sarA-lacZ reporter assay, the levels of SarA were found to be similar in strains 8325-4 and SH1000 (8325-4 rsbU(+)) and sigB mutant derivatives of these strains. This finding contrasts with previous reports that suggested that SarA expression levels are altered when they are measured transcriptionally. Inactivation of sarA in each of these strains resulted in an expected decrease in agr expression; however, the relative level of agr in SH1000 (8325-4 rsbU(+)) remained less than the relative levels in 8325-4 and the sigB mutant derivatives. We suggest that SarA is not likely to be the effector in the overall sigmaB-mediated effect on agr expression.

The 1I gene is expressed in the prespore cells of culminating Dictyostelium discoideum. The open reading frame of 1I cDNA encodes a protein of 155 amino acids with hydrophobic segments at both its NH(2)- and COOH-termini that are indicative of a glycosyl-phosphatidylinositol (GPI)-anchored protein. A hexaHis-tagged form of 1I expressed in D. discoideum cells appeared on Western blot analysis as a doublet of 27 and 24 kDa, with a minor polypeptide of 22 kDa. None of the polypeptides were released from the cell surface with bacterial phosphatidylinositol-specific phospholipase C, although all three were released upon nitrous acid treatment, indicating the presence of a phospholipase-resistant GPI anchor. Further evidence for the C-terminal sequence of 1I acting as a GPI attachment signal was obtained by replacing the GPI anchor signal sequence of porcine membrane dipeptidase with that from 1I. Two constructs of dipeptidase with the 1I GPI signal sequence were constructed, one of which included an additional six amino acids in the hydrophilic spacer. Both of the resultant constructs were targeted to the surface of COS cells and were GPI-anchored as shown by digestion with phospholipase C, indicating that the Dictyostelium GPI signal sequence is functional in mammalian cells. Site-specific antibodies recognising epitopes either side of the expected GPI anchor attachment site were used to determine the site of GPI anchor attachment in the constructs. These parallel approaches show that the C-terminal signal sequence of 1I can direct the addition of a GPI anchor.

It was previously hypothesised that the requirements for glycosyl-phosphatidylinositol (GPI) anchoring in mammalian cells and parasitic protozoa are similar but not identical. We have investigated this by converting the GPI cleavage/attachment site in porcine membrane dipeptidase to that found in the trypanosomal variant surface glycoprotein 117 and expressing the resulting mutants in COS-1 cells. Changing the entire (omega), (omega)+1 and (omega)+2 triplet in membrane dipeptidase from Ser-Ala-Ala to Asp-Ser-Ser resulted in efficient GPI anchoring of the mutant proteins, as assessed by cell-surface activity assays and susceptibility to release by phosphatidylinositol-specific phospholipase C. Immunoelectrophoretic blot analysis with antibodies recognising epitopes either side of the native (omega) residue in porcine membrane dipeptidase, and expression of a mutant in which potential alternative cleavage/attachment sites were disrupted, indicated that alternative GPI cleavage/attachment sites had not been used. These results indicate that the requirements for GPI anchoring between mammalian and protozoal cells are not as different as previously suggested, and that rules for predicting the probability of a sequence acting as a GPI cleavage/attachment site need to be applied with caution.

A fluorogenic substrate for assay of leukotriene D4 hydrolase (LTDase; EC 3.4.13.19) has been prepared and evaluated, using enzyme purified from porcine kidney. The compound is based on internal quenching of the synthetic, fluorescent amino acid d, l-2-amino-3-(7-methoxy-4-coumaryl)propanoic acid (d,l-Amp) by a 2, 4-dinitrophenyl (DNP) group. The compound is epsilon-DNP-l-Lys-d-Amp which incorporates the D-isomer of Amp to exploit the unique ability among mammalian peptidases for LTDase to hydrolyze peptides containing a d-amino acid in the C-terminal position. epsilon-DNP-l-Lys-d-Amp was found to be an excellent substrate for LTDase, with Km value of 370 microoffUnder the conditions of assay, the substrate was without noticeable quenching effect on the fluorescence of the product (d-Amp) liberated by the action of LTDase. Using porcine kidney microvillar membranes, which contain a battery of peptidases, the specific inhibitor of LTDase, cilastatin, completely inhibited the breakdown of epsilon-DNP-l-Lys-d-Amp, indicating that the substrate is selective for LTDase., (Copyright 1999 Academic Press.)