DOCSLIB.ORG

Extraction Ofan Actin-Like Protein from the Prokaryote Mycoplasma

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Extraction Ofan Actin-Like Protein from the Prokaryote Mycoplasma Proc. Natl. Acad. Scd. USA Vol. 74, No. 9, pp. 4041-4045, September 1977 Microbiology Extraction of an actin-like protein from the prokaryote Mycoplasma pneumonlae * (gliding motility/electron microscopy) HAROLD C. NEIMARK Department of Microbiology and Immunology, State University of New York, Downstate Medical Center, Brooklyn, New York 11203 Communicated by Lewis Thomas, June 29,1977 ABSTRACT An actin-like protein has been identified in pneumoniae, an important cause of respiratory disease in cell extracts from the prokaryote Mycoplasma pneumoniae. children and young adults (14), was selected for examination This protein bears a striking resemblance to actin from verte- because it posse gliding motility (8), maintains a specific cell brates: (i) the solubility of the protein during isolation is anal- and has an ogous to that of actin bound to myosin (soluble in high ionic shape, accessible cell membrane. strength salt solution and insoluble at low ionic strength), (ii) sodium dodecyl sulfate treatment of the partially purified M. MATERIALS AND METHODS pneumoniae extract produces a protein with an electrophoretic mobility very close to that of vertebrate actin in sodium dodecyl Organisms and Growth Conditions. A recently isolated sulfate/polyacrylamide gels, (iii) treatment of preparations with virulent strain of M. pneumoniae received from W. Clyde was ATP-Mg2+ allows separation of long curvilinear filaments, 5-6 used for this study. Strains passaged in the laboratory, however, nm wide, that closely resemble eukaryotic filamentous actin, and (iv) the prokaryotic filamentous actin binds vertebrate are known to retain motility (8). The media and growth con- heavy meromyosin fragments to form hybrid complexes with ditions were essentially as described (15) except that 20% horse the characteristic shape of periodic repeating arrowheads, and serum (unheated) was used. The strain was adapted to grow also no heavy meromyosin is bound in the presence of ATP. in medium containing 3% PPLO serum fraction (Difco) instead of horse serum. The final glucose concentration was 0.5%. Proteins similar to the muscle contractile proteins actin and Penicillin (300 units/ml) was added in some instances. Cell myosin occur in a wide variety of nonmuscle cells, where they proteins were labeled by growth in media supplemented with are believed to function in the fundamental cellular processes [FsSimethionine (New England Nuclear Corp.) (specific ac- of motility and maintainance of cell shape. (for a review see ref. tivity, 295 Ci/mmol; 5 ,uCi/ml). Organisms were collected 1). In nonmuscle cells, the contractility and motility processess when a confluent layer of adherent cells formed on the bottom include amoeboid movement and cytoplasmic streaming (2), of the flasks. Cells were scraped off the flasks, centrifuged phagocytosis (3), and cytokinesis (4) as well as clot retraction (23,000 X g for 45 min), and washed once with 0.145 M by platelets (5). The widespread occurrence of actomyosin-like NaCI/0.02 M potassium phosphate buffer, pH 7.5. proteins in organisms representing broadly divergent eukaryotic Preparation of the Actin-Like Protein. The isolation pro- phyla suggests that these proteins provide a general mechanism cedure was based on those developed for vertebrate platelet for cell motility and contractility and that muscle contraction actomyosin (16,17). All steps were carried out at 4°. The packed may be but a specialized case of-a very general form of cell cell pellet was resuspended in 4 volumes of 0.6 M KCI/0.015 motility (1). M Tris-HCI, pH 8.5, 25 gl of butanol was added for each mil- Among the prokaryotes are various bacteria that lack flagella liliter of cell suspension, and the cells were extracted by stirring or other recognized organelles for locomotion but are never- for 16 hr. The supernatant (SI) was separated by centrifugation theless capable of movement. Organisms in the orders Myxo- (30,000 X g for 1 hr), made to approximately 0.07 M KCI by bacterales and Cytophagales as well as most cyanobacteria diluting with 6 volumes