scholarly journals A synthetic membrane shaper for controlled liposome deformation

2021 ◽  
Author(s):  
Nicola De Franceschi ◽  
Weria Pezeshkian ◽  
Alessio Fragasso ◽  
Bart Bruininks ◽  
Sean Tsai ◽  
...  
Keyword(s):  
Dna Nanotechnology ◽  
Membrane Structures ◽  
Liposome Membrane ◽  
Synthetic Membrane ◽  
Synthetic Cell ◽  
Complex Membrane ◽  
In Vitro Reconstitution ◽  
High Yields ◽  
And Function

Shape defines the structure and function of cellular membranes. In cell division, the cell membrane deforms into a dumbbell shape, while organelles such as the autophagosome exhibit stomatocyte shapes. Bottom-up in vitro reconstitution of protein machineries that stabilize or resolve the membrane necks in such deformed liposome structures is of considerable interest to characterize their function. Here we develop a DNA-nanotechnology-based approach that we call Synthetic Membrane Shaper (SMS), where cholesterol-linked DNA structures attach to the liposome membrane to reproducibly generate high yields of stomatocytes and dumbbells. In silico simulations confirm the shape-stabilizing role of the SMS. We show that the SMS is fully compatible with protein reconstitution by assembling bacterial divisome proteins (DynaminA, FtsZ:ZipA) at the catenoidal neck of these membrane structures. The SMS approach provides a general tool for studying protein binding to complex membrane geometries that will greatly benefit synthetic cell research.

STEM-18. STROMAL EXTRACELLULAR VESICLES DRIVE GLIOMA STEM CELL REPROGRAMMING

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi24-vi25
Author(s):  
Lata Adnani ◽  
Brian Meehan ◽  
Jordan Kassouf ◽  
Cristiana Spinelli ◽  
Nadim Tawil ◽  
...  
Keyword(s):  
Molecular Subtype ◽  
Extracellular Vesicle ◽  
Host Cells ◽  
Tumour Mass ◽  
Membrane Structures ◽  
And Function ◽  
Rate Limiting ◽  
The Brain

Abstract Glioblastoma multiforme (GBM) represents the most frequent and lethal form of brain tumors originating from glioma stem cells (GSCs). GBM remains lethal because the rate limiting patho-mechanisms remain poorly understood. In this regard, few limitations involve the diversity 'between' cellular states and the molecular/cellular complexity 'within' the tumour mass. Using cell based- and mouse- models, we explored extracellular vesicle (EV) mediated interactions between cancer and stromal cells impacting phenotypes of GSCs as a function of their molecular subtype. EVs are spherical membrane structures that cells release to expel different molecular cargo (lipids, proteins, RNA, DNA), which can be transported across a distance in the brain and taken up by various ‘recipient’ cells resulting in reprogramming of the recipient cell's content and function. In vivo, GSCs altered their pattern of NOTCH signalling and molecular phenotype as a function of proximity to non-transformed host cells in the brain. In vitro stromal EVs altered GSC sphere forming capacity, proteome and expression of mesenchymal markers. Thus, EV mediated tumour-stromal interactions could represent a biological switch and a novel targeting point in the biology of GBM.

In vitro reconstitution of autophagic processes

2020 ◽  
Vol 48 (5) ◽  
pp. 2003-2014
Author(s):  
Jahangir Md. Alam ◽  
Nobuo N. Noda
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Membrane Structures ◽  
Autophagosome Formation ◽  
The Past ◽  
Atg Proteins ◽  
In Vitro Reconstitution ◽  
Complicated Process

Autophagy is a lysosomal degradation system that involves de novo autophagosome formation. A lot of factors are involved in autophagosome formation, including dozens of Atg proteins that form supramolecular complexes, membrane structures including vesicles and organelles, and even membraneless organelles. Because these diverse higher-order structural components cooperate to mediate de novo formation of autophagosomes, it is too complicated to be elaborated only by cell biological approaches. Recent trials to regenerate each step of this phenomenon in vitro have started to elaborate on the molecular mechanisms of such a complicated process by simplification. In this review article, we outline the in vitro reconstitution trials in autophagosome formation, mainly focusing on the reports in the past few years and discussing the molecular mechanisms of autophagosome formation by comparing in vitro and in vivo observations.

scholarly journals The embryonic linker histone dBigH1 alters the functional state of active chromatin

2020 ◽  
Vol 48 (8) ◽  
pp. 4147-4160 ◽  
Author(s):  
Paula Climent-Cantó ◽  
Albert Carbonell ◽  
Milos Tatarski ◽  
Oscar Reina ◽  
Paula Bujosa ◽  
...  
Keyword(s):  
Functional State ◽  
Early Embryo ◽  
Active Chromatin ◽  
Linker Histones ◽  
In Vitro Reconstitution ◽  
Transcription In Vitro ◽  
And Function ◽  
Female Specific ◽  
Zygotic Genome

Abstract Linker histones H1 are principal chromatin components, whose contribution to the epigenetic regulation of chromatin structure and function is not fully understood. In metazoa, specific linker histones are expressed in the germline, with female-specific H1s being normally retained in the early-embryo. Embryonic H1s are present while the zygotic genome is transcriptionally silent and they are replaced by somatic variants upon activation, suggesting a contribution to transcriptional silencing. Here we directly address this question by ectopically expressing dBigH1 in Drosophila S2 cells, which lack dBigH1. We show that dBigH1 binds across chromatin, replaces somatic dH1 and reduces nucleosome repeat length (NRL). Concomitantly, dBigH1 expression down-regulates gene expression by impairing RNApol II binding and histone acetylation. These effects depend on the acidic N-terminal ED-domain of dBigH1 since a truncated form lacking this domain binds across chromatin and replaces dH1 like full-length dBigH1, but it does not affect NRL either transcription. In vitro reconstitution experiments using Drosophila preblastodermic embryo extracts corroborate these results. Altogether these results suggest that the negatively charged N-terminal tail of dBigH1 alters the functional state of active chromatin compromising transcription.

