scholarly journals Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

2021 ◽  
Vol 220 (3) ◽  
Author(s):  
Michal Wieczorek ◽  
Shih-Chieh Ti ◽  
Linas Urnavicius ◽  
Kelly R. Molloy ◽  
Amol Aher ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
The Self ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Partial Cone ◽  
Ring Complex ◽  
Microtubule Networks ◽  
And Function

The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex.

scholarly journals Biochemical reconstitutions reveal principles of human γ-TuRC assembly and function

2020 ◽  
Author(s):  
Michal Wieczorek ◽  
Shih-Chieh Ti ◽  
Linas Urnavicius ◽  
Kelly R. Molloy ◽  
Amol Aher ◽  
...  
Keyword(s):  
Self Assembly ◽  
Function Analysis ◽  
The Self ◽  
Dependent Manner ◽  
Guanine Nucleotide ◽  
Partial Cone ◽  
Ring Complex ◽  
Microtubule Networks ◽  
And Function

AbstractThe formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MDa assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge”. The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a complete biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP), a ∼35S complex that nucleates microtubules in vitro. We extend our approach to generate a stable subcomplex, γ-TuRCmini-GFP, which lacks MZT1 and actin. Using mutagenesis, we show that γ-TuRCmini-GFP nucleates microtubules in a guanine nucleotide-dependent manner and proceeds with similar kinetics as reported for native γ-TuRCs. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCmini-GFP adopts a partial cone shape presenting only 8-10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our structure-function analysis suggests that the lumenal bridge facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the γ-TuRC.

Directive Effect of Chain Length in Modulating Peptide Nano-assemblies

2020 ◽  
Vol 27 (9) ◽  
pp. 923-929
Author(s):  
Gaurav Pandey ◽  
Prem Prakash Das ◽  
Vibin Ramakrishnan
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Light Scattering ◽  
Chain Length ◽  
Self Assembly ◽  
The Self ◽  
Ionic Charge ◽  
Self Assembling ◽  
Biophysical Techniques

Background: RADA-4 (Ac-RADARADARADARADA-NH2) is the most extensively studied and marketed self-assembling peptide, forming hydrogel, used to create defined threedimensional microenvironments for cell culture applications. Objectives: In this work, we use various biophysical techniques to investigate the length dependency of RADA aggregation and assembly. Methods: We synthesized a series of RADA-N peptides, N ranging from 1 to 4, resulting in four peptides having 4, 8, 12, and 16 amino acids in their sequence. Through a combination of various biophysical methods including thioflavin T fluorescence assay, static right angle light scattering assay, Dynamic Light Scattering (DLS), electron microscopy, CD, and IR spectroscopy, we have examined the role of chain-length on the self-assembly of RADA peptide. Results: Our observations show that the aggregation of ionic, charge-complementary RADA motifcontaining peptides is length-dependent, with N less than 3 are not forming spontaneous selfassemblies. Conclusion: The six biophysical experiments discussed in this paper validate the significance of chain-length on the epitaxial growth of RADA peptide self-assembly.

A Dynamic Route to Structure and Function: Recent Advances in Imine-Based Organic Nanostructured Materials

2013 ◽  
Vol 66 (1) ◽  
pp. 9 ◽  
Author(s):  
Yi Liu ◽  
Zhan-Ting Li
Keyword(s):  
Self Assembly ◽  
Reaction System ◽  
Functional Materials ◽  
Building Blocks ◽  
The Self ◽  
Interlocked Molecules ◽  
Functional Aspects ◽  
Imine Bond ◽  
And Function ◽  
Dynamic Route

The chemistry of imine bond formation from simple aldehyde and amine precursors is among the most powerful dynamic covalent chemistries employed for the construction of discrete molecular objects and extended molecular frameworks. The reversible nature of the C=N bond confers error-checking and proof-reading capabilities in the self-assembly process within a multi-component reaction system. This review highlights recent progress in the self-assembly of complex organic molecular architectures that are enabled by dynamic imine chemistry, including molecular containers with defined geometry and size, mechanically interlocked molecules, and extended frameworks and polymers, from building blocks with preprogrammed steric and electronic information. The functional aspects associated with the nanometer-scale features not only place these dynamically constructed nanostructures at the frontier of materials sciences, but also bring unprecedented opportunities for the discovery of new functional materials.

