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. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: Micron. 2017 Mar 21;98:34–38. doi: 10.1016/j.micron.2017.03.012

F-actin reorganization upon de- and rehydration in the aeroterrestrial green alga Klebsormidium crenulatum

Kathrin Blaas 1, Andreas Holzinger 1,1
PMCID: PMC5429973  EMSID: EMS72638  PMID: 28363156

Abstract

Filamentous actin (F-actin) is a dynamic network involved in many cellular processes like cell division and cytoplasmic streaming. While many studies have addressed the involvement of F-actin in different cellular processes in cultured cells, little is known on the reactions to environmental stress scenarios, where this system might have essential regulatory functions. We investigated here the de- and rehydration kinetics of breakdown and reassembly of F-actin in the streptophyte green alga Klebsormidium crenulatum. Measurements of the chlorophyll fluorescence (effective quantum yield of photosystem II [ΔF/Fm’]) via pulse amplitude modulation were performed as a measure for dehydration induced shut down of physiological activity, which ceased after 141 ± 15 min at ~84 % RH. We hypothesized that there is a link between this physiological parameter and the status of the F-actin system. Indeed, 20 min of dehydration (ΔF/Fm’ = 0) leads to a breakdown of the fine cortical F-actin network as visualized by Atto 488 phalloidin staining, and dot-like structures remained. Already 10 min after rehydration a beginning reassembly of F-actin is observed, after 25 min the F-actin network appeared similar to untreated controls, indicating a full recovery. These results demonstrate the fast kinetics of F-actin dis- and reassembly likely contributing to cellular reorganization upon rehydration.

Key index words: charophyta, desiccation, F-actin, phalloidin staining, rehydration, water potential

1. Introduction

The filamentous actin (F-actin) cytoskeleton is a dynamic network of polymeric proteins, motors and associated proteins. Among the first plant cells, where F-actin was visualized is the streptophyte green alga Nitella flexilis (Palevitz et al. 1974). F-actin contributes to the determination of cell shape in green algae (Meindl et al. 1994), serves for myosin-based cytoplasmatic streaming and was pharmacologically perturbed to study its function (eg. Foissner and Wasteneys 2007, Wasteneys et al. 1996). However, in algae little is known on the role of F-actin in environmentally relevant stress scenarios like dehydration. This is particularly interesting, as poikilohydric ancestors of land plants had to cope with fluctuating water conditions, which has been addressed in several ecophysiological studies (for summary see Holzinger and Karsten 2013). While the development of land plants from a charophyte green algal ancestor is a major event in earth´s history (e.g. Becker and Marin 2009, Leliaert et al. 2012), little is known on the cellular adaptations that allowed conquering land, and adaptation to desiccation.

We selected the basal streptophyte green alga Klebsormidium crenulatum for the present investigation, as this alga is growing on alpine soil crusts where it can be potentially exposed to desiccation in its natural habitat. Numerous data are already available on its ecophysiological performance (Karsten et al. 2010), cellular adaptations to desiccation (Holzinger et al. 2011), and osmotic stress (Kaplan et al. 2012). Moreover, investigations on transcriptomic alterations upon severe desiccation have been performed (Holzinger et al. 2014). Relative volume changes as a consequence of exposure to different relative air humidities (RHs) have been reported for this alga (Lajos et al. 2016). Analysis of the composition of the cell wall showed a remarkable proportion of callose, which increased upon desiccation stress and allowed a flexible shrinkage process (Herburger and Holzinger 2015). With the knowledge of a dynamic reaction to water loss in Klebsormdium (Karsten and Holzinger 2014, Karsten et al. 2012, 2016), it was particularly interesting, how cytoskeletal elements would react to this environmental stress. A breakdown of F-actin was observed in K. crenulatum upon severe desiccation at 5 % RH, and only dot-like batches remained (Holzinger et al. 2011). However, this treatment was not sufficient for an immediate recovery (Holzinger et al. 2014), calling for studies with a milder dehydration.

In the present study, we investigated the effects of physiologically monitored de- and rehydration on the F-actin cytosekelton, visualized by fluorescent phalloidin staining (Pflügl-Haill et al. 2000, Holzinger and Blaas 2016). We hypothesized that the breakdown of F-actin during desiccation is correlated to the reduction of the effective quantum yield (YII), which is used as physiological measure. Moreover, we wanted to investigate the kinetics of F-actin reorganization after rehydration, which was not studied before in K. crenulatum.

