A dynamically assembled cell wall synthesis machinery buffers cell growth.

Assembly of protein complexes is a key mechanism for achieving spatial and temporal coordination in processes involving many enzymes. Growth of rod-shaped bacteria is a well-studied example requiring such coordination; expansion of the cell wall is thought to involve coordination of the activity of synthetic enzymes with the cytoskeleton via a stable complex. Here, we use single-molecule tracking to demonstrate that the bacterial actin homolog MreB and the essential cell wall enzyme PBP2 move on timescales orders of magnitude apart, with drastically different characteristic motions. Our observations suggest that PBP2 interacts with the rest of the synthesis machinery through a dynamic cycle of transient association. Consistent with this model, growth is robust to large fluctuations in PBP2 abundance. In contrast to stable complex formation, dynamic association of PBP2 is less dependent on the function of other components of the synthesis machinery, and buffers spatially distributed growth against fluctuations in pathway component concentrations and the presence of defective components. Dynamic association could generally represent an efficient strategy for spatiotemporal coordination of protein activities, especially when excess concentrations of system components are inhibitory to the overall process or deleterious to the cell.

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization.

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.

Analysis of surface protein expression reveals the growth pattern of the gram-negative outer membrane.

The outer membrane (OM) of Gram-negative bacteria is a complex bilayer composed of proteins, phospholipids, lipoproteins, and lipopolysaccharides. Despite recent advances revealing the molecular pathways underlying protein and lipopolysaccharide incorporation into the OM, the spatial distribution and dynamic regulation of these processes remain poorly understood. Here, we used sequence-specific fluorescent labeling to map the incorporation patterns of an OM-porin protein, LamB, by labeling proteins only after epitope exposure on the cell surface. Newly synthesized LamB appeared in discrete puncta, rather than evenly distributed over the cell surface. Further growth of bacteria after labeling resulted in divergence of labeled LamB puncta, consistent with a spatial pattern of OM growth in which new, unlabeled material was also inserted in patches. At the poles, puncta remained relatively stationary through several rounds of division, a salient characteristic of the OM protein population as a whole. We propose a biophysical model of growth in which patches of new OM material are added in discrete bursts that evolve in time according to Stokes flow and are randomly distributed over the cell surface. Simulations based on this model demonstrate that our experimental observations are consistent with a bursty insertion pattern without spatial bias across the cylindrical cell surface, with approximately one burst of ≈ 10(-2) µm(2) of OM material per two minutes per µm(2). Growth by insertion of discrete patches suggests that stochasticity plays a major role in patterning and material organization in the OM.

Morphology and interaction between lipid domains.

Cellular membranes are a heterogeneous mix of lipids, proteins and small molecules. Special groupings enriched in saturated lipids and cholesterol form liquid-ordered domains, known as "lipid rafts," thought to serve as platforms for signaling, trafficking and material transport throughout the secretory pathway. Questions remain as to how the cell maintains small fluid lipid domains, through time, on a length scale consistent with the fact that no large-scale phase separation is observed. Motivated by these examples, we have utilized a combination of mechanical modeling and in vitro experiments to show that membrane morphology plays a key role in maintaining small domain sizes and organizing domains in a model membrane. We demonstrate that lipid domains can adopt a flat or dimpled morphology, where the latter facilitates a repulsive interaction that slows coalescence and helps regulate domain size and tends to laterally organize domains in the membrane.

Thermoelectric efficiency and compatibility.

The intensive reduced efficiency eta(r) is derived for thermoelectric power generation (in one dimension) from intensive fields and currents, giving eta(r)=(E x J) divided by (- inverted Delta)T x J(S). The overall efficiency is derivable from a thermodynamic state function, Phi=1 divided by u + alphaT, where we introduce u=J divided by kappa (inverted Delta)T as the relative current density. The method simplifies the computation and clarifies the physics behind thermoelectric devices by revealing a new materials property s=(sqrt[1+zT]-1) divided by (alphaT), which we call the compatibility factor. Materials with dissimilar compatibility factors cannot be combined by segmentation into an efficient thermoelectric generator because of constraints imposed on u. Thus, control of the compatibility factor s is, in addition to z, essential for efficient operation of a thermoelectric device, and thus will facilitate rational materials selection, device design, and the engineering of functionally graded materials.