Signal-dependent turnover of the bacterial flagellar switch protein FliM.
Proc Natl Acad Sci U S A 107:25 (2010) 11347-11351
Abstract:
Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains approximately 13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the approximately 30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.Signal-dependent turnover of the bacterial flagellar switch protein FliM
Proceedings of the National Academy of Sciences of the United States of America 107:25 (2010) 11347-11351
Abstract:
Most biological processes are performed by multiprotein complexes. Traditionally described as static entities, evidence is now emerging that their components can be highly dynamic, exchanging constantly with cellular pools. The bacterial flagellar motor contains ∼13 different proteins and provides an ideal system to study functional molecular complexes. It is powered by transmembrane ion flux through a ring of stator complexes that push on a central rotor. The Escherichia coli motor switches direction stochastically in response to binding of the response regulator CheY to the rotor switch component FliM. Much is known of the static motor structure, but we are just beginning to understand the dynamics of its individual components. Here we measure the stoichiometry and turnover of FliM in functioning flagellar motors, by using high-resolution fluorescence microscopy of E. coli expressing genomically encoded YPet derivatives of FliM at physiological levels. We show that the ∼30 FliM molecules per motor exist in two discrete populations, one tightly associated with the motor and the other undergoing stochastic turnover. This turnover of FliM molecules depends on the presence of active CheY, suggesting a potential role in the process of motor switching. In many ways the bacterial flagellar motor is as an archetype macromolecular assembly, and our results may have further implications for the functional relevance of protein turnover in other large molecular complexes.Stoichiometry and architecture of active DNA replication machinery in Escherichia coli.
Science 328:5977 (2010) 498-501
Abstract:
The multiprotein replisome complex that replicates DNA has been extensively characterized in vitro, but its composition and architecture in vivo is unknown. Using millisecond single-molecule fluorescence microscopy in living cells expressing fluorescent derivatives of replisome components, we have examined replisome stoichiometry and architecture. Active Escherichia coli replisomes contain three molecules of the replicative polymerase, rather than the historically accepted two. These are associated with three molecules of tau, a clamp loader component that trimerizes polymerase. Only two of the three sliding clamps are always associated with the core replisome. Single-strand binding protein has a broader spatial distribution than the core components, with 5 to 11 tetramers per replisome. This in vivo technique could provide single-molecule insight into other molecular machines.A novel multiple particle tracking algorithm for noisy in vivo data by minimal path optimization within the spatio-temporal volume
Proceedings - 2009 IEEE International Symposium on Biomedical Imaging: From Nano to Macro, ISBI 2009 (2009) 1158-1161
Abstract:
Automated tracking of fluorescent particles in living cells is vital for subcellular stoichoimetry analysis [1, 2]. Here, a new automatic tracking algorithm is described to track multiple particles, based on minimal path optimization. After linking feature points frame-by-frame, spatio-temporal data from time-lapse microscopy are combined together to construct a transformed 3D volume. The trajectories are then generated from the minimal energy path as defined by the solution of the time-dependent partial differential equation using a gray weighted distance transform dynamic programming method. Results from simulated and experimental data demonstrate that our novel automatic method gives sub-pixel accuracy even for very noisy images. © 2009 IEEE.Millisecond timescale slimfield imaging and automated quantification of single fluorescent protein molecules for use in probing complex biological processes.
Integr Biol (Camb) 1:10 (2009) 602-612