TY - CHAP
T1 - Measuring in vivo protein dynamics throughout the cell cycle using microfluidics
AU - de Leeuw, Roy
AU - Brazda, Peter
AU - Moolman, M. Charl
AU - Kerssemakers, J. W.J.
AU - Solano Hermosilla, B.P.
AU - Dekker, Nynke H.
PY - 2017
Y1 - 2017
N2 - Studying the dynamics of intracellular processes and investigating the interaction of individual macromolecules in live cells is one of the main objectives of cell biology. These macromolecules move, assemble, disassemble, and reorganize themselves in distinct manners under specific physiological conditions throughout the cell cycle. Therefore, in vivo experimental methods that enable the study of individual molecules inside cells at controlled culturing conditions have proved to be powerful tools to obtain insights into the molecular roles of these macromolecules and how their individual behavior influence cell physiology. The importance of controlled experimental conditions is enhanced when the investigated phenomenon covers long time periods, or perhaps multiple cell cycles. An example is the detection and quantification of proteins during bacterial DNA replication. Wide-field microscopy combined with microfluidics is a suitable technique for this. During fluorescence experiments, microfluidics offer well-defined cellular orientation and immobilization, flow and medium interchangeability, and high-throughput long-term experimentation of cells. Here we present a protocol for the combined use of wide-field microscopy and microfluidics for the study of proteins of the Escherichia coli DNA replication process. We discuss the preparation and application of a microfluidic device, data acquisition steps, and image analysis procedures to determine the stoichiometry and dynamics of a replisome component throughout the cell cycle of live bacterial cells.
AB - Studying the dynamics of intracellular processes and investigating the interaction of individual macromolecules in live cells is one of the main objectives of cell biology. These macromolecules move, assemble, disassemble, and reorganize themselves in distinct manners under specific physiological conditions throughout the cell cycle. Therefore, in vivo experimental methods that enable the study of individual molecules inside cells at controlled culturing conditions have proved to be powerful tools to obtain insights into the molecular roles of these macromolecules and how their individual behavior influence cell physiology. The importance of controlled experimental conditions is enhanced when the investigated phenomenon covers long time periods, or perhaps multiple cell cycles. An example is the detection and quantification of proteins during bacterial DNA replication. Wide-field microscopy combined with microfluidics is a suitable technique for this. During fluorescence experiments, microfluidics offer well-defined cellular orientation and immobilization, flow and medium interchangeability, and high-throughput long-term experimentation of cells. Here we present a protocol for the combined use of wide-field microscopy and microfluidics for the study of proteins of the Escherichia coli DNA replication process. We discuss the preparation and application of a microfluidic device, data acquisition steps, and image analysis procedures to determine the stoichiometry and dynamics of a replisome component throughout the cell cycle of live bacterial cells.
KW - DNA replication
KW - Escherichia coli
KW - Fluorescence imaging
KW - Microfluidics
KW - Single-molecule techniques
UR - http://www.scopus.com/inward/record.url?scp=85028616308&partnerID=8YFLogxK
U2 - 10.1007/978-1-4939-7098-8_18
DO - 10.1007/978-1-4939-7098-8_18
M3 - Chapter
AN - SCOPUS:85028616308
SN - 978-1493970971
VL - 1624
T3 - Methods in Molecular Biology
SP - 237
EP - 252
BT - The Bacterial Nucleoid
A2 - Espéli, Olivier
PB - Springer
ER -