of water, and adjusted to pH 6.3 by (blue-green algae) display a directional gliding motility on solid addition of 0.125 M sodium acetate buffer, pH 4.9. After 40 min surfaces (6, 7). The molecular basis for movement in these at 40, the precipitate (P2) containing the actin-like protein was bacteria is presently unknown. Indeed, the precise basis for collected by centrifugation (30,000 X g for 30 min) and dis- movement of bacterial flagella also is not understood. solved in a small volume of 0.6 M KCI (S3). For some prepara- Certain members of the order Mycoplasmatales are also ca- tions, solutions contained 1 mM sodium sulfite as a proteinase pable of gliding motility (8, 9), and the recently discovered inhibitor (R. Siemankowski and P. Dreizen, unpublished data) spiral-shaped mycoplasmas exhibit flexing movement (10, 11). and 5 mM dithiothreitol. Preparations were usually used within In contrast to most bacteria, the mycoplasmas lack cell walls yet 1 week. still are able to maintain specific cell shapes. In addition, my- Gel Electrophoresis and Autoradiography. Sodium dodecyl coplasmas are notable for possessing the smallest genomes of sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was any organisms known to be capable of growth on cell-free carried out as described by Laemmli (18) or by a modification media (12, 13). The relative structural simplicity of the myco- of the procedure of Weber and Osborn (19). For the latter, gel plasmas suggested that the basis for motility in these organisms buffer was either 0.075 M Tris-acetate, pH 7.9 (20), or 0.02 M could be sought directly at the molecular level and might be phosphate, pH 7.1, containing 0.1% NaDodSO4 and 0.1% 2- a contractile protein, possibly resembling actomyosin. A search mercaptoethanol; 8% gels cast the day before use were prerun for a contractile protein was therefore undertaken. Mycoplosma Abbreviations: NaDodSO4, sodium dodecyl sulfate; HMM, heavy The costs of publication of this article were defrayed in part by the meromyosin. payment of page charges. This article must therefore be hereby marked * A preliminary report of this work was presented at the Annual "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate Meeting of the American Society for Microbiology, 1976, p. 61, ab- this fact. stract D 62. 4041 Downloaded by guest on September 27, 2021 4042 Microbiology: Neimark Proc. Nati. Acad. Sci. USA 74 (1977) at 5 mA per gel for 30 min. Samples dissolved in 0.6 M KCI were dialyzed against 1% NaDodSO4/1% 2-mercaptoethanol before sample preparation. Protein samples were dissolved in gel buffer containing 1% NaDodSO4 and 1% 2-mercaptoethanol and denatured by heating in boiling water for 3 min. Gels were ... _ sliced, dried on cellophane film, and autoradiographed as de- ..f.. scribed (21). Preparation of Mycoplasma Filamentous Actin-Like Protein. The actin-like protein was obtained in a sedimentable, .. filamentous form by treatment of the preparation with ATP and Mg2+. Samples of once-precipitated and redissolved my- _r. coplasma actin preparations in 0.6 M KCI were brought to 5 ._-g'i.J.:i.' _ mM ATP, 5 mM MgCl2, and 3 mM dithiothreitol in 8 mM ..... ::_;:_ Tris-HCl buffer, pH 7.5; ATP and Mg2+ were omitted from - controls. The samples were then centrifuged at 143,000 X g for .:. :..:: :..:.i7 ._ _*:_. 1 hr, and the pellets werestaken up in a small volume of 0.1 M :: ,: _ KCI/5 mM imidazole-HCI buffer, pH 7.5, and dialyzed against the same buffer. The supernatants and pellet preparations were examined by gel electrophoresis. Pellet preparations were also _ examined by electron microscopy. I _ _ Reaction of Actin with Heavy Meromyosin (HMM), and Electron Microscopy. Pellet preparations taken up in and di- alyzed against 0.1 M KCI/5 mM imidazole.HCI buffer, pH 7.5, were mixed with rabbit muscle HMM (approximately 60 ug A B C D or 100 ,ug/ml of reaction mixture) in buffer or in buffer con- taining 10 mM ATP. Electron microscopy was done essentially FIG. 1. _NaDodSO4/polyacrylamide gels (18) of extracts of M. as described by Huxley (22). Samples placed on carbon-coated pneumoniae actin. (A) Rabbit muscle actin. (B) M. pneumoniae extract (P2) containing actin and other proteins insoluble in low ionic Formvar-covered grids were stained with 