Effect of basolateral acidification on the frog oxynticopeptic cell

1990 ◽  
Vol 259 (4) ◽  
pp. G564-G570 ◽  
Author(s):  
S. Arvidsson ◽  
K. Carter ◽  
A. Yanaka ◽  
S. Ito ◽  
W. Silen
Keyword(s):  
Electron Microscopy ◽  
Gastric Mucosa ◽  
Light And Electron Microscopy ◽  
Structure And Function ◽  
Intracellular Acidification ◽  
Intracellular Acidosis ◽  
Normal Morphology ◽  
And Function ◽  
Oxynticopeptic Cells

The effects of intracellular acidosis induced by acidification of the basolateral (nutrient) perfusate on the structure and function of the oxynticopeptic cell were studied in in vitro frog gastric mucosa. Changing the pH of the unbuffered nutrient perfusate (UNB) from 7.2 to 3.5 acidified the oxynticopeptic cell with no change in potential difference (PD) or resistance (R). Intracellular pH (pHi), PD, and R were 7.05 +/- 0.01, 16 +/- 1 mV, 165 +/- 7 omega.cm2 before and 6.44 +/- 0.01, 16 +/- 2 mV, 170 +/- 9 omega.cm2 after nutrient acidification. Acid secretion (H+) increased from 0.86 +/- 0.07 to 1.88 +/- 0.18 mu eq.cm-2.h-1. Addition of forskolin to tissues perfused with nutrient pH (pHn) 3.5 decreased PD to 2 +/- 2 mV and further increased H+ to 3.07 +/- 0.19 mu eq.cm-2.h-1. By light and electron microscopy oxynticopeptic cells perfused with UNB, pHn 3.5, appeared normal. Oxynticopeptic cells in tissues pretreated with omeprazole and then exposed to UNB, pHn 3.5, had extensive morphological damage. On increasing the pH of the nutrient perfusate from 3.5 to 7.2 there was prompt recovery of pHi in untreated and forskolin-stimulated mucosae (pHi 6.87 +/- 0.06 and 6.85 +/- 0.04) but no recovery of pHi in tissues pretreated with omeprazole or cimetidine (pHi 6.26 +/- 0.04 and 6.44 +/- 0.06, n = 6, 30 min after reexposure to UNB, pHn 7.2). We conclude that in a secreting mucosa intracellular acidification of the oxynticopeptic cell to pHi 6.4 is associated with normal morphology, PD, R, and increased H+, and that intracellular acidosis is not de facto deleterious.

scholarly journals Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Lars-Anders Carlson ◽  
Yun Bai ◽  
Sarah C Keane ◽  
Jennifer A Doudna ◽  
James H Hurley
Keyword(s):  
Self Assembly ◽  
Untranslated Region ◽  
Minimal System ◽  
The Self ◽  
Viral Rna ◽  
Large Excess ◽  
Rna Packaging ◽  
Membrane Bound ◽  
Hiv 1

HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5’ untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another.

scholarly journals Quercetin ameliorates mitochondrial dysfunction and mitigates methamphetamine-induced anxiety-like behavior

2021 ◽  
Author(s):  
Fengrong Chen ◽  
Jiaxue Sun ◽  
Yongjin Zhang ◽  
Yicong Dai ◽  
Zherui Zhang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Oxygen Consumption ◽  
Mitochondrial Dysfunction ◽  
Consumption Rate ◽  
Mitochondrial Morphology ◽  
Transmission Electron ◽  
Anxiety Behavior ◽  
And Function

