A role for glutamine 183 in the folate oxidative half-reaction of methylenetetrahydrofolate reductase from Escherichia coli.

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes a ping-pong reaction with NADH and 5,10-methylenetetrahydrofolate (CH2-H4folate) to produce NAD+ and 5-methyltetrahydrofolate (CH3-H4folate). This work focuses on the function of the invariant, active-site aminoacyl residue Gln183. X-ray structures of the enzyme complexes Ered(wild-type)•NADH and Eox(Glu28Gln)•CH3-H4folate indicate that Gln183 makes key hydrogen-bonding interactions with both NADH and folate in their respective half-reactions, suggesting roles in binding each substrate. We propose that the polarity of Gln183 may also aid in stabilizing the proposed 5-iminium cation intermediate during catalysis in the oxidative half-reaction with folate. We have prepared mutants Gln183Ala and Gln183Glu, which we hypothesize to have altered charge/polarity and hydrogen bonding properties. We have examined the enzymes by steady-state and stopped-flow kinetics and by measurement of the flavin redox potentials. In the reductive half-reaction, NADH binding affinity and the rate of flavin reduction have not been hindered by either mutation. By contrast, our results support a minor role for Gln183 in the oxidative half-reaction. The Gln183Ala variant exhibited a 6-10 fold lower rate of folate reduction and bound CH2-H4folate with 7-fold lower affinity, whereas the Gln183Glu mutant displayed catalytic constants within 3-fold of the wild-type enzyme.

Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding.

Cell surface glycosylation is thought to be involved in barrier function against microbes at mucosal surfaces. Previously we showed that the epithelium of healthy mouse corneas becomes vulnerable to Pseudomonas aeruginosa adhesion if it lacks the innate defense protein MyD88 (myeloid differentiation primary response gene 88), or after superficial injury by blotting with tissue paper. Here we explored their effect on corneal surface glycosylation using a metabolic label, tetra-acetylated N-azidoacetylgalactosamine (Ac4GalNAz). Ac4GalNAz treatment labeled the surface of healthy mouse corneas, leaving most cells viable, and bacteria preferentially associated with GalNAz-labeled regions. Surprisingly, corneas from MyD88-/- mice displayed similar GalNAz labeling to wild-type corneas, but labeling was reduced and patchy on IL-1 receptor (IL-1R)-knockout mouse corneas (P < 0.05, ANOVA). Tissue paper blotting removed GalNAz-labeled surface cells, causing DAPI labeling (permeabilization) of underlying cells. MS of material collected on the tissue paper blots revealed 67 GalNAz-labeled proteins, including intracellular proteins. These data show that the normal distribution of surface glycosylation requires IL-1R, but not MyD88, and is not sufficient to prevent bacterial binding. They also suggest increased P. aeruginosa adhesion to MyD88-/- and blotted corneas is not due to reduction in total surface glycosylation, and for tissue paper blotting is likely due to cell permeabilization.-Jolly, A. L., Agarwal, P., Metruccio, M. M. E., Spiciarich, D. R., Evans, D. J., Bertozzi, C. R., Fleiszig, S. M. J. Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding.

Kinesin-1 heavy chain mediates microtubule sliding to drive changes in cell shape.

Microtubules are typically observed to buckle and loop during interphase in cultured cells by an unknown mechanism. We show that lateral microtubule movement and looping is a result of microtubules sliding against one another in interphase Drosophila S2 cells. RNAi of the kinesin-1 heavy chain (KHC), but not dynein or the kinesin-1 light chain, eliminates these movements. KHC-dependent microtubule sliding powers the formation of cellular processes filled with parallel microtubule bundles. The growth of these cellular processes is independent of the actin cytoskeleton. We further observe cytoplasmic microtubule sliding in Xenopus and Ptk2 cells, and show that antibody inhibition of KHC in mammalian cells prevents sliding. We therefore propose that, in addition to its well established role in organelle transport, an important universal function of kinesin-1 is to mediate cytoplasmic microtubule-microtubule sliding. This provides the cell with a dedicated mechanism to transport long and short microtubule filaments and drive changes in cell shape.

