Tapping mode atomic force microscopy in liquids gives a substantial improvement in imaging quality and stability over standard contact mode. In tapping mode the probe-sample separation is modulated as the probe scans over the sample. This modulation causes the probe to tap on the surface only at the extreme of each modulation cycle and therefore minimizes frictional forces that are present when the probe is constantly in contact with the surface. This imaging mode increases resolution and reduces sample damage on soft samples. For our initial experiments we used a tapping frequency of 17 kHz to image deoxyribonucleic acid plasmids on mica in water. When we imaged the same sample region with the same cantilever, the plasmids appeared 18 nm wide in contact mode and 5 nm in tapping mode.
Fission yeast myo1 + encodes a myosin-I with all three tail homology domains (TH1, 2, 3) found in typical long-tailed myosin-Is. Myo1p tail also contains a COOH-terminal acidic region similar to the A-domain of WASp/Scar proteins and other fungal myosin-Is. Our analysis shows that Myo1p and Wsp1p, the fission yeast WASp-like protein, share functions and cooperate in controlling actin assembly. First, Myo1p localizes to cortical patches enriched at tips of growing cells and at sites of cell division. Myo1p patches partially colocalize with actin patches and are dependent on an intact actin cytoskeleton. Second, although deletion of myo1 + is not lethal, Δmyo1 cells have actin cytoskeletal defects, including loss of polarized cell growth, delocalized actin patches, and mating defects. Third, additional disruption of wsp1 + is synthetically lethal, suggesting that these genes may share functions. In mapping the domains of Myo1p tail that share function with Wsp1p, we discovered that a Myo1p construct with just the head and TH1 domains is sufficient for cortical localization and to rescue all Δmyo1 defects. However, it fails to rescue the Δmyo1 Δwsp1 lethality. Additional tail domains, TH2 and TH3, are required to complement the double mutant. Fourth, we show that a recombinant Myo1p tail binds to Arp2/3 complex and activates its actin nucleation activity.
BackgroundActin is essential for tip growth in plants. However, imaging actin in live plant cells has heretofore presented challenges. In previous studies, fluorescent probes derived from actin-binding proteins often alter growth, cause actin bundling and fail to resolve actin microfilaments.Methodology/Principal FindingsIn this report we use Lifeact-mEGFP, an actin probe that does not affect the dynamics of actin, to visualize actin in the moss Physcomitrella patens and pollen tubes from Lilium formosanum and Nicotiana tobaccum. Lifeact-mEGFP robustly labels actin microfilaments, particularly in the apex, in both moss protonemata and pollen tubes. Lifeact-mEGFP also labels filamentous actin structures in other moss cell types, including cells of the gametophore.Conclusions/SignificanceLifeact-mEGFP, when expressed at optimal levels does not alter moss protonemal or pollen tube growth. We suggest that Lifeact-mEGFP represents an exciting new versatile probe for further studies of actin's role in tip growing plant cells.
Formins are present in all eukaryotes and are essential for the creation of actin-based structures responsible for diverse cellular processes. Because multicellular organisms contain large formin gene families, establishing the physiological functions of formin isoforms has been difficult. Using RNAi, we analyzed the function of all 9 formin genes within the moss Physcomitrella patens. We show that plants lacking class II formins (For2) are severely stunted and composed of spherical cells with disrupted actin organization. In contrast, silencing of all other formins results in normal elongated cell morphology and actin organization. Consistent with a role in polarized growth, For2 are apically localized in growing cells. We show that an N-terminal phosphatase tensin (PTEN)-like domain mediates apical localization. The PTEN-like domain is followed by a conserved formin homology (FH)1-FH2 domain, known to promote actin polymerization. To determine whether apical localization of any FH1-FH2 domain mediates polarized growth, we performed domain swapping. We found that only the class II FH1-FH2, in combination with the PTEN-like domain, rescues polarized growth, because it cannot be replaced with a similar domain from a For1. We used in vitro polymerization assays to dissect the functional differences between these FH1-FH2 domains. We found that both the FH1 and the FH2 domains from For2 are required to mediate exceptionally rapid rates of actin filament elongation, much faster than any other known formin. Thus, our data demonstrate that rapid rates of actin elongation are critical for driving the formation of apical filamentous actin necessary for polarized growth.Physcomitrella patens ͉ moss ͉ profilin ͉ tip growth ͉ RNAi F ormin proteins are critical regulators of the actin cytoskeleton that drive cellular processes in all eukaryotes ranging from division and motility to cell polarity, including axonal morphogenesis (1-4). The defining features of formins are the formin homology domains (FH1 and FH2) (5). The FH1 domain is characterized by the presence of polyproline stretches known to interact with the small actin monomer binding protein, profilin (6). The FH2 domain promotes actin filament nucleation, and is located C-terminal to the FH1 domain (7,8). Structural studies reveal that the FH2 domain forms a ring-like structure, which sits at the barbed end of an actin filament (9, 10). After nucleating a filament, the FH2 domain remains at the fast-growing filament end, and influences elongation rate as it moves processively with this end as additional monomers are incorporated. In vitro, the actin nucleating and elongating characteristics of individual formins can vary quite dramatically (3, 5). Because complex eukaryotes contain large formin gene families, the in vivo significance of these differences has been difficult to assess.Plants have been particularly challenging, because most angiosperms contain many formin genes (11). For example, Arabidopsis thaliana has 21 formins that group into 2 distinct families b...
Class XI myosins are plant specific and responsible for cytoplasmic streaming. Because of the large number of myosin XI genes in angiosperms, it has been difficult to determine their precise role, particularly with respect to tip growth. The moss Physcomitrella patens provides an ideal system to study myosin XI function. P. patens has only two myosin XI genes, and these genes encode proteins that are 94% identical to each other. To determine their role in tip growth, we used RNA interference to specifically silence each myosin XI gene using 59 untranslated region sequences. We discovered that the two myosin XI genes are functionally redundant, since silencing of either gene does not affect growth or polarity. However, simultaneous silencing of both myosin XIs results in severely stunted plants composed of small rounded cells. Although similar to the phenotype resulting from silencing of other actin-associated proteins, we show that this phenotype is not due to altered actin dynamics. Consistent with a role in tip growth, we show that a functional, full-length fusion of monomeric enhanced green fluorescent protein (mEGFP) to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells.
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