Showing papers in "Journal of Cell Biology in 2009"
TL;DR: It is shown that the hypercitrullination of histones by PAD4 mediates chromatin decondensation in granulocytes/neutrophils, and citrullinations of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures.
Abstract: Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.
1,162 citations
TL;DR: The evidence is reviewed here the evidence that it is the convergence of damage developed within multiple cell types, including within neighboring nonneuronal supporting cells, which is crucial to neuronal dysfunction.
Abstract: Selective degeneration and death of one or more classes of neurons is the defining feature of human neurodegenerative disease. Although traditionally viewed as diseases mainly affecting the most vulnerable neurons, in most instances of inherited disease the causative genes are widely—usually ubiquitously—expressed. Focusing on amyotrophic lateral sclerosis (ALS), especially disease caused by dominant mutations in Cu/Zn superoxide dismutase (SOD1), we review here the evidence that it is the convergence of damage developed within multiple cell types, including within neighboring nonneuronal supporting cells, which is crucial to neuronal dysfunction. Damage to a specific set of key partner cells as well as to vulnerable neurons may account for the selective susceptibility of neuronal subtypes in many human neurodegenerative diseases, including Huntington's disease (HD), Parkinson's disease (PD), prion disease, the spinal cerebellar ataxias (SCAs), and Alzheimer's disease (AD).
941 citations
TL;DR: It is shown that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress, suggesting that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.
Abstract: Maintenance of endoplasmic reticulum (ER) function is achieved in part through Ire1 (inositol-requiring enzyme 1), a transmembrane protein activated by protein misfolding in the ER. The cytoplasmic nuclease domain of Ire1 cleaves the messenger RNA (mRNA) encoding XBP-1 (X-box-binding protein 1), enabling splicing and production of this active transcription factor. We recently showed that Ire1 activation independently induces the rapid turnover of mRNAs encoding membrane and secreted proteins in Drosophila melanogaster cells through a pathway we call regulated Ire1-dependent decay (RIDD). In this study, we show that mouse fibroblasts expressing wild-type Ire1 but not an Ire1 variant lacking nuclease activity also degrade mRNAs in response to ER stress. Using a second variant of Ire1 that is activated by a small adenosine triphosphate analogue, we show that although XBP-1 splicing can be artificially induced in the absence of ER stress, RIDD appears to require both Ire1 activity and ER stress. Our data suggest that cells use a multitiered mechanism by which different conditions in the ER lead to distinct outputs from Ire1.
893 citations
TL;DR: It is shown that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs) and angiogenin, a secreted ribonuclease, is required for stress- induced production of tiRNAs.
Abstract: Stress-induced phosphorylation of eIF2α inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2α mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-eIF2α–independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-eIF2α–independent stress response program.
741 citations
TL;DR: It is proposed that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.
Abstract: Current concepts of cell migration were established in regular two-dimensional (2D) cell culture, but the roles of topography are poorly understood for cells migrating in an oriented 3D fibrillar extracellular matrix (ECM). We use a novel micropatterning technique termed microphotopatterning (μPP) to identify functions for 1D fibrillar patterns in 3D cell migration. In striking contrast to 2D, cell migration in both 1D and 3D is rapid, uniaxial, independent of ECM ligand density, and dependent on myosin II contractility and microtubules (MTs). 1D and 3D migration are also characterized by an anterior MT bundle with a posterior centrosome. We propose that cells migrate rapidly through 3D fibrillar matrices by a 1D migratory mechanism not mimicked by 2D matrices.
706 citations
TL;DR: Glyburide, a sulfonylurea drug commonly used to treat type 2 diabetes, shuts down IL-1β secretion by preventing Cyropyrin activation.
Abstract: Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1β (IL-1β) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer's, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide's cyclohexylurea group, which binds to adenosine triphosphatase (ATP)–sensitive K+ (KATP) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking KATP subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1β secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome–associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1β secretion.
685 citations
TL;DR: Catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension, and binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range.
