Statolith positioning by microfilaments in Chara rhizoids and protonemata.

The rhizoids of the green alga Chara are tip-growing cells with a precise positive gravitropism. In rhizoids growing downwards the statoliths never sediment upon the cell wall at the very tip but keep a minimal distance of approximately 10 micrometers from the cell vertex. It has been argued that this position is attained by a force acting upon the statoliths in the basal direction and that this force is generated by an interaction between actin microfilaments and myosin on the statolith membrane. This hypothesis received experimental support from (1) effects of the actin-attacking drug cytochalasin, (2) experiments under microgravity conditions, and (3) clinostat experiments. Using video-microscopy it is now shown that this basipetal force also acts on statoliths during sedimentation. As a result, many statoliths in Chara rhizoids do not simply fall along the plumb line while sedimenting during gravistimulation, but move basipetally. This statolith movement is compared to the ones occurring in the unicellular Chara protonemata during gravistimulation. Dark-grown protonemata morphologically closely resemble the rhizoids but respond negatively gravitropic. In contrast to the rhizoids a gravistimulation of the protonemata induces a transport of statoliths towards the tip. This transport is mainly along the cell axis and not parallel to the gravity vector. It is stressed that the sedimentation of statoliths in Chara rhizoids and protonemata as well as in gravity sensing cells in mosses and higher plants is accompanied by statolith movements based on interactions with the cytoskeleton. In tip-growing cells these movements direct the statoliths to a definite region of the cell where they can sediment and elicit a gravitropic curvature. In the statocytes of higher plants the interactions of the statoliths with the cytoskeleton probably do not serve primarily to move the statoliths but to transduce mechanical stresses from the sedimenting statoliths to the plasma membrane.     

Gravisensing in single-celled systems: characean rhizoids and protonemata.

Gravitropically tip-growing cell types are attractive unicellular model systems for investigating the mechanisms and the regulation of gravitropism. Especially useful for studying the mechanisms of positive and negative gravitropic tip-growth are characean rhizoids and protonemata. They originate from the same cell type, show the same overall cell shape, cytoplasmic zonation, arrangement of actin and microtubule cytoskeleton, use statoliths for gravisensing, but show opposite gravitropism. In both cell types, actin microfilaments are complexly organized in the apical dome,where a dense spherical actin array is colocalized with spectrin-like epitopes and a unique endoplasmic reticulum aggregate, the structural center of the Spitzenk?rper. The opposite gravitropic responses seem to be based on differences in the actin-organized anchorage of the Spitzenk?rper and the actin-mediated transport of statoliths. In negatively gravitropic (upward bending) protonemata, the statoliths-induced drastic upward shift of the cell tip is preceded by a relocalization of dihydropyridine-binding calcium channels and of the apical calcium gradient to the upper flank (bending by bulging). Such relocalizations have not been observed in positively gravitropically responding (downward growing) rhizoids in which statoliths sedimentation is followed by differential flank growth (bending by bowing). This paper reviews the current knowledge and hypotheses on the mechanisms of the opposite gravitropic responses in characean rhizoids and protonemata.     

Intracellular magnetophoresis of statoliths in Chara rhizoids and analysis of cytoplasm viscoelasticity.

The statoliths in Chara rhizoids are denser and more diamagnetic than the cytoplasm, therefore they can be displaced inside a living cell by a sufficiently strong high gradient magnetic field (HGMF). An experimental setup for intracellular magnetophoresis of statoliths was developed. The movement of statoliths and rhizoid growth was measured by video microscopy either under the influence of gravity or a HGMF equivalent to about 2 g. The contribution of the cytoskeleton to statolith motility was assayed before and after depolymerizing microtubules with oryzalin and F-actin with latrunculin B. Application of latrunculin caused immediate cessation of growth, clumping of statoliths, and application of HGMF resulted in higher displacement of statoliths. Oryzalin had no effect on the behavior of statoliths. The data indicate that magnetophoresis is a useful tool to study the gravisensing system and rheology of the Chara rhizoid.     

Force sensitivity of plant gravisensing.

