Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localisation of actin microfilaments using phalloidin staining

Doris, Fiona; Steer, Martin

doi:10.1007/bf01279184

Cited by 54 publications

(42 citation statements)

References 26 publications

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“…LA binds to G-actin with a K d of 74 nM (calculated from maize pollen actin) (Gibbon et al, 1999), thus slowly dissociating from G-actin during washout, whereas CD-capped dynamic F-actin depolymerizes quickly, leading to release and efficient washout of CD. In pollen tubes, which are tip-growing cells, an actin configuration has been reported that is comparable to that in root hairs: an apical area without detectable actin filaments, followed subapically by an area with a dense actin meshwork (Doris and Steer, 1996;Miller et al, 1996;Gibbon et al, 1999;Geitmann et al, 2000). Miller et al (1996) confirmed by immunogold electron microscopic analysis after freeze substitution that the apical area is essentially free of filamentous actin.…”

Section: Discussionmentioning

confidence: 81%

Unstable F-Actin Specifies the Area and Microtubule Direction of Cell Expansion in Arabidopsis Root Hairs

2002

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Plant cells expand by exocytosis of wall material contained in Golgi-derived vesicles. We examined the role of local instability of the actin cytoskeleton in specifying the exocytosis site in Arabidopsis root hairs. During root hair growth, a specific actin cytoskeleton configuration is present in the cell's subapex, which consists of fine bundles of actin filaments that become more and more fine toward the apex, where they may be absent. Pulse application of low concentrations of the actin-depolymerizing drugs cytochalasin D and latrunculin A broadened growing root hair tips (i.e., they increased the area of cell expansion). Interestingly, recovery from cytochalasin D led to new growth in the original growth direction, whereas in the presence of oryzalin, a microtubule-depolymerizing drug, this direction was altered. Oryzalin alone, at the same concentration, had no influence on root hair elongation. These results represent an important step toward understanding the spatial and directional regulation of root hair growth.

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Section: Discussionmentioning

confidence: 81%

Unstable F-Actin Specifies the Area and Microtubule Direction of Cell Expansion in Arabidopsis Root Hairs

2002

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“…These pollen tubes, which were transformed transiently by microprojectile bombardment, elongated normally at the time of chemical fixation, which was followed by Texas red-conjugated phalloidin staining (Doris and Steer, 1996; to the disorganization of actin and growth arrest (Gibbon et al, 1999;. To examine how pollen tubes react to altered ADF levels or activities, Lat52-driven NtADF1, its S6A mutant transgene, and its S6D mutant transgene were coexpressed transiently with Lat52-GFP in microprojectile-bombarded pollen.…”

Section: Disruption Of the Pollen Adf Condition Suppresses Elongationmentioning

confidence: 99%

“…After plating, pollen tubes were allowed to recover in a moisture chamber with germination medium for 1 h. Chemical fixation procedures (Doris and Steer, 1996;) were applied to preserve pollen tubes. First, pollen tubes were treated with 300 M m-maleimidobenzoyl-N-hydroxysuccinimide ester and 271 M disuccinimidyl suberate (Pierce) in germination medium for 15 min.…”

Section: Phalloidin Stainingmentioning

confidence: 99%

The Regulation of Actin Organization by Actin-Depolymerizing Factor in Elongating Pollen Tubes[W]

