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Rapid cytoplasmic streaming in characean algae mediates communications between remote cell regions exposed to uneven irradiance. The metabolites exported from brightly illuminated chloroplasts spread along the internode with the liquid flow and cause transient changes in chlorophyll fluorescence at cell areas that are exposed to dim light or placed shortly in darkness. The largest distance to which the photometabolites can be transported has not yet been determined. Neither is it known if lateral transport has an influence on the induction of chlorophyll fluorescence. In this study, the relations between spatial connectivity of anchored chloroplasts in characean internodes and fluorescence induction curves were examined. Connectivity between remote cell parts was pronounced upon illumination of a cell spot at a distance up to 10 mm from the area of fluorescence measurement, provided the spot was located upstream in the cytoplasmic flow. Spatial interactions between distant cell sites were also manifested in strikingly different slow stages of fluorescence induction caused by narrow- and wide-field illumination. Cytochalasin D, cooling of bath solution, and inactivation of light-dependent envelope transporters were used to disturb cyclosis-mediated spatial interactions. Although fluorescence induction curves induced by narrow- and wide-field illumination differed greatly under control conditions, they became similar after the inhibition of cyclosis with cytochalasin D. The results indicate that cytoplasmic streaming not only drives the lateral translocation of photometabolites but also promotes the export of reducing power from illuminated chloroplasts due to flushing the chloroplast surface and keeping sharp concentration gradients.

Purpose To provide a biophysical basis to estimate the effect of cytoplasmic flow in neurons, and assess their contribution to the drop in the Apparent Diffusion Coefficient (ADC) in a nerve tissue following extreme conditions, such as brain injury and epileptic seizures. Methods Three mechanisms are treated using the relevant physics of hydrodynamics and electrostatics: cargo induced streaming, electroosmosis, and membrane swelling. Results We begin by discussing the lack of experimental evidence on the necessary velocities required to influence the Magnetic Resonance (MR) experiments. This is followed by demonstrating that cargo induced streaming, a widely known phenomenon in plant cells, has a minor effect on the ADC in neurons. Subsequently, we suggest and analyze two additional mechanisms that may induce fluid displacement in neurons, and are related to the electrical activity: electroosmosis and membrane swelling. Conclusion Although these mechanisms may induce interesting fluid displacements, these cannot explain the significant drop in the ADC. We conclude by outlining the criteria that any future mechanism should meet to have an influence on standard diffusion-MR measurements. Magn Reson Med, 2016. © 2016 International Society for Magnetic Resonance in Medicine

Pollen tubes usually exhibit a prominent region at their apex called the "clear zone" because it lacks light refracting amyloplasts. A robust, long clear zone often associates with fast growing pollen tubes, and thus serves as an indicator of pollen tube health. Nevertheless we do not understand how it arises or how it is maintained. Here we review the structure of the clear zone, and attempt to explain the factors that contribute to its formation. While amyloplasts and vacuolar elements are excluded from the clear zone, virtually all other organelles are present including secretory vesicles, mitochondria, Golgi dictyosomes, and the endoplasmic reticulum (ER). Secretory vesicles aggregate into an inverted cone appressed against the apical plasma membrane. ER elements move nearly to the extreme apex, whereas mitochondria and Golgi dictyosomes move less far forward. The cortical actin fringe assumes a central position in the control of clear zone formation and maintenance, given its role in generating cytoplasmic streaming. Other likely factors include the tip-focused calcium gradient, the apical pH gradient, the influx of water, and a host of signaling factors (small G-proteins). We think that the clear zone is an emergent property that depends on the interaction of several factors crucial for polarized growth.

The Drosophila formin Cappuccino (Capu) creates an actin mesh-like structure that traverses the oocyte during midoogenesis. This mesh is thought to prevent premature onset of fast cytoplasmic streaming which normally happens during late-oogenesis. Proper cytoskeletal organization and cytoplasmic streaming are crucial for localization of polarity determinants such as osk, grk, bcd, and nanos mRNAs. Capu mutants disrupt these events, leading to female sterility. Capu is regulated by another nucleator, Spire, as well as by autoinhibition in vitro. Studies in vivo confirm that Spire modulates Capu's function in oocytes; however, how autoinhibition contributes is still unclear. To study the role of autoinhibition in flies, we expressed a Capu construct that is missing the Capu Inhibitory Domain, CapuΔN. Consistent with a gain of activity due to loss of autoinhibition, the actin mesh was denser in CapuΔN oocytes. Further, cytoplasmic streaming was delayed and fertility levels decreased. Localization of osk mRNA in early stages, and bcd and nanos in late stages, were disrupted in CapuΔN-expressing oocytes. Finally, evidence that these phenotypes were due to a loss of autoinhibition comes from coexpression of the N-terminal half of Capu with CapuΔN, which suppressed the defects in actin, cytoplasmic streaming and fertility. From these results, we conclude that Capu can be autoinhibited during Drosophila oocyte development. © 2014 Wiley Periodicals, Inc.

Explanations for protoplasmic streaming began with appeals to contraction in the eighteenth century and ended with appeals to contraction in the twentieth. During the intervening years, biologists proposed a diverse array of mechanisms for streaming motions. This paper focuses on the re-emergence of contraction among the molecular mechanisms proposed for protoplasmic streaming during the twentieth century. The revival of contraction is a result of a broader transition from colloidal chemistry to a macromolecular approach to the chemistry of proteins, the recognition of the phenomena of shuttle streaming and the pulse of protoplasm, and the influential analogy between protoplasmic streaming and muscle contraction. Michael R. Dietrich

The pollen tube is fundamental for the reproduction of seed plants. Characteristically, it grows relatively quickly and uni-directionally ("polarized growth") to extend the male gametophyte to reach the female gametophyte. The pollen tube forms a channel through which the sperm cells move so that they can reach their targets in the ovule. To grow quickly and directionally, the pollen tube requires an intense movement of organelles and vesicles that allows the cell's contents to be distributed to sustain the growth rate. While the various organelles distribute more or less uniformly within the pollen tube, Golgi-released secretory vesicles accumulate massively at the pollen tube apex, that is, the growing region. This intense movement of organelles and vesicles is dependent on the dynamics of the cytoskeleton, which reorganizes differentially in response to external signals and coordinates membrane trafficking with the growth rate of pollen tubes.

