(A) Maximum-intensity projection actin filament image of the individual z-stack in a WT pollen tube as shown in (B). cortex of pollen tubes. Actin filaments appeared to be more Amprolium HCl curved in pollen tubes and this was confirmed by measurements showing that this convolutedness and the rate of switch of convolutedness of actin filaments was significantly increased in pollen tubes. This suggests that the rigidity of the actin Amprolium HCl filaments may be compromised in pollen tubes. Further, the apical cell wall composition is altered, implying that tip-directed vesicle trafficking events are impaired in pollen tubes. Thus, we found that FIM5 decorates apical actin filaments and regulates their business in order to drive polarized pollen tube growth. (Kovar mutants, suggesting that this rigidity of actin filaments is usually compromised when function is usually lost. Consequently, apical actin filaments grow in different directions within the apical cytoplasm and cannot be maintained at the cortex. The dense cortical actin structure therefore fails to form in pollen tubes. Unexpectedly, actin filaments become less dynamic in pollen tubes. The apical actin filaments are more severely disorganized in some pollen tubes than in others which may explain why pollen tubes exhibit different depolarization patterns as reported previously (Wu (Wu pollen tubes as explained previously (Wu pollen tubes were collected under a confocal laser scanning microscope, allowing the generation of high spatial resolution projection images. Consistent with our previous observation (Wu online). This implies that apically concentrated FIM5 might be important for apical actin filament business and polarized pollen tube growth. Open in a separate windows Fig. 1. FIM5-EGFP decorates apical actin filaments. Subcellular localization of FIM5 in the pollen tube was revealed by visualizing under either a confocal laser scanning microscope as explained previously (Wu pollen Amprolium HCl tubes To examine the function of FIM5 in regulating the organization of the apical actin structure, we used the null mutant which had been characterized in our previous study (Wu pollen tubes and carefully compared the staining pattern with that in WT pollen Amprolium HCl tubes. Indeed, we found that actin filaments were abnormally organized throughout the entire pollen tube in mutants and exhibited numerous patterns of disorganization (Fig. 2ACF; Wu pollen tubes (Fig. 2ACF) and the dense apical actin structure found in WT pollen tubes (Fig. 2A; reddish boxed region) was not observed in pollen tubes (Fig. Col6a3 2BCF). To uncover more details of the defects in the apical actin structure, we examined z-series optical sections from your apical regions of WT and pollen tubes (Fig. 2G, H). In WT pollen tubes, we found that most apical actin filaments are organized in a longitudinal direction, form relatively thin angles with the pollen tube growth axis, and become concentrated at the cortex of the tube (Fig. 2G). By comparison, we found that apical actin filaments appeared to develop in mixed directions in pollen tubes and did not concentrate at the cortex (Fig. 2H). The outcome of this may be that this dense cortical apical actin structure cannot form in pollen tubes. Open in a separate windows Fig. 2. Actin filament structures become disorganized in pollen tubes. Actin filaments in WT (Col-0) and pollen tubes were revealed by staining with Alexa-488 phalloidin. (A) A typical WT pollen tube. The red box indicates the apical region of interest. Scale bar=5 m for (ACF). (BCF) Several representative pollen tubes showing different distribution patterns of actin filaments. (B, C) Pollen tubes with disorganized actin filaments throughout the entire tube and very prominent actin bundles in the shank region. (D) Pollen tube with actin filaments that show relatively normal distribution in the shank, but are disorganized in the subapical and apical regions. (E) Pollen tube with actin Amprolium HCl filaments that are thinner and disorganized.