Macromolecular crowding dramatically affects cellular processes such as protein folding and assembly, regulation of metabolic pathways, and condensation of DNA. Despite increased attention, we still lack a definition for how crowded a heterogeneous environment is at the molecular scale and how this manifests in basic physical phenomena like diffusion. Here, we show by means of fluorescence correlation spectroscopy and computer simulations that crowding manifests itself through the emergence of anomalous subdiffusion of cytoplasmic macromolecules. In other words, the mean square displacement of a protein will grow less than linear in time and the degree of this anomality depends on the size and conformation of the traced particle and on the total protein concentration of the solution. We therefore propose that the anomality of the diffusion can be used as a quantifiable measure for the crowdedness of the cytoplasm at the molecular scale.
We have investigated the role for diacylglycerol (DAG) in membrane bud formation in the Golgi apparatus. Addition of propranolol to specifically inhibit phosphatidate phosphohydrolase (PAP), an enzyme responsible for converting phosphatidic acid into DAG, effectively prevents formation of membrane buds. The effect of PAP inhibition on Golgi membranes is rapid and occurs within 3 min. Removal of the PAP inhibitor then results in a rapid burst of buds, vesicles, and tubules that peaks within 2 min. The inability to form buds in the presence of propranolol does not appear to be correlated with a loss of ARFGAP1 from Golgi membranes, as knockdown of ARFGAP1 by RNA interference has little or no effect on actual bud formation. Rather, knockdown of ARFGAP1 results in an increase in membrane buds and a decrease of vesicles and tubules suggesting it functions in the late stages of scission. How DAG promotes bud formation is discussed. INTRODUCTIONFormation of buds to generate intracellular transport vesicles from membranes such as Golgi cisternae involves both coat binding and local lipid conversion (for reviews and theoretical models, see Kirchhausen, 2000;Shemesh et al., 2003;Weiss and Nilsson, 2003;Bethune et al., 2006). For COPI vesicles, formation of buds is initiated by the small GTPase ARF1 (ADP-ribosylation factor 1) which, in its GTPconferred conformation, drives coatomer recruitment from the cytosol to both Golgi and pre-Golgi membranes (Palmer et al., 1993). Indeed, ARF1 and coatomer are sufficient for both bud and vesicle formation as evidenced in in vitro experiments using liposomes forming coated vesicles in a controlled manner (Spang et al., 1998). Addition of ARF-GAP1, a GTPase-activating protein for ARF1, then yielded uncoated vesicles of the expected size of ϳ50-60 nm in diameter (Reinhard et al., 2003).The situation in biological membranes is likely more refined involving additional as well as alternative components to promote or prevent vesicle formation such that Golgi function is maintained. Here, both ARF1 and ARFGAP1 have been implicated in vesicle formation through direct or indirect modulation of lipid synthesis such that bud formation and membrane fission are promoted. For example, ARF1 stimulates the production of phosphatidic acid (PA) from phosphatidylcholine (PC; Brown et al., 1993;Cockcroft et al., 1994) through the activation of phospholipase D (PLD) in a nucleotide (GTP)-specific manner Houle et al., 1995;Ktistakis et al., 1995). Such ARF1-mediated PLD stimulation results in an increased vesicle production (Ktistakis et al., 1996;Chen et al., 1997). This ability of ARF1 to stimulate lipid formation in the Golgi apparatus offers a possibility to mechanistically link lipid conversion with coat recruitment. PA may also be converted to diacylglycerol (DAG) and the ratio between DAG and PC seems to influence protein transport through the Golgi apparatus in yeast (Rivas et al., 1999). PA can also be synthesized from lysophosphatidic acid (LPA) by a LPA acyltransferase-dependent pathway through...