Abstract: Binding of integrins to ligands provides anchorage and signals for the cell, making them prime candidates for mechanosensing molecules. How force regulates integrin–ligand dissociation is unclear. We used atomic force microscopy to measure the force-dependent lifetimes of single bonds between a fibronectin fragment and an integrin α5β1-Fc fusion protein or membrane α5β1. Force prolonged bond lifetimes in the 10–30-pN range, a counterintuitive behavior called catch bonds. Changing cations from Ca2+/Mg2+ to Mg2+/EGTA and to Mn2+ caused longer lifetime in the same 10–30-pN catch bond region. A truncated α5β1 construct containing the headpiece but not the legs formed longer-lived catch bonds that were not affected by cation changes at forces <30 pN. Binding of monoclonal antibodies that induce the active conformation of the integrin headpiece shifted catch bonds to a lower force range. Thus, catch bond formation appears to involve force-assisted activation of the headpiece but not integrin extension.
662 citations
TL;DR: It is demonstrated that cancer cells have an absolute requirement for the membrane-anchored metalloproteinase MT1-MMP for invasion, and that protease-independent mechanisms of cell migration are only plausible when the collagen network is devoid of the covalent cross-links that characterize normal tissues.
Abstract: Tissue invasion during metastasis requires cancer cells to negotiate a stromal environment dominated by cross-linked networks of type I collagen. Although cancer cells are known to use proteinases to sever collagen networks and thus ease their passage through these barriers, migration across extracellular matrices has also been reported to occur by protease-independent mechanisms, whereby cells squeeze through collagen-lined pores by adopting an ameboid phenotype. We investigate these alternate models of motility here and demonstrate that cancer cells have an absolute requirement for the membrane-anchored metalloproteinase MT1-MMP for invasion, and that protease-independent mechanisms of cell migration are only plausible when the collagen network is devoid of the covalent cross-links that characterize normal tissues.
607 citations
TL;DR: It is found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles and explained how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.
Abstract: Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150Glued bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)–LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7–RILP complex to remove p150Glued and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells.
594 citations
TL;DR: Brain-specific Drp1 knockout mice demonstrate thatDrp1-mediated organelle division is important for development, mitochondrial morphogenesis, and apoptosis.
Abstract: The dynamin-related guanosine triphosphatase Drp1 mediates the division of mitochondria and peroxisomes. To understand the in vivo function of Drp1, complete and tissue-specific mouse knockouts of Drp1 were generated. Drp1-null mice die by embryonic day 11.5. This embryonic lethality is not likely caused by gross energy deprivation, as Drp1-null cells showed normal intracellular adenosine triphosphate levels. In support of the role of Drp1 in organelle division, mitochondria formed extensive networks, and peroxisomes were elongated in Drp1-null embryonic fibroblasts. Brain-specific Drp1 ablation caused developmental defects of the cerebellum in which Purkinje cells contained few giant mitochondria instead of the many short tubular mitochondria observed in control cells. In addition, Drp1-null embryos failed to undergo developmentally regulated apoptosis during neural tube formation in vivo. However, Drp1-null embryonic fibroblasts have normal responses to apoptotic stimuli in vitro, suggesting that the apoptotic function of Drp1 depends on physiological cues. These findings clearly demonstrate the physiological importance of Drp1-mediated organelle division in mice.
571 citations
TL;DR: The parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements are focused on.
Abstract: The light microscope has long been used to document the localization of fluorescent molecules in cell biology research. With advances in digital cameras and the discovery and development of genetically encoded fluorophores, there has been a huge increase in the use of fluorescence microscopy to quantify spatial and temporal measurements of fluorescent molecules in biological specimens. Whether simply comparing the relative intensities of two fluorescent specimens, or using advanced techniques like Forster resonance energy transfer (FRET) or fluorescence recovery after photobleaching (FRAP), quantitation of fluorescence requires a thorough understanding of the limitations of and proper use of the different components of the imaging system. Here, I focus on the parameters of digital image acquisition that affect the accuracy and precision of quantitative fluorescence microscopy measurements.
