scholarly journals Shaping liposomes by cell-free expressed bacterial microtubules

2021 ◽  
Author(s):  
Christophe Danelon ◽  
Marileen Dogterom ◽  
Johannes Kattan ◽  
Anne Doerr
Keyword(s):  
Lipid Membranes ◽  
Cell Biology ◽  
De Novo ◽  
Lipid Vesicles ◽  
Translation System ◽  
Dna Templates ◽  
Cytoskeletal Network ◽  
Synthetic Cell ◽  
Directional Transport ◽  
Shape Changes

Genetic control over a cytoskeletal network inside lipid vesicles offers a potential route to controlled shape changes and DNA segregation in synthetic cell biology. Bacterial microtubules (bMTs) are protein filaments found in bacteria of the genus Prosthecobacter. They are formed by the tubulins BtubA and BtubB which polymerize in the presence of GTP. Here, we show that the tubulins BtubA/B can be functionally expressed from DNA templates in a reconstituted transcription-translation system, thus providing a cytosol-like environment to study their biochemical and biophysical properties. We found that bMTs spontaneously interact with lipid membranes and display treadmilling. When compartmentalized inside liposomes, de novo synthesized BtubA/B tubulins self-organize into cytoskeletal structures of different morphologies. Moreover, bMTs can exert a pushing force on the membrane and deform liposomes, a phenomenon that can be reversed by light-activated disassembly of the filaments. Our work establishes bMTs as a new building block in synthetic biology. In the context of creating a synthetic cell, bMTs could help shape the lipid compartment, establish polarity or directional transport, and assist the division machinery.

scholarly journals A partially self-regenerating synthetic cell

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Barbora Lavickova ◽  
Nadanai Laohakunakorn ◽  
Sebastian J. Maerkl
Keyword(s):  
Resource Competition ◽  
Living Systems ◽  
Translation System ◽  
System Function ◽  
Fundamental Function ◽  
Dna Templates ◽  
Synthetic Cell ◽  
Synthesis Activity ◽  
Robust System ◽  
Protein Components

AbstractSelf-regeneration is a fundamental function of all living systems. Here we demonstrate partial molecular self-regeneration in a synthetic cell. By implementing a minimal transcription-translation system within microfluidic reactors, the system is able to regenerate essential protein components from DNA templates and sustain synthesis activity for over a day. By quantitating genotype-phenotype relationships combined with computational modeling we find that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieve simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.

scholarly journals Molecular Transport across Lipid Membranes Controls Cell-Free Expression Level and Dynamics

2019 ◽  
Author(s):  
Patrick M. Caveney ◽  
Rosemary M. Dabbs ◽  
William T. McClintic ◽  
C. Patrick Collier ◽  
Michael L. Simpson
Keyword(s):  
Gene Expression ◽  
Membrane Transport ◽  
Molecular Species ◽  
Lipid Membranes ◽  
Molecular Transport ◽  
Lipid Vesicles ◽  
Cellular Systems ◽  
Control Of Gene Expression ◽  
External Resources ◽  
Synthetic Cell

SummaryEssential steps toward synthetic cell-like systems require controlled transport of molecular species across the boundary between encapsulated expression and the external environment. When molecular species (e.g. small ions, amino acids) required for expression (i.e. expression resources) may cross this boundary, this transport process plays an important role in gene expression dynamics and expression variability. Here we show how the location (encapsulated or external) of the expression resources controls the level and the dynamics of cell-free protein expression confined in permeable lipid vesicles. Regardless of the concentration of encapsulated resources, external resources were essential for protein production. Compared to resource poor external environments, plentiful external resources increased expression by ~7-fold, and rescued expression when internal resources were lacking. Intriguingly, the location of resources and the membrane transport properties dictated expression dynamics in a manner well predicted by a simple transport-expression model. These results suggest membrane engineering as a means for spatio-temporal control of gene expression in cell-free synthetic biology applications and demonstrate a flexible experimental platform to understand the interplay between membrane transport and expression in cellular systems.

Digression on Membrane Electroporation for Drug and Gene Delivery

2002 ◽  
Vol 1 (5) ◽  
pp. 329-339 ◽  
Author(s):  
Eberhard Neumann ◽  
Sergej Kakorin
Keyword(s):  
Lipid Membranes ◽  
Cell Biology ◽  
Cell Transformation ◽  
Single Cells ◽  
Surface Membrane ◽  
Quantitative Description ◽  
Lipid Vesicles ◽  
General Chemical ◽  
Membrane Rigidity ◽  
Medical Disciplines

