The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

The motor protein kinesin couples a temporally periodic chemical cycle (the hydrolysis of ATP) to a spatially periodic mechanical cycle (movement along a microtubule). To distinguish between different models of such chemical-to-mechanical coupling, we measured the speed of movement of conventional kinesin along microtubules in in vitro motility assays over a wide range of substrate (ATP) and product (ADP and inorganic phosphate) concentrations. In the presence and absence of products, the dependence of speed on [ATP] was well described by the Michaelis–Menten equation. In the absence of products, the K _M (the [ATP] required for half-maximal speed) was 28 ± 1 μM, and the maximum speed was 904 nm/s. P _i behaved as a competitive inhibitor with K _I = 9 ± 1 mM. ADP behaved approximately as a competitive inhibitor with K _I = 35 ± 2 μM. The data were compared to four-state kinetic models in which changes in nucleotide state are coupled to chemical and/or mechanical changes. We found that the deviation from competitive inhibition by ADP was inconsistent with models in which P _i is released before ADP. This is surprising because all known ATPases (and GTPases) with high structural similarity to the motor domains of kinesin release P _i before ADP (or GDP). Our result is therefore inconsistent with models, such as one-headed and inchworm mechanisms, in which the hydrolysis cycle takes place on one head only. However, it is simply explained by hand-over-hand models in which ADP release from one head precedes P _i release from the other.

The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

P150^glued belongs to a group of proteins accumulating at microtubule plus ends (+TIPs). It plays a key role in initiating retrograde transport by recruiting and tethering endosomes and dynein to microtubules. p150^glued contains an N-terminal microtubule-binding cytoskeleton-associated protein glycine-rich (CAP-Gly) domain that accelerates tubulin polymerization. Although this copolymerization is well-studied using light microscopic techniques, structural consequences of this interaction are elusive. Here, using electron-microscopic and spectroscopic approaches, we provide a detailed structural view of p150^glued CAP-Gly binding to microtubules and tubulin. Cryo-EM 3D reconstructions of p150^glued-CAP-Gly complexed with microtubules revealed the recognition of the microtubule surface, including tubulin C-terminal tails by CAP-Gly. These binding surfaces differ from other retrograde initiation proteins like EB1 or dynein, which could facilitate the simultaneous attachment of all accessory components. Furthermore, the CAP-Gly domain, with its basic extensions, facilitates lateral and longitudinal interactions of tubulin molecules by covering the tubulin acidic tails. This shielding effect of CAP-Gly and its basic extensions may provide a molecular basis of the roles of p150^glued in microtubule dynamics. Significance This study presents a direct visualization of the microtubule-p150glued(CAP-Gly) complex by cryo-EM and seeks to describe the molecular mechanism of the control of tubulin dynamics by p150 CAP-Gly. It highlights the neutralization of the acidic tubulin surface by the basic extensions of CAP-Gly, resulting in the activation of tubulin polymerization. In the condition where the lateral association is impeded (i.e., at low temperature), the extended CAP-Gly domain induces tubulin dimers to connect longitudinally. The two directional modes of self-association of tubulin suggest a foundation for its dynamic behavior at the tip of microtubules and its regulation.

The actin cytoskeleton is a central mediator of cellular morphogenesis, and rapid actin reorganization drives essential processes such as cell migration and cell division. Whereas several actin-binding proteins are known to be regulated by changes in intracellular pH, detailed information regarding the effect of pH on the actin dynamics itself is still lacking. Here, we combine bulk assays, total internal reflection fluorescence microscopy, fluorescence fluctuation spectroscopy techniques, and theory to comprehensively characterize the effect of pH on actin polymerization. We show that both nucleation and elongation are strongly enhanced at acidic pH, with a maximum close to the pI of actin. Monomer association rates are similarly affected by pH at both ends, although dissociation rates are differentially affected. This indicates that electrostatics control the diffusional encounter but not the dissociation rate, which is critical for the establishment of actin filament asymmetry. A generic model of protein-protein interaction, including electrostatics, explains the observed pH sensitivity as a consequence of charge repulsion. The observed pH effect on actin in vitro agrees with measurements of Listeria propulsion in pH-controlled cells. pH regulation should therefore be considered as a modulator of actin dynamics in a cellular environment.