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Strategies for cell-substratum dependent motility among protozoa
Abstract
Protozoa exhibit 2 major types of cell-substratum dependent motility, amoeboid locomotion and gliding. The latter is typified by essentially uni-directional progression at speeds in the order of micrometres per second, accompanied by neither distortion of cell shape nor overt cytoskeletal turnover. A consideration of the biophysical problems which constrain the gliding of protists identifies three fundamental requirements of this motile behaviour: (1) cell surface binding sites with which to attach to a substratum, (2) transmembrane connectors to link up with a cortical motor apparatus consisting of, (3) linear trackways along which the force-producing motor proteins may shuttle with their cargo. Simple calculations show that the energy required to power gliding in these cells is extremely small (of the order of 10-19 W in the case of the sporozoite of Plasmodium) and could be delivered by a very small number of motor proteins with the properties of myosins, kinesins or dyneins. This result tells us that the task of identifying the macromolecular assemblage of the hypothetical cell-surface linear motor (King 1988) will be a daunting one. Evidence for the presence of these essential elements of gliding motility is discussed in the case of 5 well known protistan examples - the sporozoites of Plasmodium and Eimeria, the trophozoite of Gregarina, the spindle cells of the net slime mould Labyrinthula, and the flagella of Chlamydomonas.
... Analysis of sporozoite gliding motility over consecutive small fractions of a second shows it to be a much more erratic process than it initially appears, with sporozoites speeding up and slowing down from one time interval to the next in an apparently random fashion. This also seems to be the case in Gregarina trophozoites (Preston et al, 1990) and Plasmodium sporozoites (Preston & King, 1996). ...
... A low Re (<1.0) indicates that viscous forces predominate whereas at high Re (>100) the forces of inertia are the more important consideration. Preston & King (1996) calculated that for a gliding Plasmodium sporozoite 10/xm long gliding at l/xms * through an aqueous medium the Re would be 10'^, and for a gliding Eimeria sporozoite 12/im long gliding at 20pims * through an aqueous medium the Re would be ~ 2 x lO '*. It is clear therefore that sporozoite motility is dependent on overcoming viscous forces. ...
... For a Plasmodium sporozoite travelling at 1/xms ' F ' = 7.5 x lO '^N (Preston & King, 1996). ...
Eimeria tenella is an obligate intracellular parasite within the phylum Apicomplexa. It is the causative agent of coccidiosis in domesticated chickens and under modem farming conditions can have a considerable economic impact. Motility is employed by the sporozoite to effect release from the sporocyst and enable invasion of appropriate host cells and occurs at an average speed of 16.7 6 ms-1. Frame by frame video analysis of gliding motility shows it to be an erratic non-substrate specific process and this observation was confirmed by studies of bead translocation across the cell surface occurring at an average speed of 16.9 7.6 ums-1. Incubation with cytochalasin D, 2,3-butanedione monoxime and colchicine, known inhibitors of the motility associated proteins actin, myosin and tubulin respectively, indicated that it is an actomyosin complex which generates the force to power sporozoite motility. Western blotting analysis confirmed the presence of actin with an apparent molecular weight of 43kDa and an unconventional myosin with an apparent molecular weight of 93kDa. Antibodies against the actin binding proteins spectrin, vinculin, filamin, -actinin, cofilin and tropomyosin failed to recognise any polypeptides in whole cell extracts. Immunofluoresence studies showed actin was found predominantly in the anterior third of the sporozoite. Myosin appeared to have a more widespread distribution, with a strong signal found at the margins of the cell. Genomic DNA samples were prepared and two degenerate primers against highly conserved regions of the myosin head were used in a polymerase chain reaction (PCR) to probe for the presence of myosin genes. These PCR products were inserted into suitable plasmids followed by amplification in bacteria. Selection of appropriate bacterial colonies and subsequent DNA sequence analysis identified a clone with significant homology to a Homo sapiens myosin II gene previously described.
... In the present study, we report that gliding movement in P. trichophorum is generated by cell surface motility on the anterior flagellum. Surface motility is one of the most mysterious movements among unicellular organisms; extracellular materials are translocated along the cell surface with no distinct change in cell shape [Bloodgood, 1990;Preston and King, 1996]. For example, in members of apicomplexan parasites such as Toxoplasma and Plasmodium, actomyosin-dependent surface motility is used for the cell invasion process into the host tissue [Dobrowolski et al., 1977;Sultan et al., 1997]. ...
