PMC

Tip-localized actin filaments and cytosolic Ca²⁺ gradient participate in controlling oscillatory RIC4/ROP activity and tip growth. The effects of LatB or LaCl₃ treatment were tested for the oscillatory tip growth and GFP-RIC4ΔC localization to the tip. For GFP-RIC4ΔC control, 10 pollen tubes with oscillatory tip growth were used for analysis. Six pollen tubes with oscillatory tip growth for each treatment of LaCl₃ or LatB were analyzed. For GFP-RIC4-expressing tubes, 4 and 3 tubes were analyzed for with and without LatB, respectively. Combined results are presented in A, B, and C. (A) Mean growth rate and mean period length of oscillatory growing pollen tubes with or without treatment of LatB or LaCl₃. (B) Relative amplitude of growth rate oscillation versus mean growth rate of individual growth pulses from oscillatory growing pollen tubes with or without treatment of LatB or LaCl₃. Relative amplitude of growth rate is calculated as peak growth rate over basal (interpulse) growth rate. LaCl₃-treated tubes were not significantly different from control tubes of similar mean growth rate. (C) Relative amplitude of RIC4 oscillation versus mean growth rate of individual RIC4 oscillations from oscillatory growing pollen tubes with or without treatment of LatB or LaCl₃. Relative amplitude of RIC4 (I-avg-pm) oscillation is calculated as peak value over basal (interpulse) I-avg-pm. LaCl₃- and LatB-treated tubes were significantly different from control tubes of similar mean growth rate. (D) Representative oscillations of tip growth rate and tip-localized RIC4 for control tubes without treatment, LaCl₃-treated tubes (LaCl3), and LatB-treated tubes (LatB).

Ficolin-2 levels vary by blood collection tube type. (A) Serum ficolin-2 binding to serotype 11A pneumococci for serum samples collected from 7 healthy human adult donors using glass tubes (black bars), coated glass tubes (white bars), plastic SSTs (gray bars), or plastic tubes (striped bars). MFI, mean fluorescence intensity. The inset shows representative flow cytometry histograms for ficolin-2 binding to serotype 11A pneumococci (black line) for serum samples collected from glass tubes (G), coated glass tubes (CG), plastic serum separator tubes (SST), or plastic tubes (P) from donor B. The gray-shaded areas represent bacteria stained with an isotype control antibody. (B) Serum ficolin-2 levels (μg/ml) for 7 healthy human donors using glass tubes (black bars), coated glass tubes (white bars), plastic SSTs (gray bars), or plastic tubes (striped bars). (C) Western blots for ficolin-2 (35 kDa) in serum samples collected in glass tubes (G), coated glass tubes (CG), plastic SSTs (SST), or plastic tubes (P). M indicates molecular mass markers (lane 1). *, not determined; serum was unavailable for the coated glass tube type from donor F. In panel A, data represent the means ± SEM for an experiment run in triplicate. In panel B, data represent the averages of duplicate results, and error bars represent the ranges.

X-axis: sample numbers; Y-axis: 25OHD results in VACUETTE 4-mL tubes with gel and clot activator minus 25OHD results in VACUETTE 4-mL additive-free tubes.

The Actin Bundle Phenotype is Alleviated within the Shank Region of fim5 Pollen Tubes When the Expression of PLIM2a and/or PLIM2b is Abolished
(A) Actin filaments in pollen tubes derived from WT, fim5, plim2a plim2b, and fim5 plim2a plim2b mutants of different lengths. Heavy actin bundle and fine actin structures are indicated by green and blue arrowheads in WT and plim2a plim2b pollen tubes, respectively. Uniformly sized but disorganized actin bundles in fim5 and fim5 plim2a plim2b pollen tubes are indicated by yellow arrowheads.
(B) Images showing actin filaments within the apical region (10 μm from the tip) and shank region (30–40 μm from the tip). Apical actin filaments (left columns) are indicated by magenta arrows in WT and plim2a plim2b pollen tubes and yellow arrows in fim5 and fim5 plim2a plim2b pollen tubes. Actin filaments are arranged into bright actin structures within the apical region of WT and plim2a plim2b pollen tubes, whereas they are arranged into uniformly sized but disorganized actin bundles with a moderate extent of bundling within the apical region of fim5 and fim5 plim2a plim2b pollen tubes. The middle regions with less abundant actin filaments are indicated by yellow asterisks within the apical region of WT and plim2a plim2b pollen tubes. Within the shank region (right columns), thick and thin actin bundles of WT and plim2a plim2b pollen tubes are indicated with green and blue arrowheads, respectively, whereas the intermediate-sized actin bundles in fim5 and fim5 plim2a plim2b pollen tubes are indicated by yellow arrowheads. It suggests that the alignment of actin filaments is slightly recovered in fim5 plim2a plim2b pollen tubes compared with that in fim5 pollen tubes.
(C) Histograms of the angles formed between the shank-localized actin bundles and the growth axis of pollen tubes. The values of average angles of each genotype are indicated in the image. At least three independent experiments were performed and one typical result was shown. More than 700 actin bundles were measured for each genotype.
(D) Transverse sections of pollen tubes within the shank region. The distances of the transverse sections from the pollen tube tip are indicated in images.
(E) Quantification of the area and fluorescence intensity of actin structures within transverse sections from the shank region of pollen tubes. The inset image shows the method of measuring the area and fluorescence intensity of actin structures (different colored circles) and their distances to the center (indicated by the red plus sign). The fluorescence intensity was plotted versus the area of actin structures. Red, blue, and green dashed lines indicate actin filament areas of 0.2, 0.5, and 0.8 μm², respectively. At least three independent experiments were performed and one typical result was shown. More than 170 actin bundles were measured for each genotype.
(F) Histogram of the distances between actin structures and the center of cross sections derived from the shank region of pollen tubes. The method of distance measurement was described in (E). As the size of pollen tubes varies, the measured distances were normalized to the radius of pollen tubes before generating the plot. The image (left panel) is the schematic diagram showing that the area of the inner circle (white colored) is equal to that in the outside annulus (purple colored). The radius of the inner circle is 70.7, whereas the radius of the transverse section is normalized to 100. Yellow and red colored dots indicate actin structures, and the value of “d” indicates the distance of actin structures to the center. The histogram was generated via plotting the count versus the values of “d” of actin structures. The green dashed line indicates the radius at 70.7. Green arrows indicate the peaks in the histogram, and blue arrows indicate the fluorescence intensity of actin structures in mutant pollen tubes that are obviously different from that in WT pollen tubes. In terms of the role of actin in driving cytoplasmic streaming in angiosperm pollen tubes, actin structures within the outside annule (corresponding to cortical actin structures in longitudinal sections) and actin structures in inner circle (corresponding to middle actin structures in longitudinal sections) are more relevant to the tipward and backward movement of vesicles, respectively. At least three independent experiments were performed and one typical result was shown. More than 170 actin bundles were measured for each genotype.
(G) Schematic describing the distribution of actin structures in the shank region of WT (a), fim5 (b), plim2a plim2b (c), and fim5 plim2a plim2b (d) pollen tubes. Left and right panels are longitudinal and transverse sections of pollen tubes, respectively. In the shank region of WT pollen tubes, both thin and thick actin exist, and they are aligned longitudinally. The shank region of fim5 pollen tubes is filled with uniformly sized but disorganized actin bundles, and they are comparatively more concentrated at the cortex. Compared with fim5 pollen tubes, fim5 plim2a plim2b pollen tubes have fewer actin bundles at the cortex and they are comparatively straight. Scale bars, 5 μm in all images.