Protein–protein interaction at the organelle level can be analyzed by using tagged proteins and assessing Forster resonance energy transfer (FRET) between fluorescent donor and acceptor proteins. Such studies are able to uncover partners in the regulation of proteins and enzymes. However, any organelle movement is an issue for live FRET microscopy, as the observed organelle must not change position during measurement. One of the mobile organelles in plants is the Golgi apparatus following cytoplasmic streaming. It is involved in the decoration of proteins and processing of complex glycan structures for the cell wall. Understanding of these processes is still limited, but evidence is emerging that protein–protein interaction plays a key role in the function of this organelle. In the past, mobile organelles were usually immobilized with paraformaldehyde (PFA) for FRET-based interaction studies. Here, we show that the actin inhibitor Cytochalasin D (CytD) is superior to PFA for immobilization of Golgi stacks in plant cells. Two glycosyltransferases known to interact were tagged with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, coexpressed in Nicotiana benthamiana leaves and analyzed using confocal microscopy and spectral imaging. Fixation with PFA leads to reduced emission intensity when compared to CytD treatment. Furthermore, the calculated FRET efficiency was significantly higher with CytD than with PFA. The documented improvements are beneficial for all methods measuring FRET, where immobilization of the investigated molecules is necessary. It can be expected that FRET measurement in organelles of animal cells will also benefit from the use of inhibitors acting on the cytoskeleton. © 2013 International Society for Advancement of Cytometry

Summary Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter-compartmental and inter-hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome-derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild-type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α-glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.

Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4-64 and charged nanogold. Time-lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co-localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin-dependent and actin-indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin-dependent secretory activity occurs in the shank of pollen tubes.

Pulsed electric fields (PEF) nonthermally induce cell membrane permeabilization and thereby improve dehydration and extraction efficiencies in food plant materials. Effects of electrical field strength and number of pulses on plant tissue integrity have been studied extensively. Two previous studies on the effect of pulse frequency, however, did not provide a clear view: one study suggested no effect of frequency, while the other found a greater impact on tissue integrity at lower frequency. This study establishes the effect of pulse frequency on integrity of onion tissues. Changes in electrical characteristics, ion leakage, texture parameters, and percent weight loss were quantified for a wide range of pulse frequencies under conditions of fixed field strength and pulse number. Optical microscopy and viable-cell staining provided direct visualization of effects on individual cells. The key finding is that lower frequencies (f1 Hz) cause more damage to tissue integrity than higher frequencies (f = 1 to 5000 Hz). Intriguingly, the optical microscopy observations demonstrate that the speed of intracellular convective motion (that is, cytoplasmic streaming) following PEF application is strongly correlated with PEF frequency. We provide the first in situ visualization of the intracellular consequence of PEF at different frequencies in a plant tissue. We hypothesize that cytoplasmic streaming plays a significant role in moving conductive ionic species from permeabilized cells to the intercellular space between plant cells, making subsequent pulses more efficacious at sufficiently low frequencies. The results suggest that decreasing the pulse frequency in PEF may minimize the number of pulses needed to achieve a desired amount of permeabilization, thus lowering the total energy consumption. Practical Application: PEF cause pores to be formed in plant cell membranes, thereby improve moisture removal and potential extraction of desirable components. This study used in situ microscopic evaluation of onion cells, as they were pulsed with electric fields at different frequencies, to determine whether frequency was an important parameter. We illustrate that membranes were more effectively broken at lower frequencies as compared to higher frequencies. Application of this information will allow for improved design of PEF systems for more energy efficient dehydration or extraction of plant tissues.

Transversely oriented cortical microtubules in elongating cells typically reorient themselves towards longitudinal directions at the end of cell elongation. We have investigated the reorientation mechanism along the outer epidermal wall in maturing leek (Allium porrum L.) leaves using a GFP-MBD microtubule reporter gene and fluorescence microscopy. Incubating leaf segments for 14-18 h with the anti-actin or anti-actomyosin agents, 20 microm cytochalasin D or 20 mM 2,3-butanedione monoxime, inhibited the normal developmental reorientation of microtubules to the longitudinal direction. Observation of living cells revealed a small subpopulation of microtubules with their free ends swinging into oblique or longitudinal directions, before continuing to assemble in the new direction. Electron microscopy confirmed that longitudinal microtubules are partly detached from the plasma membrane. Incubating leaf segments with 0.2% 1 degree-butanol, an activator of phospholipase D, which has been implicated in plasma membrane-microtubule anchoring, promoted the reorientation, presumably by promoting microtubule detachment from the membrane. Stabilizing microtubules with 10 microm taxol also promoted longitudinal orientation, even in the absence of cytoplasmic streaming. These results were consistent with confocal microscopy of live cells before and after drug treatments, which also revealed that the slow (days) global microtubule reorientation is superimposed over short-term (hours) regional cycling in a clockwise and an anti-clockwise direction. We propose that partial detachment of transverse microtubules from the plasma membrane in maturing cells exposes them to hydrodynamic forces of actomyosin-driven cytoplasmic streaming, which bends or shifts pivoting microtubules into longitudinal directions, and thus provides an impetus to push microtubule dynamics in the new direction.

SUMMARY Light microscopic study of the giant-celled, marine green alga Callipsygma wilsonis J. Agardh (Udoteaceae, Bryopsidales) revealed numerous birefringent crystalline inclusions in the terminal segments of the assimilatory axes. The inclusions were thin plates with a triangular shape in face view, a base up to 75 m mi n length, and a height that was one-seventh the length of the base. Crystals of various sizes commonly were stacked face-to-face with one or more edges coinciding, but removal of organic material by treatment in sodium hypochlorite resulted in disaggregation. The crystals were soluble in dilute hydrochloric acid without effervescence but were insoluble in acetic acid. These diagnostic chemical solubility tests and a positive reaction to the Yasue staining reaction indicated that the crystals were composed of calcium oxalate. Scanning electron microscopy showed that most crystals had smoothly curving edges, but some had truncate or beveled margins. Calcium oxalate crystals have been reported to occur in the large central vacuoles of several bryopsidalean species, but the crystals in C. wilsonis were present in the parietal cytoplasm, which was evident from the presence of crystals in streaming cytoplasm. Calcium oxalate crystals, amyloplasts, chloroplasts, and other cytoplasmic constituents moved along cytoskeletal cables at rates of approximately 2.8 m ms -1 . These findings add to a growing body of evidence that calcium oxalate crystals in diverse algae may be present in cellular compartments other than the central vacuole.