... In haptophyceae such as Chrysochromulina, surface motility is observed on the haptonema, which is an organelle unique to this group of flagellates [Leadbeater, 1971]. In all of these gliding protozoans, translocation of microsphere particles is also observed on the plasma membrane as a manifestation of the same force-generating mechanism [Bloodgood, 1995;Kawachi et al., 1991;Preston and King, 1996]. Also in P. trichophorum, polystyrene beads were found to be translocated on the anterior flagellum, and this phenomenon is discussed in relation to the feeding mechanism of this flagellate. ...
... In Plasmodium, bead translocation is faster than cell gliding. In Peranema, on the contrary, velocity of bead translocation varies, but is always slower than cell gliding. 1 Bloodgood, [1977]; 2 Poulsen et al. [1999]; 3 Suzaki and Shigenaka [1982]; 4 Preston and King [1996]; 5 King [1981]; 6 This study; 7 Dobrowolski et al. [1997]; 8 Lapidus and Berg [1982]; 9 Ridgway and Lewin [1988]; 10 Piper et al. [1987]; 11 Spormann and Dale [1995]; 12 Choi et al. [1999]. ND, not determined. ...
A colorless euglenoid flagellate Peranema trichophorum shows unique unidirectional gliding cell locomotion on the substratum at velocities up to 30 micro m/s by an as yet unexplained mechanism. In this study, we found that (1) treatment with NiCl(2) inhibited flagellar beating without any effect on gliding movement; (2) water currents applied to a gliding cell from opposite sides caused detachment of the cell body from the substratum. With only the anterior flagellum adhering to the substratum, gliding movement continued along the direction of the anterior flagellum; (3) gentle pipetting induced flagellar severance into various lengths. In these cells, gliding velocity was proportional to the flagellar length; and (4) Polystyrene beads were translocated along the surface of the anterior flagellum. All of these results indicate that a cell surface motility system is present on the anterior flagellum, which is responsible for cell gliding in P. trichophorum.
... Thus far, the actin bundles are the only components of this system that have been identi®ed (Edgar and Zavortink 1983). Several other protists display adhesion-mediated gliding, without a distortion of cell shape, notably the sporozoites of Plasmodium and Eimeria (Preston and King 1996). The model for protozoan parasite gliding (Preston and King 1996) is essentially the same as the model of diatom gliding (Edgar and Pickett-Heaps 1984), although no components of this system have been identi®ed. ...
... Several other protists display adhesion-mediated gliding, without a distortion of cell shape, notably the sporozoites of Plasmodium and Eimeria (Preston and King 1996). The model for protozoan parasite gliding (Preston and King 1996) is essentially the same as the model of diatom gliding (Edgar and Pickett-Heaps 1984), although no components of this system have been identi®ed. These models share some features with the apparatus proposed to bind animal cells to substrata. ...
Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and > 200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion.
... Afterwards, a force applied to the transmembrane protein (e.g., putative myosin)/actin connectors, parallel to the actin bundle, produces movement of the transmembrane proteins through the cell, and consequent movement of the cell in the direction opposite to the force (Aumeier and Menzel, 2012). A similar mechanism has been proposed to explain the locomotion of other protists that exhibit substrate-adherence mediated gliding (Preston and King, 1996;Dobrowolski et al., 1997;Pinder et al., 1998). A variant of the Edgar model has been proposed to explain the locomotion of Navicula sp., postulating that it is done via two or more pseudopods or stalks projected out of the frustules. ...