Methamphetamine (MA) abuse results in neurotoxic outcomes, including increased anxiety and depression, during both MA use and withdrawal. Although numerous studies have reported an association between MA exposure and anxiety, the underlying mechanism remains elusive. In this study, escalating dose of MA was used to establish an MA-treated mouse model presenting anxiety behavior. RNA seq was then performed to profile the gene expression patterns in the hippocampus (HIPP). Differentially expressed genes (DEGs) were identified and function enrichment analysis was conducted to explore the underlying mechanisms. Quercetin as an mitochondria protector was used in vivo and in vitro. The C57BL/6J mice were co-treated with 50 mg/kg Quercetin and escalating MA. Anxiety behavior was evaluated by utilizing the elevated plus maze and the open field test. Transmission electron microscopy and immunohistochemistry were conducted to study the pathology of MA-inducced anxiety . The effects of MA and Quercetin on astrocytes were investigated by fluorescence staining, transmission electron microscopy, flow cytometry, and oxygen consumption rate. Western blot and qPCR were performed to analyze altered protein and gene levels of HIPP in mice and astrtocytes. The results demonstrated that forteen upregulated differentially expressed genes were identified and significantly enriched in signaling pathways related to psychiatric disorders and mitochondrial function. Interestingly, we found that quercetin was able to alleviate MA-induced anxiety-like behavior by improving neuron number and mitochondria injury. Mechanistically, quercetin can mitigate aberrant mitochondrial morphology and mitochondrial dysfunction not only by decreasing the levels of total cytoplasmic reactive oxygen species (ROS), mitochondria-derived ROS (mtROS), and mitochondrial membrane potential (MMP), but also increasing the oxygen consumption rate (OCR) and mitochondrial ATP production in vitro, indicating Quercetin ameliorated MA-induced anxiety-like behavior by modifying mitochondrial morphology and function. Furthermore, quercetin reversed OPA1 and DRP1 expression in astrocytes, and mitigated astrocyte activation and the release of inflammatory factors, which can trigger neuronal apoptosis and synaptic loss. Taken together, we provided evidence showing that MA can induce anxiety-like behavior via the induction of oxidative stress and mitochondrial dysfunction. Quercetin exerted antipsychotic activity through mitochondrial modulation, suggesting its potential for further therapeutic development in MA-induced anxiety.

scholarly journals Self-assembled peptide nanostructures for electrical, optical, and magnetic applications

2018 ◽  
Author(s):  
◽  
Soma Khanra
Keyword(s):  
Electron Microscopy ◽  
Self Assembly ◽  
Nonlinear Optical ◽  
Electronic Devices ◽  
Longitudinal Component ◽  
The Self ◽  
Assembly Process ◽  
Experimental Characterization ◽  
X Ray ◽  
Self Assembled

