Understanding the kinetic roles of the inducer heparin and of rod-like protofibrils during amyloid fibril formation by Tau protein

The aggregation of the natively disordered protein, Tau, to form lesions called neurofibrillary tangles is a characteristic feature of several neurodegenerative tauopathies. The polyanion, heparin, is commonly used as an inducer in studies of Tau aggregation in vitro, but there is surprisingly no comprehensive model describing, quantitatively, all aspects of the heparin-induced aggregation reaction. In this study, rate constants and extents of fibril formation by the four repeat domain of Tau (Tau4RD) have been reproducibly determined over a full range of heparin and protein concentrations. The kinetic role of heparin in the nucleation-dependent fibril formation reaction is shown to be limited to participation in the initial rate-limiting steps; a single heparin molecule binds two Tau4RD molecules, forming an aggregation-competent protein dimer, which then serves as a building block for further fibril growth. Importantly, the minimal kinetic model that is proposed can quantitatively account for the characteristic bell-shaped dependence of the aggregation kinetics on the stoichiometry of protein to heparin. Very importantly, this study also identifies for the first time short and thin, rod-like protofibrils that are populated transiently, early during the time course of fibril formation. The identification of these protofibrils as bona fide off-pathway species has implications for the development of therapies for tauopathies based on driving fibril formation as a means of protecting the cell from smaller, putatively toxic aggregates.     

荧光漂白后恢复技术及其在活细胞分子机制研究中的应用

荧光漂白后恢复（FRAP）是一项利用荧光探针研究活体细胞中各类分子迁移特性的技术。简要介绍了FRAP技术的原理和具体实施要求,列出了动态比和扩散系数的计算公式,并例举了近几年FRAP技术在细胞分子机制研究中的应用。     

Anomalously slow mobility of fluorescent lipid probes in the plasma membrane of the yeastSaccharomyces cerevisiae

Summary We measured the lateral mobility of two fluorescent lipid probes dioctadecylindocarbocyanine (dil) and tetramethyl rhodamine phosphatidylethanolamine (R-PE) in the plasma mem branesof Saccharomyces cerevisiae inol andopi 3 spheroplasts. These are well-characterized strains with mutations in the inositol and phosphatidylcholine biosynthetic pathways. Membrane phospholipid composition was altered by growing these mutants in the presence or absence of inositol and choline. Lateral mobil ity was measured by fluorescence recovery after photobleaching (FRAP). Microscopic fluorescence polarization employing CCD digital imaging produced an ordered orientation distribution of the lipid probe dil, confirming that at least one of the probes was largely incorporated into the bilayer membrane. Our results demonstrated anomalously slow mobility of both lipid probes for both mutants, regardless of whether the lipid composition was near normal or dramatically altered in relative composition of phosphatidylinositol and phosphatidylcholine. Trypsinization of the spheroplasts to remove surface proteins resulted in markedly increased lateral mobility. However, even in trypsinized sphero plasts, mobility was still somewhat lower than the mobility ob served in the membrane of mammalian cells, such as rat smooth muscle culture cells tested here for comparison.     

Parvalbumin is freely mobile in axons, somata and nuclei of cerebellar Purkinje neurones

The Ca(2+) -binding protein (CaBP) parvalbumin (PV) is strongly expressed in cerebellar Purkinje neurones (PNs). It is considered a pure Ca(2+) buffer, lacking any Ca(2+) sensor function. Consistent with this notion, no PV ligand was found in dendrites of PNs. Recently, however, we observed for a related CaBP that ligand-targeting differs substantially between dendrites and axons. Thus, here we quantified the diffusion of dye-labelled PV in axons, somata and nuclei of PNs by two-photon fluorescence recovery after photobleaching (FRAP). In all three compartments the fluorescence rapidly returned to baseline, indicating that no large or immobile PV ligand was present. In the axon, FRAP was well described by a one-dimensional diffusion equation and a diffusion coefficient (D) of 12 (IQR 6-20) micro m(2)/s. For the soma and nucleus a three-dimensional model yielded similar D values. The diffusional mobility in these compartments was approximately 3 times smaller than in dendrites. Based on control experiments with fluorescein dextrans, we attributed this reduced mobility of PV to different cytoplasmic properties rather than to specific PV interactions in these compartments. Our findings support the notion that PV functions as a pure Ca(2+) buffer and will aid simulations of neuronal Ca(2+) signalling.     