1% uranyl acetate strength salt solution (0.07 M KCl). (C) Soluble proteins remaining and examined in a Siemens Elmiskop IA. in low-salt solution after removal by centrifugation of insoluble pro- Biochemical Determinations. ATPase (ATP phosphohy- teins (supernatant was dialyzed, lyophilized, and reconstituted in gel drolase, EC 3.6.1.3) activity was determined as described (18) buffer). Note low protein content in region ofactin band (arrow). (D) by measuring release of inorganic phosphate in reaction mix- Proteins (S3) soluble in high ionic strength salt solution (0.6 M KCl) tures incubated at 370 for 30 min. Protein concentration was after precipitation in low-salt solution. a, Actin band; d, position of estimated by the method of Lowry et al. (23). the tracking dye. Materials. Chicken muscle myosin, rabbit muscle myosin (24), and column-purified rabbit muscle myosin (25) were gifts than 15 nmol of inorganic phosphate released per mg of protein from E. McGowan, P. Dreizen, and C. Moos, respectively. per 30 min) could be demonstrated in the partially purified Rabbit muscle actin was prepared by the method of Spudich preparation. and Watt (26) and HMM was prepared by the procedure of The actin content of M. pneumoniae, estimated by densi- Lowey et al. (27) from partially purified myosin (24) or col- tometry of Coomassie brilliant blue-stained gels, was near 6% No actin of the total stainable proteins soluble in NaDodSO4 sample umn-purified myosin (25).
Load more

Recommended publications
  • A Viable Therapy Against Metastatic Breast Cancer
    Graduate Theses, Dissertations, and Problem Reports 2017 Dual Targeting of the Mesenchymal and Amoeboid Pathways: A Viable Therapy against Metastatic Breast Cancer Brandon C. Jones Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Jones, Brandon C., "Dual Targeting of the Mesenchymal and Amoeboid Pathways: A Viable Therapy against Metastatic Breast Cancer" (2017). Graduate Theses, Dissertations, and Problem Reports. 5905. https://researchrepository.wvu.edu/etd/5905 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Dual Targeting of the Mesenchymal and Amoeboid Pathways: A Viable Therapy against Metastatic Breast Cancer Brandon C. Jones Dissertation submitted to the School of Medicine at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry & Molecular Biology Elena Pugacheva, Ph.D., Chair J. Michael Ruppert, M.D., Ph.D. Maxim Sokolov, Ph.D. Michael Schaller, Ph.D. Scott Weed, Ph.D.
    [Show full text]
  • Membrane Potentials in Amoeba Proteus
    J. Exp. Biol. (1966), 45, 251-267 251 With 10 text-figures Printed in Great Britain MEMBRANE POTENTIALS IN AMOEBA PROTEUS BY M. S. BINGLEY R.A.F. Institute of Aviation Medicine, Farnborough, Hants. {Received 16 February 1966) INTRODUCTION The possibility that the initiation of pseudopods together with the direction of cytoplasmic streaming may be induced by local depolarization of the membrane has been advanced from time to time for many years past. But is it difficult to find a direct statement to this effect in the literature. Amici (1818) suggested an electrical theory to account for streaming in plant cells and more recently Kitching (1961) considers the possibility of depolarization of the cell surface initiating contraction. When he discussed Hahnert's work (Hahnert, 1932) on the response of amoebae to electricity he pointed out that movement of the pseudopods towards the cathode may be an enhancement of 'local currents associated with local excitation'. It is one thing to put forward a hypothesis but another to obtain convincing measurements which are free from the suspicion of doubt and artifact. Membrane potentials were first seriously measured in Amoeba proteus by Telkes in 1931. Using large electrodes, she obtained low values of membrane potential but produced extremely valuable information on various depolarizing agents such as potassium and sodium chloride. Buchtal & Peterfi (1936) obtained low values of potential for A. proteus. Wolfson (1943) produced convincing values for membrane potential in Chaos chaos and, using electrodes of large diameter, he obtained potentials as high as workers using more modern micro- electrodes. More recently Riddle (1962) working on PeUomyxa caroHnensis recorded values of — 90 mV.