The physiology of the diel movements of epipelic microphytobenthic diatoms is not fully understood. As well, the evolutionary pressures that led to migratory behavior and the ecological role of vertical migrations remain unknown. The behavioral photoprotection hypothesis, according to which the diatoms move along the vertical light gradient to find their optimal light environment, is the most generally accepted. However, the motion is associated with an energy cost that has not been fully acknowledged before. To throw light on this issue, we looked at the mechanisms of diatom locomotion and reviewed their patterns of movement. Making use of published data, we estimated an energy cost of 0.12 pJ for a typical diatom cell to move upward (or downward) in a 400 μm photic zone. This amounts to 3.93 ×10⁻¹⁸ mol of ATP, which are released by the oxidation of 1.31 ×10⁻¹⁹ mol of glucose. This represents only 0.0001% of the daily net photosynthetic production of a typical microphytobenthic diatom cell, showing that diel vertical migrations have a negligible impact on cell and ecosystem energy budget. Even though the migration energy cost of individual cells may depart almost two orders of magnitude from the central value presented for a typical diatom (depending on cell size, velocity of displacement, and viscosity of the medium), the maximum value calculated is still negligible from the metabolic and ecologic point of view. Results show that behavioral photoprotection might be an energetically cheap mechanism, offering competitive advantages when compared with structural/physiological photoprotection.
... Adhesion-mediated gliding, without a change in overall cell shape, is found in the sporozoites of the protists Plasmodium and Eimeria, where Preston and King (1996) proposed a model for protozoan gliding that is virtually the same as the model proposed for diatoms (Edgar and Pickett-Heaps 1984). With the exception of actin, none of the linker molecules of these model systems has been identified, although these models share certain features with the mechanism proposed to attach animal cells to substrata. ...
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... A similar mechanism has been proposed for other protists that display adhesion-mediated gliding [King, 1988;Preston and King, 1996;Dobrowolski et al., 1997;Pinder et al., 1998]. These models suggest that actin filaments localized beneath the plasma membrane act as a scaffold for myosins coupled to transmembrane proteins that interact via adhesion molecules with a substratum to transfer the mechanical force generated by the actinmyosin complex. ...
Diatoms are a group of unicellular microalgae that are encased in a highly ornamented siliceous cell wall, or frustule. Pennate diatoms have bilateral symmetry and many genera possess an elongated slit in the frustule called the raphe, a feature synonymous with their ability to adhere and glide over a substratum, a process little understood. We have used cytoskeleton-disrupting drugs to investigate the roles of actin, myosin, and microtubules in diatom gliding or motility. No effect on diatom gliding was observed using the cytochalasins, known actin inhibitors, or the microtubule-inhibitors oryzalin and nocodazole. The latrunculins are a new group of anti-actin drugs, and we show here that they are potent inhibitors of diatom gliding, resulting in the complete disassociation of the raphe-associated actin cables. The recovery of actin staining and motility following latrunculin treatment was extremely fast. Cells exposed to latrunculin for 12 h recovered full function and actin staining within 5 sec of the drug being removed, demonstrating that the molecular components required for this motility system are immediately available. Butanedione monoxime (BDM), a known myosin inhibitor, also reversibly inhibited diatom gliding in a manner similar to the latrunculins. This work provides evidence that diatom gliding is based on an actin/myosin motility system.
... The strategies selected by these parasites for either gliding onto a substratum or for invading their host cells depend on the dynamics of their actin cytoskeleton (King, 1988;Dobrowolski and Sibley, 1996;Preston and King, 1996). However, although it has been established that actin dynamics is required for the progression of the parasite's life cycle, studies on the molecular basis of parasite actin dynamics have been hampered by the transient and discrete nature of actin cytoskeleton remodeling. ...
Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.
... Forward gliding is thought to result from secretion of substrate-binding factors at the parasite anterior pole, followed by their redistribution to the posterior pole. Gliding is exceptionally rapid, 1 to 20 m/s in vitro (Preston and King, 1996). This is 1 to several order(s) of magnitude faster than the speed of the most rapid crawling cells, such as keratocytes (Small et al., 1999), amoebae (Van Duijn and Inouye, 1991 ), and polymorphonuclear cells (Mitchinson and Cramer, 1996). ...
Actin polymerization in Apicomplexa protozoa is central to parasite motility and host cell invasion. Toxofilin has been characterized as a protein that sequesters actin monomers and caps actin filaments in Toxoplasma gondii. Herein, we show that Toxofilin properties in vivo as in vitro depend on its phosphorylation. We identify a novel parasitic type 2C phosphatase that binds the Toxofilin/G-actin complex and a casein kinase II-like activity in the cytosol, both of which modulate the phosphorylation status of Toxofilin serine53. The interplay of these two molecules controls Toxofilin binding of G-actin as well as actin dynamics in vivo. Such functional interactions should play a major role in actin sequestration, a central feature of actin dynamics in Apicomplexa that underlies the spectacular speed and nature of parasite gliding motility.