2. Material and Methods

2.1. Algal cultures and desiccation experiments

Klebsormidium crenulatum (SAG 2415) was isolated previously from an alpine soil crust at mount Schönwieskopf (Tyrol, Austria; Karsten et al. 2010), purified and established as unialgal culture. Stock cultures were grown on 1.5 % agar plates containing modified Bold’s Basal Medium at low light conditions (30-35 µmol photons m-2s-1, light dark regime 16:8 h, at 20°C, Karsten et al. 2010).

Desiccation and rehydration experiments were performed according to Karsten et al. (2014) in a transparent 735 mL polystyrol box filled at its bottom with 150 mL saturated KCl solution (Merck, Darmstadt, Germany) or A. bidest., respectively. The temperature inside the chamber during the desiccation experiment was between 22°C and 23.6°C. Small amounts of algal biomass (20–28 d old) were placed by tweezers in 10 µL drops of A. bidest. (n = 8) on Parafilm ® stripes. After all their uniform appearance, quarters of Whatman GF/C glass microfiber filters (Whatman, Dassel, Germany; ø 47 mm) were placed onto the algal samples which were soaked up immediately. Filter pieces were positioned on coverslips (Menzel, Braunschweig, Germany; 24 x 24 mm) in the measuring box around the PCEMSR145S-TH mini data logger (PCE Instruments, Meschede, Germany). The distance between the samples and the inner side of the chamber lid was 3 mm. RH, temperature and ΔF/Fm’ (PAM 2500, Heinz Walz GmbH, Effeltrich, Germany) were determined in intervals of 5 min during desiccation. After signal remained for 20 min at YII = 0, samples were rehydrated by adding 180 µL A. bidest. to each sample and the KCl solution was replaced by A. bidest and measurements were continued. Three different conditions for the subsequent F-actin staining were chosen: untreated cells (n=4), desiccated cells with YII = 0 for 20 min (n=4) and cells rehydrated for different periods: 3 s (n=3), 10 min (n=3) and 25 min (n=4).

2.2. F-actin visualization

Atto-phalloidin staining of F-actin followed a modified protocol after Pflügl-Haill et al. (2000). Briefly, cells were incubated for 15 min at room temperature in fixative comprising 0.60 mM M-maleimidobenzoyl N-hydroysuccinimide ester (MBS; Pierce; 300 mM stock solution in DMSO) for cross-linking F-actin, 3.70 % formaldehyde and 0.07 % glutaraldehyde in PIPES buffer (12.50 mM piperazine-N,N'-bis 2-ethane sulfonic acid [PIPES], 2.50 mM EGTA, 1.25 mM MgCl2·6H2O; pH 7). After a washing step in PIPES buffer the cells were labeled for 75 min (in darkness, at room temperature) with 2.44 % Atto 488 Phalloidin (Sigma; stock solution: 10 nmol in 500 µL methanol) and 0.50 % Triton X-100 (15 % stock solution) in PIPES buffer. After a second washing step, cells were mounted in PIPES buffer. Samples were analyzed microscopically immediately after the staining experiment.

Stained F-actin was visualized by confocal laser scanning microscopy (CLSM) on an inverted Zeiss Axiovert 200M microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a Zeiss Pascal system. Samples were excited with an argon laser beam (488 nm) and emission was collected in two separate channels at 505–550 nm and long pass 560 nm. Using a Zeiss 63x immersion oil objective (NA = 1.4), z-stacks of confocal images (512 × 512 pixel, mean of two images) with slide-height of 1.0 µm were generated. Raw images, covering the whole width of samples, were further processed with the visualization software package ImageJ (Rasband 1997-2016). Raw images (512 x 512 pixel) were imported as 2-channel z-stacks, z-projections for each channel were created (projection type: standard deviation) and then split in order to have one z-projection per channel. Further, z-projections were thresholded against the backgrounds (method: RenyiEntrop) and brightness and contrast in z-projection from channel 560 nm (long pass; chloroplasts respectively chlorophyll) were adjusted manually. In z-projections from channel 505–550 nm (Atto-phalloidin stained F-actin) the minimal gray values – as they were responsible for the noisy background below the thin filamentous structures – were measured and then subtracted. The (cortical) slide of the raw z-stack where F-actin in top view was captured, was duplicated, thresholded and with background-minimizing (like described above) a slide with cortical F-actin was created that was then added to the z-projection of this channel (tool: image calculator). Afterwards the channel colors were adjusted: 505–550 nm false-colored yellow and 560 nm long pass false-colored blue, images were then merged. Stacks were created from the singular images and prior making the montages, stacks were aligned and canvas sizes were adjusted (mostly to 200 x 310 pixel).