    [Show full text]
  • Cytoplasmic Streaming and Microtubules in the Coenocytic Marine Alga, Caulerpa Prolifera
    J. Cell Sci. 2, 465-472 (1967) 465 Printed in Great Britain CYTOPLASMIC STREAMING AND MICROTUBULES IN THE COENOCYTIC MARINE ALGA, CAULERPA PROLIFERA D. D. SABNIS AND W. P. JACOBS Biology Department, Princeton University, Princeton, N.J., U.S.A. SUMMARY Two distinct patterns of cytoplasmic streaming in the leaf of Caulerpa prolifera are described. Broad, longitudinally running, two-way streams are restricted to the endoplasm of one leaf surface. Also present are large numbers of narrow, two-way streams that coil helically through- out the endoplasm surrounding the central vacuole. Numerous unique bundles of aggregated, evenly spaced, oriented microtubules are distributed within the inner cytoplasm some distance from the cell wall. Cortical microtubules, as described for other plant material, have been only very infrequently encountered in Caulerpa and appear to be sparsely distributed. Apart from the prominent bundles of oriented microtubules, no other significant ultrastructural differences were noted between the stationary ectoplasm and streaming endoplasm. The possible cyto- skeletal role of the oriented microtubules in the development and maintenance of asymmetries in organ differentiation is discussed in relation to their direct or indirect influence on the directional migration of cytoplasmic components. INTRODUCTION Although there have been numerous reports of the occurrence of microtubular and microfibrillar elements in the cytoplasm of a variety of cell types, only a limited number of publications has described these structures in algal cells (Berkaloff, 1966; Nagai & Rebhun, 1966). The possible functions of cytoplasmic microtubules and microfilaments in the plant cell have been the subjects of some considerable con- jecture and controversy. Microtubules have been considered to play a role in the laying down of secondary walls in differentiating tissue (Wooding & Northcote, 1964), and cell-plate formation in dividing cells (Pickett-Heaps & Northcote, 1966).
    [Show full text]
  • Emergence of Self-Organized Amoeboid Movement in a Multi-Agent Approximation of Physarum Polycephalum
    Emergence of Self-Organized Amoeboid Movement in a Multi-Agent Approximation of Physarum polycephalum Jeff Jones and Andrew Adamatzky Centre for Unconventional Computing, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK. [email protected] , [email protected] Abstract: The giant single-celled slime mould Physarum polycephalum exhibits complex morphological adaptation and amoeboid movement as it forages for food and may be seen as a minimal example of complex robotic behaviour. Swarm computation has previously been used to explore how spatio- temporal complexity can emerge from, and be distributed within, simple component parts and their interactions. Using a particle based swarm approach we explore the question of how to generate collective amoeboid movement from simple non-oscillatory component parts in a model of P. polycephalum . The model collective behaves as a cohesive and deformable virtual material, approximating the local coupling within the plasmodium matrix. The collective generates de-novo and complex oscillatory patterns from simple local interactions. The origin of this motor behaviour is distributed within the collective rendering is morphologically adaptive, amenable to external influence, and robust to simulated environmental insult. We show how to gain external influence over the collective movement by simulated chemo-attraction (pulling towards nutrient stimuli) and simulated light irradiation hazards ( pushing from stimuli). The amorphous and distributed properties of the collective are demonstrated by cleaving it into two independent entities and fusing two separate entities to form a single device, thus enabling it to traverse narrow, separate or tortuous paths. We conclude by summarising the contribution of the model to swarm based robotics and soft-bodied modular robotics and discuss the future potential of such material approaches to the field.