This supplement brings together the contents and authors indexes of Acta Protozoologica throughout the 40 volumes that have been published since 1963. The journal gradually became established through the support of a number famous scientists who used it to publish their original articles and reviews. With the publication of our 40-th volume, we are indebted to the famous Polish scientists, Professor Zdzisław Raabe from the Warsaw University, and Professor Stanisław Dryl from the Nencki Institute of Experimental Biology, Polish Academy of Sciences, for their efforts and initiative in establishing the international journal Acta Protozoologica in 1963. We believe that this Cumulative Indexes will be an important contribution to the field of protistology and a useful tool for retrieving suitable references.
Cytoskeleton elements of aLabyrinthula isolate from the Falkland Islands were studied. The most important characteristic of the genusLabyrinthula is a colourless branched plasmatic network of pseudopodia-like tubes with sliding spindle-shaped uninuclear plasma portions (cell bodies). After fluorescent staining tubulin appears to be uniformly and diffusely distributed throughout the whole network and to form a reticulate structure in the cell bodies. The inhibitor colchicine has no influence on the sliding motility of the cell bodies nor on the movement of the network. Actin is frequently found in the network, partly in the form of microfilament bundles, which are longitudinally arranged. Actin is also present in the cortical region of cell bodies, or of cell body groups. It was difficult to distinguish single cell bodies within groups by fluorescence. The inhibitors cytochalasin B and D stop the movement of cell bodies and network. Myosin is present in the cortical region of each cell body, and the central portions of each individual cell body contain accumulations of this protein. We could not observe any fluorescence in the network after myosin staining with the antibodies we used. An actin-myosin complex is probably responsible for the sliding movement of cell bodies in the Labyrinthula network, because actin is found in the pseudopodia-like tubes, and the cortex of the cell bodies is rich in actin and myosin. This actin-myosin complex seems to differ from another actin-myosin complex that has been postulated to be responsible for the locomotion of pseudopodia-like tubes. We propose that two actin-myosin complexes exist. One of them is responsible for locomotory phenomena of the network, and the second for cell body sliding in the pseudopodia-like tubes. In each case the myosin is probably anchored in the inner matrix membrane of the pseudopodia-like tubes. A model for actin-myosin interaction inLabyrinthula spp. is presented.
Actin polymerization and actin-myosin coupling activity most likely provide the driving force that the protozoan parasite Toxoplasma gondii has to exert to propulse itself during gliding and host cell entry. Nevertheless, little information is available on T. gondii tachyzoite actin dynamics, and in particular, the presence of actin filaments remains largely uncharacterized. Here, we report that the marine sponge peptide jasplakinolide, known to bind to filamentous actin, does indeed stabilize a pool of a parasite detergent-insoluble actin. This pool is likely to be formed by a dynamic assembled actin complex: first, it is competent for assembly/disassembly and secondly, it is sensitive to nucleotide phosphate concentration. In addition, T. gondii tachyzoites contain molecules which inhibit actin assembly and destabilize actin filaments. Thus, these activities could account for the remarkably low amount of the myosin-containing F-actin pool we describe here. Furthermore, when parasites are treated with cell-permeant jasplakinolide, they display a significant loss of both motility and host cell invasiveness. These data suggest that in vivo, the detergent-insoluble pool of actin is dynamic.
The main conclusion from the present study is that T. parva sporozoite entry is dependent on a functional host cell actin cytoskeleton and is not driven by the parasite. Treating lymphocytes with cytochalasin D resulted in a dose-dependent reduction in the levels of host cell infection. However, the primary effect was to block sporozoite binding and only at the highest concentration (20 microM) was sporozoite internalization significantly reduced. In fact at lower concentrations (1-10 microM) cytochalasin treatment lead to a relative increase in sporozoite internalization. The results are consistent with sporozoite entry being primarily a passive process and with a functional host cell actin cytoskeleton that is required only to maintain the molecular integrity of the surface membrane. Thus T. parva sporozoite entry differs from the process in other apicomplexans, although the results are consistent with a number of features of sporozoite biology. Treatment of lymphocytes with either the microtubule-destabilizing agent, nocodazole, or taxol, which induces microtubule polymerization, had no significant effect on sporozoite binding or entry. As both reagents had the expected effects on the lymphocyte microtubule system, it is unlikely that host cell microtubules are essential for successful sporozoite invasion or establishment.
