磷酸肌醇激酶FAB1调控拟南芥根毛伸长

FAB1/PIKfyve是介导PI(3,5)P₂ (磷脂酰肌醇3,5-二磷酸)生物合成的磷酸肌醇激酶。在动物和酵母(Saccharomyces cerevisiae)中, PI(3,5)P₂参与调控胞内膜运输, 但在植物中的研究较少。该文通过分析拟南芥(Arabidopsis thaliana) FAB1的T-DNA插入突变体的表型解析PI(3,5)P₂的生物学功能。拟南芥FAB1基因家族包含FAB1A、FAB1B、FAB1C和FAB1D四个基因。研究发现, fab1a/b呈现雄配子体致死的表型。利用遗传杂交获得fab1b/c/d三突变体, 发现FAB1B、FAB1C和FAB1D功能缺失导致根毛相比野生型变短, 经FAB1特异性抑制剂YM201636处理后的野生型中也观察到相似的短根毛表型。此外, fab1b/c/d三突变体中DR5转录水平降低。同时, 外源施加生长素类似物2,4-D和NAA能部分恢复fab1b/c/d植株短根毛的表型, 但fab1b/c/d突变体对生长素转运抑制剂(1-NOA和TIBA)的敏感性与野生型相似。此外, FAB1B/C/D功能缺失使根毛中ROS的含量减少且影响肌动蛋白的表达。上述结果表明, FAB1B/C/D通过调控生长素分布、ROS含量和肌动蛋白的表达影响拟南芥根毛伸长。     

A comparative Proteomics Analysis Identified Differentially Expressed Proteins in Pancreatic Cancer–Associated Stellate Cell Small Extracellular Vesicles

Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor-derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Plant action: Multiple levels of regulation

This lecture is devoted to the relative contribution of various levels of regulation of the actin cytoskeleton functioning in the cell. Regulation at the levels of gene expression, mRNA and protein synthesis and stability, processes of actin polymerization/depolymerization and actin structures reorganization is briefly considered. Novel information about the pathways of signal transduction to the actin cytoskeleton with the involvement of Arp2/3 complex and RIC proteins is highlighted.     

Simulation of protein misfolding using a lattice model

The structure of the tightly bound complex of the globular myosin head with F-actin is the key to understanding important details of the mechanism of how the actin-myosin motor functions. The current notion on this complex is based on the docking of known atomic structures of constituent proteins into low-resolution electron-density maps. The atomic structure of the complex was refined by the molecular mechanics method, which consists in minimizing the energy of molecular interaction and which makes it possible to optimize not only the relative position of protein backbones as rigid bodies, but also the position of side chains on the protein interface. The structure calculated using ICM-Pro software, on the one hand, is close to the model obtained using electron microscopy; on the other hand, it ensures the best calculated interaction energy and accounts for the results of mutagenesis experiments. On the basis of the structure obtained, we can suggest the molecular mechanisms underlying the actin-activated release of ATP hydrolysis products from myosin and the decrease in the affinity of myosin for actin upon binding of nucleotides.     

A novel regulatory role of TRAPPC9 in L-plastin-mediated osteoclast actin ring formation

Trafficking protein particle complex 9 (TRAPPC9) is a major subunit of the TRAPPII complex. TRAPPC9 has been reported to bind nuclear factor κB kinase subunit β (IKKβ) and NF-kB-inducing kinase (NIK) where it plays a role in the canonical and noncanonical of nuclear factor-κB (NF-kB) signaling pathways, receptively. The role of TRAPPC9 in protein trafficking and cytoskeleton organization in osteoclast (OC) has not been studied yet. In this study, we examined the mRNA expression of TRAPPC9 during OC differentiation. Next, we examined the colocalization of TRAPPC9 with cathepsin-K, known to mediate OC resorption suggesting that TRAPPC9 mediates the trafficking pathway within OC. To identify TRAPPC9 protein partners important for OC-mediated cytoskeleton re-organization, we conducted immunoprecipitation of TRAPPC9 in mature OCs followed by mass spectrometry analysis. Our data showed that TRAPPC9 binds various protein partners. One protein with high recovery rate is L-plastin (LPL). LPL localizes at the podosomes and reported to play a crucial role in actin aggregation thereby actin ring formation and OC function. Although the role of LPL in OC-mediated bone resorption has not fully reported in detail. Here, first, we confirmed the binding of LPL to TRAPPC9 and, then, we investigated the potential regulatory role of TRAPPC9 in LPL-mediated OC cytoskeleton reorganization. We assessed the localization of TRAPPC9 and LPL in OC and found that TRAPPC9 is colocalized with LPL at the periphery of OC. Next, we determined the effect of TRAPPC9 overexpression on LPL recruitment to the actin ring using a viral system. Interestingly, our data showed that TRAPPC9 overexpression promotes the recruitment of LPL to the actin ring when compared with control cultures. In addition, we observed that TRAPPC9 overexpression reorganizes actin clusters/aggregates and regulates vinculin recruitment into the OC periphery to initiate podosome formation.     