    [Show full text]
  • Protozoa : Locomotion and Nutrition
    Protozoa : locomotion and Nutrition Lecturer : P. V. Deokar Department of Zoology R.K.M.M. Ahmednagar Locomotion in Protozoa • The following points highlight the three main types of locomotion exhibited by protozoans. The types of locomotion are: • 1. Amoeboid Movement • 2. Flagellar Movement • 3. Ciliary Movement. Protozoans: Type of Locomotion # 1. Amoeboid Movement: • movement of the animal is made by the throwing of pseudopodium, called amoeboid movement • In the direction of movement of Amoeba a new pseudopodium is formed and the pseudopodium at the opposite side gradually disappears. • Types of pseudopodia: • According to form, structure and activity four different kinds of pseudopodia are recognised • These are: • (a) Lobopodium • (b) Filopodium • (c) Reticulopodium or Rhizopodium • (d) Axopodium or Actinopodium (a) Lobopodium [Gk. lobes = lobe; podium = foot]: • It is a short, finger or tongue-like projection which is accompanied by a flow of endoplasm and ectoplasm. • The pseudopodium is broad with rounded or blunt tips. • The ectoplasmmic area is distinctly clear, called the hyaline cap. • It is the characteristic of many amoebas such as Amoeba. (b) Filopodium [L.filo = a thread; podium = foot]: • The filopodium is a slender, thread-like or filamentous projection. • It is formed by the ectoplasm alone and without a hyaline cap. • The filaments are narrow and may be branched but do not anastomose, Filopodium is the characteristic in Filosea (e.g., Euglypha ). (c) Reticulopodium or Rhizopodium [L. reticulos = a net, podium = foot]: • Similar in structure to that of filopodium but the branches anastomose. • The numerous branched and anastomosed pseudopodia form a dense network, help primarily in capturing the prey and the secondary function is locomotion.
    [Show full text]
  • Differential Organelle Movement on the Actin Cytoskeleton in Lily Pollen Tubes
    Cell Motility and the Cytoskeleton (2007) Differential Organelle Movement on the Actin Cytoskeleton in Lily Pollen Tubes Alenka Lovy-Wheeler,1 Luis Ca´rdenas,2 Joseph G. Kunkel,1 and Peter K. Hepler1* 1Department of Biology and Plant Biology Graduate Program, Morrill Science Center III, University of Massachusetts, Amherst, Massachusetts 2Departamento de Biologı´a Molecular de Plantas, Instituto de Biotechnologı´a, Cuernavaca, Morelos, Me´xico We have examined the arrangement and movement of three major compart- ments, the endoplasmic reticulum (ER), mitochondria, and the vacuole during oscillatory, polarized growth in lily pollen tubes. These movements are de- pendent on the actin cytoskeleton, because they are strongly perturbed by the anti-microfilament drug, latrunculin-B, and unaffected by the anti-microtubule agent, oryzalin. The ER, which has been labeled with mGFP5-HDEL or cyto- chalasin D tetramethylrhodamine, displays an oscillatory motion in the pollen tube apex. First it moves apically in the cortical region, presumably along the cortical actin fringe, and then periodically folds inward creating a platform that transects the apical domain in a plate-like structure. Finally, the ER reverses its direction and moves basipetally through the central core of the pollen tube. When subjected to cross-correlation analysis, the formation of the platform precedes maximal growth rates by an average of 3 s (35–408). Mitochondria, labeled with Mitotracker Green, are enriched in the subapical region, and their movement closely resembles that of the ER. The vacuole, labeled with car- boxy-dichlorofluorescein diacetate, consists of thin tubules arranged longitudi- nally in a reticulate network, which undergoes active motion.
