FH5-GFP is localized to the pollen-tube tip. (A) A schematic representation of angiosperm pollen-tube actin cytoskeleton and the apical inverted cone region (or “clear zone”) of vesicles. Arrows trace the “reverse-fountain” cytoplasmic streaming pattern. (B–F) Localization of formin homology-5 (FH5) promoter (FH5p) and the pollen-specific Lat52 promoter-expressed FH5-GFP to the apical and subapical membrane of Arabidopsis and tobacco elongating pollen tubes. (B) Emerging to elongating (Left to Right) Arabidopsis tubes. (D and E) Whole-tube projection of Arabidopsis tubes (Upper) by confocal imaging; three medial sections are shown (with increased γ to reveal signal from the shank, where no cell membrane-associated GFP-signal was detected). (F) Typical FH5-GFP localization pattern of prominent association with the apical and subapical, tip-focused collection of vesicles, and vesicular congregates that streamed along the cytoplasm (Movie S1). (G) A reorienting tobacco tube. Arrowheads highlight the apical flank and subapical regions; arrows indicate inverted cone region; asterisk in G indicates where the tip was at 0 s. See Fig. S2 for additional details and FH5p:GUS expression in transformed Arabidopsis pollen. Arabidopsis tubes were from stably transformed pollen. Tobacco tubes were transiently transformed by either 2 μg of Lat52::FH5-GFP or 10 μg of FH5p::FH5-GFP; elongating pollen tubes showed comparable FH5-GFP signals. (Scale bars, 10 μm.)

FH5 regulates the prominence of the subapical actin structure. Data are from transiently transformed tobacco pollen tubes. GFP-ADF was used as the actin reporter as it most efficiently reveals the subapical actin structure and least adversely affects tip growth among several plant actin reporters (3, 8, 36). Pollen tubes were either transformed by the actin reporter alone (control) or cotransformed by Lat52::FH5 (+FH5). (A) Growth response to FH5 transgene dosage (indicated in data bars). Data are the mean ± SE of the average tube lengths from triplicate samples (with 20–30 tubes per replicate) in one representative experiment. Difference between the +FH5 and control samples were significant (at 4 h, P = 5.8 × 10⁻⁵, 4.7 × 10⁻³, and 10⁻³ for the +0.25, +0.5, and +1 μg FH5 samples, respectively; at 7 h, P = 1.1 × 10⁻³, 2.9 × 10⁻³ and 3.2 × 10⁻⁴, respectively). Pollen tube-width comparison, which negatively correlates with growth quality (7, 17, 25), showed 0.25 μg FH5-transformed pollen tubes being slightly but significantly narrower than the controls (Fig. S3B), consistent with enhance growth quality. (B) Wide-field images showing the relatively subtle subapical actin structure in a control (arrow) and the substantially augmented structure in a +FH5 tube. The structures were elongating at 35 and 41.3 nm/s, respectively, over a 10-min period. The subapical actin structure signal intensity (SAC):distal cytoplasm signal intensity (Cyt) ratio was used as a measure for the prominence of the subapical actin structure. The SAC:Cyt ratio was 1.1 and 1.63 for the control and +FH5 tube, respectively. Fig. S3C shows details on SAC and Cyt analyses. (C) Percentage of tubes showing a prominent subapical actin structure (SAC:Cyt >1.15). Data shows average ± SD (n = 73–145 tubes analyzed for each sample). Differences from controls were significant (P = 10⁻⁴ and 2 × 10⁻³ at 4 h, 1.4 × 10⁻⁴, and 2.4 × 10⁻³ by 7 h for the +0.25 and 0.5 μg samples, respectively). The reduced percentage observed for the 7-h +0.5 μg FH5 sample reflected that many of the transformed tubes were already growth-inhibited and their subapical actin structure obliterated. Analyses in A to C established that 0.25 μg of Lat52::FH5 was optimum in obtaining the largest number of rapidly elongating transformed pollen tubes over the best growth period for observations (4–8 h after transformation) (3, 8, 36). (D) Wide-field coimaging of a FH5-Ch and actin reporter cotransformed pollen tube showing the spatial relationship between the subapical actin structure and the FH5-Ch–occupied apical dome membrane. (E) Whole-tube projections from confocal z-stack images of a control and a +FH5 tube. The frontal view (90°) and orthogonal sections of the +FH5 tube (1–6 taken at *, Right to Left) revealed a basket-shaped subapical actin structure anchored at the apical flank cortex, projecting centripetally to span the central cytoplasm at its base. (F) Medial sections of a +FH5 tube taken from a confocal time series (Movie S2). White lines show distance grown by a control tube for a comparable period (Movie S3), showing more rapid growth in the +FH5 tube (26.8 nm/s vs. 18.9 nm/s in the control). (G) Growth-rate comparison. Growth rates were averaged from control (n = 31) and +FH5 (n = 33) tubes individually monitored for durations of between 3 min and 1 h (with intermittent image acquisition) in several independent comparative experiments. Difference was significant (P = ∼5 × 10⁻³) and resulted from a higher percentage of FH5-transformed pollen tubes elongating at higher growth rates than controls (Fig. S3D). (H) SAC:Cyt intensity ratio comparison shows significantly more prominent subapical actin structures in FH5 transformed tube compared with controls (P = ∼4.7 × 10⁻⁷). Data were average ± SDs from the same samples analyzed in G. (Scale bars: 5 μm in E, 10 μm in all others.)

