Self-Assembly of Poly(L-glutamate)-block-poly(2-(diethylamino)ethyl methacrylate) in Aqueous Solutions

2011 ◽  
Vol 64 (9) ◽  
pp. 1247 ◽  
Author(s):  
Angela M. H. Leung ◽  
Vanessa Ng ◽  
Yoon K. Ho ◽  
Lihan Zhou ◽  
Heng P. Too ◽  
...  
Keyword(s):  
Self Assembly ◽  
Hydrodynamic Radius ◽  
Neuroblastoma Cells ◽  
Acid Group ◽  
Low Ph ◽  
The Self ◽  
Solution Ph ◽  
High Ph ◽  
Ethyl Methacrylate

The self-assembly and gene delivery applications of poly(l-glutamate)-block-poly(2-(diethylamino)ethyl methacrylate) (PLG18-b-PDEAEMA37) were investigated. Owing to the functional groups on the polymer, an amine and a carboxylic acid group, the self-assembly process is dependent on the solution pH, where the critical micelle concentration was determined to be 61 and 320 μg mL–1 at high and low pH, respectively. The block copolymer forms positively charged vesicles at low pH with a hydrodynamic radius of 90 nm and negatively charged vesicles at high pH with a hydrodynamic radius of 50 nm. At the isoelectric point of 4.9, PLG-b-PDEAEMA was found to form larger micellar aggregates with a hydrodynamic radius of 180 nm because of the presence of both positive and negative charges. The complexation between PLG-b-PDEAEMA and DNA was studied at physiological pH as well as at low and high pH. DNA is condensed most effectively at pH 3, at an N/P ratio of 5, whereas at pH 7 and 10, an N/P ratio of 20 is required. However, in-vitro studies at physiological pH using neuroblastoma cells did not show substantial gene expression.

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 Self-Assembled Metal–Organic Biohybrids (MOBs) Using Copper and Silver for Cell Studies

Nanomaterials ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 1282 ◽  
Author(s):  
Neha Karekar ◽  
Anik Karan ◽  
Elnaz Khezerlou ◽  
Neela Prajapati ◽  
Chelsea D. Pernici ◽  
...  
Keyword(s):  
Self Assembly ◽  
Neuroblastoma Cells ◽  
The Novel ◽  
Culture Methods ◽  
Synthesis Conditions ◽  
Tissue Constructs ◽  
Silver Sulfate ◽  
Significant Toxicity ◽  
Metal Organic

The novel synthesis of metal-containing biohybrids using self-assembly methods at physiological temperatures (37 °C) was compared for copper and silver using the amino acid dimer cystine. Once assembled, the copper containing biohybrid is a stable, high-aspect ratio structure, which we call CuHARS. Using the same synthesis conditions, but replacing copper with silver, we have synthesized cystine-capped silver nanoparticles (AgCysNPs), which are shown here to form stable colloid solutions in contrast to the CuHARS, which settle out from a 1 mg/mL solution in 90 min. Both the copper and silver biohybrids, as synthesized, demonstrate very low agglomeration which we have applied for the purpose of applications with cell culture methods, namely, for testing as anti-cancer compounds. AgCysNPs (1000 ng/mL) demonstrated significant toxicity (only 6.8% viability) to glioma and neuroblastoma cells in vitro, with concentrations as low as 20 ng/mL causing some toxicity. In contrast, CuHARS required at least 5 μg/mL. For comparative purposes, silver sulfate at 100 ng/mL decreased viability by 52% and copper sulfate at 100 ng/mL only by 19.5% on glioma cells. Using these methods, the novel materials were tested here as metal–organic biohybrids (MOBs), and it is anticipated that the functionalization and dynamics of MOBs may result in building a foundation of new materials for cellular applications, including cell engineering of both normal and diseased cells and tissue constructs.

scholarly journals A natural product inhibits the initiation of α-synuclein aggregation and suppresses its toxicity

2017 ◽  
Vol 114 (6) ◽  
pp. E1009-E1017 ◽  
Author(s):  
Michele Perni ◽  
Céline Galvagnion ◽  
Alexander Maltsev ◽  
Georg Meisl ◽  
Martin B. D. Müller ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Natural Product ◽  
Self Assembly ◽  
Lipid Membranes ◽  
Neuroblastoma Cells ◽  
Lipid Vesicles ◽  
Human Neuroblastoma

The self-assembly of α-synuclein is closely associated with Parkinson’s disease and related syndromes. We show that squalamine, a natural product with known anticancer and antiviral activity, dramatically affects α-synuclein aggregation in vitro and in vivo. We elucidate the mechanism of action of squalamine by investigating its interaction with lipid vesicles, which are known to stimulate nucleation, and find that this compound displaces α-synuclein from the surfaces of such vesicles, thereby blocking the first steps in its aggregation process. We also show that squalamine almost completely suppresses the toxicity of α-synuclein oligomers in human neuroblastoma cells by inhibiting their interactions with lipid membranes. We further examine the effects of squalamine in a Caenorhabditis elegans strain overexpressing α-synuclein, observing a dramatic reduction of α-synuclein aggregation and an almost complete elimination of muscle paralysis. These findings suggest that squalamine could be a means of therapeutic intervention in Parkinson’s disease and related conditions.