Bio-nanotechnology has become a widespread exciting field of research as the basic biological structure of bio-inspired materials and nanotechnology share the common length scale. Bio-nanotechnology, which is mainly based on bio-inspired nanostructured materials, has potential applications in nanomedicine, drug delivery, bio-sensors, and bio-degradable electronic devices. The nanostructures obtained from biomolecules are attractive due to their biocompatibility for molecular recognition, ease of chemical modification, and the ability to scaffold other organic and inorganic materials. Peptide nanostructures formed through the self-assembly process of the basic building block of diphenylalanine show promising applications in biodegradable electronic devices, drug delivery, catalysis agent, waveguide, and frequency converter. This research focusses on the self-assembly process in a dipeptide, L, L diphenylalanine (FF) and exploring its electronic, optical, and magnetic properties. The role of solvents in the self-assembly process of FF is explored by combining density functional theory (DFT) along with experimental characterization techniques such as electron microscopy, Raman scattering, and x-ray diffraction (XRD). One of the objectives of this work was to explore the nonlinear optical (NLO) properties of FF nanostructures via second harmonic generation (SHG). The ratio of the nonlinear optical coefficients was obtained from individual FF nanotubes as a function of the tube diameter and thermal annealing conditions. The ratio of the shear to the longitudinal component (d15/d33) of the NLO coefficient increases with the diameter of the tubes. One of the transverse components, d31, of the NLO coefficient is found to be negative, and its magnitude with respect to the longitudinal component (d33) increases with the tube diameter. Thermal treatment of individual FF tubes has a similar effect as increasing the diameter of the tubes in SHG polarimetry. The functionalization of FF micro-nanostructures (FF-MNS) with nanomaterials was studied. FF-MNS with Ag or Au nanoparticles were explored in surface-enhanced Raman scattering (SERS). Such self-assembled nanostructures provide a natural template for tethering Au and Ag nanoparticles (Nps) due to its fractal surface. The FF-MNS undergo an irreversible phase transition from hexagonal packing (hex) to an orthorhombic (ort) structure at [about] 150 [degree]C. The metal Nps form chains on hex FF-MNS as inferred from transmission electron microscopy images and a uniform non-aggregated distribution in the ort phase. The SERS spectra obtained from R6G bound to FF-MNSs with AuNps show a higher enhancement for the ort phase compared with the hex phase. The experimental results agree well with our calculated Raman spectra of model systems using DFT. Our results indicate that FF-MNS both in the hex and ort phase can be used as substrates for SERS analysis with different metal Nps, opening up a novel class of optically active bio-based substrates. The use of magnetic nanoparticles with biomolecules offers a versatile path for tuning the functionality of the composite material for several applications. The functionalization of FF-MNS with cobalt ferrite (CFO) magnetic nanoparticles was achieved. The interaction between CFO nanoparticles and FF-MNS was investigated by optical spectroscopy, x-ray photoelectron spectroscopy (XPS), and magnetization measurements. The changes in the XPS data from pristine FF-MNS and CFO:FF-MNS are indicative of a charge transfer process from CFO to FF-MNS, changing the electronic states of the Fe2+ and Co2+ ions. A comparison of the magnetic characterization from CFO nanoparticles and CFO:FF-MNS shows a higher saturation magnetization from the nanocomposite sample, which is attributed to a change in the cationic distribution in CFO upon binding with the peptide. We were further successful in demonstrating the application of FF-MNS as a bio-degradable active layer in an organic light emitting diode (OLED). FF-MNS were functionalized with two blue-emitting conducting polymers: di-octyl-substituted polyfluorene (PF8) and ethyl-hexyl polyfluorene (PF2/6), and used as an active layer in an OLED architecture. A combination of molecular dynamics and experimental characterization techniques reveals a stronger binding mechanism for PF8 compared to PF2/6 with FF-MNS. Biodegradability tests from FF-MNS:PF8 nanocomposite films show more than 80% weight loss in 2 h by enzymatic action compared to PF8 pristine films, which do not degrade. Self-assembled FF-MNS with organic semiconductors open up a new generation of biocompatible and biodegradable materials in organic electronics.

scholarly journals Reconstructed Skin by the Self‐Assembly Approach: Is the Stem Cell Niche Present In Vitro?

2008 ◽  
Vol 22 (S1) ◽  
Author(s):  
Amélie Lavoie ◽  
Paquet Claudie ◽  
Danielle Larouche ◽  
Claudia Fugère ◽  
Israel Martel ◽  
...  
Keyword(s):  
Stem Cell ◽  
Self Assembly ◽  
Stem Cell Niche ◽  
The Self ◽  
Reconstructed Skin ◽  
Cell Niche

scholarly journals Biophysical and biochemical properties of Deup1 self-assemblies: a potential driver for deuterosome formation during multiciliogenesis