Nervous system development in the fairy shrimp Branchinella sp. (Crustacea: Branchiopoda: Anostraca): Insights into the development and evolution of the branchiopod brain and its sensory organs

Using immunohistochemical labeling against acetylated a‐tubulin and serotonin in combination with confocal laser scanning microscopy and 3D‐reconstruction, we investigated the temporary freshwater pond inhabitant Branchinella sp. (Crustacea: Branchiopoda: Anostraca) for the first time to provide detailed data on the development of the anostracan nervous system. Protocerebral sense organs such as the nauplius eye and frontal filament organs are present as early as the hatching stage L0. In the postnaupliar region, two terminal pioneer neurons grow from posterior to anterior to connect the mandibular neuromeres. The first protocerebral neuropil to emerge is not part of the central complex but represents the median neuropil, and begins to develop from L0+ onwards. In stage L3, the first evidence of developing compound eyes is visible. This is followed by the formation of the visual neuropils and the neuropils of the central complex in the protocerebrum. From the deutocerebral lobes, the projecting neuron tract proceeds to both sides of the lateral protocerebrum, forming a chiasma just behind the central body. In the postnaupliar region, the peripheral nervous system, commissures and connectives develop along an anterior–posterior gradient after the fasciculation of the terminal pioneer neurons with the mandibular neuromere. The peripheral nervous system in the thoracic segments consists of two longitudinal neurite bundles on each side which connect the intersegmental nerves, together with the ventral nervous system forming an orthogon‐like network. Here, we discuss, among other things, the evidence of a fourth nauplius eye nerve and decussating projecting neuron tract found in Branchinella sp., and provide arguments to support our view that the crustacean frontal filament (organ) and onychophoran primary antenna are homologous. J. Morphol. 277:1423–1446, 2016. © 2016 Wiley Periodicals, Inc.     

The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt,Notophthalmus viridescens,during limb regeneration

Summary Previous work has shown that the monoclonal antibody 22/18 identifies progenitor cells (blastemal cells) which depend on the nerve for their division in the early stages of limb regeneration in the newt,Notophthalmus viridescens. This antibody also reacts with cultured cells derived from the newt limb, and the intensity of immunoreactivity appears related to cell density and differentiation into myotubes. We report here that the monoclonal antibody 22/18 recognizes a polypeptide (22/18 antigen) which is intracellular and filamentous. Double staining of cells with 22/18 monoclonal antibody and antibodies against various cytoskeletal components indicates that the epitope is expressed on an intermediate filament component. Although this antibody is specific for blastemal cells in cryostat sections of the regenerating limb, its reactivity on immunoblots is not confined to this tissue. The 22/18 antigen is differentially affected by aldehyde fixatives distinguished by the spacing of their reactive groups. While formaldehyde fixation impairs detection of the antigen, ethylene glycol-bis[succinic acid n-hydroxysuccinimide ester] reveals the antigen in sections of normal and regenerating limbs in a distribution that is consistent with the one obtained from immunoblots. We suggest that the 22/18 monoclonal antibody detects a change in protein conformation, probably related to changes in the physiological state of the cell, that occurs transiently during regeneration and possibly during development.     

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.