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After budding out from the ER membrane, some COPII vesicles lose their coat and fuse with one another to form larger vesicles and interconnected tubules called vesicular tubular clusters or VTCs. These clusters constitute a compartment at the ER-Golgi interface known as ERGIC (Endoplasmic Reticulum Golgi Intermediate Compartment). The ERGIC is a mobile membrane-bound cargo transport system that sorts proteins secreted from ER and delivers them to the Golgi.
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Secretory vesicles, also known as dense core vesicles (DCVs), are membrane-bound vesicles that transport secretory proteins, such as hormones or neurotransmitters. Regulated secretory vesicles transport proteins from the trans-Golgi network to the exterior of the cell. Proteins present in regulated secretory vesicles are required to be rapidly exocytosed in large amounts upon a specific stimulus.
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Vesicle budding is orchestrated by distinct cytosolic proteins such as adaptor proteins, coat proteins, and GTPases. To initiate vesicle budding, membrane-bending proteins containing crescent-shaped BAR domains bind to the lipid heads in the bilayer and distort the membrane to form a protein-coated vesicle bud. Adaptors proteins such as AP2 for clathrin-coated vesicles can nucleate on the deformed membrane. Finally, coat proteins such as clathrin or COPI and COPII assemble into a coat forming...
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In eukaryotes, transcription and translation are compartmentalized; an mRNA is first synthesized in the nucleus and then selectively transported to the cytoplasm for protein synthesis. Before transport, a pre-mRNA undergoes several steps of post-transcriptional modifications including splicing, 5' capping, and the addition of a poly-adenine tail. Various proteins bind to the pre-mRNA during these modifications. The mRNA transport takes place with the help of multiple proteins playing...
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Related Experiment Video

Updated: Aug 25, 2025

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics
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Crowding-Induced Spatial Organization of Gene Expression in Cell-Sized Vesicles.

Gaurav Chauhan1, S Elizabeth Norred2,3, Rosemary M Dabbs3

  • 1Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, Knoxville, Tennessee37996, United States.

ACS Synthetic Biology
|October 19, 2022
PubMed
Summary

Macromolecular crowding controls gene expression in cell-sized vesicles by organizing DNA and mRNA. This method allows researchers to study spatial gene regulation and improve cell-free protein synthesis applications.

Keywords:
cell-free protein synthesiscomputer simulationsdepletion interactionsgene expressionmacromolecular crowdingtranslational efficiency

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Synthetic Biology

Background:

  • Cell-free protein synthesis (CFPS) is vital for gene expression studies and applications.
  • Spatial organization of transcription and translation regulates gene expression within cells.
  • Current CFPS methods lack control over molecular component organization, limiting insights into spatial effects.

Purpose of the Study:

  • To investigate macromolecular crowding as a method to control spatial organization and translational efficiency in cell-sized vesicles.
  • To explore how crowding influences the localization of gene expression components.
  • To understand the impact of spatial organization on protein synthesis in a cell-mimicking environment.

Main Methods:

  • Utilized complementary computational (simulations) and experimental (imaging, CFPS) approaches.
  • Employed cell-sized vesicles to mimic cellular environments.
  • Varied macromolecular crowding levels to observe effects on component localization and protein production.

Main Results:

  • Increased crowding led to DNA plasmid localization at vesicle inner surfaces.
  • Ribosomes remained uniformly distributed, showing differential organization of gene expression components.
  • High crowding caused mRNA localization near vesicle surfaces, decreased translational efficiency, and reduced protein abundance.
  • Observed anomalous scaling of protein abundance with vesicle size at high crowding levels.

Conclusions:

  • Macromolecular crowding effectively controls spatial organization of gene expression in cell-sized vesicles.
  • Crowding influences diffusion rates and spatial distribution of gene expression machinery.
  • This approach provides a straightforward method to probe spatial gene regulation and enhance CFPS.