Bidirectional intracellular transport: utility and mechanism.

Bidirectional transport of intracellular cargo along microtubule tracks is the subject of intense debate in the motility field. In the present review, we provide an overview of the models describing the possible mechanisms driving intracellular saltatory transport, taking into account current experimental results that may at first seem contradictory. We examine the phenomenon of saltatory motion, in an attempt to interpret the mechanistic debate in terms of the utility of saltatory motion.

A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells.

Long-distance intracellular transport of organelles, mRNA, and proteins ("cargo") occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

Pseudomonas aeruginosa-induced bleb-niche formation in epithelial cells is independent of actinomyosin contraction and enhanced by loss of cystic fibrosis transmembrane-conductance regulator osmoregulatory function.

UNLABELLED: The opportunistic pathogen Pseudomonas aeruginosa can infect almost any site in the body but most often targets epithelial cell-lined tissues such as the airways, skin, and the cornea of the eye. A common predisposing factor is cystic fibrosis (CF), caused by defects in the cystic fibrosis transmembrane-conductance regulator (CFTR). Previously, we showed that when P. aeruginosa enters epithelial cells it replicates intracellularly and occupies plasma membrane blebs. This phenotype is dependent on the type 3 secretion system (T3SS) effector ExoS, shown by others to induce host cell apoptosis. Here, we examined mechanisms for P. aeruginosa-induced bleb formation, focusing on its relationship to apoptosis and the CFTR. The data showed that P. aeruginosa-induced blebbing in epithelial cells is independent of actin contraction and is inhibited by hyperosmotic media (400 to 600 mOsM), distinguishing bacterially induced blebs from apoptotic blebs. Cells with defective CFTR displayed enhanced bleb formation upon infection, as demonstrated using bronchial epithelial cells from a patient with cystic fibrosis and a CFTR inhibitor, CFTR(Inh)-172. The defect was found to be correctable either by incubation in hyperosmotic media or by complementation with CFTR (pGFP-CFTR), suggesting that the osmoregulatory function of CFTR counters P. aeruginosa-induced bleb-niche formation. Accordingly, and despite their reduced capacity for bacterial internalization, CFTR-deficient cells showed greater bacterial occupation of blebs and enhanced intracellular replication. Together, these data suggest that P. aeruginosa bleb niches are distinct from apoptotic blebs, are driven by osmotic forces countered by CFTR, and could provide a novel mechanism for bacterial persistence in the host. IMPORTANCE: Pseudomonas aeruginosa is an opportunistic pathogen problematic in hospitalized patients and those with cystic fibrosis (CF). Previously, we showed that P. aeruginosa can enter epithelial cells and replicate within them and traffics to the membrane blebs that it induces. This "bleb-niche" formation requires ExoS, previously shown to cause apoptosis. Here, we show that the driving force for bleb-niche formation is osmotic pressure, differentiating P. aeruginosa-induced blebs from apoptotic blebs. Either CFTR inhibition or CFTR mutation (as seen in people with CF) causes P. aeruginosa to make more bleb niches and provides an osmotic driving force for blebbing. CFTR inhibition also enhances bacterial occupation of blebs and intracellular replication. Since CFTR is targeted for removal from the plasma membrane when P. aeruginosa invades a healthy cell, these findings could relate to pathogenesis in both CF and healthy patient populations.

Cytoplasmic microtubule sliding: An unconventional function of conventional kinesin.

There are well known examples in nature of microtubules dramatically changing their function by re-organizing their structure. Most interphase animal cells rely on the radial organization of the microtubule network for precise cargo delivery. Dividing cells re-organize microtubules with the help of motor proteins to form the spindle and drive the segregation of chromosomes into daughter cells. These examples present a kind of dichotomy: microtubules can be utilized as stationary tracks along which motor proteins move, or they can perform work themselves by utilizing the power of motor proteins. While both occur during mitosis, our recent findings demonstrate that both functions may occur simultaneously in interphase cells as well. We find that kinesin-1 (a motor known for its role in transporting cargo along microtubule tracks) powers microtubule sliding in non-dividing cells and this mechanism is used to form cellular protrusions.