基于激光共聚焦同步双扫描技术的LC3脱乙酰化修饰调控自噬的机理研究

激光共聚焦同步双扫描(simultaneous,SIM)技术在常规扫描单元的基础上,引入一个同步扫描单元(SIM scanner),该技术独立控制了两个激光束,一个用于激光光刺激,另一个用于同步成像。本实验中,采用激光共聚焦同步双扫描系统的405 nm和488 nm激光分别对细胞的特定部位进行刺激和同步成像,实时检测了LC3复合物的形成,记录并分析了乙酰化前后LC3的光动力学变化过程,证实了LC3的脱乙酰化修饰是自噬性降解所必须的,本实验体系为激光共聚焦双扫描技术的推广提供了一个很好的平台。SIM技术的应用,解决了刺激过程无法成像的问题,为漂白后荧光恢复(fluorescence recovery after photobleaching,FRAP)、漂白后荧光损失(fluorescence loss in photobleaching,FLIP)和光诱导激活等研究提供了最佳的解决方案,可作为光刺激的一种实验模式在很多实验设计中进行延伸应用。     

Mutations in the B1 domain of protein G that delay the onset of amyloid fibril formation in vitro

We previously reported that under certain experimental conditions, many variants of the B1 domain of IgG-binding protein G from Streptococcus form fibrils reproducibly. The variant I6T53 was the focus of the present study because the lag phase in the kinetics of fibril formation by this variant is significantly longer than that of other variants. This lag phase is distinguished by changes in both intrinsic fluorescence intensity and in light scattering of the protein. NMR diffusion measurements suggest that the soluble protein during the lag phase is monomeric. The kinetic profiles of fibril formation are found to depend on experimental conditions. The first kinetic phase diminishes almost completely when the reaction is seeded with preformed amyloid fibrils.     

Connexin Type and Fluorescent Protein Fusion Tag Determine Structural Stability of Gap Junction Plaques

Gap junctions (GJs) are made up of plaques of laterally clustered intercellular channels and the membranes in which the channels are embedded. Arrangement of channels within a plaque determines subcellular distribution of connexin binding partners and sites of intercellular signaling. Here, we report the discovery that some connexin types form plaque structures with strikingly different degrees of fluidity in the arrangement of the GJ channel subcomponents of the GJ plaque. We uncovered this property of GJs by applying fluorescence recovery after photobleaching to GJs formed from connexins fused with fluorescent protein tags. We found that connexin 26 (Cx26) and Cx30 GJs readily diffuse within the plaque structures, whereas Cx43 GJs remain persistently immobile for more than 2 min after bleaching. The cytoplasmic C terminus of Cx43 was required for stability of Cx43 plaque arrangement. We provide evidence that these qualitative differences in GJ arrangement stability reflect endogenous characteristics, with the caveat that the sizes of the GJs examined were necessarily large for these measurements. We also uncovered an unrecognized effect of non-monomerized fluorescent protein on the dynamically arranged GJs and the organization of plaques composed of multiple connexin types. Together, these findings redefine our understanding of the GJ plaque structure and should be considered in future studies using fluorescent protein tags to probe dynamics of highly ordered protein complexes.     

Isolation of a single carboxyl-carboxylate proton binding site in the pore of a cyclic nucleotide-gated channel.

The pore of the catfish olfactory cyclic nucleotide-gated (CNG) channel contains four conserved glutamate residues, one from each subunit, that form a high-affinity binding site for extracellular divalent cations. Previous work showed that these residues form two independent and equivalent high-pKa (approximately 7.6) proton binding sites, giving rise to three pH-dependent conductance states, and it was suggested that the sites were formed by pairing of the glutamates into two independent carboxyl-carboxylates. To test further this physical picture, wild-type CNG subunits were coexpressed in Xenopus oocytes with subunits lacking the critical glutamate residue, and single channel currents through hybrid CNG channels containing one to three wild-type (WT) subunits were recorded. One of these hybrid channels had two pH-dependent conductance states whose occupancy was controlled by a single high-pKa protonation site. Expression of dimers of concatenated CNG channel subunits confirmed that this hybrid contained two WT and two mutant subunits, supporting the idea that a single protonation site is made from two glutamates (dimer expression also implied the subunit makeup of the other hybrid channels). Thus, the proton binding sites in the WT channel occur as a result of the pairing of two glutamate residues. This conclusion places these residues in close proximity to one another in the pore and implies that at any instant in time detailed fourfold symmetry is disrupted.     

Dynamics of putative raft-associated proteins at the cell surface

Lipid rafts are conceptualized as membrane microdomains enriched in cholesterol and glycosphingolipid that serve as platforms for protein segregation and signaling. The properties of these domains in vivo are unclear. Here, we use fluorescence recovery after photobleaching to test if raft association affects a protein's ability to laterally diffuse large distances across the cell surface. The diffusion coefficients (D) of several types of putative raft and nonraft proteins were systematically measured under steady-state conditions and in response to raft perturbations. Raft proteins diffused freely over large distances (> 4 microm), exhibiting Ds that varied 10-fold. This finding indicates that raft proteins do not undergo long-range diffusion as part of discrete, stable raft domains. Perturbations reported to affect lipid rafts in model membrane systems or by biochemical fractionation (cholesterol depletion, decreased temperature, and cholesterol loading) had similar effects on the diffusional mobility of raft and nonraft proteins. Thus, raft association is not the dominant factor in determining long-range protein mobility at the cell surface.     