3. Results

3.1. Desiccation effects on physiology

Desiccation of K. crenluatum at (~84 % RH) caused a decrease of YII from 0.54 ± 0.03 in the first 141 ± 15 min to 0 within 15 ± 4 min. (n = 32), suggesting complete inhibition of PS II. Upon rehydration, YII values recovered immediately and increased steadily towards the initial YII values at ~95 % RH. After 3 s rehydration, YII had recovered to about a third of the initial YII (n = 5), 10 min rehydration enabled to recover to 63 ± 11 % (n = 24), after 25 min rehydration 79 ± 12 % were reached (n = 14). Fig. 1 shows a representative curve of one experiment (n=4).

Fig. 1.

Fig. 1

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Effective quantum yield (YII) measurements during monitored desiccation and rehydration in Klebsormidium crenulatum. Saturated KCl solution served as desiccant, for recovery cells were rehydrated with A. bidest. (n = 4, mean values ± SD).

3.2. F-actin reorganization

In control cells, prior to treatment, Atto-phalloidin staining showed an intricate cortical network of F-actin (Fig. 2 A) that rapidly disappeared upon desiccation (YII = 0 for 20 min) and was replaced by dot-like structures (Fig. 2 B). The cells remained desiccated for 20 min. While after 3 s rehydration, YII values had already increased by about one third of the initial value, this period was not sufficient for a recovery of the F-actin cytoskeleton (Fig. 2 C). However, 10 min after rehydration beginning of the reassembly of F-actin was observed (Fig. 2 D), 25 min after rehydration the F-actin network had a similar appearance with long filaments when compared to control cells (Fig. 2 E). Fig. 2 F-J shows the F-actin cytoskelton (emission 505-550 nm) and Fig. 2 K-O shows the chlorophyll autofluorescence (emission > 560 nm) of the respective treatments.

Fig. 2.

Fig. 2

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F-actin staining of Klebsormidium crenulatum by atto-phalloidin. (A-E) merged images of both channels, (F-J) F-actin (505-550 nm), (K-O) chlorophyll autofluorescence (560 nm long pass), (A, F, K) Undesiccated control, (B, G, L) desiccated filaments (YII = 0 for 20 min) and rehydrated samples: (C, H, M) 3 s, (D; I, N) 10 min, cell starting reorganization of the F-actin system (arrow), (E, J, O) 25 min. scale bar = 10 µm.

4. Discussion

In the present study, we have shown dehydration effects on the F-actin cytoskeleton in K. crenulatum exposed to ~84 % RH. As a measure for physiological activity, YII values were recorded for the samples that were subjected to F-actin visualization. These values were stable for the first ~140 min, decreased then rapidly and were kept for 20 min at zero before rewetting. Despite we do not have data on the relative water content of the cells during dehydration, we consider the YII values of zero as a clear indication of a physiological ‘shut down’ due to water loss. During the dehydration event, the F-actin network broke down totally, only dot-like remnants were visible. Upon rewetting, the YII values increased immediately (already after 3s), while the reorganization of the F-actin network started after 10 min, and it had a similar appearance as the controls after 25 min.

It was shown that the slow dehydration rate over saturated KCl solution (~84 % RH) was beneficial for a fast and persistent recovery of YII in K. crenulatum. In an earlier study, K. crenulatum was air-dried without monitoring the actual RH during the dehydration process (Karsten et al. 2010). This lead to a strong decrease of the maximum PSII quantum efficiency (Fv/Fm) after about 180 min, a partial recovery in Fv⁄Fm was observed already after 10 min after rehydration, however full recovery was reached only after 2 h. In contrast, when silica gel was used as desiccant, the reduction of the effective quantum yiel was observed much earlier, i.e. already after ~40 min, and immediate recovery of K. crenulatum was not observed (Holzinger et al. 2014). Analysis of the relative volume reduction (between ~ 46-76%) showed a continuous increase during equilibration to declining external RHs in K. crenulatum (Lajos et al. 2016). In the range of ~65% RH to ~ 85% RH, the values for relative volume reduction were not significantly different, which helps to explain the similar drying rates in the present study (at 84%) when compared to the ambient-air drying by Karsten et al. (2010). The recovery rate is likely related to the duration of the dehydration period and the strength of dehydration, which corroborates findings of Herburger and Holzinger (2015), who demonstrated by imaging PAM a rapid recovery of YII in K. crenulatum after short term desiccation.

