Distinct cytosolic complexes containing the type III secretion system ATPase resolved by 3D single-molecule tracking in liveYersinia enterocolitica

Abstract

The membrane-embedded injectisome, the structural component of the virulence-associated type III secretion system (T3SS), is used by gram-negative bacterial pathogens to inject species-specific effector proteins into eukaryotic host cells. The cytosolic injectisome proteins are required for export of effectors and display both stationary, injectisome-bound populations as well as freely-diffusing cytosolic populations. How the cytosolic injectisome proteins interact with each other in the cytosol and associate with membrane-embedded injectisomes remains unclear. Here, we utilize 3D single-molecule tracking to resolve distinct cytosolic complexes of injectisome proteins in living Yersinia enterocolitica cells. Tracking of the eYFP-labeled ATPase, YeSctN, and its regulator, YeSctL, reveals that these proteins form a cytosolic complex with each other and then further with YeSctQ. YeSctNL and YeSctNLQ complexes can be observed in both in wild type cells and in ΔsctD mutants, which cannot assemble injectisomes. In ΔsctQ mutants, the relative abundance of the YeSctNL complex is considerably increased. These data indicate that distinct cytosolic complexes of injectisome proteins can form prior to injectisome binding - a feature that could contribute to injectisome functional regulation.

Dimerization of iLID Optogenetic Proteins Observed Using 3D Single-Molecule Tracking in Live Bacterial Cells

“…Because single-molecule trajectories in living cells are limited in duration by fluorophore photobleaching, information from thousands of trajectories are often pooled and statistical data analysis approaches are employed to resolve different diffusive states [16][17][18][19][20][21][22][23] . Indeed, different diffusive states can be resolved in living cells due to homo-and heterooligomeric complex formation among interacting proteins [23][24][25] or proteins binding to quasistationary structures, such as DNA [26][27][28][29][30][31][32][33][34][35][36] . However, to assign diffusive states to specific biomolecular complexes of different protein compositions, additional experimental perturbation is needed.…”

“…Because single-molecule trajectories in living cells are limited in duration by fluorophore photobleaching, information from thousands of trajectories are pooled and statistical data analysis approaches employed to resolve different diffusive states and determine their relative abundances and diffusion coefficients [11][12][13][14][15][16][17] . Using this approach, homo-and heterooligomeric complex formation among interacting proteins [18][19][20][21] or proteins binding to quasistationary structures (such as DNA) [22][23][24][25][26][27][28][29][30][31][32][33] have been successfully resolved in living cells.…”

“…Additional experimental perturbations are often needed to conclusively assign diffusive states to biomolecular complexes of specific protein composition. Single-point mutations or genetic deletion mutants of potential interacting partners can be used to disrupt complex formation and observe the resulting changes in diffusive behavior 11,12,14,[16][17][18][19]21,[23][24][25][26][27][28][29][30][31][32][33]40,41 . A drawback of such genetic approaches is that the permanent disruption of an interaction interface or absence of an essential binding partner can interfere with cellular processes in ways that are difficult to predict or control, and may compromise cell viability 42,43 .…”