et al. 2002

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Pollen tube elongation is a polarized cell growth process that transports the male gametes from the stigma to the ovary for fertilization inside the ovules. Actomyosin-driven intracellular trafficking and active actin remodeling in the apical and subapical regions of pollen tubes are both important aspects of this rapid tip growth process. Actin-depolymerizing factor (ADF) and cofilin are actin binding proteins that enhance the depolymerization of microfilaments at their minus, or slow-growing, ends. A pollen-specific ADF from tobacco, NtADF1, was used to dissect the role of ADF in pollen tube growth. Overexpression of NtADF1 resulted in the reduction of fine, axially oriented actin cables in transformed pollen tubes and in the inhibition of pollen tube growth in a dose-dependent manner. Thus, the proper regulation of actin turnover by NtADF1 is critical for pollen tube growth. When expressed at a moderate level in pollen tubes elongating in in vitro cultures, green fluorescent protein (GFP)-tagged NtADF1 (GFP-NtADF1) associated predominantly with a subapical actin mesh composed of short actin filaments and with long actin cables in the shank. Similar labeling patterns were observed for GFP-NtADF1-expressing pollen tubes elongating within the pistil. A Ser-6-to-Asp conversion abolished the interaction between NtADF1 and F-actin in elongating pollen tubes and reduced its inhibitory effect on pollen tube growth significantly, suggesting that phosphorylation at Ser-6 may be a prominent regulatory mechanism for this pollen ADF. As with some ADF/cofilin, the in vitro actin-depolymerizing activity of recombinant NtADF1 was enhanced by slightly alkaline conditions. Because a pH gradient is known to exist in the apical region of elongating pollen tubes, it seems plausible that the in vivo actin-depolymerizing activity of NtADF1, and thus its contribution to actin dynamics, may be regulated spatially by differential H ؉ concentrations in the apical region of elongating pollen tubes.

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“…Early studies with chemical fixation and fluorescent phalloidin-labeling techniques have suggested the presence of a dense actin network at the extreme tip (Derksen et al, 1995). In disagreement with this result, rapid freezing, freeze substitution, and electron and light microscopy techniques revealed only thin actin bundles and short individual actin microfilaments in random orientation (Lancelle et al, 1987;Doris and Steer, 1996). Also, no actin network was observed in living lily pollen tubes stained by injected fluorescencelabeled phalloidin .…”

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confidence: 99%

Ca2+-Permeable Channels in the Plasma Membrane of Arabidopsis Pollen Are Regulated by Actin Microfilaments

et al. 2004

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Cytosolic free Ca 21 and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca 21 channels, as well as channel-mediated Ca 21 influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca 21 in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca 21 channel blockers La 31 or Gd 31 and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca 21 . Ca 21 fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca 21 levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca 21 increase induced by CD or CB was abolished by addition of Ca 21 channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca 21 currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca 21 -permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca 21 -permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca 21 channel activity and may consequently regulate cytoplasmic Ca 21 .The primary function of pollen and pollen tubes is to deliver sperms to egg apparatus for double fertilization that is required for sexual reproduction of flowering plants. Pollen germination and pollen tube growth are a continuous and highly polarized process characteristic of tip growth; thus pollen and pollen tubes provide an ideal model system for the study of cell polarity control and tip growth. It is well known that extracellular Ca 21 is required for pollen germination and tube growth (for review, see Steer and Steer, 1989;Taylor and Hepler, 1997;Malhó , 1998;Franklin-Tong, 1999), which indicates a possible involvement of Ca 21 influx in pollen germination and tube growth. Upon pollen hydration and germination, cytoplasmic Ca 21 concentration ([Ca 21 ] i ) at the germinal aperture where the pollen tube emerges increases to a higher level than other regions, and a tip-focused Ca 21 gradient is then established and sustained while a pollen tube grows forward (Rathore et al., 1991;Pierson et al., 1994;Feijó et al., 1995). Disruption or modification of the Ca 21 gradient inhibits pollen tube growth (Miller et al., 1992) or changes its growth direction (Malhó and Trewavas, 1996). The tip-focused Ca 21 gradient oscillates in the pollen tubes of lily (Lilium longiflorum) and other species (Holdaway-Clarke et al., 1997;Messerli and Robinson, 1997;Messerli et al., 2000). Ca 21 mobilization from intracellular Ca...

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Effects of fixatives and permeabilisation buffers on pollen tubes: implications for localisation of actin microfilaments using phalloidin staining

Cited by 54 publications

References 26 publications

Unstable F-Actin Specifies the Area and Microtubule Direction of Cell Expansion in Arabidopsis Root Hairs

Unstable F-Actin Specifies the Area and Microtubule Direction of Cell Expansion in Arabidopsis Root Hairs

The Regulation of Actin Organization by Actin-Depolymerizing Factor in Elongating Pollen Tubes[W]

Ca2+-Permeable Channels in the Plasma Membrane of Arabidopsis Pollen Are Regulated by Actin Microfilaments

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