BackgroundApicomplexan parasites employ a unique form of movement, termed gliding motility, in order to invade the host cell. This movement depends on the parasite’s actomyosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system, since mutants for core components of the gliding machinery, such as parasite actin or subunits of the MyoA-motor complex (the glideosome), remain motile and invasive, albeit at significantly reduced efficiencies. While compensatory mechanisms and unusual polymerisation kinetics of parasite actin have been evoked to explain these findings, the actomyosin system could also play a role distinct from force production during parasite movement.ResultsIn this study, we compared the phenotypes of different mutants for core components of the actomyosin system in Toxoplasma gondii to decipher their exact role during gliding motility and invasion. We found that, while some phenotypes (apicoplast segregation, host cell egress, dense granule motility) appeared early after induction of the act1 knockout and went to completion, a small percentage of the parasites remained capable of motility and invasion well past the point at which actin levels were undetectable. Those act1 conditional knockout (cKO) and mlc1 cKO that continue to move in 3D do so at speeds similar to wildtype parasites. However, these mutants are virtually unable to attach to a collagen-coated substrate under flow conditions, indicating an important role for the actomyosin system of T. gondii in the formation of attachment sites.ConclusionWe demonstrate that parasite actin is essential during the lytic cycle and cannot be compensated by other molecules. Our data suggest a conventional polymerisation mechanism in vivo that depends on a critical concentration of G-actin. Importantly, we demonstrate that the actomyosin system of the parasite functions in attachment to the surface substrate, and not necessarily as force generator.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0343-5) contains supplementary material, which is available to authorized users.

Within the base excision repair (BER) pathway, the DNA N-glycosylases are responsible for locating and removing the majority of oxidative base damages. Endonuclease III (Nth), formamidopyrimidine DNA glycosylase (Fpg) and endonuclease VIII (Nei) are members of two glycosylase families: the helix–hairpin–helix (HhH) superfamily and the Fpg/Nei family. The search mechanisms employed by these two families of glycosylases were examined using a single molecule assay to image quantum dot (Qdot)-labeled glycosylases interacting with YOYO-1 stained λ-DNA molecules suspended between 5 µm silica beads. The HhH and Fpg/Nei families were found to have a similar diffusive search mechanism described as a continuum of motion, in keeping with rotational diffusion along the DNA molecule ranging from slow, sub-diffusive to faster, unrestricted diffusion. The search mechanism for an Fpg variant, F111A, lacking a phenylalanine wedge residue no longer displayed slow, sub-diffusive motion compared to wild type, suggesting that Fpg base interrogation may be accomplished by Phe111 insertion.

For pancreatic β-cells to secrete insulin in response to elevated blood glucose, insulin granules retained within the subplasmalemmal space must be transported to sites of secretion on the plasma membrane. Using a combination of super-resolution STORM imaging and live cell TIRF microscopy we investigate how the organization and dynamics of the actin and microtubule cytoskeletons in INS-1 β-cells contribute to this process. GFP-labeled insulin granules display 3 different modes of motion (stationary, diffusive-like, and directed). Diffusive-like motion dominates in basal, low glucose conditions. Upon glucose stimulation no gross rearrangement of the actin cytoskeleton is observed but there are increases in the 1) rate of microtubule polymerization; 2) rate of diffusive-like motion; and 3) proportion of granules undergoing microtubule-based directed motion. By pharmacologically perturbing the actin and microtubule cytoskeletons, we determine that microtubule-dependent granule transport occurs within the subplasmalemmal space and that the actin cytoskeleton limits this transport in basal conditions, when insulin secretion needs to be inhibited.

DNA glycosylases are enzymes that perform the initial steps of base excision repair, the principal repair mechanism that identifies and removes endogenous damages that occur in an organism's DNA. We characterized the motion of single molecules of three bacterial glycosylases that recognize oxidized bases, Fpg, Nei, and Nth, as they scan for damages on tightropes of λ DNA. We find that all three enzymes use a key "wedge residue" to scan for damage because mutation of this residue to an alanine results in faster diffusion. Moreover, all three enzymes bind longer and diffuse more slowly on DNA that contains the damages they recognize and remove. Using a sliding window approach to measure diffusion constants and a simple chemomechanical simulation, we demonstrate that these enzymes diffuse along DNA, pausing momentarily to interrogate random bases, and when a damaged base is recognized, they stop to evert and excise it.DNA repair | search mechanisms O xidative DNA damage is produced endogenously during normal cellular metabolism or exogenously by chemical agents and ionizing radiation (1, 2). Oxidatively induced DNA damage resulting from attack by reactive oxygen species accounts for approximately one-half of all DNA base damages (3). Some oxidative base damages, such as thymine glycol, are blocks to DNA polymerases and thus potentially lethal; however, the majority of oxidative base lesions mispair with noncognate bases and are potentially mutagenic (4). Therefore, damaged bases must be repaired to maintain the cell's genomic integrity. With substantial in vivo steady-state levels of oxidative damages, alkylation damages, and apurinic/apyrimidinic (AP) sites among the nearly six billion normal bases, how DNA repair enzymes locate these damages in the sea of undamaged bases has been the subject of much speculation.The DNA repair mechanism responsible for the removal of the majority of endogenous DNA damages is the base excision repair (BER) pathway (4-6). The critical first step in BER is carried out by a DNA glycosylase that, fueled only by thermal energy, locates a damaged base and cleaves the N-glycosyl bond, thus removing the base lesion from the sugar phosphate backbone. Glycosylases are small monomeric proteins that are found in all living organisms and can be separated into different families based on substrate specificity and structural motifs. In Escherichia coli, there are three glycosylases, Nth, Fpg, and Nei, that directly remove oxidized DNA bases, and all three have an associated lyase activity that cleaves the DNA backbone. These glycosylases are members of two structural families, the helixhairpin-helix (HhH) or Nth superfamily and the helix-two turnshelix (H2TH) or Fpg/Nei family (7-9) (Fig. 1A). Interestingly, the HhH superfamily member, endonuclease III (Nth), and Fpg/ Nei family member, endonuclease VIII (Nei), primarily catalyze the removal of oxidized pyrimidines, such as 5,6-dihydroxy-5, 6-dihydrothymine (Tg), whereas formamidopyrimidine DNA glycosylase (Fpg) primarily removes oxidized purines...