Xylella fastidiosa is a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plant's vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered that X. fastidiosa migrates via type IV-pilus-mediated twitching motility at speeds up to 5 m min ؊1 against a rapidly flowing medium (20,000 m min ؊1 ). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 m) and short type I pili (0.4 to 1.0 m). We further demonstrated that two knockout mutants (pilB and pilQ mutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions in pilB or pilQ (possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion in fimA (possessing only type IV pili) is biofilm deficient.

Xylella fastidiosa, an important phytopathogenic bacterium, causes serious plant diseases including Pierce's disease of grapevine. It is reported here that type I and type IV pili of X. fastidiosa play different roles in twitching motility, biofilm formation and cell-cell aggregation. Type I pili are particularly important for biofilm formation and aggregation, whereas type IV pili are essential for motility, and also function in biofilm formation. Thirty twitching-defective mutants were generated with an EZ : : TN transposome system, and several type-IV-pilus-associated genes were identified, including fimT, pilX, pilY1, pilO and pilR. Mutations in fimT, pilX, pilO or pilR resulted in a twitch-minus phenotype, whereas the pilY1 mutant was twitching reduced. A mutation in fimA resulted in a biofilm-defective and twitching-enhanced phenotype. A fimA/pilO double mutant was twitch minus, and produced almost no visible biofilm. Transmission electron microscopy revealed that the pili, when present, were localized to one pole of the cell. Both type I and type IV pili were present in the wild-type isolate and the pilY1 mutant, whereas only type I pili were present in the twitch-minus mutants. The fimA mutant produced no type I pili. The fimA/pilO double mutant produced neither type I nor type IV pili.

Xylella fastidiosa, a bacterium responsible for Pierce's disease in grapevines, possesses both type I and type IV pili at the same cell pole. Type IV pili facilitate twitching motility, and type I pili are involved in biofilm development. The adhesiveness of the bacteria and the roles of the two pili types in attachment to a glass substratum were evaluated using a microfluidic flow chamber in conjunction with pilus-defective mutants. The average adhesion force necessary to detach wild-type X. fastidiosa cells was 147 ؎ 11 pN. Mutant cells possessing only type I pili required a force of 204 ؎ 22 pN for removal, whereas cells possessing only type IV pili required 119 ؎ 8 pN to dislodge these cells. The experimental results demonstrate that microfluidic flow chambers are useful and convenient tools for assessing the drag forces necessary for detaching bacterial cells and that with specific pilus mutants, the role of the pilus type can be further assessed.

Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve a subcellular level, neardiffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.

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Understanding how biomineralization occurs in the extracellular matrix (ECM) of bone cells is crucial to the understanding of bone formation and the development of a successfully engineered bone tissue scaffold. It is still unclear how ECM mechanical properties affect protein–mineral interactions in early stages of bone mineralization. We investigated the longitudinal mineralization properties of MC3T3-E1 cells and the elastic modulus of their ECM using shear modulation force microscopy, synchrotron grazing incidence X-ray diffraction (GIXD), scanning electron microscopy, energy dispersive X-ray spectroscopy, and confocal laser scanning microscopy (CLSM). The elastic modulus of the ECM fibers underwent significant changes for the mineralizing cells, which were not observed in the nonmineralizing cells. On substrates conducive to ECM network production, the elastic modulus of mineralizing cells increased at time points corresponding to mineral production, whereas that of the nonmineralizing cells did not vary over time. The presence of hydroxyapatite in mineralizing cells and the absence thereof in the nonmineralizing ones were confirmed by GIXD, and CLSM showed that a restructuring of actin occurred only for mineral-producing cells. These results show that the correct and complete development of the ECM network is required for osteoblasts to mineralize. This in turn requires a suitably prepared synthetic substrate for bone development to succeed in vitro.

We report that the nanometer-scale architecture of polymer chains plays a crucial role in its protein resistant property over surface chemistry. Protein-repellent (noncharged), few nanometer thick polymer layers were designed with homopolymer chains physisorbed on solids. We evaluated the antifouling property of the hydrophilic or hydrophobic adsorbed homopolymer chains against bovine serum albumin in water. Molecular dynamics simulations along with sum frequency generation spectroscopy data revealed the selforganized nanoarchitecture of the adsorbed chains composed of inner nematiclike ordered segments and outer brush-like segments across homopolymer systems with different interactions among a polymer, substrate, and interfacial water. We propose that this structure acts as a dual barrier against protein adsorption.

Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatiotemporal levels. Fig. 13. Imaging Golgi-derived membrane vesicles in living COS-7 cells at a volume acquisition time of 5 ms using HR-LFM. (a) Reconstructed 3D images of vesicles in living COS-7 cells labeled with mEmerald-Golgi-7 acquired at a volume acquisition time of 5 ms. The left and right are reconstructed 3D images at t = 0.005s and 5.000s of a 1000-time-point series, respectively. The depth-information is color-coded according to the color scale bar. (b) Zoomedin images of the corresponding boxed region in (a) at t = 0.005s, 3.000s, 3.500s, 4.000s, 4.500s and 5.000s, respectively. White arrows indicate vesicles moving during time-points. (c) Zoomed-in image (leftmost) of the boxed region in (b) at t = 0.005s and its z-stack images from z = 0.2 µm to +1.3 µm at an axial step size of 100 nm, resolving several nearby vesicles. (d) Zoomed-in images of the corresponding boxed region in (a) at t = 0.005s, 0.500s, 1.000s, 1.500s, 2.000s, 2.500s, 3.000s and 3.500s, respectively. Scale bars: 10 µm (a), 1 µm (b-d).