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 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 Human Organ-Specific 3D Cancer Models Produced by the Stromal Self-Assembly Method of Tissue Engineering for the Study of Solid Tumors

2020 ◽  
Vol 2020 ◽  
pp. 1-23 ◽  
Author(s):  
Vincent Roy ◽  
Brice Magne ◽  
Maude Vaillancourt-Audet ◽  
Mathieu Blais ◽  
Stéphane Chabaud ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Tumor Microenvironment ◽  
Self Assembly ◽  
The Self ◽  
Human Organ ◽  
Assembly Method ◽  
Cancer Models ◽  
Organ Specific

Cancer research has considerably progressed with the improvement of in vitro study models, helping to understand the key role of the tumor microenvironment in cancer development and progression. Over the last few years, complex 3D human cell culture systems have gained much popularity over in vivo models, as they accurately mimic the tumor microenvironment and allow high-throughput drug screening. Of particular interest, in vitrohuman 3D tissue constructs, produced by the self-assembly method of tissue engineering, have been successfully used to model the tumor microenvironment and now represent a very promising approach to further develop diverse cancer models. In this review, we describe the importance of the tumor microenvironment and present the existing in vitro cancer models generated through the self-assembly method of tissue engineering. Lastly, we highlight the relevance of this approach to mimic various and complex tumors, including basal cell carcinoma, cutaneous neurofibroma, skin melanoma, bladder cancer, and uveal melanoma.

Polymorphism of tubulin assembly in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 479-488
Author(s):  
PAULA KARECLA ◽  
ELIZABETH HIRST ◽  
PETER BAYLEY
Keyword(s):  
Self Assembly ◽  
Basic Structure ◽  
Limited Range ◽  
The Self ◽  
Tubulin Dimer ◽  
Thin Sectioning ◽  
Associated Proteins ◽  
Bona Fide ◽  
Polymorphic Forms

Polymorphism in the self-assembly of tubulin dimer and microtubule protein (tubulin plus the microtubule-associated proteins) has been investigated as a function of systematic variation of solution composition (i.e. buffer ion, [glycerol] and [Mg2+]). The nature of the assembly product was examined using negative staining and thin sectioning electron microscopy. The morphology of the product of assembly of tubulin dimer was found to be strongly influenced by the concentration of glycerol and Mg2+ in Pipes and Mes buffers; the effects are less marked in phosphate buffer. Formation of bona fide microtubules in O.l M-Pipes occurs for a limited range of solution conditions (e.g. with [glycerol] <2 M and [Mg2+]<1mM). Conditions of elevated [glycerol] and [Mg2+], which enhance the rate and extent of assembly, have the adverse effect of strongly promoting the formation of polymorphic forms in addition to, and in place of, the normal microtubule morphology. In both Pipes and Mes buffers, increasing [glycerol] from 1 to 3 M favours the formation of extended multiply curved sheets, apparently made up from a basic structure with an S-like crosssection. By contrast, increasing [Mg2+] promotes the formation of junctions between microtubule walls, giving products whose cross-section shows multiple hook-like appendages, attached to closed microtubules. The assembly of tubulin dimer in a typical ‘dimer assembly buffer’ (e.g. 0.05-0.1 MMes, with 1–3.4M-glycerol and 2–7mM-Mg2+), invariably produces substantial proportions of nonmicrotubule structures such as open sheets, ribbons, and hooked structures. We conclude that the self-assembly of tubulin dimer exclusively into bona fide microtubules occurs over a very restricted range of solution conditions in the normally used Pipes- and Mes-based buffers. Deviation from these conditions readily promotes the formation of mixtures of polymorphic forms. Many buffer systems used for the assembly and disassembly of microtubules composed of tubulin dimer appear likely to promote the formation of structures related to, but significantly different from, normal microtubules. This represents a cautionary factor in the interpretation of in vitro assembly and disassembly properties of microtubules

The Origin of the Eukaryotic Cell Based on Conservation of Existing Interfaces

2006 ◽  
Vol 12 (4) ◽  
pp. 513-523 ◽  
Author(s):  
Albert D. G. de Roos
Keyword(s):  
Plasma Membrane ◽  
Protein Interactions ◽  
Self Assembly ◽  
Evolutionary Model ◽  
Lipid Vesicles ◽  
Eukaryotic Cell ◽  
The Self ◽  
Lipid Protein Interactions

Current theories about the origin of the eukaryotic cell all assume that during evolution a prokaryotic cell acquired a nucleus. Here, it is shown that a scenario in which the nucleus acquired a plasma membrane is inherently less complex because existing interfaces remain intact during evolution. Using this scenario, the evolution to the first eukaryotic cell can be modeled in three steps, based on the self-assembly of cellular membranes by lipid-protein interactions. First, the inclusion of chromosomes in a nuclear membrane is mediated by interactions between laminar proteins and lipid vesicles. Second, the formation of a primitive endoplasmic reticulum, or exomembrane, is induced by the expression of intrinsic membrane proteins. Third, a plasma membrane is formed by fusion of exomembrane vesicles on the cytoskeletal protein scaffold. All three self-assembly processes occur both in vivo and in vitro. This new model provides a gradual Darwinistic evolutionary model of the origins of the eukaryotic cell and suggests an inherent ability of an ancestral, primitive genome to induce its own inclusion in a membrane.

scholarly journals Yeast proteins associated with microtubules in vitro and in vivo.

1992 ◽  
Vol 3 (1) ◽  
pp. 29-47 ◽  
Author(s):  
G Barnes ◽  
K A Louie ◽  
D Botstein
Keyword(s):  
Self Assembly ◽  
Synthetic Peptides ◽  
Immunofluorescence Microscopy ◽  
The Self ◽  
Microtubule Associated Proteins ◽  
Amino Terminal ◽  
Novel Proteins ◽  
Associated Proteins

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

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