The disintegration of F-actin upon desiccation in K. crenulatum has been visualized previously, but only after strong desiccation (5% RH, 24 h), which altered the F-actin network to dot-like batches (Holzinger et al. 2011); however the kinetics of this breakdown and a possible reestablishment remained unknown. The staining protocol, that allowed to visualize dense F-actin network in control cells of K. crenulatum, was modified from Pflügl-Haill et al. (2000). We observed positive effects on the F-actin visualization by pretreatments with the crosslinker MBS, which was not reported for e.g. in Micrasterias denticulata (Pflügl-Haill et al. 2000) and pollen tubes (Doris and Steer 1996). Particularly for analyzing recovery of the F-actin network after a previous break down, a reliable method is essential. In the present study, we were able to gain full recovery of the F-actin appearance after 25 min rehydration. The F-actin system showed a very dynamic response, while it broke down rapidly upon water loss, its reestablishment was already complete in this relatively short time. As an intact F-actin network is crucial for cytoplasmic streaming, it is likely that this is important for reestablishment of cellular functions that were abolished during the dehydrated state.

F-actin alterations in response to experimental turgor reduction have been studied in GFP-tagged Arabidopis lines (Lang et al. 2014). Rearrangements of the F-actin were found upon plasmolysis, and the F-actin appeared to reorganize quickly upon shrinking of the protoplast during plasmolysis; however, the cytological situation in a vegetative vascular plant cell is different with a prominent central vacuole. While during the plasmolytic process, water is removed from the vacuole, the cells remain in a hydrated state, and a total breakdown of the F-actin was not observed (Lang et al. 2014). In contrast, changes of the F-actin as a consequence of de- and rehydration were analyzed in the desiccation tolerant moss protonemas of Funaria hygrometrica (Pressel and Duckett 2010). These studies are likely more comparable to what we observe in the cytoplasm rich K. crenulatum after dehydration. Upon slow drying in F. hygrometrica, by placing protonemal colonies in closed but not sealed Petri dishes for 2–3 d, a disintegration of F-actin to granular and diffuse structures was found. In fast dried samples more randomly distributed fragments of F-actin were observed. In barley (Hordeum vulgare), two cultivars with differences in drought tolerance showed remarkable changes in configuration of F-actin, and an increased amount of F-actin was observed in the drought-tolerant cultivar (Śniegowska-Świerk et al. 2015).

Moreover, a proteomics study in desiccation tolerant bryophytes by Cruz de Carvalho et al. (2014) confirmed a general down regulation of cytoskeleton components and proteins associated with photosynthesis after slow dehydration (95 % RH, Ψ of −6 MPa, 72 h; as compared to fast dehydration: 50 % RH, Ψ of −100 MPa, 3 h). Subsequent rehydration caused upregulation of these proteins. However, during slow dehydration the actin protein started to increase in abundance and after rehydration an almost twofold elevation was observed. These studies, while indicating changes in cytoskeletal components, do not allow to judge on the polymerization status of actin.

Summarizing our results, we could show a rapid recovery of YII upon desiccation in K. crenulatum, and a beginning recovery of the F-actin system already after 10 min rehydration reaching a full recovery after 25 min. This ability is likely to contribute to desiccation tolerance in this aeroterrestrial alga frequently exposed to desiccation in its natural habitat.

Acknowledgements

This study was supported by Austrian Science Fund (FWF) grant P 24242-B16 and I 1951-B16 to AH.

Abbreviations

3N MBBM

Bold’s Basal Medium with triple nitrate concentration

ΔF/Fm’

effective quantum yield of photosystem II (= YII)

EGTA

ethylene glycol tetraacetic acid

F-actin

filamentous actin

MBS

m-maleimidobenzoyl n-hydroysuccinimide ester

PAM

pulse amplitude modulation

PIPES

piperazine-N,N'-bis 2-ethane sulfonic acid

RH

relative air humidity

YII

effective quantum yield of photosystem II (= ΔF/Fm’)

Footnotes

The authors declare that they have no conflict of interests.

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