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins

AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.     

The formation and stability of DC-SIGN microdomains require its extracellular moiety

Dendritic cell-specific intercellular adhesion molecule (ICAM)-3-grabbing non-integrin (DC-SIGN) is a Ca(2+) -dependent transmembrane lectin that binds a large variety of pathogens and facilitates their uptake for subsequent antigen presentation. This receptor is present in cell surface microdomains, but factors involved in microdomain formation and their exceptional stability are not clear. To determine which domain/motif of DC-SIGN facilitates its presence in microdomains, we studied mutations at key locations including truncation of the cytoplasmic tail, and ectodomain mutations that resulted in the removal of the N-linked glycosylation site, the tandem repeats and the carbohydrate recognition domain (CRD), as well as modification of the calcium sites in the CRD required for carbohydrate binding. Confocal imaging and fluorescence recovery after photobleaching measurements showed that the cytoplasmic domain and the N-linked glycosylation site do not affect the ability of DC-SIGN to form stable microdomains. However, truncation of the CRD results in complete loss of visible microdomains and subsequent lateral diffusion of the mutants. Apart from cell adhesions, membrane domains are thought to be localized primarily via the cytoskeleton. By contrast, we propose that interactions between the CRD of DC-SIGN and the extracellular matrix and/or cis interactions with transmembrane scaffolding protein(s) play an essential role in organizing these microdomains.     

Oligopeptide-mediated acceleration of amyloid fibril formation of amyloid beta(Abeta) and alpha-synuclein fragment peptide (NAC).

The effects of oligopeptides on the secondary structures of Abeta and NAC, a fragment of alpha-synuclein protein, were studied by circular dichroism (CD) spectra. The effects of oligopeptides on the amyloid fibril formation were also studied by fluorescence spectra due to thioflavine-T. The oligopeptides were composed of a fragment of Abeta or NAC and were interposed by acidic or basic amino acid residues. The peptide, Ac-ELVFFAKK-NH2, which involved a fragment Leu-Val-Phe-Phe-Ala at Abeta(17-21), had no effect on the secondary structures of Abeta(1-28) in 60% or 90% trifluoroethanol (TFE) solutions at both pH 3.2 and pH 7.2. However, it showed pronounced effects on the secondary structure of Abeta(1-28) at pH 5.4. The Ac-ELVFFAKK-NH2 reduced the alpha-helical content, while it increased the beta-sheet content of Abeta(1-28). In phosphate buffer solutions at pH 7.0, Ac-ELVFFAKK-NH2 had little effect on the secondary structures of Abeta(1-28). However, it accelerated amyloid fibril formation when monitored by fluorescence spectra due to thioflavine-T. On the other hand, LPFFD, a peptide known as a beta-sheet breaker, caused neither an appreciable extent of change in the secondary structure nor amyloid fibril formation in the same buffer solution. The peptide, Ac-ETVK-NH2, which involved a fragment Thr-Val at NAC(21-22), had no effect on the secondary structure of NAC in 90% TFE and in isotonic phosphate buffer. However, Ac-ETVK-NH2 in water with small amounts of NaN3 and hexafluoroisopropanol greatly increased the beta-sheet content of NAC after standing the solution for more than 1 week. Interestingly, in this solution. Ac-ETVK-NH2, accelerated the fibril formation of NAC. It was concluded that an oligopeptide that involves a fragment of amyloidogenic proteins could be a trigger for the formation of amyloid plaques of the proteins even when it had little effect on the secondary structures of the proteins as monitored by CD spectra for a short incubation time.     

Elimination of radiation-induced gamma-H2AX foci in mammalian nucleus can occur by histone exchange

Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX (gamma-H2AX) and formation of large nuclear gamma-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of gamma-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced gamma-H2AX foci. Maximal number of gamma-H2AX foci is observed one hour after irradiation, when approximately 20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of gamma-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of gamma-H2AX. This indicates that elimination of gamma-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange.     