The acquisitions of mitochondria and plastids were important events in the evolution of the eukaryotic cell, supplying it with compartmentalized bioenergetic and biosynthetic factories. Ancient invasions by eubacteria through symbiosis more than a billion years ago initiated these processes. Advances in geochemistry, molecular phylogeny, and cell biology have offered insight into complex molecular events that drove the evolution of endosymbionts into contemporary organelles. In losing their autonomy, endosymbionts lost the bulk of their genomes, necessitating the evolution of elaborate mechanisms for organelle biogenesis and metabolite exchange. In the process, symbionts acquired many host-derived properties, lost much of their eubacterial identity, and were transformed into extraordinarily diverse organelles that reveal complex histories that we are only beginning to decipher.
Intracellular parasites use various strategies to invade cells and to subvert cellular signaling pathways and, thus, to gain a foothold against host defenses. Efficient cell entry, ability to exploit intracellular niches, and persistence make these parasites treacherous pathogens. Most intracellular parasites gain entry via host-mediated processes, but apicomplexans use a system of adhesion-based motility called "gliding" to actively penetrate host cells. Actin polymerization-dependent motility facilitates parasite migration across cellular barriers, enables dissemination within tissues, and powers invasion of host cells. Efficient invasion has brought widespread success to this group, which includes Toxoplasma, Plasmodium, and Cryptosporidium.
In contrast to crawling movement (e.g. in amoebae and tissue cells) the other major class of substratum-associated motility in eukaryotes, gliding, has received relatively little attention. The net slime mold Labyrinthula provides a useful laboratory model for studying this process since it exhibits a particular kind of gliding in its plasmodial stage. Here nucleated spindle cells glide along self-established cytoplasmic trackways in a predominantly unidirectional manner, at 1-2 microm/s. These trackways, upon which gliding is dependent, are held by filopodial tethers some distance off the well-developed reticulopodial mesh anchoring the plasmodium onto the substratum. Reflection interference microscopy resolves this matrix in live plasmodia. The axially disposed cytoskeletal elements of the trackways are revealed by rhodamine-labelled phalloidin to be rich in F-actin. A weft of peripheral, rapidly extending filopodia (50 microm/min) typifies the expanding regions of the plasmodium. Here spindle cells are recruited before emigrating into newly differentiated trackways. Immunoblotting whole plasmodia or a sucrose-soluble cytoplasmic extract reveals a single actin-positive band of Mr 48 kDa. Polyclonal antibodies to two distinct myosin peptide sequences identify a single myosin HC (Mr 96 kDa) in immunoblots. Gliding was reversibly blocked by 10 mM 2,3-butanedione-2-monoxime, a myosin ATPase inhibitor, but it was insensitive to the actin-binding drugs cytochalasin D and phalloidin. We suggest that the force (>50 pN) for gliding motility results from interaction of myosin molecules, associated with the spindle cells, with trackway F-actin via the bothrosomes.
Gliding is a form of substrate-dependent cell locomotion exploited by a variety of disparate cell types. Cells may glide at rates well in excess of 1 microm/sec and do so without the gross distortion of cellular form typical of amoeboid crawling. In the absence of a discrete locomotory organelle, gliding depends upon an assemblage of molecules that links cytoplasmic motor proteins to the cell membrane and thence to the appropriate substrate. Gliding has been most thoroughly studied in the apicomplexan parasites, including Plasmodium and Toxoplasma, which employ a unique assortment of proteins dubbed the glideosome, at the heart of which is a class XIV myosin motor. Actin and myosin also drive the gliding locomotion of raphid diatoms (Bacillariophyceae) as well as the intriguing form of gliding displayed by the spindle-shaped cells of the primitive colonial protist Labyrinthula. Chlamydomonas and other flagellated protists are also able to abandon their more familiar swimming locomotion for gliding, during which time they recruit a motility apparatus independent of that driving flagellar beating.