    [Show full text]
  • On the Physiology of Amoeboid Movement
    ON THE PHYSIOLOGY OF AMOEBOID MOVEMENT. IV.—THE ACTION OF MAGNESIUM. BY C. F. A. PANTIN. {The Marine Biological Laboratory, Plymouth?) (Received January 6th, 1926.) CONTENTS 1. Maintenance of the cell-surface . 297 6. The action of Ce'" in the pres- 2. The action of barium . 300 ence of Ca" .... 306 3. The action of cerium . 301 4. Action of Mg" in the presence 7. Discussion 306 of Ca" 302 8. Summary 310 5. Interaction of the ions of sea- 9. References 311 water 303 THE previous paper of this series (Pantin, 1926) described the action of certain ions upon a species of marine amoeba, and especially the relation of calcium to amoeboid movement. The same paper detailed the method of preparing isotonic salt solutions and of determining their effect on amoeboid movement. The essential feature is that the average velocity is taken as a measure of the effect of a solution upon the power of movement, apart from other effects produced upon the cell. Unless otherwise stated, the CH of the solutions was maintained at /H 7.0 to 7.2. 1. Maintenance of the cell-surface. It was shown previously that whereas cytolysis occurred rapidly in pure isotonic NaCl or KCl (at about />H 7), amoebae remained alive for a longer time in isotonic CaCl= and MgClj. Movement was inhibited in these solutions, but reversibly, for, provided immersion had been brief, recovery occurred on return to natural sea-water. In mixtures of two salts, it was found that movement only occurred if Ca were 297 C. F. A.
    [Show full text]
  • Hydrodynamic Flow in the Cytoplasm of Plant Cells
    Journal of Microscopy, Vol. 231, Pt 2 2008, pp. 274–283 Received 14 June 2008; accepted 28 March 2008 Hydrodynamic flow in the cytoplasm of plant cells A. ESSELING-OZDOBA∗,‡,D.HOUTMAN†, A.A.M. VAN LAMMEREN∗, E. EISER† & A.M.C. EMONS∗ ∗Laboratory of Plant Cell Biology, Department of Plant Sciences, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands †van’t Hoff Institute for Molecular Sciences (HIMS), Universiteit van Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands ‡Current address: Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences (NCMLS), Geert Grooteplein zuid 28, 6500 HB Nijmegen, The Netherlands Key words. Cytoplasmic streaming, FRAP, GFP, hydrodynamic flow, lipid vesicles, micro-injection, tobacco BY-2 suspension cultured cells, Tradescantia virginiana. Summary that synthetic lipid (DOPG) vesicles and ‘stealth’ vesicles with PEG phospholipids moved in the cytoplasm. Plant cells show myosin-driven organelle movement, called cytoplasmicstreaming.Solublemolecules,suchasmetabolites do not move with motor proteins but by diffusion. However, is all of this streaming active motor-driven organelle transport? Introduction Our recent simulation study (Houtman et al., 2007) shows The cytoplasm of eukaryotic cells consists of all cell material that active transport of organelles gives rise to a drag in the between the nucleus and the plasma membrane and cytosol, setting up a hydrodynamic flow, which contributes to contains membrane-bounded structures, organelles, which a fast distribution of proteins and nutrients in plant cells. Here, are embedded in the cytosol consisting of water, salts and we show experimentally that actively transported organelles organic molecules, including sugars, proteins and many produce hydrodynamic flow that significantly contributes to enzymes that catalyze reactions.
    [Show full text]
  • Pressure Sensing Through Piezo Channels Controls Whether Cells Migrate with Blebs Or Pseudopods
    Pressure sensing through Piezo channels controls whether cells migrate with blebs or pseudopods Nishit Srivastavaa,c,d, David Traynorb,1, Matthieu Pielc,d, Alexandre J. Kablaa, and Robert R. Kayb,2 aDepartment of Engineering, University of Cambridge, Cambridge CB2 1PZ, United Kingdom; bLaboratory of Molecular Biology, Medical Research Council, Cambridge CB20QH, United Kingdom; cInstitut Curie, Université Paris Sciences et Lettres, CNRS, UMR 144, 75005 Paris, France; and dInstitut Pierre-Gilles de Gennes, Université Paris Sciences et Lettres, 75005 Paris, France Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved December 24, 2019 (received for review April 4, 2019) Blebs and pseudopods can both power cell migration, with blebs Dictyostelium amoebae move through varied environments often favored in tissues, where cells encounter increased mechan- during their life cycle. As single cells, they hunt bacteria through ical resistance. To investigate how migrating cells detect and the interstices of the soil, and when starved and developing, they respond to mechanical forces, we used a “cell squasher” to apply chemotax to cyclic AMP and move in coordinated groups that uniaxial pressure to Dictyostelium cells chemotaxing under develop into stalked fruiting bodies, with cell sorting playing a soft agarose. As little as 100 Pa causes a rapid (<10 s), sustained key role (33, 34). We found previously that Dictyostelium cells shift to movement with blebs rather than pseudopods. Cells are prefer pseudopods when moving under buffer, but blebs under a flattened under load and lose volume; the actin cytoskeleton is stiff agarose overlay (35). In both cases, the cells move on the reorganized, with myosin II recruited to the cortex, which may same glass substratum, but under agarose they must also break pressurize the cytoplasm for blebbing.