Biology Open ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. bio056432 ◽  
Author(s):  
Shohei Yamamoto ◽  
Ryoichi Yabuki ◽  
Daiju Kitagawa
Keyword(s):  
Self Assembly ◽  
Large Scale ◽  
Molecular Mechanisms ◽  
Coiled Coil ◽  
Biochemical Properties ◽  
The Self ◽  
Centrosomal Protein ◽  
Biochemical Analyses ◽  
Stable Structures

ABSTRACTThe deuterosome is a non-membranous organelle involved in large-scale centriole amplification during multiciliogenesis. Deuterosomes are specifically assembled during the process of multiciliogenesis. However, the molecular mechanisms underlying deuterosome formation are poorly understood. In this study, we investigated the molecular properties of deuterosome protein 1 (Deup1), an essential protein involved in deuterosome assembly. We found that Deup1 has the ability to self-assemble into macromolecular condensates both in vitro and in cells. The Deup1-containing structures formed in multiciliogenesis and the Deup1 condensates self-assembled in vitro showed low turnover of Deup1, suggesting that Deup1 forms highly stable structures. Our biochemical analyses revealed that an increase of the concentration of Deup1 and a crowded molecular environment both facilitate Deup1 self-assembly. The self-assembly of Deup1 relies on its N-terminal region, which contains multiple coiled coil domains. Using an optogenetic approach, we demonstrated that self-assembly and the C-terminal half of Deup1 were sufficient to spatially compartmentalize centrosomal protein 152 (Cep152) and polo like kinase 4 (Plk4), master components for centriole biogenesis, in the cytoplasm. Collectively, the present data suggest that Deup1 forms the structural core of the deuterosome through self-assembly into stable macromolecular condensates.This article has an associated First Person interview with the first author of the paper.

scholarly journals Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum

1996 ◽  
Vol 109 (6) ◽  
pp. 1479-1495 ◽  
Author(s):  
L.A. Temesvari ◽  
J.M. Rodriguez-Paris ◽  
J.M. Bush ◽  
L. Zhang ◽  
J.A. Cardelli
Keyword(s):  
Morphological Changes ◽  
Specific Inhibitor ◽  
Fluid Phase ◽  
Contractile Vacuole ◽  
Dependent Manner ◽  
Concanamycin A ◽  
Latex Beads ◽  
And Function

We have investigated the effects of Concanamycin A (CMA), a specific inhibitor of vacuolar type H(+)-ATPases, on acidification and function of the endo-lysosomal and contractile vacuole (CV) systems of D. discoideum. This drug inhibited acidification and increased the pH of endo-lysosomal vesicles both in vivo and in vitro in a dose dependent manner. Treatment also inhibited endocytosis and exocytosis of fluid phase, and phagocytosis of latex beads. This report also confirms our previous conclusions (Cardelli et al. (1989) J. Biol. Chem. 264, 3454–3463) that maintenance of acidic pH in lumenal compartments is required for efficient processing and targeting of a lysosomal enzyme, alpha-mannosidase. CMA treatment compromised the function of the contractile vacuole complex as amoebae exposed to a hypo-osmotic environment in the presence of CMA, swelled rapidly and ruptured. Fluorescence microscopy revealed that CMA treatment induced gross morphological changes in D. discoideum cells, characterized by the formation of large intracellular vacuoles containing fluid phase. The reticular membranes of the CV system were also no longer as apparent in drug treated cells. Finally, this is the first report describing cells that can adapt in the presence of CMA; in nutrient medium, D. discoideum overcame the effects of CMA after one hour of drug treatment even in the absence of protein synthesis. Upon adaptation to CMA, normal sized endo-lysosomal vesicles reappeared, endo-lysosomal pH decreased, and the rate of endocytosis, exocytosis and phagocytosis returned to normal. This study demonstrates that the V-H(+)-ATPase plays an important role in maintaining the integrity and function of the endo-lysosomal and CV systems and that D. discoideum can compensate for the loss of a functional V-H(+)-ATPase.

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