Membrane-bound organelles move bidirectionally along microtubules in the freshwater ameba, Reticulomyxa. We have examined the nucleotide requirements for transport in a lysed cell model and compared them with kinesin and dynein-driven motility in other systems. Both anterograde and retrograde transport in Reticulomyxa show features characteristic of dynein but not of kinesin-powered movements: organelle transport is reactivated only by ATP and no other nucleoside triphosphates; the Km and Vmax of the ATP-driven movements are similar to values obtained for dynein rather than kinesin-driven movement; and of 15 ATP analogues tested for their ability to promote organelle transport, only 4 of them did. This narrow specificity resembles that of dynein-mediated in vitro transport and is dissimilar to the broad specificity of the kinesin motor (Shimizu, T., K. Furusawa, S. Ohashi, Y. Y. Toyoshima, M. Okuno, F. Malik, and R. D. Vale. 1991. J. Cell Biol. 112: 1189-1197). Remarkably, anterograde and retrograde organelle transport cannot be distinguished at all with respect to nucleotide specificity, kinetics of movement, and the ability to use the ATP analogues. Since the "kinetic fingerprints" of the motors driving transport in opposite directions are indistinguishable, the same type of motor(s) may be involved in the two directions of movement.
We describe the use of interferometric microscopy coupled with a novel application of Sénarmont compensation for detecting and quantifying the distribution of dry matter in cultured cells. In conjunction with video techniques and digital image processing, a two-dimensional, calibrated map of the dry mass distribution in an isolated cell can be obtained and digitally recorded. We have called the technique Digitally Recorded Interferometric Microscopy with Analyser Shift (DRIMAS). The method greatly facilitates the automatic recognition of cells by computer. Recorded time-lapse sequences can be used to establish a database of the growth and motility of specific cells in given experimental conditions.
Databases of this type can be analysed to reveal the patterns of growth and locomotory behaviour of individual cells. We describe a systematic method of obtaining parameters of cell size, shape, spreading, intracellular motility and translocation. Autocorrelations and cross-correlations between these parameters can be detected and quantified using time series analysis, revealing potential cause/effect relationships in the mechanisms of growth and motility.
Besides characterizing the overall pattern of cell behaviour, these data can also yield information about the instantaneous pattern of intracellular motility. We describe the use of finite element analysis to reveal the dynamics of the intracellular transport of dry matter. This yields the pattern of the minimum flow of dry matter required to account for the changes in its distribution. Most of this flux is not associated with the movement of visible structures and possibly represents the transport of dissociated components of the cytoskeleton. In chick heart fibroblasts, surprisingly high velocities of nearly 2·0 μm s−1 were detected during the period of increased motility following tail detachment. The total kinetic energy associated with the dry mass flux is a single parameter, which characterizes the instantaneous motility of the cell. We found that the kinetic energy of intracellular motility can be several hundred times greater than the kinetic energy of translocation. Kinetic energy may prove to be a very informative single measure of intracellular motility for assessing the effects of malignant transformation, genetic manipulations, and other experimental treatments on the locomotory machinery of the cell.
The locomotory patterns of Protozoa have been compared, in an attempt to draw a general picture of how these protists solved the problem of relating properly with their environment. Locomotion seems to have evolved according to a progressive complexification not only of the engines (from prokaryotes to eukaryotes) but also of the systems that control how they work (different beating patterns of the eukaryotic flagella and cilia under a sophisticated control of the electrophysiological potentials of the cell). Among ci‐liates, the Hypotrichida with their ciliary engines (cirri) and electric control (cell membrane more complex and sophisticated), have reached the highest peak of specialization. The evolutionary leap to a multicellular organization typical of Metazoa did not play any relevant role in locomotion: it was only with the appearance of the most complex phyla (Mollusca, Annelida) that a new locomotory asset was gained (muscular layers or bundles) and perfected by the coupling of muscles and levers (art), especially by Arthropoda and Chordata.