Arctic and alpine plants like Oxyria digyna have to face enhanced environmental stress. This study compared leaves from Oxyria digyna collected in the Arctic at Svalbard (78 degrees N) and in the Austrian Alps (47 degrees N) at cellular, subcellular, and ultrastructural levels. Oxyria digyna plants collected in Svalbard had significantly thicker leaves than the samples collected in the Austrian Alps. This difference was generated by increased thickness of the palisade and spongy mesophyll layers in the arctic plants, while epidermal cells had no significant size differences between the two habitats. A characteristic feature of arctic, alpine, and cultivated samples was the occurrence of broad stroma-filled chloroplast protrusions, 2 - 5 microm broad and up to 5 microm long. Chloroplast protrusions were in close spatial contact with other organelles including mitochondria and microbodies. Mitochondria were also present in invaginations of the chloroplasts. A dense network of cortical microtubules found in the mesophyll cells suggested a potential role for microtubules in the formation and function of chloroplast protrusions. No direct interactions between microtubules and chloroplasts, however, were observed and disruption of the microtubule arrays with the anti-microtubule agent oryzalin at 5 - 10 microM did not alter the appearance or dynamics of chloroplast protrusions. These observations suggest that, in contrast to studies on stromule formation in Nicotiana, microtubules are not involved in the formation and morphology of chloroplast protrusions in Oxyria digyna. The actin microfilament-disrupting drug latrunculin B (5 - 10 microM for 2 h) arrested cytoplasmic streaming and altered the cytoplasmic integrity of mesophyll cells. However, at the ultrastructural level, stroma-containing, thylakoid-free areas were still visible, mostly at the concave sides of the chloroplasts. As chloroplast protrusions were frequently found to be mitochondria-associated in Oxyria digyna, a role in metabolite exchange is possible, which may contribute to an adaptation to alpine and arctic conditions.

To understand how auxin regulates root growth, we quantified cell division and elemental elongation, and examined actin organization in the primary root of Arabidopsis thaliana. In treatments for 48 h that inhibited root elongation rate by 50%, we find that auxins and auxin-transport inhibitors can be divided into two classes based on their effects on cell division, elongation and actin organization. Indole acetic acid (IAA), 1-naphthalene acetic acid (NAA) and tri-iodobenzoic acid (TIBA) inhibit root growth primarily through reducing the length of the growth zone rather than the maximal rate of elemental elongation and they do not reduce cell production rate. These three compounds have little effect on the extent of filamentous actin, as imaged in living cells or by chemical fixation and immuno-cytochemistry, but tend to increase actin bundling. In contrast, 2,4-dichlorophenoxy-acetic acid (2,4-D) and naphthylphthalamic acid (NPA) inhibit root growth primarily by reducing cell production rate. These compounds remove actin and slow down cytoplasmic streaming, but do not lead to mislocalization of the auxin-efflux proteins, PIN1 or PIN2. The effects of 2,4-D and NPA were mimicked by the actin inhibitor, latrunculin B. The effects of these compounds on actin were also elicited by a 2 h treatment at higher concentration but were not seen in two mutants, eir1-1 and aux1-7, with deficient auxin transport. Our results show that IAA regulates the size of the root elongation zone whereas 2,4-D affects cell production and actin-dependent processes; and, further, that elemental elongation and localization of PINs are appreciably independent of actin.

Summary • Actin organization and dynamics are essential for cell division, growth and cytoplasmic streaming. Here we analyse the effects of the overexpression of Actin Interacting Protein 1 (AIP1) on Arabidopsis development. • Arabidopsis plants were transformed with an ethanol-inducible AIP1 construct and the characteristics of these plants were analysed after induction. • When AIP1 was increased to approx. 90% above wild-type values, root hair development and actin organization in all cell types examined were disrupted. • Our data demonstrate that AIP1 is a key regulator of actin organization and that its regulation is essential for normal plant cell morphogenesis.

The sedimentation of amyloplasts in inverted statocytes of the flower stalk of dandelion (Taraxacum officinale Weber) was followed using light microscopy. Much variety in sedimentation pattern was evident with complete sedimentation occurring in as little as 3–4 min to as much as a few h and individual amyloplasts showing barely perceptible sedimentation or sedimenting at speeds of up to 40 μm min−1. It was also apparent that amyloplasts were subjected to considerable movement after they had sedimented. Large vacuoles and cytoplasmic streaming in statocytes were confirmed and are considered to greatly influence amy-loplast sedimentation pattern. The implications of these observations for the starch-statolith hypothesis of geotropism are discussed.

We investigated the mechanism underlying the perception of extracellular changes in osmotic pressure in Vallisneria gigantea Graebner and transgenic Arabidopsis thaliana (L.) Heynh. expressing cytoplasmic aequorin. Hypertonic and hypotonic treatments of A. thaliana leaves each rapidly induced a Ca2+ transient. Both responses were essentially dependent on the presence of extracellular Ca2+ and were sensitive to Gd3+ a potential blocker of stretch-activated Ca2+ channels. Immediately after plasmolysis caused by hypertonic treatment and subsequent deplasmolysis caused by hypotonic treatment, the cells did not respond to a second hypertonic treatment and exhibited an impaired adhesion of the plasma membrane (PM) to the cell wall (CW). Recovery of the responsiveness required about 6 h. By contrast, no refractory phenomenon was observed in response to hypotonic treatment. Pretreatment with cellulase completely inhibited the Ca2+ transient induced by hypertonic treatment, but it did not affect the response to hypotonic treatment. V. gigantea mesophyll cells pretreated with cellulase exhibited an impaired adhesion of the PM to the CW. The leaf cells of multicellular plants can respond to both hypertonic and hypotonic treatments through the stretch-activated Ca2+ channels, whereas cellulase-sensitive adhesion of the PM to the CW is involved only in the response to hypertonic treatment.

Growth of secondary plasmodia of the clubroot pathogen Plasmodiophora brassicae was studied in dual culture of P. brassicae and turnip suspension cells. Suspension culture of P. brassicae-infected turnip cells was achieved by using P. brassicae-infected callus in Murashige and Skoog medium supplemented with 0·1 mg 2,4-D L−1 and 0·02 mg kinetin L−1. The shape of secondary plasmodia in suspension cells was spherical-to-subspherical. A few young plasmodia divided and became numerous spherical, small plasmodia which eventually formed a plasmodial cluster. The plasmodia fused and became vegetative plasmodia. Infected cells were significantly larger than noninfected cells. Secondary plasmodia moved within transformed turnip suspension host cells by cytoplasmic streaming of the host cells. Secondary plasmodia divided in synchrony with the transformed turnip cells.

Mycelial fungi play a central role in element cycling in nature by degrading dead organic material such as wood. Fungal colonization of a substrate starts with the invasion of exploring hyphae. These hyphae secrete enzymes that convert the organic material into small molecules that can be taken up by the fungus to serve as nutrients. Using green fluorescent protein (GFP) as a reporter, we show for the first time that exploring hyphae of Aspergillus niger differentiate with respect to enzyme secretion; some strongly express the glucoamylase gene glaA, while others hardly express it at all. When a cytoplasmic GFP was used, 27% of the exploring hyphae of a 5-day-old colony belonged to the low expressing hyphae. By fusing GFP to glucoamylase and by introducing an ER retention signal, this number increased to 50%. This difference is due to cytoplasmic streaming of the reporter in the former case, as was shown by using a photo-activatable GFP. Our findings indicate that a fungal mycelium is highly differentiated, especially when taking into account that hyphae in the exploration zone were exposed to the same nutritional conditions.