TL;DR: The role of various feedback loops that regulate cyclin B–Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells are discussed.
Abstract: The decision to enter mitosis is mediated by a network of proteins that regulate activation of the cyclin B–Cdk1 complex. Within this network, several positive feedback loops can amplify cyclin B–Cdk1 activation to ensure complete commitment to a mitotic state once the decision to enter mitosis has been made. However, evidence is accumulating that several components of the feedback loops are redundant for cyclin B–Cdk1 activation during normal cell division. Nonetheless, defined feedback loops become essential to promote mitotic entry when normal cell cycle progression is perturbed. Recent data has demonstrated that at least three Plk1-dependent feedback loops exist that enhance cyclin B–Cdk1 activation at different levels. In this review, we discuss the role of various feedback loops that regulate cyclin B–Cdk1 activation under different conditions, the timing of their activation, and the possible identity of the elusive trigger that controls mitotic entry in human cells.
TL;DR: This work generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates, and identified many myofibrillar components in denervated muscle that were decreased by a mechanism not requiring Mu RF1.
Abstract: Loss of myofibrillar proteins is a hallmark of atrophying muscle. Expression of muscle RING-finger 1 (MuRF1), a ubiquitin ligase, is markedly induced during atrophy, and MuRF1 deletion attenuates muscle wasting. We generated mice expressing a Ring-deletion mutant MuRF1, which binds but cannot ubiquitylate substrates. Mass spectrometry of the bound proteins in denervated muscle identified many myofibrillar components. Upon denervation or fasting, atrophying muscles show a loss of myosin-binding protein C (MyBP-C) and myosin light chains 1 and 2 (MyLC1 and MyLC2) from the myofibril, before any measurable decrease in myosin heavy chain (MyHC). Their selective loss requires MuRF1. MyHC is protected from ubiquitylation in myofibrils by associated proteins, but eventually undergoes MuRF1-dependent degradation. In contrast, MuRF1 ubiquitylates MyBP-C, MyLC1, and MyLC2, even in myofibrils. Because these proteins stabilize the thick filament, their selective ubiquitylation may facilitate thick filament disassembly. However, the thin filament components decreased by a mechanism not requiring MuRF1.
TL;DR: The cleavage by OMA1 causes an accumulation of the short OPA1 variants, and the role ofm-AAA proteases in ensuring a balance of long and short Opa1 isoforms is investigated.
Abstract: Mitochondrial fusion depends on the dynamin-like guanosine triphosphatase OPA1, whose activity is controlled by proteolytic cleavage. Dysfunction of mitochondria induces OPA1 processing and results in mitochondrial fragmentation, allowing the selective removal of damaged mitochondria. In this study, we demonstrate that two classes of metallopeptidases regulate OPA1 cleavage in the mitochondrial inner membrane: isoenzymes of the adenosine triphosphate (ATP)-dependent matrix AAA (ATPase associated with diverse cellular activities [m-AAA]) protease, variable assemblies of the conserved subunits paraplegin, AFG3L1 and -2, and the ATP-independent peptidase OMA1. Functionally redundant isoenzymes of the m-AAA protease ensure the balanced accumulation of long and short isoforms of OPA1 required for mitochondrial fusion. The loss of AFG3L2 in mouse tissues, down-regulation of AFG3L1 and -2 in mouse embryonic fibroblasts, or the expression of a dominant-negative AFG3L2 variant in human cells decreases the stability of long OPA1 isoforms and induces OPA1 processing by OMA1. Moreover, cleavage by OMA1 causes the accumulation of short OPA1 variants if mitochondrial DNA is depleted or mitochondrial activities are impaired. Our findings link distinct peptidases to constitutive and induced OPA1 processing and shed new light on the pathogenesis of neurodegenerative disorders associated with mutations in m-AAA protease subunits.
TL;DR: CHOP turns on ERO1-α to release calcium via IP3R and trigger cell death in response to ER stress.