Acanthamoeba castellanii (CCAP 1534/3) was found to bind avidly the common soil bacterium Pseudomonas fluorescens. This adhesion was mediated not by pili nor by the general bacterial surface but by the polar flagella. Because of the nature of the flagellar rotary motor, the cell bodies of the attached bacteria could be seen rotating clearly. While initially bacterial binding occurred uniformly over the cell membrane of Acanthamoeba. the bacteria were soon swept posteriorly to form a cap and either endocytosed or sloughed off, still agglutinated by their flagella. Such capped amoebae would not bind Pseudomonas if challenged immediately, indicating a depletion of flagella-binding sites. The bacteria could not bind to amoebae pre-treated with concanavalin A (Con A) even after the lectin had been capped to the uroid. However, capping of flagella-binding sites did not co-cap all the Con A-binding sites on the surface of the amoeba. The flagella-binding sites were not affected by pre-treatment with Pronase (1 mg ml-1) or znti-Acanthamoeba surface antibody. Proteus mirabilis also bound avidly by its flagella to Acanthamoeba and, furthermore, competition experiments suggested that Proteus and Pseudomonas adhere to a common surface site on the amoeba. The presence of sites on the cell membrane of A. castellanii that are specific for flagellin would enhance strongly the adsorption of motile bacteria prior to endocytosis. This would represent an excellent feeding strategy for a soil-dwelling phagotroph.
Colonies of Labyrinthula, a colonial marine protist, expand by protrusive movements of the specialized slimeways. The movements recorded in time-lapse films are of two types – filopodial and lamellipodial – and occur at rates equivalent to those of cell translocation.
Evidence is presented that Ca2+ regulates the contraction of the actomyosin system of filaments present in the slimeways of Labyrinthula. In glycerinated models or in colonies exposed to ionophore A23187 contraction is evidenced by the occurrence of periodic contractions of the slimeways, giving them the appearance of strings of beads. Glycerinated slimeways contract on the addition of Ca2+ and ATP while slimeways provided with ionophore A23187 contract on addition of Ca2+ alone. The concentration required is 1.1 × 10−7 M Ca2+ while concentrations of 6.2 × 10−8 or lower were ineffective. Rates of contraction were measured in time-lapse films which provide evidence that contractions and beading occur everywhere in the slimeway system. When beading occurs, the 6-nm filaments transform from an array of parallel single filaments into an interwoven meshwork.
We have identified by pyroantimonate-OsO4 fixation, as possible Ca2+ reservoirs, deposits of Ca2+ in bothrosomes – structures through which cell secretions pass into the slimeways. The electron-dense deposits are located at the base of the bothrosome and disappear after incubation with EGTA. We propose that the translocation of cells as well as the movements of slimeways may be regulated by the cells through the local measured liberation of Ca2+ from the bothrosome where it is sequestered.
A myosin-like protein (Mr 175,000) was detected in the parasitic protozoan Gregarina blaberae, by both immunofluorescence and immunoblotting of one- and two-dimensional electrophoresis gels using anti-myosin antibodies. This protein was present in the trophozoite ghost but not in the cytoplasmic extract, nor in extract from the sexual stage, suggesting a protein-stage-dependent expression. the protein tightly bound to the cortical membranes was insoluble at low ionic strength, or in detergent solutions, but could be extracted from Gregarina ghosts by 6 M urea in high ionic strength solution (0.5 M NaCI) and in the presence of reducing agents (20 mM DTT). the protein was localized by indirect immunofluorescence in the cortex of the epimerite, in the fibrillar disc (the socalled septum) separating the proto- and the deutomerite segments, in the contractile ring or sphincter at the top of the protomerite, and as longitudinal lines underlying the G. blaberae epicyte folds. the presence of both actin-like and myosin-like proteins would be consistent with a role in gliding and other cell motility processes of this parasite.
The amoeboid locomotion of the soil protozoon Naegleria gruberi has been studied using reflexion-interference microscopy. Two types of contact are made with a planar glass substrate. One, formed at a considerable distance from the substrate in deionized water (congruent to 100 nm) has been termed 'associated contact' and usually involves a considerable surface area (of the order of 100 micrometer2), i.e. about a third of the cell profile. From this broad platform filopodia are produced which form close contacts ('focal contacts'). In locomotion the area of associated contact is very mobile, in contrast to the focal contacts which, once established, are stable. Focal contact sites are left behind on the glass surface ('footprints') when the amoeba moves away. The cell-substrate gap in the associated contact is greatly affected by the ionic strength of the medium and particularly the valency of the cation component. This suggests that long-range forces of attraction play an important role in keeping the amoeba close to a substrate and thus allow the production of filopodia from the ventral surface to form focal contacts.