Actin polymerization is important in the control of pollen tube growth. Thus, treatment of pollen tubes with low concentrations of latrunculin B (Lat-B), which inhibits actin polymerization, permits streaming but reversibly blocks oscillatory growth. In the current study, we employ Jasplakinolide (Jas), a sponge cyclodepsipeptide that stabilizes actin microfilaments and promotes polymerization. Uniquely, Jas (2 microM) blocks streaming in the shank of the tube, but induces the formation of a toroidal-shaped domain in the swollen apex, of which longitudinal optical sections exhibit circles of motion. The polarity of this rotary motion is identical to that of reverse fountain motility in control pollen tubes, with the forward direction occurring at the edge of the cell and the rearward direction in the cell interior. Support for the idea that actin polymerization in the apical domain contributes to the formation of this rotary motility activity derives from the appearance therein of aggregates and flared cables of F-actin, using immunofluorescence, and by the reduction in G-actin as indicated with fluorescent DNAse. In addition, Jas reduces the tip-focused Ca2+ gradient. However, the alkaline band appears in the swollen apex and is spatially localized with the reverse fountain streaming activity. Taken together, our results support the idea that actin polymerization promotes reversal of streaming in the apex of the lily pollen tube.

Amoeba proteus may drive a current through themselves, and this current may determine the direction of cytoplasmic streaming. Cytoplasm streams from the more contracted tail or uroid region to the extending pseudopods. The positions of the uroid and pseudopods change as the amoeba changes direction. The membrane potential of the amoeba may change with location in the cell. This voltage gradient may regulate the magnitude and direction of cytoplasmic streaming. Bingley and Thompson (1962), recorded from amoebae using intracellular microelectrodes and reported that the membrane potential of the uroid region was more negative than that of the pseudopods. In contrast, Nuccitelli et al. (1977), using a vibrating probe, observed that current entered the uroid region and exited through the pseudopods. They concluded that the uroid region was more depolarized than the pseudopods. We revisited this issue by recording membrane potential simultaneously from two regions of the amoeba and by recording ionic currents using the two-electrode voltage clamp. Our data support the findings of Nuccitelli et al. Cytoplasm may stream from more depolarized to more hyperpolarized regions. Moreover, overshooting depolarizations of the cell may produce global cytoplasmic contractions and rounding of the amoeba.

Life cycle stages of the heterotrophic dinoflagellate Protoperidinium depressum were determined for a culture isolated from the west coast of Ireland. Division, sexuality, DNA content, gamete morphology, and identity of morphologically distinct life-history stages were revealed for the first time using a suite of morphological and molecular tools. Asexual division occurred by eleutheroschisis within a temporary, immotile cyst, yielding two daughter cells. Morphological features, including apical and antapical horns, were not fully formed upon emergence from the division cyst, but developed some time after cell division. Daughter cells were half to two-thirds the size of parent cells and swam attached in an epitheca-to-hypotheca orientation before separating. Stages indicating a completed life cycle, including gametes, planozygotes, and nuclear cyclosis were observed. Small cells, believed to be gametes, formed in repeated clonal and non-clonal re-isolations of the culture. In addition to their small size (40–50 μm), blunt apical horn, small antapical horns, and lack of pink pigmentation, these putative gametes had a thecal plate morphology different from that described for P. depressum. The LSU rDNA sequence (D1–D3 region) of small cells was, however, the same as that of P. depressum. Gametes were never witnessed to feed, but did form fusing, isogamous pairs. When isolated to well plates, gamete pairs failed to complete fusion to form a planozygote. Thus, the morphology of the initial planozygote remains uncertain. Large cells believed to be planozygotes had “typical”P. depressum morphology, but possessed two longitudinal flagella and had 2C DNA based on DAPI. Nuclear cyclosis was documented in one such tri-flagellated, motile cell. The cell remained motile after the completion of nuclear cyclosis, but did not progress to the next life stage. Dormant hypnozygotes were not observed.

The role and regulation of specific plant myosins in cyclosis is not well understood. In the present report, an affinity-purified antibody generated against a conserved tail region of some class XI plant myosin isoforms was used for biochemical and immunofluorescence studies of Zea mays. Myosin XI co-localized with plastids and mitochondria but not with nuclei, the Golgi apparatus, endoplasmic reticulum, or peroxisomes. This suggests that myosin XI is involved in the motility of specific organelles. Myosin XI was more than 50% co-localized with tailless complex polypeptide-1α (TCP-1α) in tissue sections of mature tissues located more than 1.0 mm from the apex, and the two proteins co-eluted from gel filtration and ion exchange columns. On Western blots, TCP-1α isoforms showed a developmental shift from the youngest 5.0 mm of the root to more mature regions that were more than 10.0 mm from the apex. This developmental shift coincided with a higher percentage of myosin XI /TCP-1α co-localization, and faster degradation of myosin XI by serine protease. Our results suggest that class XI plant myosin requires TCP-1α for regulating folding or providing protection against denaturation. Cell Motil. Cytoskeleton 57:218–232, 2004. © 2004 Wiley-Liss, Inc.

Radiocaesium enters the food chain when plants absorb it from soil, in a process that is strongly dependent on soil properties and plant and microbial species. Among the microbial species, arbuscular mycorrhizal (AM) fungi are obligate symbionts that colonize the root cortex of many plants and develop an extraradical mycelial (ERM) network that ramifies in the soil. Despite the well-known involvement of this ERM network in mineral nutrition and uptake of some heavy metals, only limited data are available on its role in radiocaesium transport in plants. We used root-organ culture to demonstrate that the ERM of the AM fungus Glomus lamellosum can take up, possibly accumulate and unambiguously translocate radiocaesium from a 137Cs-labelled synthetic root-free compartment to a root compartment and within the roots. The accumulation of 137Cs by hyphae in the root-free compartment may be explained by sequestration in the hyphae or by a bottleneck effect resulting from a limited number of hyphae crossing the partition between the two compartments. Uptake and translocation resulted from the incorporation of 137Cs into the fungal hyphae, as no 137Cs was detected in mycorrhizal roots treated with formaldehyde. The importance of the translocation process was indicated by the correlation between 137Cs measured in the roots and the total hyphal length connecting the roots with the labelled compartment. 137Cs may be translocated via a tubular vacuolar system or by cytoplasmic streaming per se.

