In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and super-resolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion, Bap1 allowed the developing biofilm to adhere to surfaces, and heterogeneous mixtures of Vibrio polysaccharide (VPS), RbmC, and Bap1 formed dynamic, flexible and ordered envelopes that encased the cell clusters.

Protein synthesis requires the accurate positioning of mRNA and tRNA in the peptidyl-tRNA site of the ribosome. Here we describe x-ray crystal structures of the intact bacterial ribosome from Escherichia coli in a complex with mRNA and the anticodon stem-loop of P-site tRNA. At 3.5-Å resolution, these structures reveal rearrangements in the intact ribosome that clamp P-site tRNA and mRNA on the small ribosomal subunit. Binding of the anticodon stem-loop of P-site tRNA to the ribosome is sufficient to lock the head of the small ribosomal subunit in a single conformation, thereby preventing movement of mRNA and tRNA before mRNA decoding.

SUMMARY Telomeres, repetitive DNA sequences at chromosome ends, are shielded against the DNA damage response (DDR) by the shelterin complex. To understand how shelterin protects telomere ends, we investigated the structural organization of telomeric chromatin in human cells using super-resolution microscopy. We found that telomeres form compact globular structures through a complex network of interactions between shelterin subunits and telomeric DNA, and not by DNA methylation, histone deacetylation or histone trimethylation at telomeres and subtelomeric regions. Mutations that abrogate shelterin assembly or removal of individual subunits from telomeres cause up to a 10-fold increase in telomere volume. Decompacted telomeres become more accessible to telomere-associated proteins and accumulate DDR signals. Recompaction of telomeric chromatin using an orthogonal method displaces DDR signals from telomeres. These results reveal the chromatin remodeling activity of shelterin and demonstrate that shelterin-mediated compaction of telomeric chromatin provides robust protection of chromosome ends against the DDR machinery.

In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion, Bap1 allowed the developing biofilm to adhere to surfaces, and heterogeneous mixtures of Vibrio polysaccharide (VPS), RbmC, and Bap1 formed dynamic, flexible and ordered envelopes that encased the cell clusters. Microbes within biofilms are more resistant to antibiotics, to immune clearance, and to osmotic, acid and oxidative stresses compared to planktonic cells (1-7). Despite advances in identifying the polysaccharide and proteinaceous constituents of the biofilm extracellular matrix, the mechanisms by which these factors yield a mechanically defined and spatially organized biofilm are largely unknown (8-10). The small size of most microbes has precluded multi-scale optical investigation of living biofilms. Vibrio cholerae biofilm formation involves the production of Vibrio polysaccharide (VPS) and three matrix proteins (RbmA, RbmC, and Bap1) predicted to contain carbohydrate-binding domains (fig. S1A) (11-13). To investigate the molecular mechanisms of biofilm development, we used a V. cholerae rugose variant with increased capacity to form biofilms (11). We inserted Myc, FLAG, and HA (Human influenza hemagglutinin) epitopes into its genome at the 3' ends of * To whom correspondence should be addressed.

No abstract

Experimental work on mixing in microfluidic devices has been of growing importance in recent years. Interest in probing reaction kinetics faster than the minute or hour time scale has intensified research in designing microchannel devices that would allow the reactants to be mixed on a time scale faster than that of the reaction. Particular attention has been paid to the design of microchannels in order to enhance the advection phenomena in these devices. Ultimately, in vitro studies of biological reactions can now be performed in conditions that reflect their native intracellular environments. Liau et al. (Anal. Chem., vol. 77, 2005, p. 7618) have demonstrated a droplet-based microfluidic mixer that induces improved chaotic mixing of crowded solutions in milliseconds due to protrusions (‘bumps’) on the microchannel walls. Liau et al. (2005) have shown it to be possible to mix rapidly plugs of highly concentrated protein solutions such as bovine hemoglobin and bovine serum albumin. The present work concerns an analysis of the underlying mechanisms of shear stress transfer at liquid–liquid interfaces and associated enhanced mixing arising from the protrusions along the channel walls. The role of non-Newtonian rheology and surfactants is also considered within the mixing framework developed by Aref, Ottino and Wiggins in several publications. Specifically, we show that proportional thinning of the carrier fluid lubrication layer at the bumps leads to greater advection velocities within the plugs, which enhances mixing. When the fluid within the plugs is Newtonian, mixing will be enhanced by the bumps if they are sufficiently close to one another. Changing either the rheology of the fluid within the plugs (from Newtonian to non-Newtonian) or modifying the mechanics of the carrier fluid-plug interface (by populating it with insoluble surfactants) alters the mixing enhancement.