A high-speed supernatant extract was obtained from infective oocysts of Eimeria tenella homogenised in a sucrose-low ionic strength buffer. Immunoblotting showed this soluble, micropore-filtered preparation (designated E1) to be rich in actin. E1 underwent superprecipitation on addition of ATP but not its non-hydrolysable analogue AMP.PMP--behaviour typical of an actomyosin solution. The superprecipitate fluoresced strongly in the presence of rhodamine-phalloidin (indicative of the presence of F-actin) and electron microscopy of negatively-stained preparations of this flocculent matter confirmed the abundance of filamentous material within it. This is the first demonstration of a functional actomyosin isolated from a member of the economically important phylum Apicomplexa.
Actin was identified in the motile trophozoites of Gregarina by indirect immunofluorescence microscopy. Ultrastructurally actin was found by immunogold labelling to be associated with the internal cytomembranes of the ectoplasmic folds and the general ectoplasmic region below the folds. Western blotting with antibodies to actin identified polypeptides with molecular weights around 43kD and 98kD in non-ionic detergent extracted trophozoites. The possibility of two distinct forms of cytoplasmic actin (43kD and 98kD) was considered.
Gregarines, parasitic protozoa of invertebrates, possess a highly differentiated cell surface, with three cortical membranes and associated structures. Transmission electron microscopy and freezefracture reveal the presence of two cytomembranes lying uniformly under the plasma membrane. The density and the distribution of the intramembraneous particles (IMPs) in the plasma membrane of Gregarina blaberae are similar to those reported for other eukaryotic cells. The IMP density is lower in the cytomembranes than in the plasma membrane. The distribution of IMPs in the different fracture faces of the two cytomembranes suggests that they are in topological continuity, forming either side of a flattened vesicle or cisterna. The sizes of the cytomembrane IMPs show a high variability. The nature of the IMPs, both for the plasma membrane and the cytomembranes, is discussed with regard to the integral proteins and glycoproteins of the ghost.
The cell surface of G. blaberae exhibits numerous longitudinal folds with three types of cortical membrane-associated structures: 12 nm filaments, an internal lamina, and homogeneous structures described as ‘rippled dense structures’. The 12 nm filaments, running under the cytomembranes along the longitudinal axis of each fold, exhibit the properties of intermediate filaments. Their distribution in mature cells and during the growth process suggests a participation in cell surface morphogenesis. The internal lamina, also localized under the cytomembranes, would stabilize each fold and assure a scaffolding function between the numerous folds. The rippled dense structures, settled on the external cytomembrane, show a regular distribution at the top of each fold. The membrane-associated structures are discussed with regard to the gliding movement mechanism.
Russell D. G. and Sinden R. E. 1982. Three-dimensional study of the intact cytoskeleton of coccidian sporozoites. International Journal for Parasitology12: 221–226. Critical-point dried sporozoites of Eimeria acervulina were studied by transmission electron microscopy. The three dimensional organisation of the microtubular cytoskeleton and its close association with the trimembraneous pellicle was visualised. The information revealed on the structure and substructure of the sporozoite cytoskeleton and its importance to the understanding of coccidian motility were discussed together with other possible applications of this technique.
The bacterial pathogen Listeria monocytogenes displays the remarkable ability to reorganize the actin cytoskeleton within host cells as a means for promoting cell-to-cell transfer of the pathogen, in a manner that evades humoral immunity. In a series of events commencing with the biosynthesis of the bacterial surface protein ActA, host cell actin and many actin-associated proteins self-assemble to form rocket-tail structures that continually grow at sites proximal to the bacterium and depolymerize distally. Widespread interest in the underlying molecular mechanism of Listeria locomotion stems from the likelihood that the dynamic remodeling of the host cell actin cytoskeleton at the cell's leading edge involves mechanistically analogous interactions. Recent advances in our understanding of these fundamental cytoskeletal rearrangements have been achieved through a clearer recognition of the central role of oligo-proline sequence repeats present in ActA, and these findings provide a basis for inferring the role of analogous host cell proteins in the force-producing and position-securing steps in pseudopod and lamellipod formation at the peripheral membrane.