High velocity cytoplasmic streaming is found in various plant cells from algae to angiosperms. We characterized mechanical and enzymatic properties of a higher plant myosin purified from tobacco bright yellow-2 cells, responsible for cytoplasmic streaming, having a 175 kDa heavy chain and calmodulin light chains. Sequence analysis shows it to be a class XI myosin and a dimer with six IQ motifs in the light chain-binding domains of each heavy chain. Electron microscopy confirmed these predictions. We measured its ATPase characteristics, in vitro motility and, using optical trap nanometry, forces and movement developed by individual myosin XI molecules. Single myosin XI molecules move processively along actin with 35 nm steps at 7 micro m/s, the fastest known processive motion. Processivity was confirmed by actin landing rate assays. Mean maximal force was approximately 0.5 pN, smaller than for myosin IIs. Dwell time analysis of beads carrying single myosin XI molecules fitted the ATPase kinetics, with ADP release being rate limiting. These results indicate that myosin XI is highly specialized for generation of fast processive movement with concomitantly low forces.

The distributing of materials throughout a symplastic domain must involve at least two classes of transport steps: plasmodesmatal and cytoplasmic. To underpin the latter, the most obvious candidate mechanisms are cytoplasmic streaming and diffusion. The thesis will be here advanced that, although both candidates clearly do transport cytoplasmic entities, the cytoplasmic streaming per se is not of primary importance in symplastic transport but that its underlying molecular motor activity (of which the streaming is a readily visible consequence) is. Following brief tutorials on low Reynolds number flow, diffusion, and targeted intracytoplasmic transport, the hypothesis is broached that macromolecular and vesicular transport along actin trackways is both the cause of visible streaming and the essential metabolically driven cytoplasmic step in symplastic transport. The concluding discussion highlights four underdeveloped aspects of the active cytoplasmic step: (i) visualization of the real-time transport of messages and metabolites; (ii) enumeration of the entities trafficked; (iii) elucidation of the routing of the messages and metabolites within the cytoplasm; and (iv) transference of the trafficked entities from cytoplasm into plasmodesmata.

Myosin was detected on Western blots of Micrasterias denticulata extracts by use of antibodies from different sources. Inhibitors with different targets of the actomyosin system, such as the myosin ATPase-blockers N-ethylmaleimide (NEM) and 2,3-butanedione monoxime (BDM), or the myosin light chain kinase inhibitor 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexhydro-1,4-diazapine (ML7), had similar effects on intracellular motility during cell development in the green alga Micrasterias, thus pointing towards a participation of myosin in these processes. The drugs markedly altered the mode of postmitotic nuclear migration, slowed down cytoplasmic streaming, changed cell pattern development and prevented normal chloroplast distribution and spreading into the growing semicell. In addition, an increase and dilatations in ER cisternae and marked morphological changes of the Golgi system were observed by transmission electron microscopy after exposure of growing cells to BDM. Neither BDM nor ML7 exhibited any effect on the distribution or arrangement of the cortical F-actin network nor on the F-actin basket around the nucleus, characteristic of untreated growing Micrasterias cells (J Cell Sci 107 (1994) 1929). This is particularly interesting since BDM caused disintegration of the microtubule system co-localized to the F-actin cage during normal nuclear migration. Together with the fact that other microtubules not connected to the F-actin system remained uninfluenced by BDM, this observation is evidence of an integrative function of myosin between the cytoskeleton elements.

In Characean cells endoplasmic streaming stops upon membrane depolarization accompanied by Ca2+ entry. We investigated the mechanism of this cessation of endoplasmic streaming by reconstituting the vesicle movement in vitro. In a living cell of Chara corallina, there are a number of vesicles moving along actin cables. Vesicles in the endoplasm squeezed out of the cell into a medium containing Mg-ATP showed directional movements under a dark field microscope. When the extracted endoplasm was treated with 20 nM okadaic acid, vesicles showed only movements like the Brownian motion. When it was treated with 50 nM staurosporine, directional movements of vesicles were activated. These movements were analyzed by image processing of videomicroscopic records. Vesicle movements along F-actin filaments were also observed by merging both images of the same field by dark field microscopy and fluorescence microscopy, indicating that myosin on the vesicle surface was responsible for vesicle movements. We also examined the effects of okadaic acid and staurosporine on in vitro sliding of F-actin on Chara myosin. When Chara myosin was treated with 20 nM okadaic acid in the cell extract, the number of sliding F-actin filaments was greatly reduced. In contrast, it increased when Chara myosin was treated with 50 nM staurosporine. In addition, Chara myosin treated with protein kinase C greatly diminished its motility. These results suggest that inactivation of Chara myosin via its phosphorylation is responsible for cessation of endoplasmic streaming. Cell Motil. Cytoskeleton 53:66–76, 2002. © 2002 Wiley-Liss, Inc.

Summary In this report, we describe a highly reproducible femtosecond near-infrared (NIR) laser-based nanoprocessing technique that can be used both for non-invasive intra-tissue nanodissection of plant cell walls as well as selective destruction of a single plastid or part thereof without compromising the viability of the cells. The ultra-precise intra-tissue nanoprocessing is achieved by the generation of high light intensity (1012 W cm−2) by diffraction-limited focusing of the radiation of an NIR (λ = 740 and 800 nm) femtosecond titanium-sapphire laser to a sub-femtolitre volume and subsequent highly localized instantaneous plasma formation. Following nanosurgery, electron microscopical analysis of the corresponding cellular target areas revealed clean non-staggering lesions across the cell wall with a cut width measuring less than 400 nm. To our knowledge, this is the smallest cut made non-invasively within a plant tissue. Further evidence, including two-photon imaging of chlorophyll fluorescence, revealed that a single target chloroplast or part thereof can be completely knocked out using intense ultra-fast NIR pulses without any visible deleterious effect on the adjacent plastids. The vitality of the cells after nanoprocessing has been ascertained by exclusion of propidium iodide from the cells as well as by the presence of cytoplasmic streaming. The potential applications of this technical advance include developmental biology applications, particularly studies addressing spatio-temporal control of ontogenetic events and cell–cell interactions, and gravitational biology applications.

A new method is described for monitoring hyphal P-32 transport in compartmented, monoxenic mycorrhizal root cultures. Nondestructive time-course measurements of P transport in hyphae were obtained by capturing digital autoradiograms on P-imaging screens, and comparing with growth observed by optical scanning. P-32 distribution measured by densitometry on the day of harvest closely agreed with values obtained by liquid scintillation counting after destructive harvest. Virtually all labeled PO4 was absorbed by arbuscular mycorrhizal (AM) hyphae, but transfer to the roots appeared to be incomplete. P transport was not unidirectional towards the roots, as P-32 was also transported from the root compartment to the hyphal compartment. Net P flux rates were calculated for hyphae crossing between compartments, taking bidirectional flow into account. Amounts of transported P were poorly correlated with extra-radical hyphal length and root d. wt, but highly correlated with the number of hyphae crossing the barrier separating the two compartments. Such correlations were highest when only hyphae with detectable protoplasmic streaming were considered. The method was tested using radiolabeled P sources, H2PO4- and cytidine triphosphate (CTP), and the AM fungi, Glomus intraradices and G. proliferum. Fungal transport of P-32 from CTP was much slower than from PO4 for both fungi. (C) New Phytologist (2002) 154: 809-819.