Abstract: Endoplasmic reticulum (ER) stress–induced apoptosis is involved in many diseases, but the mechanisms linking ER stress to apoptosis are incompletely understood. Based on roles for C/EPB homologous protein (CHOP) and ER calcium release in apoptosis, we hypothesized that apoptosis involves the activation of inositol 1,4,5-triphosphate (IP3) receptor (IP3R) via CHOP-induced ERO1-α (ER oxidase 1 α). In ER-stressed cells, ERO1-α is induced by CHOP, and small interfering RNA (siRNA) knockdown of ERO1-α suppresses apoptosis. IP3-induced calcium release (IICR) is increased during ER stress, and this response is blocked by siRNA-mediated silencing of ERO1-α or IP3R1 and by loss-of-function mutations in Ero1a or Chop. Reconstitution of ERO1-α in Chop−/− macrophages restores ER stress–induced IICR and apoptosis. In vivo, macrophages from wild-type mice but not Chop−/− mice have elevated IICR when the animals are challenged with the ER stressor tunicamycin. Macrophages from insulin-resistant ob/ob mice, another model of ER stress, also have elevated IICR. These data shed new light on how the CHOP pathway of apoptosis triggers calcium-dependent apoptosis through an ERO1-α–IP3R pathway.
TL;DR: This work has shown that increasing the size of the ER by lipid synthesis helps the cell deal with ER stress.
Abstract: Cells constantly adjust the sizes and shapes of their organelles according to need In this study, we examine endoplasmic reticulum (ER) membrane expansion during the unfolded protein response (UPR) in the yeast Saccharomyces cerevisiae We find that membrane expansion occurs through the generation of ER sheets, requires UPR signaling, and is driven by lipid biosynthesis Uncoupling ER size control and the UPR reveals that membrane expansion alleviates ER stress independently of an increase in ER chaperone levels Converting the sheets of the expanded ER into tubules by reticulon overexpression does not affect the ability of cells to cope with ER stress, showing that ER size rather than shape is the key factor Thus, increasing ER size through membrane synthesis is an integral yet distinct part of the cellular program to overcome ER stress
TL;DR: This work finds that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy, and enhances viral yields by using the early stages while inhibiting the late stages of autophagic activity.
Abstract: Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.
TL;DR: Accumulation of autophagosomes because of impaired autophagy during valosin-containing protein (VCP)–linked dementia is explained by the absence or reduced activity of VCP.
Abstract: Mutations in valosin-containing protein (VCP) cause inclusion body myopathy (IBM), Paget's disease of the bone, and frontotemporal dementia (IBMPFD). Patient muscle has degenerating fibers, rimmed vacuoles (RVs), and sarcoplasmic inclusions containing ubiquitin and TDP-43 (TARDNA-binding protein 43). In this study, we find that IBMPFD muscle also accumulates autophagosome-associated proteins, Map1-LC3 (LC3), and p62/sequestosome, which localize to RVs. To test whether VCP participates in autophagy, we silenced VCP or expressed adenosine triphosphatase–inactive VCP. Under basal conditions, loss of VCP activity results in autophagosome accumulation. After autophagic induction, these autophagosomes fail to mature into autolysosomes and degrade LC3. Similarly, IBMPFD mutant VCP expression in cells and animals leads to the accumulation of nondegradative autophagosomes that coalesce at RVs and fail to degrade aggregated proteins. Interestingly, TDP-43 accumulates in the cytosol upon autophagic inhibition, similar to that seen after IBMPFD mutant expression. These data implicate VCP in autophagy and suggest that impaired autophagy explains the pathology seen in IBMPFD muscle, including TDP-43 accumulation.
TL;DR: The cross talk between autophagy and intracellular compartments is discussed, highlighting recent exciting data about the role and regulation of the Vps34 class III phosphatidylinositol (PI) 3-kinase in autophophagy.