Rotation at 4, 10, 50 and 100 rpm on a horizontal clinostat and in microgravity exerts limited effects on the morphogenesis of lettuce and cress root statocytes and statoliths if compared with the vertical control or 1 g spaceflight reference centrifuge. However, the average distance of statoliths from the distal wall increases. The pattern of plastid location of microgravity-grown and that of clino-rotated samples has been determined at 10, 50, and 100 rpm. Experiments on the centrifuge-clinostat and spaceflight centrifuge (acceleration forces of 0.005 to 1 g) revealed that the average statolith location depends on the amplitude of acropetally or basipetally directed mass acceleration. Decreasing the acropetally directed force from 1 g to 0.4 g dislocates statoliths towards the cell center possibly mediated by the elastic forces of the cytoskeleton. In statocytes formed on the clinostat or in microgravity, the majority of statoliths are located at the center of the cell. To force the statoliths from the center of the statocyte towards one of its poles, a threshold mass acceleration of 0.01 g is required. Statocytes with centrally-located statoliths are considerably more effective in transducing a gravistimulus than those with distally-located plastids. The latent time of the graviresponse is shorter and the response itself is enhanced in roots grown on the clinostat compared to vertically grown samples. The early phases of graviperception are independent of root growth conditions since presentation time and g-threshold are similar for roots grown stationary and those on a clinostat. We propose a sequence of events in gravitropic stimulation that considers not only the lateral displacement of statoliths, as predicted by the starch-statolith hypothesis, but also its longitudinal motion, together with differential gravisensitivity of mechanotransducing structures along the lower-most longitudinal cell wall.     

Gravisensitivity of cress roots.

The minimum dose (stimulus x time [gs]) eliciting a visible gravitropic response, has been determined using continuous and intermittent stimulation and two different accelerations at 1 g and 0.l g. The minimum dose of 20-30 gs estimated for microgravity roots and of 50-60 gs for roots grown on a 1 g-centrifuge indicated a higher sensitivity of microgravity roots. Applying intermittent stimuli to microgravity-grown roots, gravitropic responses were observed after two stimuli of 13.5 gs separated by a stimulus free interval of 118 s. The curvature of microgravity-grown roots to lateral stimulation by 0.1 g was remarkably smaller than by 1g in spite of the same doses which were applied to the seedlings. Microscopic investigations corresponding to stimulations in the range of the threshold values, demonstrated small displacement (< 2 micrometers) of statoliths in root statocytes. Accepting the statolith theory, one can conclude that stimulus transformation has to occur in the cytoplasm in close vicinity to the statoliths and that this transformation system was affected during seedling cultivation in microgravity.     

Microinjection--a tool to study gravitropism.

Despite extensive studies on plant gravitropism this phenomenon is still poorly understood. The separation of gravity sensing, signal transduction and response is a common concept but especially the mechanism of gravisensing remains unclear. This paper focuses on microinjection as powerful tool to investigate gravisensing in plants. We describe the microinjection of magnetic beads in rhizoids of the green alga Chara and related subsequent manipulation of the gravisensing system. After injection, an external magnet can control the movement of the magnetic beads. We demonstrate successful injection of magnetic beads into rhizoids and describe a multitude of experiments that can be carried out to investigate gravitropism in Chara rhizoids. In addition to examining mechanical properties, bead microinjection is also useful for probing the function of the cytoskeleton by coating beads with drugs that interfere with the cytoskeleton. The injection of fluorescently labeled beads or probes may reveal the involvement of the cytoskeleton during gravistimulation and response in living cells.     

Function of the cytoskeleton in gravisensing during spaceflight.

Since astronauts and cosmonauts have significant bone loss in microgravity we hypothesized that there would be physiological changes in cellular bone growth and cytoskeleton in the absence of gravity. Investigators from around the world have studied a multitude of bone cells in microgravity including Ros 17/2.8, Mc3T3-E1, MG-63, hFOB and primary chicken calvaria. Changes in cytoskeleton and extracellular matrix (ECM) have been noted in many of these studies. Investigators have noted changes in shape of cells exposed to as little as 20 seconds of microgravity in parabolic flight. Our laboratory reported that quiescent osteoblasts activated by sera under microgravity conditions had a significant 60% reduction in growth (p<0.001) but a paradoxical 2-fold increase in release of the osteoblast autocrine factor PGE2 when compared to ground controls. In addition, a collapse of the osteoblast actin cytoskeleton and loss of focal adhesions has been noted after 4 days in microgravity. Later studies in Biorack on STS-76, 81 and 84 confirmed the increased release of PGE2 and collapse of the actin cytoskeleton in cells grown in microgravity conditions, however flown cells under 1 g conditions maintained normal actin cytoskeleton and fibronectin matrix. The changes seen in the cytoskeleton are probably not due to alterations in fibronectin message or protein synthesis since no differences have been noted in microgravity. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity, suggesting a common root cause for the change in cell architecture. The inability of the O g grown osteoblast to respond to sera activation suggests that there is a major alteration in anabolic signal transduction under microgravity conditions, most probably through the growth factor receptors and/or the associated kinase pathways that are connected to the cytoskeleton. Cell cycle is dependent on the cytoskeleton. Alterations in cytoskeletal structure can block cell growth either in G1 (F-actin microfilament collapse), or in G2/M (inhibition of microtubule polymerization during G2/M-phase). We therefore hypothesize that microgravity would inhibit growth in either G1, or G2/M.     

Possible use of a 3-D clinostat to analyze plant growth processes under microgravity conditions.

A three-dimensional (3-D) clinostat equipped with two rotation axes placed at right angles was constructed, and various growth processes of higher plants grown on this clinostat were compared with ground controls, with plants grown on the conventional horizontal clinostat, and with those under real microgravity in space. On the 3-D clinostat, cress roots developed a normal root cap and the statocytes showed the typical polar organization except a random distribution of statoliths. The structural features of clinostatted statocytes were fundamentally similar to those observed under real microgravity. The graviresponse of cress roots grown on the 3-D clinostat was the same as the control roots. On the 3-D clinostat, shoots and roots exhibited a spontaneous curvature as well as an altered growth direction. Such an automorphogenesis was sometimes exaggerated when plants were subjected to the horizontal rotation, whereas the curvature was suppressed on the vertical rotation. These discrepancies in curvature between the 3-D clinostat and the conventional ones appear to be brought about by the centrifugal force produced. Thus, the 3-D clinostat was proven as a useful device to simulate microgravity.     

Organization of cytoskeleton during differentiation of gravisensitive root sites under clinorotation.

Key role in cell gravisensing is attributed to the actin cytoskeleton which acts as a mediator in signaling reactions, including graviperception. Despite of increased attention to the actin cytoskeleton, major gaps in our understanding of its functioning in plant gravisensing still remain. To fill these gaps, we propose a novel approach focused on the investigation of actin involvement in the development of columella cells and cells in the transition zone of roots submitted to clinorotation. Both statocytes and cells in the transition zone represent the postmitotic cells which take origin in root meristems and are specified into graviperceptive (root cap) and gravireacting (transition zone) root tissues. The aim of the research was to investigate and compare the microfilament arrangements in root cap statocytes and peripheral root tissues (epidermis and cortex cells) in the transition zone and to find out how the actin cytoskeleton is involved in their specification under clinostat conditions. So far, our experiments have shown that under clinorotation the cytoplasmic microfilament network in the cortex cells in the transition zone is significantly enhanced. It is suggested that more abundant cytoplasmic microfilaments could strengthen the cortical actin cytoskeleton arranged parallel with the cortical microtubules, which are found to be partially disorganized in this area. Due to microtubule disorganization, the functioning of cellulose-synthesizing machinery and proper deposition of cell wall might be affected and could cause the alterations in the growth mode. But, in our case growth of the cells in the transition zone under clinorotation was rather stable. Due to our opinion, general stability of cell growth under clinorotation is promoted by mutual functional interrelation between actin and tubulin cytoskeletons. It is suggested that a strengthened cortical actin cytoskeleton restricts the cell growth instead of disorganized microtubules.     

Gravisensing in plants and fungi.

The principle of establishing and maintaining a gravitropic set point angle depends on gravisensing and a subsequent cascade of events that result in differential elongation of the responsive structures. Since gravity acts upon masses, the gravisensing mechanisms of all biological systems must follow the same principle, namely the sensing of some force due to differential acceleration of the perceiving entity and a reference structure. This presentation will demonstrate that gravisensing can be accomplished by various means, ranging from cytoskeletal organization, mechano-elastic stress to perturbation of electric signals. However, several arguments indicate that sedimentation of either dense plastids (statoliths), the entire protoplast, or a combination of these represents the primary step in graviperception in plants. In fungi, nuclei and cytoskeletal proteins are believed to form a network capable of gravisensing but sedimenting organelles that may function as statoliths have been identified. Theoretical and practical limitations of gravisensing and detection of acceleration forces necessitate microgravity experiments to identify the primary perceptor, subsequent biochemical mechano-transduction, and biological response processes.     

Kinetics of amyloplast movement in cress root statocytes under different gravitational loads.

In order to investigate the movement of a statolith complex along the longitudinal axis of root cap statocytes under different mass accelerations, a series of experiments with Lepidium sativum L. in an automatically operating centrifuge during the Bion-11 satellite flight and on a centrifuge-clinostat have been performed. During spaceflight, roots were grown for 24 h under root-tip-directed centrifugal 1-g acceleration, then exposed to microgravity for 6, 12 and 24 min and chemically fixed. During the first 6 min of microgravity, the statoliths moved towards the cell center with a mean velocity of 0.31 +/- 0.04 micrometers/min, which decreased to 0.12 +/- 0.01 micrometers/min within subsequent 12-24 min period. The mean relative position of the statolith complex in respect to the distal cell wall (% of total cell length) increased from 24.0 +/- 0.5% in 1 g-grown roots to 38.8 +/- 0.8% in roots exposed for 24 min to microgravity, but remained smaller than in roots grown continuously in microgravity (48.0 +/- 0.7%). The properties of the statolith movement away from the distal pole of the statocyte were studied in roots grown for 24 h vertically under 1 g and then placed for 6 min on a fast rotating clinostat (50 rpm) or 180 degrees inverted. After 2 min of both treatments, the mean relative position of the statoliths increased by about 10% versus its initial position. Later on, the proximal displacement of amyloplasts slowed down under simulated weightlessness, while it proceeded at a constant velocity under 1 g inversion. In roots grown on the clinostat and then exposed to 1 g in the longitudinal direction, amyloplast sedimentation away from the central region of statocyte was similar at the beginning of distal and proximal 6-min 1-g stimulation. However, at the end of this period statolith displacement was more pronounced in proximal direction as compared to distal. It is proposed that statolith position in the statocyte of a vertical root is controlled by the force of gravity, however, the intracellular forces, first of all those generated by the network of the cytoskeleton, are manifested when an usual orientation of the organ is changed or the statocytes are exposed to microgravity and clinorotation.     

Effects of altered gravity on the swimming behaviour of fish.

Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness-phenomena (SMS, a kinetosis). It has been argued that SMS during PAFs might not be based on microgravity alone but rather on changing accelerations from 0 g to 2 g. We test here the hypothesis that PAF-induced kinetosis is based on asymmetric statoliths (i.e., differently weighed statoliths on the right and the left side of the head), with asymmetric inputs to the brain being disclosed at microgravity. Since fish frequently reveal kinetotic behaviour during PAFs (especially so-called spinning movements and looping responses), we investigated (1) whether or not kinetotically swimming fish at microgravity would have a pronounced inner ear otolith asymmetry and (2) whether or not slow translational and continuously changing linear (vertical) acceleration on ground induced kinetosis. These latter accelerations were applied using a specially developed parabel-animal-container (PAC) to stimulate the cupular organs. The results suggest that the fish tested on ground can counter changing accelerations successfully without revealing kinetotic swimming patterns. Kinetosis could only be induced by PAFs. This finding suggests that it is indeed microgravity rather than changing accelerations, which induces kinetosis. Moreover, we demonstrate that fish swimming kinetotically during PAFs correlates with a higher otolith asymmetry in comparison to normally behaving animals in PAFs.     

The promotive effect of latrunculin B on maize root gravitropism is concentration dependent.

The cytoskeleton has been proposed to be a key player in the gravitropic response of higher plants. A major approach to determine the role of the cytoskeleton in gravitropism has been to use inhibitors to disrupt the cytoskeleton and then to observe the effect that such disruption has on organ bending. Several investigators have reported that actin or microtubule inhibitors do not prevent root gravitropism, leading to the conclusion that the cytoskeleton is not involved in this process. However, there are recent reports showing that disruption of the actin cytoskeleton with the actin inhibitor, latrunculin B, promotes the gravitropic response of both roots and shoots. In roots, curvature is sustained during prolonged periods of clinorotation despite short periods of gravistimulation. These results indicate that an early gravity-induced signal continues to persist despite withdrawal of the constant gravity stimulus. To investigate further the mechanisms underlying the promotive effect of actin disruption on root gravitropism, we treated maize roots with varying concentrations of latrunculin B in order to determine the lowest concentration of latrunculin B that has an effect on root bending. After a 10-minute gravistimulus, treated roots were axially rotated on a one rpm clinostat and curvature was measured after 15 hours. Our results show that 100 nM latrunculin B induced the strongest promotive effect on the curvature of maize roots grown on a clinostat. Moreover, continuously gravistimulated roots treated with 100 nM latrunculin B exhibited stronger curvature responses while decapped roots treated with this concentration of latrunculin B did not bend during continuous gravistimulation. The stronger promotive effect of low concentrations of latrunculin B on the curvature of both clinorotated and continuously gravistimulated roots suggests that disruption of the finer, more dynamic component of the actin cytoskeleton could be the cause of the enhanced tropic responses of roots to gravity.     

Structure of cress root statocytes in microgravity (Bion-10 mission).

Experiments on primary roots of Lepidium sativum L. have been performed on board the Bion-10 satellite. The experimental set-up was extremely miniaturized and completely automatic. The results demonstrate the effectiveness of the instrumentation. The spatial orientation, growth, root cap differentiation and statocyte structure of roots grown under microgravity (MG) have been compared with control roots grown on the ground (GC) and in a 1G-reference centrifuge in space (RC). Root length and cap shape did not differ between MG and control samples. Under MG, the mean distance of the statoliths from the distal cell wall of the statocytes increased significantly, the mean distance of the mitochondria decreased and the nucleus did not change its position in comparison to both controls. The number and the shape of the amyloplasts (statoliths) were not influenced by the space flight factors, but their size as well as their relative area in the cell decreased. The number of starch grains per statolith as well as their size and shape changed under MG. In MG and RC samples the number of lipid bodies in the statocytes was higher and the relative area larger than in GC samples. The relative area occupied by vacuoles was greater in RC statocytes than in GC and MG statocytes. These results partly confirm and, in addition, extend the data from earlier experiments in space.     

Gravisensing in roots.

The mode of gravisensing in higher plants is not yet elucidated. Although, it is generally accepted that the amyloplasts (statoliths) in the root cap cells (statocytes) are responsible for susception of gravity. However, the hypothesis that the whole protoplast acts as gravisusceptor cannot be dismissed. The nature of the sensor that is able to transduce and amplify the mechanical energy into a biochemical factor is even more controversial. Several cell structures could potentially serve as gravireceptors: the endoplasmic reticulum, the actin network, the plasma membrane, or the cytoskeleton associated with this membrane. The nature of the gravisusceptors and gravisensors is discussed by taking into account the characteristics of the gravitropic reaction with respect to the presentation time, the threshold acceleration, the reciprocity rule, the deviation from the sine rule, the movement of the amyloplasts, the pre-inversion effect, the response of starch free and intermediate mutants and the effects of cytochalasin treatment. From this analysis, it can be concluded that both the amyloplasts and the protoplast could be the gravisusceptors, the former being more efficient than the latter since they can focus pressure on limited areas. The receptor should be located in the plasma membrane and could be a stretch-activated ion channel.     

Cell fusion in space: plasma membrane fusion in human fibroblasts during short term microgravity.

During short-term microgravity in sounding rocket experiments (6 min.) the cytoskeleton undergoes changes and therefore it is possible that cell processes which are dependent on the structure and function of the cytoskeleton are influenced. A cell fusion experiment, initiated by a short electric pulse, was chosen as a model experiment for this sounding rocket experiment. Confluent monolayers of primary human skin fibroblasts, grown on coverslips, were mounted between two electrodes (distance 0.5 cm) and fused by discharging a capacitor (68 micro F; 250 V; 10 msec) in a low conductive medium. During a microgravity experiment in which nearly all the requirements for an optimal result were met (only the recovery of the payload was delayed) results were found that indicated that microgravity during 6 minutes did not influence cell fusion since the percentage of fused products did not change during microgravity. Within the limits of discrimination using morphological assays microgravity has no influence on the actin/cortical cytoskeleton just after electrofusion.     

Role of calcium in gravity perception of plant roots

Calcium ions may play a key role in linking graviperception by the root cap to the asymmetric growth which occurs in the elongation zone of gravistimulated roots. Application of calcium-chelating agents to the root cap inhibits gravitropic curvature without affecting growth. Asymmetric application of calcium to one side of the root cap induces curvature toward the calcium source, and gravistimulation induces polar movement of applied ⁴⁵Ca²⁺ across the root cap toward the lower side. The action of calcium may be linked to auxin movement in roots since 1) auxin transport inhibitors interfere both with gravitropic curvature and gravi-induced polar calcium movement and 2) asymmetric application of calcium enhances auxin movement across the elongation zone of gravistimulated roots. Indirect evidence indicates that the calcium-modulated regulator protein, calmodulin, may be involved in either the transport or action of calcium in the gravitropic response mechanism of roots.     

Protein crystals in Phycomyces sporangiophores are involved in graviperception.

The sporangiophores of the zygomycete fungus Phycomyces blakesleeanus contain octahedral crystals with diameters of up to 5 micrometers in their vacuole. The crystals are associated with the intracellular membrane system. In tilted or horizontally placed sporangiophores, the crystals sediment to the respective lower face of the vacuole with a velocity of up to 100 micrometers per minute. The sedimentation is completed within about 2 minutes, well within the latency period for the negative gravitropic response of Phycomyces. Crystal-lacking mutant strains display a smaller maximal bending angle and a reduced gravitropic bending rate in comparison to the wild type. We therefore conclude that the crystals serve as statoliths for gravitropism in Phycomyces.     

Gravitropism in cut flower stalks of snapdragon.

The negative gravitropic response of cut flower stalks is a complex multistep process that requires the participation of various cellular components acting in succession or in parallel. The process was particularly characterized in snapdragon (Antirrhinum majus L.) spikes with regard to (1) gravity stimulus perception associated with amyloplast reorientation; (2) stimulus transduction mediated through differential changes in the level, action and related genes of auxin and ethylene and their possible interaction; (3) stimulus response associated with differential growth leading to stalk curvature; (4) involvement of cytosolic calcium and actin cytoskeleton. Results show that the gravity-induced amyloplast reorientation, differential over-expression of two early auxin responsive genes and asymmetrical distribution of free IAA are early events in the bending process. These precede the asymmetrical ethylene production and differential stem growth, which was derived from initial shrinkage of the upper stem side and a subsequent elongation of the lower stem side. Results obtained with various calcium- and cytoskeleton-related agents indicate that cytosolic calcium and actin filaments may play essential roles in gravitropism-related processes of cut flower stalks. Therefore, modulators of these two physiological mediators may serve as means for controlling any undesired gravitropic bending.     

An active role of the amyloplasts and nuclei of root statocytes in graviperception.

Three main phases are discerned in the gravitropic reaction: perception of a gravitational stimulus, its transduction, and fixation of the reaction resulting in bending of an organ. According to the starch-statolith hypothesis of Nemec and Haberlandt, amyloplasts in the structurally and functionally specialized graviperceptive cells (statocytes) sediment in the direction of a gravitational vector in the distal part of a cell while a nucleus is in the proximal one. If amyloplasts appear to act as gravity sensors, the receptors, which interact with sedimented amyloplasts, and next signaling are still unclear. An analysis of the structural-functional organization of cells in different root cap layers of such higher plants as pea, Arabidopsis thaliana, and Brassica rapa grown under 1 g, on the clinostats, and in microgravity, allows us to support the hypothesis that amyloplasts function as statoliths in statocytes, but they may not be only the passive statolithic mass. We propose that amyloplasts fulfill a more complex function by interacting with a receptor, which is a nucleus, in transduction of some signal to it. Gravity-induced statolith movement in certain order leads to a new functional connection between gravity susceptors--amyloplasts and a receptor--a nucleus receiving some signal presumedly of a mechanical or biochemical nature from the amyloplasts. During gravitropism, sugar signaling could induce expression of genes encoding auxin transport proteins in a nucleus giving the nucleus an intermediate role in signal trunsduction following perception.