scholarly journals A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly

2018 ◽  
Author(s):  
Sagardip Majumder ◽  
Patrick T. Willey ◽  
Maxwell S. DeNies ◽  
Allen P. Liu ◽  
G.W. Gant Luxton
Keyword(s):  
Synthetic Biology ◽  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Experimental System ◽  
Free Expression ◽  
Supported Lipid Bilayers ◽  
Linc Complex ◽  
Complex Assembly ◽  
Complex Core

ABSTRACTThe linker of nucleoskeleton and cytoskeleton (LINC) is a conserved nuclear envelope-spanning molecular bridge that is responsible for the mechanical integration of the nucleus with the cytoskeleton. LINC complexes are formed by a transluminal interaction between the outer and inner nuclear membrane KASH and SUN proteins, respectively. Despite recent structural insights, our mechanistic understanding of LINC complex assembly remains limited by the lack of an experimental system for its in vitro reconstitution and manipulation. Here, we describe artificial nuclear membranes (ANMs) as a synthetic biology platform based on mammalian cell-free expression for the rapid reconstitution of SUN proteins in supported lipid bilayers. We demonstrate that SUN1 and SUN2 are oriented in ANMs with solvent-exposed C-terminal KASH-binding SUN domains. We also find that SUN2 possesses a single transmembrane domain, while SUN1 possesses three. Finally, SUN protein-containing ANMs bind synthetic KASH peptides, thereby reconstituting the LINC complex core. This work represents the first in vitro reconstitution of KASH-binding SUN proteins in supported lipid bilayers using cell-free expression, which will be invaluable for testing proposed models of LINC complex assembly and its regulation.

scholarly journals Cell-Free Synthetic Biology Platform for Engineering Synthetic Biological Circuits and Systems

2019 ◽  
Vol 2 (2) ◽  
pp. 39 ◽  
Author(s):  
Dohyun Jeong ◽  
Melissa Klocke ◽  
Siddharth Agarwal ◽  
Jeongwon Kim ◽  
Seungdo Choi ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Free Expression ◽  
Dna Nanostructures ◽  
Expression Systems ◽  
Natural Systems ◽  
The Past ◽  
Current Cell ◽  
Recent Developments ◽  
Biological Circuits

Synthetic biology integrates diverse engineering disciplines to create novel biological systems for biomedical and technological applications. The substantial growth of the synthetic biology field in the past decade is poised to transform biotechnology and medicine. To streamline design processes and facilitate debugging of complex synthetic circuits, cell-free synthetic biology approaches has reached broad research communities both in academia and industry. By recapitulating gene expression systems in vitro, cell-free expression systems offer flexibility to explore beyond the confines of living cells and allow networking of synthetic and natural systems. Here, we review the capabilities of the current cell-free platforms, focusing on nucleic acid-based molecular programs and circuit construction. We survey the recent developments including cell-free transcription–translation platforms, DNA nanostructures and circuits, and novel classes of riboregulators. The links to mathematical models and the prospects of cell-free synthetic biology platforms will also be discussed.

scholarly journals A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly

2018 ◽  
Vol 132 (4) ◽  
pp. jcs219451 ◽  
Author(s):  
Sagardip Majumder ◽  
Patrick T. Willey ◽  
Maxwell S. DeNies ◽  
Allen P. Liu ◽  
G. W. Gant Luxton
Keyword(s):  
Synthetic Biology ◽  
Linc Complex ◽  
Complex Assembly

scholarly journals Simultaneous monitoring of transcription and translation in mammalian cell-free expression in bulk and in cell-sized droplets

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Shue Wang ◽  
Sagardip Majumder ◽  
Nicholas J Emery ◽  
Allen P Liu
Keyword(s):  
Synthetic Biology ◽  
Real Time ◽  
Protein Dynamics ◽  
Mammalian Cell ◽  
Locked Nucleic Acid ◽  
Free Expression ◽  
Reporter Protein ◽  
Bottom Up ◽  
Lna Probe

Abstract Transcription and translation are two critical processes during eukaryotic gene expression that regulate cellular activities. The development of mammalian cell-free expression (CFE) systems provides a platform for studying these two critical processes in vitro for bottom-up synthetic biology applications such as construction of an artificial cell. Moreover, real-time monitoring of the dynamics of synthesized mRNA and protein is key to characterize and optimize gene circuits before implementing in living cells or in artificial cells. However, there are few tools for measurement of mRNA and protein dynamics in mammalian CFE systems. Here, we developed a locked nucleic acid (LNA) probe for monitoring transcription in a HeLa-based CFE system in real-time. By using this LNA probe in conjunction with a fluorescent reporter protein, we were able to simultaneously monitor mRNA and protein dynamics in bulk reactions and cell-sized single-emulsion droplets. We found rapid production of mRNA transcripts that decreased over time as protein production ensued in bulk reactions. Our results also showed that transcription in cell-sized droplets has different dynamics compared to the transcription in bulk reactions. The use of this LNA probe in conjunction with fluorescent proteins in HeLa-based mammalian CFE system provides a versatile in vitro platform for studying mRNA dynamics for bottom-up synthetic biology applications.