Neurodegenerative diseases such as Alzheimer’s disease are characterized by the progressive spreading and accumulation of hyper-phosphorylated tau protein in the brain. Anti-tau antibodies have been shown to reduce tau pathology in in vivo models and antibody-mediated clearance of tau exerted by microglia has been proposed as a contributing factor. By subjecting primary microglia cultured in vitro to anti-phospho-tau antibodies in complex with pathological tau, we show that microglia internalise and degrade tau in a manner that is dependent on FcγR interaction and functional lysosomes. It has recently been discussed if anti-tau antibody effector-functions are required for induction of tau clearance. Using antibodies with compromised FcγR binding and non-compromised control antibodies we show that antibody effector functions are required for induction of microglial clearance of tau. Understanding the inflammatory consequences of targeting microglia using therapeutic antibodies is important when developing these molecules for clinical use. Using RNA sequencing, we show that treatment with anti-tau antibodies increases transcription of mRNA encoding pro-inflammatory markers, but that the mRNA expression profile of antibody-treated cells differ from the profile of LPS activated microglia. We further demonstrate that microglia activation alone is not sufficient to induce significant tau clearance.
Immunotherapy is a promising strategy for the treatment of Alzheimer’s disease (AD). Antibodies directed against Amyloid Beta (Aβ) are able to successfully clear plaques and reverse cognitive deficits in mouse models. Excitement towards this approach has been tempered by high profile failures in the clinic, one key issue has been the development of inflammatory side effects in the brain (ARIAs). New antibodies are entering the clinic for Alzheimer’s disease; therefore, it is important to learn all we can from the current generation. In this study, we directly compared 3 clinical candidates in the same pre-clinical model, with the same effector function, for their ability to clear plaques and induce inflammation in the brain. We produced murine versions of the antibodies: Bapineuzumab (3D6), Crenezumab (mC2) and Gantenerumab (chGantenerumab) with an IgG2a constant region. 18-month transgenic APP mice (Tg2576) were injected bilaterally into the hippocampus with 2 µg of each antibody or control. After 7 days, the mice tissue was analysed for clearance of plaques and neuroinflammation by histology and biochemical analysis. 3D6 was the best binder to plaques and in vitro, whilst mC2 bound the least strongly. This translated into 3D6 effectively clearing plaques and reducing the levels of insoluble Aβ, whilst chGantenerumab and mC2 did not. 3D6 caused a significant increase in the levels of pro-inflammatory cytokines IL-1β and TNFα, and an associated increase in microglial expression of CD11B and CD68. chGantenerumab increased pro-inflammatory cytokines and microglial activation, but minimal changes in CD68, as an indicator of phagocytosis. Injection of mC2 did not cause any significant inflammatory changes. Our results demonstrate that the ability of an antibody to clear plaques and induce inflammation is dependent on the epitope and affinity of the antibody.
Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.
IntroductionThe abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies.MethodsHighly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays.ResultsThe antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau.DiscussionTargeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.
Nerve growth factor-β (NGF) is essential for the correct development of the nervous system. NGF exists in both a mature form and a pro-form (proNGF). The two forms have opposing effects on neurons: NGF induces proliferation, whereas proNGF induces apoptosis via binding to a receptor complex of the common neurotrophin receptor (p75NTR) and sortilin. The overexpression of both proNGF and sortilin has been associated with several neurodegenerative diseases. Insights into the conformational differences between proNGF and NGF are central to a better understanding of the opposing mechanisms of action of NGF and proNGF on neurons. However, whereas the structure of NGF has been determined by X-ray crystallography, the structural details for proNGF remain elusive. Here, using a sensitive MS-based analytical method to measure the hydrogen/deuterium exchange of proteins in solution, we analyzed the conformational properties of proNGF and NGF. We detected the presence of a localized higher-order structure motif in the pro-part of proNGF. Furthermore, by comparing the hydrogen/deuterium exchange in the mature part of NGF and proNGF, we found that the presence of the pro-part in proNGF causes a structural stabilization of three loop regions in the mature part, possibly through a direct molecular interaction. Moreover, using tandem MS analyses, we identified two -linked and two-linked glycosylations in the pro-part of proNGF. These results advance our knowledge of the conformational properties of proNGF and NGF and help provide a rationale for the diverse biological effects of NGF and proNGF at the molecular level.
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