left column, beginning on line 10, ''Although gacA has been implicated in biofilm formation in PAO1 (37), we observed a very mild PVC-attachment defect for both the PA14NR Set gacA mutant and for a strain carrying a nonpolar deletion of the gacA gene (19),'' should instead read: ''Although gacA has been implicated in biofilm formation in PAO1 (37), we observed a very mild PVC-attachment defect for both the PA14NR Set gacA mutant and for a strain carrying a transposon insertion in the gacA gene (19, 20, 38).'' The related references appear below. This error does not affect the conclusions of the article.

body in a specific direction. What is unclear is if the cell is capable of forming stable adhesions with the collagen matrix and how the force is generated. Using the modulation tracking imaging method we can follow the changes in shape of the cellular protrusion and also image separately various proteins, including actin in the cytoplasm and in the membrane. In the thin long protrusion we observe both fast diffusing actin molecules and also relatively immobile species, presumably part of the actin cytoskeleton. We are developing a method to directly measure the movement of the entire actin bundle inside the very thin cells protrusions. The method is conceptually similar to speckle imaging; however, it works in 3D. 3338-Pos Board B443Super-Resolution Imaging of Chromosomal DNA in Cells Paul D. Simonson, Eli Rothenberg, Paul R. Selvin. Super-resolution imaging is achieved by localizing diffraction-limited spots with high accuracy. Here we combined two powerful approaches to image the chromosomal DNA inside cells. In one method, the accumulated, stochastic binding of fluorescent labels to an imaging target are localized, while in the other the fluorophore transitions between dark and bright states (compatible with binding, photobleaching, photo-activation, blinking, etc.), even when fluorophore images overlap, are localized. Combining the two techniques results in a robust microscopy that is faster than what is possible with either technique alone, requires less optimization, and corrects for cell autofluorescence. In addition, background noise due to fluorescent labels in solution can be virtually eliminated by using labels that fluoresce only when bound to the target. Many DNA-specific dyes show dramatic fluorescence enhancement upon binding to DNA, including SYTO, LOLO, and YOYO dyes. We used nanomolar concentrations of SYTO and LOLO to image lambda DNA attached to poly-L-lysine coated glass and chromosomal DNA in fixed HEK 293 and HeLa cells. We found average single-fluorophore localization errors of 36 nm and 24 nm on glass and in cells, respectively. These imaging techniques may prove useful in future studies of chromosomal DNA in cells, including chromatin structure and defects. This approach was further applied to imaging microtubules in vitro. We used commercial Oregon Green 488 paclitaxel to achieve a 10 nm average fluorophore localization error, and streptavidin S45A to transiently label biotinylated microtubules with Atto647N, resulting in an average of 18 nm fluorophore localization error. Future work will involve simultaneous imaging of DNA and proteins to answer important biological questions. 3339-Pos Board B444Investigation of Lysosomes as Enzyme Storage Vesicles using Single Particle Tracking Fluorescence Microscopy William H. Humphries IV, Christine K. Payne. Intracellular, vesicle-mediated, degradation of extracellular cargo is an essential cellular function. Of particular interest is the population of vesicles responsible for degradation of extracellular cargo. Previous work using lowdensity li...