Chara longifolia is a salt-tolerant Charophyte which regulates its turgor inresponse to osmotic stress. Membrane depolarization, in creased membrane conductance, and cessation of cytoplasmic streaming (due to increase in cytoplasmic Ca2+) precede regulation in response to hypotonic stress. Measurements of these three parameters are presented here with simultaneous turgor measurements. Variability in the occurrence, rate and extent of turgor regulation in individual cells was correlated with magnitude of the stress. Hypertonic stress showed the same slow time course as was found previously, requiring several days for complete regulation. Fifty μM nifedipine, a Ca2+ channel blocker, inhibited turgor regulation. In the presence of 5 μM nifedipine, turgor regulation was delayed. An increase in conductance preceded regulation, but membrane depolarization was less and no detectable change in cytoplasmic streaming was observed, requiring modifications to a previously presented model for turgor regulation. There was no significant difference in 45Ca2+ influx under control and stress conditions. However, the control flux was insensitive to nifedipine, whereas under stress the flux is inhibited 54% by nifedipine. We suggest that osmotic stress results in a rapid increase in a nifedipine-sensitive Ca2+ entry mechanism, followed very quickly by a decrease in the control entry mechanism.

The role of the cytoskeleton in the regulation of chloroplast motility and positioning has been investigated by studying: (1) structural relationship of actin microfilaments, microtubules, and chloroplasts in cryofixed and freeze-substituted leaf cells of Arabidopsis; and (2) the effects of anti-actin (Latrunculin B; LAT-B) and anti-microtubule (Oryzalin) drugs on intracellular distribution of chloroplasts. Immunolabeling of leaf cells with two plant-actin specific antibodies, which react equivalently with all the expressed Arabidopsis actins, revealed two arrangements of actin microfilaments: longitudinal arrays of thick actin bundles and randomly oriented thin actin filaments that extended from the bundles. Chloroplasts were either aligned along the actin bundles or closely associated with the fine filaments. Baskets of actin microfilaments were also observed around the chloroplasts. The leaf cells labeled with an anti-tubulin antibody showed dense transverse arrays of cortical microtubules that exhibited no apparent association with chloroplasts. The application of LAT-B severely disrupted actin filaments and their association with chloroplasts. In addition, LAT-B induced aberrant aggregation of chloroplasts in the mesophyll and bundle sheath cells. Double labeling of LAT-B treated cells with anti-actin and anti-tubulin antibodies revealed that the microtubules in these cells were unaffected. Moreover, depolymerization of microtubules with Oryzalin did not affect the distribution of chloroplasts. These results provide evidence for the involvement of actin, but not tubulin, in the movement and positioning of chloroplasts in leaf cells. We propose that using motor molecules, some chloroplasts migrate along the actin cables directly, while others are pulled along the cables by the fine actin filaments. The baskets of microfilaments may anchor the chloroplasts during streaming and allow control over proper three-dimensional orientation to light.

Cells of the salt-tolerant charophyte Lamprothamnium respond differently to hypotonic challenge according to their position on the plant (i.e. cell age). Differences in electrophysiological response are coupled with differences in cell fine structure, and the presence or absence of extracellular mucilage. (1) Young, apical (fast-regulating, FR) cells respond with sudden cessation of cyclosis, depolarization to –50 mV (in some cells by more than 100 mV) and increase in membrane conductance by up to an order of magnitude. Intracellular [K+]v, [Na+]v and [Cl–]v decrease 1 h after hypotonic challenge. Patch-clamping cytoplasmic droplets reveals two types of K+ channel, 150 pS and 35 pS, and a small conductance Cl– channel, 35 pS (conductances at estimated tonoplast resting potential between zero and 20 mV). Extracellular mucilage is thin (< 5 μm thick) or lacking, similar to freshwater Chara. Unlike freshwater charophytes these cells have a canalicular vacuolar system of large surface area and compartment the fluorochrome 6 carboxyfluorescein in the cytoplasm rather than the vacuolar system. (2) Older basal (slow-regulating, SR) cells do not cease streaming on hypotonic challenge and depolarize only slightly (by approximately 20 mV) with small or no change in membrane conductance. After 1 h the intracellular [K+]v, [Na+]v and [Cl–]v scarcely change. Patch-clamping cytoplasmic droplets reveals two types of K+ channel, medium conductance 90 pS and low conductance (as in FR cells). The large conductance K+ channel was not observed. The Cl– channel was more active in SR cells. The cells were coated with extracellular mucilage more than 10 μm thick. In a similar manner to freshwater Chara, these cells compartment 6 carboxyfluorescein in a large central vacuole. In the older cells, making up the bulk of any given plant, the simultaneous development of extracellular mucilage and a large central vacuole which compartments 6 carboxyfluorescein is associated with a minimal electrophysiological response to hypotonic challenge. The significance of these findings for salt-tolerance is discussed.

Paramecia demonstrate rotational cytoplasmic streaming, in which some cytoplasmic granules and organelles, including symbiotic algae, flow in a constant direction. To elucidate the mechanism of this streaming, we examined the effects of cytochalasins (cytochalasin B and D, and dihydrocytochalasin B) and nocodazole, which are reagents affecting microfilament and microtubule networks, respectively, in the cell. In previous reports, paramecia have been compressed with a coverslip to facilitate observation of cytoplasmic streaming. Here we found that the cytoplasmic streaming of paramecia was suppressed by such compression and then observed the process without compression in this work. In the presence of cytochalasins, cytoplasmic streaming was not affected. In contrast, treatment with nocodazole (10 μg/ml) resulted in discontinuation of cytoplasmic streaming in paramecia. Immunofluorescent microscopic observations by confocal microscopy revealed that the number of intracellular microtubules in nocodazole-treated cells was markedly decreased compared to that of controls. Electron microscopic observations confirmed the decrease. These results suggest that cytoplasmic microtubules play an important role in the cytoplasmic streaming of paramecia. Cell Motil. Cytoskeleton 43:85–98, 1999. © 1999 Wiley-Liss, Inc.

An interactive computer-assisted video microscopy method has been developed for the acquisition of extensive data on the sequential positions of pollen tube organelles, which cannot be automatically tracked using geometric or motion patterns. The method consists of video microscopy, analog and digital contrast enhancement, digital time-lapsing of the images, and interactive selection of positions in a coordinate system corresponding to the cell shape and real size. Data on 15,000 positions acquired with this method have been used to make quantitative analyses of the movement patterns of the organelles. From these analyses and the reconstruction of 900 trajectories, it appears that movements are random in the tip of the pollen tube but become more directed in distal regions of the cell, indicating an increase in axial arrangement of the actin filaments. (All custom-made software is available from the authors on request.) Cytometry 32:263–267, 1998. © 1998 Wiley-Liss, Inc.