Active Galpha(q) subunits and M3 acetylcholine receptors promote distinct modes of association of RGS2 with the plasma membrane

RGS proteins accelerate the GTPase activity of heterotrimeric G proteins at the plasma membrane. Association of RGS proteins with the plasma membrane can be mediated by interactions with other membrane proteins and by direct interactions with the lipid bilayer. Here we use fluorescence recovery after photobleaching (FRAP) to characterize interactions between RGS2 and M3 acetylcholine receptors (M3Rs), Galpha subunits and the lipid bilayer. Active Galpha(q) and M3Rs both recruited RGS2-EGFP to the plasma membrane. RGS2-EGFP remained bound to the plasma membrane between interactions with active Galpha(q), but rapidly exchanged between membrane-associated and cytosolic pools when recruited by M3Rs.     

Retention and mobility of the mammalian lamin B receptor in the plant nuclear envelope

Background information. In a previous study, we showed that GFP (green fluorescent protein) fused to the N‐terminal 238 amino acids of the mammalian LBR (lamin B receptor) localized to the NE (nuclear envelope) when expressed in the plant Nicotiana tabacum. The protein was located in the NE during interphase and migrated with nuclear membranes during cell division. Targeting and retention of inner NE proteins requires several mechanisms: signals that direct movement through the nuclear pore complex, presence of a transmembrane domain or domains and retention by interaction with nuclear or nuclear‐membrane constituents. Results. Binding mutants of LBR—GFP were produced to investigate the mechanisms for the retention of LBR in the NE. FRAP (fluorescence recovery after photobleaching) analysis of mutant and wild‐type constructs was employed to examine the retention of LBR—GFP in the plant NE. wtLBR—GFP (wild‐type LBR—GFP) was shown to have significantly lower mobility in the NE than the lamin‐binding domain deletion mutant, which showed increased mobility in the NE and was also localized to the endoplasmic reticulum and punctate structures in some cells. Modification of the chromatin‐binding domain resulted in the localization of the protein in nuclear inclusions, in which it was immobile. Conclusions. As expression of truncated LBR—GFP in plant cells results in altered targeting and retention compared with wtLBR—GFP, we conclude that plant cells can recognize the INE (inner NE)‐targeting motif of LBR. The altered mobility of the truncated protein suggests that not only do plant cells recognize this signal, but also have nuclear proteins that interact weakly with LBR.     

Unrestricted Diffusion of Exogenous and Endogenous PIP2 in Baby Hamster Kidney and Chinese Hamster Ovary Cell Plasmalemma

We used two approaches to characterize the lateral mobility of phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasmalemma of baby hamster kidney and Chinese hamster ovary fibroblasts. First, nitrobenzoxadiazole-labeled C6-phosphatidylcholine and C16-PIP₂ were incorporated into plasma membrane “lawns” (∼20 × 30 μm) from these cells and into the outer monolayer of intact cells. Diffusion coefficients determined by fluorescence recovery after photobleaching were similar for the two lipids and were higher in lawns, ∼0.3 μm²/s, than on the cell surface, ∼0.1 μm²/s. For membrane lawns, the fractional recoveries (75–90%) were close to those expected from the fraction of total membrane bleached, and labeling by the probes was several times greater than for intact cells. Second, we analyzed cells expressing M1 muscarinic receptors and green fluorescent protein fused with PIP₂-binding pleckstrin-homology domains, Tubby domains or diacylglycerol (DAG)-binding C1 domains. On-cell gigaseal patches were formed with pipette tips >5 μm in diameter. When the agonist carbachol (0.3 mm) was applied either within or outside of the pipette, lipid signals crossed the pipette barrier rapidly in both directions and membrane blebbing occurred on both membrane sides. Accurate simulations of lipid gradients required diffusion coefficients >1 μm²/s. Exogenous DAG also crossed the pipette barrier rapidly. In summary, we found no evidence for restricted diffusion of signaling lipids in these cells. The lower mobility and incorporation of phospholipid at the extracellular leaflet may reflect a more ordered and condensed extracellular monolayer, as expected from previous studies. An erratum to this article can be found at     

Monitoring fibril formation of the N-terminal domain of PABPN1 carrying an alanine repeat by tryptophan fluorescence and real-time NMR

Intranuclear fibrils due to poly-alanine expansions in the N-terminal domain of the poly(A) binding protein PABPN1 correlate with the disease oculopharyngeal muscular dystrophy (OPMD). For monitoring fibril formation by fluorescence and real-time NMR spectroscopy, tryptophans were introduced either into the middle or C-terminal of the poly-alanine segment. The kinetics of fibril formation which were monitored by fluorescence spectroscopy were matched by real-time NMR kinetics. Our results show that fibril formation is concomitant with the burial of the tryptophans in the fibrillar core. Since no soluble pre-fibrillar intermediate(s) was detected, fibril formation of this domain may be regarded as a two state conversion from an unfolded soluble into folded insoluble species.