Abstract: Autophagy or “self-eating” is a highly conserved pathway that enables cells to degrade pieces of themselves in autolysosomes to enable their survival in times of stress, including nutrient deprivation. The formation of these degradative compartments requires cytosolic proteins, some of which are autophagy specific, as well as intracellular organelles, such as the ER and Golgi, and the endosome–lysosome system. Here we discuss the cross talk between autophagy and intracellular compartments, highlighting recent exciting data about the role and regulation of the Vps34 class III phosphatidylinositol (PI) 3-kinase in autophagy.
TL;DR: A proteolytic cascade ensures that OMA1 cleaves and inactivates mitochondrial fusion protein OPA1 in times of stress, preventing damaged mitochondria from fusing with healthy organelles.
Abstract: The mammalian mitochondrial inner membrane fusion protein OPA1 is controlled by complex patterns of alternative splicing and proteolysis. A subset of OPA1 isoforms is constitutively cleaved by YME1L. Other isoforms are not cleaved by YME1L, but they are cleaved when mitochondria lose membrane potential or adenosine triphosphate. In this study, we show that this inducible cleavage is mediated by a zinc metalloprotease called OMA1. We find that OMA1 small interfering RNA inhibits inducible cleavage, helps retain fusion competence, and slows the onset of apoptosis, showing that OMA1 controls OPA1 cleavage and function. We also find that OMA1 is normally cleaved from 60 to 40 kD by another as of yet unidentified protease. Loss of membrane potential causes 60-kD protein to accumulate, suggesting that OMA1 is attenuated by proteolytic degradation. We conclude that a proteolytic cascade controls OPA1. Inducible cleavage provides a mechanism for quality control because proteolytic inactivation of OPA1 promotes selective removal of defective mitochondrial fragments by preventing their fusion with the mitochondrial network.
TL;DR: Given the large numbers of long noncoding transcripts currently being discovered through whole transcriptome analysis, paraspeckles may be a paradigm for a class of subnuclear bodies formed around longnoncoding RNA.
Abstract: Paraspeckles are ribonucleoprotein bodies found in the interchromatin space of mammalian cell nuclei. These structures play a role in regulating the expression of certain genes in differentiated cells by nuclear retention of RNA. The core paraspeckle proteins (PSF/SFPQ, P54NRB/NONO, and PSPC1 [paraspeckle protein 1]) are members of the DBHS (Drosophila melanogaster behavior, human splicing) family. These proteins, together with the long nonprotein-coding RNA NEAT1 (MEN-ϵ/β), associate to form paraspeckles and maintain their integrity. Given the large numbers of long noncoding transcripts currently being discovered through whole transcriptome analysis, paraspeckles may be a paradigm for a class of subnuclear bodies formed around long noncoding RNA.
TL;DR: The function of Bruchpilot seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca 2+ channel domains.
Abstract: Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.
TL;DR: It is shown that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of T PC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release byNAADP.
Abstract: Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.
TL;DR: It is demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect.
Abstract: End binding proteins (EBs) are highly conserved core components of microtubule plus-end tracking protein networks. Here we investigated the roles of the three mammalian EBs in controlling microtubule dynamics and analyzed the domains involved. Protein depletion and rescue experiments showed that EB1 and EB3, but not EB2, promote persistent microtubule growth by suppressing catastrophes. Furthermore, we demonstrated in vitro and in cells that the EB plus-end tracking behavior depends on the calponin homology domain but does not require dimer formation. In contrast, dimerization is necessary for the EB anti-catastrophe activity in cells; this explains why the EB1 dimerization domain, which disrupts native EB dimers, exhibits a dominant-negative effect. When microtubule dynamics is reconstituted with purified tubulin, EBs promote rather than inhibit catastrophes, suggesting that in cells EBs prevent catastrophes by counteracting other microtubule regulators. This probably occurs through their action on microtubule ends, because catastrophe suppression does not require the EB domains needed for binding to known EB partners.
TL;DR: An immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae is devised and it is demonstrated that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretoryVesicles.
Abstract: The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.