Through the injection of f-aequorin (a calcium-specific luminescent reporter), and the use of an imaging photon detector, transient localized elevations of free cytosolic calcium in the forming blastodisc (BD) and animal hemisphere cortex were visualized that correlated with ooplasmic segregation. The introduction of an appropriate concentration of the weak (KD = 1.5 micromol/L) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits cytoplasmic streaming and the subsequent formation of a BD at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: > or = 1.3 mmol/L blocks ooplasmic streaming; < 1.3 mmol/L eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (KD = 0.15 micromol/L) to a final concentration of 1.5 mmol/L as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish. Furthermore, a hypothetical model is presented linking these calcium transients to the contraction of a cortically located actin microfilament network as a possible mechanism providing the driving force for segregation.

Summary Maize actin-depolymerizing factor (ADF) binds both monomeric and filamentous actin and increases actin dynamics in vitro. To test its effects in vivo, recombinant pollen ADF1 was expressed in bacteria and microinjected into Tradescantia stamen hair cells. Initially, all cytoplasmic streaming ceased and the central, longitudinal transvacuolar strands were disrupted. After 20–45 min, streaming resumed but in the form of conspicuous transverse pathways of movement in the cortex. Staining the actin filaments by a second injection of fluorescein-conjugated phalloidin showed that the longitudinal actin cables seen in controls had been replaced by a thickening of the transverse cortical arrays, whose orientation matched the new pattern of streaming. Microinjection of rhodamine–tubulin confirmed that the microtubules also formed a transverse cortical array and it is suggested that the spatial cues for re-modelling the actin after ADF1 injection may be provided by the microtubular system.

It is generally thought that sedimenting plastids are responsible for gravity sensing in higher plants. We directly tested the model generated by the current statolith hypothesis that the gravity sensing that leads to gravitropism results from an interaction between the plastids and actin microfilaments. We find that the primary roots of rice, corn, and cress undergo normal gravitropism and growth even when exposed to cytochalasin D, a disruptor of actin microfilaments. These results indicate that an interaction between amyloplasts and the actin cytoskeleton is not critical for gravity sensing in higher plants and weaken the current statolith hypothesis.

Gravity induces a polarity of cytoplasmic streaming in vertical internodal cells of Chara such that the downwardly directed stream moves faster than the upwardly directed stream. In order to determine whether the statolith theory (in which intracellular sedimenting particles are responsible for gravity sensing) or the gravitational pressure theory (in which the entire protoplast acts as the gravity sensor) best explain the gravity response in Chara internodal cells, we controlled the physical properties of the external medium, including density and osmolarity, with impermeant solutes and examined the effect on the polarity of cytoplasmic streaming. As the density of the external medium is increased, the polarity of cytoplasmic streaming decreases and finally disappears when the density of the external medium is equal to that of the cell (1015 kg/m3). A further increase in the density of the external medium causes a reversal of the gravity response. These results are consistent with the gravitational pressure theory of gravity sensing since the buoyancy of the protoplast is dependent on the difference between the density of the protoplast and the external medium, and are inconsistent with the statolith theory since the buoyancy of intracellular particles are unaffected by changes in the external medium.

SUMMARY Living epidermal cells of cowpea (Vigna unguiculata (L.) Walp.) were examined by contrast-enhanced video microscopy during penetration by invasion hyphae of the monokaryotic stage of the cowpea rust fungus (Uromyces vignae Barclay race 1). In resistant or susceptible host cvs, the plant nucleus migrated to the penetration site before the fungus had fully penetrated the plant wall and, at sites of unsuccessful infection, remained during the formation of a callose-containing papilla. Nuclear migration was also induced by applying hemicellulase, but not H2O2, to localized sites of wall damage. Hemicellulase-induced migration was inhibited by calcium chelators and a protein kinase inhibitor, but not catalase. In both resistant and susceptible cvs, the plant nucleus migrated away from successful infection sites at about the time that the fungal penetration peg made contact with the plant plasma membrane, and the epidermal cell showed no further cytological responses to the growth of the fungal intra-epidermal vesicle. In the susceptible cv., the nucleus migrated back to the fungus when the latter initiated tip growth. Inhibitors of transcription or translation did not affect this migration and only slightly reduced fungal growth. In the resistant cv. in which the invaded cell exhibited a hypersensitive response (HR), the plant nucleus changed its appearance before the cessation of cytoplasmic streaming and usually did not migrate to the fungus, even if the latter initiated tip growth. Nuclear DNA cleavage usually followed the subsequent cessation of cytoplasmic streaming. Treatments that delayed cell death and increased fungal growth also increased the frequency of nuclear migration to the fungus. It is argued that these and other data suggest that U. vignae negates nonspecific, penetration-induced, defence responses upon entering cells of both susceptible and resistant cultivars. The results also suggest that effects on the plant nucleus are one of the earliest signs of the HR in this system, often preceding the cessation of cytoplasmic streaming and detectable changes in plasma membrane permeability.

Specific inhibitors of the secretory pathway represent important tools for investigation of cell wall synthesis and tip growth in pollen tubes. Brefeldin A completely inhibits germination of Nicotiana tabacum pollen tubes at 2.2 μM. Ultrastructural investigation of pollen tube cytoplasm showed that brefeldin A caused the appearance of reticular structures and “brefeldin A compartments” containing unesterified pectins. Monensin caused inhibition of pollen tube germination at 80 nM. The drug induced swelling of the Golgi cisternae, many of which contained methyl-esterified pectins. Cytochalasin D was effective at 1 μg/ml, the inhibition of germination being fully reversible. Application of the drug caused accumulation of secretory vesicles containing methyl-esterified pectin around the dictyosomes. In contrast to brefeldin A and monensin, cytochalasin D caused a slowdown of cytoplasmic streaming. Monensin, but not the other drugs, caused a considerable decrease in pollen tube diameter. The characterization and quantification of the effects of the drugs on pollen tubes represents a necessary prerequisite for their application in physiological studies.

The inhibitory effects of benomyl on pollen tube growth have received little attention, particularly at the microscopic and immunohistochemical levels. Pollen germination and tube growth in the presence of benomyl were evaluated in Tradescantia virginiana to investigate the effects of this fungicide on pollen germination rate, tube growth and morphology, and microtubule (Mt) organization. Benomyl was incorporated in germination media at 0, 480, 600 or 720 mg l -1 . Inhibition of pollen germination, cytoplasmic streaming, and tube elongation were associated with benomyl treatments. Benomyl also induced abnormal pollen tube morphology and Mt organization. Compared to controls, Mts in the treated tubes were characteristically fewer in number, fragmented, sinuous and increasingly disorganized. At the two highest benomyl concentrations, Mts were considerably fewer or absent in apical/subapical regions of the pollen tubes. This work verifies that benomyl incorporated into germination media at concentrations lower than recommended field rates inhibit pollen germination and tube growth, and that the effects are associated with alterations of Mt organization.