Membrane electroporation (ME) defines an electrical technique to render lipid membranes porous and permeable, transiently and reversibly, by external voltage pulses. Although there are numerous applications of ME to manipulate cells, organelles and tissues in cell biology, biotechnology and medicine, yet the molecular mechanism of ME is only slowly being understood. A general chemical-thermodynamical approach for the quantitative description of cell membrane electroporation has been developed to provide the framework to quantitatively rationalize electroporative cell transformation and electroporative uptake of drug-like dyes into cells, as well as electrolyte efflux from salt-filled electroporated vesicles. Mechanistically, the electroporative transfer of gene and drug-like dyes involves the coupling between an interactive contact formation of the permeates with the cell surface membrane and the structural electroporation-resealing cycle [Formula: see text] where C is the closed and (P) represents a number of different porated membrane states, respectively. The experimentally accessible concentration fraction fp = [(P)] /([C] + [(P)]) of porous states is related to thermodynamic and electro-mechanic parameters such as temperature and the electric field strength, membrane rigidity or curvature. The results of the theoretical approach, mainly based on electrooptical data of lipid vesicles, have been successfully used to analyze single cells and to specify conditions for the practical purpose of direct electroporative gene transfer and drug delivery, in particular in the new medical disciplines of electroporative chemotherapy and electroporative gene vaccination.

scholarly journals A self-regenerating synthetic cell model

2020 ◽  
Author(s):  
Barbora Lavickova ◽  
Nadanai Laohakunakorn ◽  
Sebastian J. Maerkl
Keyword(s):  
Resource Competition ◽  
Cell Model ◽  
Translation System ◽  
System Function ◽  
Fundamental Function ◽  
Dna Templates ◽  
Synthetic Cell ◽  
Synthesis Activity ◽  
Robust System ◽  
Protein Components

AbstractSelf-regeneration is a fundamental function of all living systems. Here we demonstrate molecular self-regeneration in a synthetic cell model. By implementing a minimal transcription-translation system within microfluidic reactors, the system was able to regenerate essential protein components from DNA templates and sustained synthesis activity for over a day. By mapping genotype-phenotype landscapes combined with computational modeling we found that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieved simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.

scholarly journals Formation of Giant Lipid Vesicles in the Presence of Nonelectrolytes—Glucose, Sucrose, Sorbitol and Ethanol

Processes ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 945
Author(s):  
Qiong Wang ◽  
Ning Hu ◽  
Jincan Lei ◽  
Qiurong Qing ◽  
Jing Huang ◽  
...  
Keyword(s):  
Sodium Chloride ◽  
Cell Membrane ◽  
Lipid Membranes ◽  
Volume Fraction ◽  
Hydrodynamic Force ◽  
Lipid Vesicles ◽  
Ethanol Solution ◽  
Sugar Solution ◽  
Ethanol Content ◽  
Membrane Models

Lipid vesicles, especially giant lipid vesicles (GLVs), are usually adopted as cell membrane models and their preparation has been widely studied. However, the effects of some nonelectrolytes on GLV formation have not been specifically studied so far. In this paper, the effects of the nonelectrolytes, including sucrose, glucose, sorbitol and ethanol, and their coexistence with sodium chloride, on the lipid hydration and GLV formation were investigated. With the hydration method, it was found that the sucrose, glucose and sorbitol showed almost the same effect. Their presence in the medium enhanced the hydrodynamic force on the lipid membranes, promoting the GLV formation. GLV formation was also promoted by the presence of ethanol with ethanol volume fraction in the range of 0 to 20 percent, but higher ethanol content resulted in failure of GLV formation. However, the participation of sodium chloride in sugar solution and ethanol solution stabilized the lipid membranes, suppressing the GLV formation. In addition, the ethanol and the sodium chloride showed the completely opposite effects on lipid hydration. These results could provide some suggestions for the efficient preparation of GLVs.

scholarly journals CALINCA—A Novel Pipeline for the Identification of lncRNAs in Podocyte Disease

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 692
Author(s):  
Sweta Talyan ◽  
Samantha Filipów ◽  
Michael Ignarski ◽  
Magdalena Smieszek ◽  
He Chen ◽  
...  
Keyword(s):  
Cell Biology ◽  
Mammalian Cells ◽  
De Novo ◽  
Depth Information ◽  
Gene Products ◽  
Classical Analysis ◽  
Protein Coding ◽  
Bioinformatic Pipeline ◽  
Non Coding Rnas ◽  
Filtration Unit

Diseases of the renal filtration unit—the glomerulus—are the most common cause of chronic kidney disease. Podocytes are the pivotal cell type for the function of this filter and focal-segmental glomerulosclerosis (FSGS) is a classic example of a podocytopathy leading to proteinuria and glomerular scarring. Currently, no targeted treatment of FSGS is available. This lack of therapeutic strategies is explained by a limited understanding of the defects in podocyte cell biology leading to FSGS. To date, most studies in the field have focused on protein-coding genes and their gene products. However, more than 80% of all transcripts produced by mammalian cells are actually non-coding. Here, long non-coding RNAs (lncRNAs) are a relatively novel class of transcripts and have not been systematically studied in FSGS to date. The appropriate tools to facilitate lncRNA research for the renal scientific community are urgently required due to a row of challenges compared to classical analysis pipelines optimized for coding RNA expression analysis. Here, we present the bioinformatic pipeline CALINCA as a solution for this problem. CALINCA automatically analyzes datasets from murine FSGS models and quantifies both annotated and de novo assembled lncRNAs. In addition, the tool provides in-depth information on podocyte specificity of these lncRNAs, as well as evolutionary conservation and expression in human datasets making this pipeline a crucial basis to lncRNA studies in FSGS.

