Actin nano-architecture of phagocytic podosomes.

Podosomes are actin-enriched adhesion structures important for multiple cellular processes, including migration, bone remodeling, and phagocytosis. Here, we characterize the structure and organization of phagocytic podosomes using interferometric photoactivated localization microscopy, a super-resolution microscopy technique capable of 15-20 nm resolution, together with structured illumination microscopy and localization-based super-resolution microscopy. Phagocytic podosomes are observed during frustrated phagocytosis, a model in which cells attempt to engulf micropatterned IgG antibodies. For circular patterns, this results in regular arrays of podosomes with well-defined geometry. Using persistent homology, we develop a pipeline for semi-automatic identification and measurement of podosome features. These studies reveal an hourglass shape of the podosome actin core, a protruding knob at the bottom of the core, and two actin networks extending from the core. Additionally, the distributions of paxillin, talin, myosin II, α-actinin, cortactin, and microtubules relative to actin are characterized.

Biosensors based on peptide exposure show single molecule conformations in live cells.

We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

Attempted use of PACE for riboswitch discovery generates three new translational theophylline riboswitch side products.

OBJECTIVE: The purpose of this project was to use an in vivo method to discover riboswitches that are activated by new ligands. We employed phage-assisted continuous evolution (PACE) to evolve new riboswitches in vivo. We started with one translational riboswitch and one transcriptional riboswitch, both of which were activated by theophylline. We used xanthine as the new target ligand during positive selection followed by negative selection using theophylline. The goal was to generate very large M13 phage populations that contained unknown mutations, some of which would result in new aptamer specificity. We discovered side products of three new theophylline translational riboswitches with different levels of protein production. RESULTS: We used next generation sequencing to identify M13 phage that carried riboswitch mutations. We cloned and characterized the most abundant riboswitch mutants and discovered three variants that produce different levels of translational output while retaining their theophylline specificity. Although we were unable to demonstrate evolution of new riboswitch ligand specificity using PACE, we recommend careful design of recombinant M13 phage to avoid evolution of "cheaters" that short circuit the intended selection pressure.