Characean internodal cells readily form wound walls upon local membrane damage. In the present study we documented the dynamics of vesicles involved in wound wall secretion and compared them with actin organization in equivalent cells using immunofluorescence. Single exocytotic events (spreading of vesicle contents) could be visualized using image enhancement by video microscopy. In control unwounded cells vesicles moved unidirectionally along parallel actin bundles and rarely contacted the plasma membrane. The wound response started with (1) local inhibition of active cytoplasmic streaming (unidirectional movements) due to inactivation, depolymerization, or mechanical displacement of the subcortical actin bundles. Accordingly, vesicles performed only oscillating motions and moved slowly with the same velocity and direction as passive endoplasmic flow. (2) Several minutes after wounding, vesicles started to perform random saltatory movements with frequently changing velocities, punctuated by oscillating motion and periods of immobility (docking) at the plasma membrane. Vesicle trajectories correlated with a fine-meshed actin network at the wound site. (3) Several hours after wounding, vesicles moved again unidirectionally along regenerated subcortical actin bundles. Spreading of vesicles (vesicle contents) was observed during wound wall formation, i.e., during the period of saltatory movements when vesicles had access to the plasma membrane. Dependent on the type of wound wall being secreted, three variants could be distinguished: (1) slow and continuous spreading over a time period of several seconds up to 30 min near the plasma membrane, (2) fast spreading within 80 ms inside an already formed wound wall, and/or (3) fast spreading at the plasma membrane. We conclude from our study that wounding-induced changes in vesicle dynamics are due to transient reorganization of the actin cytoskeleton from parallel bundles to a fine-meshed network. Furthermore, our results indicate that spreading of vesicle contents varies considerably with time and may be delayed by vesicle docking and/or discharge.

The salt-tolerant alga Chara longifolia (Robinson) is capable of regulating its turgor in response to hypotonic stress resulting from a decrease in the osmotic pressure of the medium. This regulatory process takes only 40 min in small cells (length ≤ 10 mm), but requires 3 d in large cells (length ≥ 30 mm). Turgor regulation in small cells is comprised of two phases, a fast phase reducing the increased turgor by about 25 % in the first 5 min, and a second phase reducing the turgor to near the original value within 40 min. The second phase is inhibited by reducing the concentration of Ca 2+ in the external medium from 4.6 to 0.01 mol m −3 ; the first phase is less affected by the reduction of Ca 2+ . In the first 5 min of stress, the membrane depolarizes in a voltage-dependent fashion, electrical conductance of the membrane increases transiently and cytoplasmic streaming is inhibited. When the external Ca 2+ concentration is lowered, conductance does not increase and streaming continues unaffected. In a low ionic strength medium, Ca 2+ is not required in the medium for turgor regulation. To test the hypothesis that there is increased Ca 2+ entry from the medium during turgor regulation, we measured the influx of 45 Ca 2+ into the cell. We found an increased influx of Ca 2+ , from 18 to 36 nmol m −2 s −1 during the first 30 to 90 s following osmotic stress. This increase was evident only in cells below about 7 mm in length, and was more marked in smaller cells

A striking characteristic of plant cells is that their organelles can move rapidly through the cell. This movement, commonly referred to as cytoplasmic streaming, has been observed for over 200 years, but we are only now beginning to decipher the mechanisms responsible for it. The identification of the myosin motor proteins responsible for these movements allows us to probe the regulatory events that coordinate organelle displacement with normal cell physiology. This review will highlight several recent developments that have provided new insight into the regulation of organelle movement, both at the cellular level and at the molecular level. [

The locomotion of human polymorphonuclear leukocytes (PMNs) was studied with two complementary methods: Three-dimensional shapes were reconstructed from time series of optical sectioning microscopy using differential interference contrast (DIC) optics, and the diffusion of cytoplasm granules within individual cells was measured using quasielastic laser light scattering (QELS). The three-dimensional cell edges outlined in the optical sections were analyzed qualitatively in time-lapse film strips and quantitatively from morphometry. The fastest locomotion occurred in chemotactic gradient with cell velocity that oscillated between 10 and 30 microns/min with a period of 50-55 seconds. Within the periodic bursts of speed, a fibroblast-like locomotory cycle was observed, with leading lamella extended and contacts formed with the substrate surface, followed by rapid motion of the cell body and nucleus over the immobile contacts. Consistent with this apparent staged motion, correlation analysis revealed a phase lag of 2-3 seconds in velocities between the bottom (ventral) and the top layers of the cell. In addition there was a tendency to a lower cell profile at times of higher velocity. The diffusion of natural cytoplasmic granules within resting PMNs was not affected by cytoskeleton disrupting drugs. During the stage of most rapid motion, when cytoplasmic streaming could be seen, diffusion of the granules decreased two- to 2.5-fold, and then returned to resting levels. These observations suggest that PMN locomotion consists of extensions near the surface to form forward contacts and then stiffening or possibly contraction of the cytoskeleton when the body of the cell is moved forward. Three-dimensional movies of PMN cells are included in the video supplement.

Copyright © 2011 The Author(s). Organelle motility is an essential cellular function that is regulated by molecular motors, and their adaptors and activators. Here we established a new method that allows more direct investigation of the function of these peripheral membrane proteins in organelle motility than is possible by analysis of the organelle movement alone. This method uses multi-channel time-lapse microscopy to record the movement of organelles and associated fluorescent proteins, and automatic organelle tracking, to compare organelle movement parameters with the association of membrane proteins. This approach allowed large-scale, unbiased analysis of the contribution of organelle-associated proteins and cytoskeleton tracks in motility. Using this strategy, we addressed the role of membrane recruitment of Rab GTPases and effectors in organelle dynamics, using the melanosome as a model. We found that Rab27a and Rab32/38 were mainly recruited to sub-populations of slow-moving/static and fast-moving melanosomes, respectively. The correlation of Rab27a recruitment with slow movement/docking was dependent on the effector melanophilin. Meanwhile, using cytoskeleton-disrupting drugs, we observed that this speed:Rab content relationship corresponded to a decreased frequency of microtubule (MT)-based transport and an increased frequency of actin-dependent slow movement/docking. Overall, our data indicate the ability of Rab27a and effector recruitment to switch melanosomes from MT- to actin-based tethering and suggest that a network of Rab signalling may integrate melanosome biogenesis and transport.