scholarly journals Correction: A synthetic biology platform for the reconstitution and mechanistic dissection of LINC complex assembly (doi:10.1242/jcs.219451)

2019 ◽  
Vol 132 (10) ◽  
pp. jcs234153
Author(s):  
Sagardip Majumder ◽  
Patrick T. Willey ◽  
Maxwell S. DeNies ◽  
Allen P. Liu ◽  
G. W. Gant Luxton
Keyword(s):  
Synthetic Biology ◽  
Linc Complex ◽  
Complex Assembly

scholarly journals Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

2016 ◽  
Vol 113 (46) ◽  
pp. E7185-E7193 ◽  
Author(s):  
Rahul Grover ◽  
Janine Fischer ◽  
Friedrich W. Schwarz ◽  
Wilhelm J. Walter ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Single Molecule ◽  
Gliding Motility ◽  
Supported Lipid Bilayers ◽  
Transport Efficiency ◽  
Collective Transport ◽  
Wide Range ◽  
Motor Density

In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors’ anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted “membrane-anchored” gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in the presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect that we directly observed using single-molecule fluorescence microscopy. Our results illustrate the importance of motor–cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.

scholarly journals Transport efficiency of membrane-anchored kinesin-1 motors depends on motor density and diffusivity

2016 ◽  
Author(s):  
Rahul Grover ◽  
Janine Fischer ◽  
Friedrich W. Schwarz ◽  
Wilhelm J. Walter ◽  
Petra Schwille ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Gliding Motility ◽  
Supported Lipid Bilayers ◽  
Transport Efficiency ◽  
Collective Transport ◽  
Wide Range ◽  
Biological Substrates ◽  
Motor Density

AbstractIn eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to non-biological substrates. However, the collective transport by membrane-anchored motors, i.e. motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors anchorage to a lipid bilayer on the collective transport characteristics. We reconstituted ‘membrane-anchored’ gliding motility assays using truncated kinesin-1 motors with a streptavidin-binding-peptide tag that can attach to streptavidin-loaded, supported lipid bilayers. We found that the diffusing kinesin-1 motors propelled the microtubules in presence of ATP. Notably, we found the gliding velocity of the microtubules to be strongly dependent on the number of motors and their diffusivity in the lipid bilayer. The microtubule gliding velocity increased with increasing motor density and membrane viscosity, reaching up to the stepping velocity of single-motors. This finding is in contrast to conventional gliding motility assays where the density of surface-immobilized kinesin-1 motors does not influence the microtubule velocity over a wide range. We reason, that the transport efficiency of membrane-anchored motors is reduced because of their slippage in the lipid bilayer, an effect which we directly observed using singlemolecule fluorescence microscopy. Our results illustrate the importance of the motor-cargo coupling, which potentially provides cells with an additional means of regulating the efficiency of cargo transport.

scholarly journals Direct reconstitution and study of SUN protein interactions in vitro using mammalian cell-free expression

2021 ◽  
Author(s):  
Sagardip Majumder ◽  
Yen-Yu Hsu ◽  
Allen P Liu
Keyword(s):  
Membrane Proteins ◽  
Mammalian Cell ◽  
Protein Function ◽  
Mammalian Cells ◽  
Expression System ◽  
Full Length ◽  
Free Expression ◽  
Lipid Bilayer Membranes ◽  
Linc Complex

SUN proteins are an integral part of LINC (Linker of Nucleoskeleton and Cytoskeleton) complex which spans the nuclear envelope and acts as a physical tether between the cytoskeletal filaments and the nuclear lamina. Several human diseases associated with nuclear deformation are primarily caused by impaired functioning of SUN proteins. Studies in yeast and mammalian cells have illustrated the detrimental effects of different SUN mutants in nuclear positioning and movement. While cell-based studies provide physiological relevance to the functioning of a protein, in vitro reconstitution of isolated proteins is useful in mechanistically dissecting protein function in a biochemically defined environment. In this study, we used a mammalian cell-free expression system to synthesize and reconstitute SUN proteins in artificial lipid bilayer membranes. Building on our previous work demonstrating directional reconstitution of full-length SUN proteins, we deciphered the mechanism of such protein reconstitution and leveraged it to test several theories/models of LINC complex assembly. By using a simple fluorescence-based assay, we revealed the importance of cations such as calcium and the presence of disulfide bonds in the formation of LINC complexes. Through sequential reconstitutions of SUN proteins and soluble luminal domains of SUN proteins, we found that coiled coil domains of SUN proteins are necessary for homomeric and heteromeric interactions of reconstituted SUN proteins. Overall, our results provide mechanistic insights on LINC complex formation and how this might impact cellular mechanotransduction. The facile approach for reconstituting full-length membrane proteins can be extended to study other difficult-to-study membrane proteins in vitro.

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