TL;DR: The protein mutated in Huntington's disease is phosphorylated by the inflammatory kinase IKK, which promotes other post-translational modifications, and protein degradation.
Abstract: Expansion of the polyglutamine repeat within the protein Huntingtin (Htt) causes Huntington's disease, a neurodegenerative disease associated with aging and the accumulation of mutant Htt in diseased neurons. Understanding the mechanisms that influence Htt cellular degradation may target treatments designed to activate mutant Htt clearance pathways. We find that Htt is phosphorylated by the inflammatory kinase IKK, enhancing its normal clearance by the proteasome and lysosome. Phosphorylation of Htt regulates additional post-translational modifications, including Htt ubiquitination, SUMOylation, and acetylation, and increases Htt nuclear localization, cleavage, and clearance mediated by lysosomal-associated membrane protein 2A and Hsc70. We propose that IKK activates mutant Htt clearance until an age-related loss of proteasome/lysosome function promotes accumulation of toxic post-translationally modified mutant Htt. Thus, IKK activation may modulate mutant Htt neurotoxicity depending on the cell's ability to degrade the modified species.
TL;DR: The stages of invadopodium assembly and maturation are dissected and it is shown thatInvadopodia use cortactin phosphorylation as a master switch during these processes to regulate cofilin and Arp2/3 complex–dependent actin polymerization.
Abstract: Invadopodia are matrix-degrading membrane protrusions in invasive carcinoma cells. The mechanisms regulating invadopodium assembly and maturation are not understood. We have dissected the stages of invadopodium assembly and maturation and show that invadopodia use cortactin phosphorylation as a master switch during these processes. In particular, cortactin phosphorylation was found to regulate cofilin and Arp2/3 complex–dependent actin polymerization. Cortactin directly binds cofilin and inhibits its severing activity. Cortactin phosphorylation is required to release this inhibition so cofilin can sever actin filaments to create barbed ends at invadopodia to support Arp2/3-dependent actin polymerization. After barbed end formation, cortactin is dephosphorylated, which blocks cofilin severing activity thereby stabilizing invadopodia. These findings identify novel mechanisms for actin polymerization in the invadopodia of metastatic carcinoma cells and define four distinct stages of invadopodium assembly and maturation consisting of invadopodium precursor formation, actin polymerization, stabilization, and matrix degradation.
TL;DR: It is shown that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filipodia tip and root, and that perturbation of these key steps in actin dynamics results in altered synaptic transmission.
Abstract: Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.
TL;DR: Results show that focal adhesion disassembly occurs through a targeted mechanism involving MTs, clathrin, and specificClathrin adaptors and that direct endocytosis of integrins from focal adhesions mediates their disassembly in migrating cells.
Abstract: Focal adhesion disassembly is regulated by microtubules (MTs) through an unknown mechanism that involves dynamin. To test whether endocytosis may be involved, we interfered with the function of clathrin or its adaptors autosomal recessive hypercholesteremia (ARH) and Dab2 (Disabled-2) and found that both treatments prevented MT-induced focal adhesion disassembly. Surface labeling experiments showed that integrin was endocytosed in an extracellular matrix–, clathrin-, and ARH- and Dab2-dependent manner before entering Rab5 endosomes. Clathrin colocalized with a subset of focal adhesions in an ARH- and Dab2-dependent fashion. Direct imaging showed that clathrin rapidly accumulated on focal adhesions during MT-stimulated disassembly and departed from focal adhesions with integrin upon their disassembly. In migrating cells, depletion of clathrin or Dab2 and ARH inhibited focal adhesion disassembly and decreased the rate of migration. These results show that focal adhesion disassembly occurs through a targeted mechanism involving MTs, clathrin, and specific clathrin adaptors and that direct endocytosis of integrins from focal adhesions mediates their disassembly in migrating cells.
TL;DR: mTor, acting mainly via mTORC1, controls dystrophin transcription in a raptor- and rictor-independent mechanism.
Abstract: Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death. mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent.