Gravity has been a pervasive factor throughout evolutionary history, and biological systems are assumed to have used this external clue for orientation rather early to find and stay in optimal living conditions, which offer ecological advantages. Consequently, already early during evolution, unicellular organisms developed organelles for active movement and sensors for diverse environmental stimuli. This chapter summarizes some aspects and ideas concerning evolution and refinement in gravisensing, and focuses on the fundamental question concerning common origins in the underlying signal transduction chains.
Development of gravisensing during evolution
As discussed in several chapters: To introduce a detectable change in the cell, gravity has to interact with a mass. Consequently, size matters! To be more precise: size and specific density difference matters. Published values for the specific density of prokaryotes are scarce. Guerrero and coworkers (1984) published a value of 1.16 g ml—1 for Allochromatium vinosum, a purple sulfur bacterium. This value might vary drastically due to living conditions, feeding and metabolic status, etc. The sedimentation rate is only 0.12 µms—1. In contrast, sedimentation rates in eukaryotes are generally one to two orders of magnitude higher. Size makes the difference. Prokaryotes are typically at least one order of magnitude smaller than eukaryotes. Brownian motion, as well as low Reynolds numbers, prohibit any active steering mechanism in free-swimming species. But, there is a way out: limiting diffusion dimensions.
Microorganisms respond to a multitude of external stimuli in their habitat to select a suitable niche for survival and reproduction. Light and gravity are probably the most important cues for most motile microorganisms. Several types of light-induced behavior can be distinguished in microorganisms, including phototaxis, photophobic responses, and photokinesis. Chemical gradients, such as oxygen or carbon dioxide, are sensed by many organisms. Bacteria recognize and follow gradients of attractants (e.g., nutrients such as sugars) or avoid sources of toxins (e.g., phenol). Heterotrophic eukaryotic microorganisms also use chemical gradients to find their food. Pheromones are produced and emitted to attract gametes of the opposite sex. Some prokaryotic and eukaryotic organisms are capable of sensing extremely small thermal gradients very close to the physical limits. Almost all motile organisms, from bacteria to vertebrates, recognize and use the magnetic field of the Earth. Responses to electrical fields are not easy to explain, because these stimuli are not expected in nature. The responses to multiple stimuli may be additive or connected in a complex network of signal transduction chains. In other cases, responses to certain stimuli may override those to others.
Microorganisms respond to a host of stimuli in their environment to search for and stay at favorable habitats optimal for their growth and survival, as well as reproduction. Their responses to these environmental clues may vary depending on their developmental stage.
Motility and orientation of motile microorganisms can be quantified basically by two approaches: population methods and individual tracking. Individual tracking analyzes the movement parameters of single organisms and subsequently averages over the behavior of a statistically significant number of individuals to evaluate the behavior of a population. Population methods operate on the assumption that the movement of the individual organisms will lead to a translocation of the whole population. Both methods have their advantages and drawbacks. Individual tracking is tedious and can be prone to error or bias, and may only be applicable for restricted path segments. Reaching a significant conclusion for a whole population may strain the patience of an experimenter. On the other hand, population methods may measure something different than the experimenter assumes. For example, a population may seem to be moving in a certain direction and end up, for instance, at the top of a water column, which may be interpreted as the result of negative gravitactic orientation. However, the behavioral result may be due to the organisms moving in random directions, but become immobile near the water surface. Other phenomena leading to a displacement of a population, erroneously interpreted as the result of gravitaxis, include sedimentation, phobic responses, kinetic effects, or the response to other stimuli — including light, magnetic field lines, or chemical gradients (such as oxygen, carbon dioxide, or nutrients).
Ciliates can be regarded as swimming sensory cells. Their ion channels in the cell membrane have been extensively studied, and a direct correlation between particular ion currents, the membrane potential, the control of ciliary activity, and the swimming behavior of the cells was established. Conclusively, changes in ion fluxes can be identified by corresponding changes in swimming velocity and swimming direction. Thus, ciliates represent suitable model systems to study with noninvasive methods the effects of changes of environmental stimuli on the cellular level. By studying distinct graviresponses (gravitaxis and gravikinesis) under different gravitational stimulations, new results were found indicating that different mechanisms for graviperception have been developed. Uniquely, in the ciliate family Loxodidae, specialized gravireceptor organelles exist, whereas in other species, common cell structures seem to be responsible for gravisensing. Based on the fact that in many ciliates mechanosensitive ion channels are arranged in a bipolar manner and thus ideally suited for perception of the linear stimulus gravity, the old “statocyst hypothesis” was renewed. In the current hypothesis, gravity (e.g., in Paramecium) is perceived by sensing the mass of the cell body via distinct stimulation of mechanosensitive ion channels. Signal amplification by cytoskeletal elements, as well as involvement of the ubiquitous second messenger cAMP, seems likely.
Whether a pure physical mechanism is sufficient to describe gravi-related motility phenomena is a question discussed for more than 100 years. The scope of this chapter is not the energetic considerations (cf. Chapter 8 for an in-depth discussion of energetics), but the description of historical and recent models explaining gravitaxis and gravikinesis in ciliates and flagellates. In general, it can be stated that, in most systems, where enough information for a detailed model is available, most likely gravi-related behavior is a combination of both: a physical component and a physiological component.
Since the first discovery of gravitational effects on motile, free-swimming, unicellular organisms, scientists discussed the underlying mechanisms and principles (Schwarz, 1884; Verworn, 1889b; Jennings, 1906; cf. Section 1.1). In general, since the early days, two schools claimed to understand gravity-related phenomena. The “physics” group tried to explain gravitaxis — to the best of our knowledge no physical model for gravikinesis exists — first detected by Dembowski (1929b) as a pure physical phenomenon. The “physiology” group thought of gravitaxis as a typical signal transduction-based cellular response. As usual, the truth will be somewhere in the middle, as we will see later.
Schwarz (1884) was the first who expressed these as a first-glance contra-dictory hypotheses based on his results with Euglena viridis (a close relative of Euglena gracilis).
In this chapter, we discuss energetic considerations regarding the mechanisms thought to underlie the orientation of single cells in the gravitational field. Although it is still under discussion, whether gravireactions are the result of a physical or physiological mechanisms or a combination of both, it is undisputed that the basis must be an interaction of gravity with a mass. This chapter compares potential mechanisms for gravidetection with physical and energetic limits set by nature. Several models consider mechanosensitive channels as an important part of gravity perception in single cells. The weak energy supplied by the gravity—mass interaction will be shown to be sufficient to at least potentially allow to activate such channels. As a model, the hearing system of the inner ear will be compared with the conditions in single cells.
Past and recent discussions were centered around the question of whether the reorientational movements in single cells are the result of a pure physical or a pure physiological mechanism or a combination of both (cf. Chapter 9). Independent of such considerations, the most basic event of the related movement reactions (gravitaxis, gravikinesis) will be an interaction between a cellular entity and gravity. This is the gravity stimulus perception. Depending on the model, we are looking at the following steps, including a receptor (typically a protein, but not necessarily a membrane protein), a receptor-signaling-state change (i.e., the point where a stimulus such as light or gravity is transformed into a chemical, biochemical, or electrical signal), and a signal transduction chain (that may or may not include an amplification).
To vary the influence of the unique stimulus gravity, different experimental and technical approaches have been followed and developed. Today, we are in the ideal situation to perform gravitational biological experiments on the ground — by means of, for example, clinostats and centrifuges — and in real microgravity using different facilities in dependence of the time of free fall needed. Comparative studies between simulated and real microgravity reveal similar results, though the response in actual microgravity appears to be more pronounced and faster. The methods that are used to answer questions in gravitational biology are presented, explained, and discussed within this chapter.
In their natural environment, swimming microorganisms are confronted with a large number of interacting stimuli, which, after a complex signal processing, result in behavioral responses. To understand the impact of a single stimulus, such as gravity, the behavior has to be studied under controlled and defined conditions. Experimenters had to learn that the behavior of microorganisms also depends on parameters such as geometry of the observation chamber, cell density, thermo-convection, time of measurement with respect to circadian rhythm, and that the responses are “full of surprises” (Kessler, 1985b). To study the responses with respect to gravity, an observation chamber should be completely filled and air bubbles should be excluded to avoid chemotactic responses (cf. Section 7.3) and shearing forces.
The phenomenon that some free-swimming unicellular organisms tend to swim to the top of a tube and gather there — independent of whether the tube is open or closed — has been observed more than 100 years ago. This behavior was termed geotaxis (orientation with respect to the gravity vector of the Earth) — negative geotaxis if the organisms orient upward and positive geotaxis if they swim downward (cf. Section 1.2). Nowadays, this term has been replaced by gravitaxis. Many early and detailed studies between 1880–1920 provided descriptive observations limited by optical and analytical means. This led to the establishment of various hypotheses that have been reviewed by different authors (Bean, 1984; Davenport, 1908; Dryl, 1974; Haupt, 1962b; Hemmersbach et al., 1999b; Jennings, 1906; Kuznicki, 1968; Machemer & Bräucker, 1992).
The results were rather conflicting and led to controversial interpretations. While Stahl (1880) stated that Euglena and Chlamydomonas do not orient with respect to gravity, Schwarz (1884) concluded from his observations that Euglena moves upward by an active orientational movement and is not passively driven (e.g., by currents in the water or attracted by oxygen at the surface). He found that the force of gravity could be replaced by centrifugal force and that Euglena could move upward against forces of up to 8.5 × g. The author also concluded that Euglena belongs to the negative geotactic organisms.
There comes a point in the career of a scientist when he or she should write a book about his or her subject of interest. Two of us always wondered when and how this was going to happen. Now we know: by pure accident. And, here is one word of advice: You are often warned not to get involved in the book business. Please consider those who are warning you as your best friends; they know what they are talking about. However, one day, we received an e-mail (actually much longer ago than we would have anticipated) asking whether we would be willing to write a book about the effects of gravity on single cells. One of us knew what that meant; he warned us, but we agreed anyway. Finally, all three of us completed the project, and we learned a lot in the process. So, thank you, Peter Barlow and Cambridge University Press for keeping your faith in us.
Those who teach about gravity effects on living systems, including single cells, quickly realize that this weak force seems to have escaped human attention. Although we all had strong fights with gravity, especially during the early phase of our lives, it seems that afterward, we have almost completely forgotten about it. However, for all living organisms in our world, it is the one parameter most steadily encountered. Gravity is so basic for all of us that it is almost hardwired into our interpretation of reality. Gravity is not only related to living organisms; convection and the weather are two other subjects that come to mind when thinking about gravity.
This chapter summarizes our knowledge from gravitational biological experiments performed with “other” organisms, which means other than ciliates and flagellates. Amoeba, cellular and acellular slime molds, swimming reproductive stages — such as zoospores and sperm cells — and bacteria have been exposed to altered gravitational stimulation to analyze the impact on behavior and, in few cases, on biochemical processes. In all examples given, a clear hypothesis on the mechanism of graviperception is still missing and should be a task for the future.
Amoeboid cells are characterized by their actin- and myosin-driven (amoeboid) movement along surfaces (for a review, see Hausmann & Hülsmann, 1996). A weak tendency for negative gravitaxis in Amoeba has been stated (Klopocka, 1983). Cultivation of Amoeba proteus at 40 × g for 36 days did not induce detectable changes in cell form or function (Montgomery et al., 1965). Cultivation of Pelomyxa carolinesis in microgravity on Biosatellite II for 2 days indicated a slightly increased division rate (Ekberg et al., 1971), whereas another experiment stated no effect on growth rate and morphology (Abel et al., 1971). The mechanism of graviperception of amoeba needs to be investigated.
The cellular slime mold Dictyostelium discoideum is characterized by a life cycle alternating between a multicellular pseudoplasmodium (slug) stage and a unicellular amoeboid stage.
Many photosynthetic or heterotrophic flagellates from various taxonomic origins investigated so far have been found to be capable of gravitactic orientation and to orient themselves in the water column by positive, negative, or transversal gravitaxis. Two species can be regarded as model systems, Chlamydomonas and Euglena, since in these organisms gravitaxis has been studied in more detail. Earlier hypotheses assumed that gravitactic orientation is mediated by a buoy effect, where the cell is tail-heavy, and the flagellum — emerging from the anterior end — pulls the organism upward. A number of observations are in contradiction to this model. Rather, at least in Euglena, an active, physiological graviperception mechanism seems to be responsible for the observed orientation. For this organism, the whole cell body is assumed to function as a statolith and exert pressure on the lower membrane. This force is thought to activate mechanosensitive calcium ion channels. During each rotation around its long axis, more calcium enters the cell until a concentration threshold is reached — upon which the flagellum swings out and induces a course correction. Other elements of the sensory transduction chain include changes in the membrane potential, cAMP as secondary messenger, and possibly additional elements. In Euglena and other flagellates, gravitaxis is controlled by an endogenous rhythm that also affects the cell form, cAMP concentration, and other physiological parameters.
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