Measuring In Vivo Protein Dynamics Throughout the Cell Cycle Using Microfluidics.

Area of Science:

• Cell Biology
• Molecular Biology
• Microscopy

Background:

• Studying macromolecule dynamics in live cells is crucial for understanding cell physiology.
• Controlled culturing conditions are essential for long-term in vivo experiments, especially for processes like bacterial DNA replication.
• Wide-field microscopy and microfluidics offer a powerful combination for observing molecular behavior within cells.

Purpose of the Study:

• To present a protocol for combining wide-field microscopy and microfluidics.
• To enable the study of proteins involved in the Escherichia coli DNA replication process.
• To determine the stoichiometry and dynamics of a replisome component throughout the cell cycle.

Main Methods:

• Utilizing a microfluidic device for cellular orientation, immobilization, and medium control.
• Employing wide-field microscopy for fluorescence experiments in live bacterial cells.
• Implementing specific data acquisition and image analysis procedures.

Main Results:

• The combined technique allows for well-defined cellular conditions and high-throughput experimentation.
• The protocol facilitates the detection and quantification of proteins during bacterial DNA replication.
• Stoichiometry and dynamics of a specific replisome component were determined throughout the cell cycle.

Conclusions:

• The integration of wide-field microscopy and microfluidics provides a robust method for studying intracellular dynamics.
• This approach offers valuable insights into the molecular mechanisms of bacterial DNA replication.
• The protocol is effective for long-term, high-resolution observation of macromolecular behavior in live cells.