RNAi suppression of Nt-FH5 diminished the subapical actin structure and compromised tip-focused vesicular organization and growth. Data are from tobacco pollen tubes transiently transformed by marker GFP with or without RNAi Nt-FH5 (control). (A and B) Pollen tubes expressing GFP (control) (A) or coexpressing GFP and NtFH5 RNAi (B) at ∼10 h of in vitro growth when RNAi-induced phenotype was prevalent. An average of 80.3 ± 14% of Nt-FH5 RNAi transformed and 2.47 ± 0.75% of control pollen tubes developed severe meandering (p ∼3 × 10⁻⁴): n = 3 independent experiments, each with 40 to 55 control and >50 Nt-FH5 RNAi tubes each. (C–E) Actin cytoskeleton (C), apical vesicular accumulation pattern (D), and Golgi localization pattern (E) in control and Nt-FH5 RNAi transformed pollen tubes at ∼6 h of growth when the meandering phenotype was just developing. GFP-ADF was used as actin reporter. GFP-Rab11 and GFP-Rab2 was used as vesicle and Golgi reporters, respectively (24, 26). Insets in D and E show a longer profile of the meandering tubes. Arrowheads indicate subapex. Arrow in E indicates apical clear zone. (Scale bars: 50 μm in A and B Center and 10 μm in all others.) Fig. S4 shows control and Nt-FH5 antisense transformed tubes.

FH5 nucleates assembly of the subapical actin structure. Pollen tubes shown were transiently cotransformed by FH5 and actin reporter. (A and B) Subapical actin structure and growth recovery after Latb (12.5 nM) treatment. (A) Selected images from a confocal time series (Upper) and growth rates (Lower). (B) Subapical emergence of actin filaments (arrowheads), and their merging to form a structure spanning the central cytoplasm throughout the entire recovery period. More individual still frames and the entire time series are shown in Fig. S5 and Movie S6. (C) Selected images from a wide-field time series (after deconvolution) from a tube observed (∼5 min after Latb washout) when apical actin assembly was recovering from the apical membrane (Movie S7). Arrows, emanating actin filaments; arrowheads, dislodging actin fragments.

FH5-nucleated actin filaments support subapex-targeted vesicular trafficking. (A) Wide-field images monitoring growth of a tobacco pollen tube cotransformed by FH5-Ch and actin marker. (Upper) A representative image in individual channels and merged. Arrow, subapical actin structure; arrowhead, apical membrane and inverted cone region. The red signal was stretched equally in all merged images to illustrate the FH5-Ch labeled vesicular zone. Fig. S6 A and B show analysis details for the prominence of the subapical actin structure (SAC:Cyt) and of the apical vesicular zone (AV:Cyt) during transition from rapid to decelerated growth for this and other similarly transformed tubes. (B) A FH5-induced apical actin rim during the growth transition period. (C) A FH5-induced subapical vesicle targeting pattern (arrowheads) during the growth transition period. Two consecutive medial confocal sections are shown (7.5 s apart). (D) Subapex-focused actin filaments during a FH5-Ch induced growth transition. (E) Reaccumulation of vesicles during recovery from Latb-induced (+Latb) vesicle dispersal. Growth rate accelerated from < 7 nm/s to > 21 nm/s before and after 178 s. Arrowheads indicate subapical region. (Scale bars, 10 μm.)

Formin-regulated apical actin assembly and the tip growth process. Elongating actin filaments are shown with their barbed end toward the membrane where formins are located. Short F-actins in the core cytoplasm where actin restructuring is expected are shown in random orientation. F-actins flowing out of the subapical structure are shown with their barbed end in the direction of the retroflow (37). Vesicles are shown only in the upper half of the tube. (A) Recently deposited apical dome membrane-associated formins most actively nucleates actin assembly from the subapical membrane as the previous subapical structure disassembles and actin cables flow rearward in the central cytoplasm. (B and C) Membrane-anchored actin filaments elongate centripetally to merge in the central cytoplasm; some are fragmented and released to the subapical cytoplasm providing nuclei for assembly into the subapical actin structure. These elongating nascent actin filaments, with their plus ends linked to membrane-anchored formins, support subapex-targeted vesicle trafficking. The morphological dynamics and variations observed for this subapical structure during growth reflect the rapid reconfiguration, disassembly and reassembly of this subapical collection of actin filaments. To maintain the spatial relationship with the extending tube tip, the process is reiterative, and as the actin cables from the assembled structure funnel into retro-streams of actin, a new round of subapical actin assembly ensues (C–A). (D) A confocal stack of an elongating, FH5-transformed pollen tube that were also coexpressing actin and vesicle markers, showing an apical vesicular zone (in red) closely subtended by the pronounced subapical actin structure trailed by retro-streams of actin bundles in the core cytoplasm (yellow).