MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

Bacteria use homologs of eukaryotic cytoskeletal filaments to conduct many different tasks, controlling cell shape, division, and DNA segregation. These filaments, combined with factors that regulate their polymerization, create emergent self-organizing machines. Here, we summarize the current understanding of the assembly of these polymers and their spatial regulation by accessory factors, framing them in the context of being dynamical systems. We highlight how comparing the in vivo dynamics of the filaments with those measured in vitro has provided insight into the regulation, emergent behavior, and cellular functions of these polymeric systems.

Rhodamine-phalloidin-labeled actin filaments were visualized gliding over a skeletal heavy meromyosin (HMM)-coated surface. Experiments at low filament densities showed that when two filaments collided, their paths were affected in a manner that depended on collision angle. Some collisions resulted in complete alignment of the filament paths; in others, the filaments crossed over one another. Filament crossover or alignment was equally probable at ∼40° contact angle. Filaments often underwent significant bending during collision and analysis of filament shape indicated an energy requirement of ∼13 kBT. Experiments were performed over a wide range of HMM surface density and actin filament bulk concentration. Actin filament gliding speed and path persistence plateaued above a critical HMM surface density, and at high (micromolar) actin filament concentrations, filament motion became dramatically aligned in a common direction. Spatiotemporal features of alignment behavior were determined by correlation analysis, supported by simulations. The thermal drift of individual filament tracks was suppressed as the population became more oriented. Spatial correlation analysis revealed that long-range alignment was due to incremental recruitment rather than fusion of locally ordered seed domains. The global alignment of filament movement, described by an "order parameter," peaked at optimal actin concentrations and myosin surface densities, in contrast to previous predictions of a critical phase transition. Either hydrodynamic coupling or exchange of filaments between the surface bound and adjacent bulk phase layers might degrade order at high actin filament concentration, and high HMM surface densities might decrease alignment probability during collisions. Our results are compatible with generation of long-range order from mechanical interaction between individual actin filaments. Furthermore, we show that randomly oriented myosin motors align relatively short, submicrometer actin filaments into motile surface domains that extend over many tens of micrometers and these patterns persist for several minutes.

24MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments 25 move around the rod circumference, helping to insert cell wall in the radial direction to 26 reinforce rod shape. To understand how oriented MreB motion arises, we altered the 27 shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is 28 restored when rod shape is externally imposed. Stationary filaments orient within 29 protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. 30Together, this demonstrates MreB orients along the greatest principal membrane 31 curvature, a conclusion supported with biophysical modeling. We observed that 32 spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly 33 propagating rods emerge from small bulges, exhibiting oriented MreB motion and 34 increased glycan crosslinking. We propose that the coupling of MreB filament alignment 35 to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing 36 mechanism allowing the rapid establishment and stable maintenance of emergent rod 37 shape.

No abstract

MreB is essential for rod shape in many bacteria. Membrane-associated MreB filaments move around the rod circumference, helping to insert cell wall in the radial direction to reinforce rod shape. To understand how oriented MreB motion arises, we altered the shape of Bacillus subtilis. MreB motion is isotropic in round cells, and orientation is restored when rod shape is externally imposed. Stationary filaments orient within protoplasts, and purified MreB tubulates liposomes in vitro, orienting within tubes. Together, this demonstrates MreB orients along the greatest principal membrane curvature, a conclusion supported with biophysical modeling. We observed that spherical cells regenerate into rods in a local, self-reinforcing manner: rapidly propagating rods emerge from small bulges, exhibiting oriented MreB motion. We propose that the coupling of MreB filament alignment to shape-reinforcing peptidoglycan synthesis creates a locally-acting, self-organizing mechanism allowing the rapid establishment and stable maintenance of emergent rod shape.

Dendritic filopodia are actin-filled highly dynamic subcellular structures that sprout densely on neuronal dendrites during early brain development. A fraction of filopodia undergo a transition to dendritic spines that later mature into synapses -a process crucial for memory formation and learning, and deficient in neurodevelopmental and neurodegenerative diseases. The dynamics of dendritic filopodia is also different from that of conventional filopodia: the former exhibit sustained length fluctuations and cease their dynamic behavior after transition into spines. While actin retrograde flow in dendritic filopodia was measured previously, there has been no detailed theoretical description of how the flow is maintained and how length oscillations are sustained in dendritic filopodia. We apply mathematical modeling and experimental techniques to dissect and analyze the components of actin-based motility in dendritic filopodia, and suggest its role in the subsequent transition into the spine shape. The simulations demonstrate that the movement of actin network influenced by myosin contractility and viscous shear stresses lead to the myosin build up at the base of the filopodium and its consequent retraction. When myosin is inactive, the filopodium grows with the rate of actin polymerization. However, when myosin is active, the processes of polymerization at the tip, and cytoskeletal contraction due to myosin, and resistance to the flow out of the filopod at the base, conspire to produce an actin flow gradient along the filopodial axis. We have measured average rates of growth and retraction of filopodia in cultured hippocampal neurons using a custom tip-tracking algorithm. The simulated length fluctuations and actin retrograde flow compare well with the experimental data. We estimate the resistive force at the base of filopodia necessary to maintain the pattern of growth and shrinking and suggest new experiments to test the proposed mechanism.