scholarly journals Acetylation-Dependent Binding Analysis of the Yeast Gcn5 Bromodomain Protein

2008 ◽  
Vol 7 (1) ◽  
Author(s):  
Jesús Garzón ◽  
Christopher Kupitz ◽  
Joshua Bailey ◽  
Martin Thompson
Keyword(s):  
Biologically Active ◽  
Histone Acetyltransferases ◽  
Chromatin Binding ◽  
Expression And Purification ◽  
Acetylation State ◽  
One Step ◽  
High Yields ◽  
And Function

The 439 amino acid yeast Gcn5 protein contains a C-terminal bromodomain, which is required for SAGA (Spt-Ada-Gcn5-Acetyltransferase) mediated nucleosomal acetylation and transcriptional coactivation. Bromodomains are acetyl-lysine binding modules found in many chromatin binding proteins and histone acetyltransferases. Recently, both in vivo and in vitro studies indicate that bromodomains are able to discriminate the acetylation state of lysine side-chains within histone proteins. Here, the cloning, expression and bioactivity of a recombinant bromodomain from the yeast Gcn5 protein is described. The bromodomain from Gcn5 was cloned from yeast genomic DNA enabling effective one-step purification by affinity chromatography. Steady-state fluorescence anisotropy was used to quantify the interaction of Gcn5 with acetylated histone H3. The present cloning, expression, and purification procedure enabled the preparation of large quantity and high yields of biologically active recombinant Gcn5 bromodomain for in vitro structure and function studies.

scholarly journals Mechanism of how augmin directly targets the γ-tubulin ring complex to microtubules

2018 ◽  
Vol 217 (7) ◽  
pp. 2417-2428 ◽  
Author(s):  
Jae-Geun Song ◽  
Matthew R. King ◽  
Rui Zhang ◽  
Rachel S. Kadzik ◽  
Akanksha Thawani ◽  
...  
Keyword(s):  
Cell Shape ◽  
Insect Cells ◽  
The Other ◽  
Negative Stain ◽  
In Vitro Reconstitution ◽  
Ring Complex ◽  
Negative Stain Electron Microscopy ◽  
The Right ◽  
And Function

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin’s architecture and function, we purified Xenopus laevis augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs.

scholarly journals Polyamines Disrupt the KaiABC Oscillator by Inducing Protein Denaturation

Molecules ◽  
2019 ◽  
Vol 24 (18) ◽  
pp. 3351 ◽  
Author(s):  
Jinkui Li ◽  
Lingya Zhang ◽  
Junwen Xiong ◽  
Xiyao Cheng ◽  
Yongqi Huang ◽  
...  
Keyword(s):  
Protein Denaturation ◽  
Structure And Function ◽  
Clock Proteins ◽  
In Vitro Reconstitution ◽  
Living Organisms ◽  
And Function ◽  
Cellular Components ◽  
The Impact

Polyamines are positively charged small molecules ubiquitously existing in all living organisms, and they are considered as one kind of the most ancient cellular components. The most common polyamines are spermidine, spermine, and their precursor putrescine generated from ornithine. Polyamines play critical roles in cells by stabilizing chromatin structure, regulating DNA replication, modulating gene expression, etc., and they also affect the structure and function of proteins. A few studies have investigated the impact of polyamines on protein structure and function previously, but no reports have focused on a protein-based biological module with a dedicated function. In this report, we investigated the impact of polyamines (putrescine, spermidine, and spermine) on the cyanobacterial KaiABC circadian oscillator. Using an established in vitro reconstitution system, we noticed that polyamines could disrupt the robustness of the KaiABC oscillator by inducing the denaturation of the Kai proteins (KaiA, KaiB, and KaiC). Further experiments showed that the denaturation was likely due to the induced change of the thermal stability of the clock proteins. Our study revealed an intriguing role of polyamines as a component in complex cellular environments and would be of great importance for elucidating the biological function of polyamines in future.

Molecular architecture and functions of the neuronal cytoskeleton

1990 ◽  
Vol 48 (3) ◽  
pp. 2-3
Author(s):  
Nobutaka Hirokawa
Keyword(s):  
Major Elements ◽  
Molecular Structures ◽  
Organelle Transport ◽  
Molecular Architecture ◽  
Neuronal Morphogenesis ◽  
Microtubule Associated Proteins ◽  
Associated Proteins ◽  
And Function ◽  
Small Clusters

In this symposium I will present our studies about the molecular architecture and function of the cytomatrix of the nerve cells. The nerve cell is a highly polarized cell composed of highly branched dendrites, cell body, and a single long axon along the direction of the impulse propagation. Each part of the neuron takes characteristic shapes for which the cytoskeleton provides the framework. The neuronal cytoskeletons play important roles on neuronal morphogenesis, organelle transport and the synaptic transmission. In the axon neurofilaments (NF) form dense arrays, while microtubules (MT) are arranged as small clusters among the NFs. On the other hand, MTs are distributed uniformly, whereas NFs tend to run solitarily or form small fascicles in the dendrites Quick freeze deep etch electron microscopy revealed various kinds of strands among MTs, NFs and membranous organelles (MO). These structures form major elements of the cytomatrix in the neuron. To investigate molecular nature and function of these filaments first we studied molecular structures of microtubule associated proteins (MAP1A, MAP1B, MAP2, MAP2C and tau), and microtubules reconstituted from MAPs and tubulin in vitro. These MAPs were all fibrous molecules with different length and formed arm like projections from the microtubule surface.

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