scholarly journals The interplay between lipids and dopamine on α-synuclein oligomerization and membrane binding

2013 ◽  
Vol 33 (5) ◽  
Author(s):  
Chi L. L. Pham ◽  
Roberto Cappai
Keyword(s):  
Lipid Membranes ◽  
Amyloid Fibrils ◽  
Lipid Membrane ◽  
Hydrophobic Interactions ◽  
Membrane Integrity ◽  
Lipid Vesicles ◽  
Helical Conformation ◽  
Unfolded Protein ◽  
Natively Unfolded Protein ◽  
Natively Unfolded

The deposition of α-syn (α-synuclein) as amyloid fibrils and the selective loss of DA (dopamine) containing neurons in the substantia nigra are two key features of PD (Parkinson's disease). α-syn is a natively unfolded protein and adopts an α-helical conformation upon binding to lipid membrane. Oligomeric species of α-syn have been proposed to be the pathogenic species associated with PD because they can bind lipid membranes and disrupt membrane integrity. DA is readily oxidized to generate reactive intermediates and ROS (reactive oxygen species) and in the presence of DA, α-syn form of SDS-resistant soluble oligomers. It is postulated that the formation of the α-syn:DA oligomers involves the cross-linking of DA-melanin with α-syn, via covalent linkage, hydrogen and hydrophobic interactions. We investigate the effect of lipids on DA-induced α-syn oligomerization and studied the ability of α-syn:DA oligomers to interact with lipids vesicles. Our results show that the interaction of α-syn with lipids inhibits the formation of DA-induced α-syn oligomers. Moreover, the α-syn:DA oligomer cannot interact with lipid vesicles or cause membrane permeability. Thus, the formation of α-syn:DA oligomers may alter the actions of α-syn which require membrane association, leading to disruption of its normal cellular function.

Adhesion of Lipid Membranes Mediated by Electrostatic and Specific Interactions

1997 ◽  
Vol 489 ◽  
Author(s):  
Christian W. Maier ◽  
Almuth Behrisch ◽  
Annette Kloboucek ◽  
Rudolf Merkel
Keyword(s):  
Lipid Membranes ◽  
Cell Adhesion Molecule ◽  
Lipid Vesicles ◽  
Membrane Properties ◽  
Specific Cell ◽  
Receptor Aggregation ◽  
Host Membrane ◽  
Aspiration Technique ◽  
Membrane Adhesion ◽  
Positively Charged

AbstractWe used the micropipet aspiration technique for a study of biomembrane adhesion. Adhesion was caused by contact site A, a highly specific cell adhesion molecule, reconstituted in lipid vesicles of DOPC with 5 %(mol/mol) DOPE-PEG2000. We found adhesion and subsequent receptor aggregation in the contact zone. Additionally, electrostatic modulation of membrane adhesion was studied. Whereas addition of the negatively charged lipid SOPS to the lecithin (SOPC) host membrane suppressed adhesion due to electrostatic repulsion, a positively charged lipid (DOTAP) was surprisingly ineffective. This might be due to either phase separation of the mixture or DOTAP changing other membrane properties as bending stiffness and the Hamaker constant.

Synthetic cell biology

2001 ◽  
Vol 12 (6) ◽  
pp. 638-644 ◽  
Author(s):  
Adam P. Arkin
Keyword(s):  
Cell Biology ◽  
Synthetic Cell

ELECTROFUSION OF MODEL LIPID MEMBRANES VIEWED WITH HIGH TEMPORAL RESOLUTION

2006 ◽  
Vol 01 (04) ◽  
pp. 387-400 ◽  
Author(s):  
KARIN. A. RISKE ◽  
NATALYA BEZLYEPKINA ◽  
REINHARD LIPOWSKY ◽  
RUMIANA DIMOVA
Keyword(s):  
Electric Fields ◽  
Temporal Resolution ◽  
Lipid Membranes ◽  
Cell Biology ◽  
Digital Camera ◽  
Extensive Study ◽  
High Temporal Resolution ◽  
Giant Vesicles ◽  
Model Lipid Membranes ◽  
Millisecond Time Scale

The interaction of electric fields with lipid membranes and cells has been extensively studied in the last decades. The phenomena of electroporation and electrofusion are of particular interest because of their widespread use in cell biology and biotechnology. Giant vesicles, being of cell size and convenient for microscopy observations, are the simplest model of the cell membrane. However, optical microscopy observation of effects caused by electric DC pulses on giant vesicles is difficult because of the short duration of the pulse. Recently this difficulty has been overcome in our lab. Using a digital camera with high temporal resolution, we were able to access vesicle fusion dynamics on a sub-millisecond time scale. In this report, we present some observations on electrodeformation and –poration of single vesicles followed by an extensive study on the electrofusion of vesicle couples. Finally, we suggest an attractive approach for creating multidomain vesicles using electrofusion and present some preliminary results on the effect of membrane stiffness on the fusion dynamics.

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