    [Show full text]
  • On the Physiology of Amoeboid Movement.*
    ON THE PHYSIOLOGY OF AMOEBOID MOVEMENT.* II.—THE EFFECT OF TEMPERATURE. BY C. F. A. PANTIN. (Assistant Physiologist at the Marine Biological Laboratory, Plymouth.) IT was shown in the first paper of this series1S that amoeboid activity was affected by certain changes in the conditions of the medium in the same way as certain other forms of con- tractility. This suggested that some fundamental mechanism of contractility was similar in all these cases. Like the majority of biological processes, contractility is affected in a characteristic manner by temperature, and if there really is a fundamental similarity between amoeboid and other forms of contractility, the effect of temperature should be similar in both cases. i. Material, Methods, etc. Marine Amoebae, obtained from the laboratory tanks, were used for the experiments. A full description of the Amoebae, their habitat and mode of progression, has been given in a previous paper.16 The Amoebae were of the " Umax " form, that is, they progressed by the continuous protrusion of a single anterior pseudopodium. Two species were used, one relatively "fluid" (type A), and one with relatively "solid," consistent, protoplasm (type B). In the absence of external stimuli the Amoebae tend to move in a straight line. And if the conditions of the medium are constant the velocity of an individual Amoeba is constant to within from i per cent, to 5 per cent, for at least twenty-four hours. This holds true even if the conditions of the medium have been varied and then brought back to the original state, provided the variation has not been great enough to damage the organism.
    [Show full text]
  • Mechanics and Control of the Cytoskeleton in Amoeba Proteus
    Mechanics and control of the cytoskeleton in Amoeba proteus Micah Dembo Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 ABSTRACT Many models of the cyto- accounts for the kinematics of the Several dynamical factors are crucial skeletal motility of Amoeba proteus can cytoskeleton: the detailed velocity field to the success of the minimal model be formulated in terms of the theory of of the forward flow of the endoplasm, and are likely to be general features of reactive interpenetrating flow (Dembo the contraction of the ectoplasmic cytoskeletal mechanics and control in and Harlow, 1986). We have devised tube, and the inversion of the flow in the amoeboid cells. These are: a constitu- numerical methodology for testing such fountain zone. The model also gives a tive law for the viscosity of the contrac- models against the phenomenon of satisfactory account of measurements tile network that includes an automatic steady axisymmetric fountain flow. The of pressure gradients, measurements process of gelation as the network simplest workable scheme revealed by of heat dissipation, and measurements density gets large; a very vigorous such tests (the minimal model) is the of the output of useful work by amoe- cycle of network polymerization and main preoccupation of this study. All ba. Finally, the model suggests a very depolymerization (in the case of A. parameters of the minimal model are promising (but still hypothetical) con- proteus, the time constant for this reac- determined from available data. Using tinuum formulation of the free boundary tion is z12 s); control of network con- these parameters the model quantita- problem of amoeboid motion.
    [Show full text]
  • The Cell-Motility-Lowering Effects of Microtubule Depolymerization in Amoebae Proteus
    The cell-motility-lowering effects of Microtubule depolymerization in Amoebae Proteus Bio219 Home The cell-motility-lowering effects of Microtubule depolymerization in Amoebae Proteus. Robert Borkowski Wheaton College Norton, MA 02766 (860) 539-4969 Posted on 25 November 2003, Revised 9 December 2003 Introduction Materials Results Discussion Bibliography I. Introduction In a phenomenom known as amoeboid movement, certain organisms, and certain cell types, move in a fairly slow manner by extendeding appendages known as pseudopodia (Fukui, 2002). These psuedopodia form primarily from the actions of actin filaments, and mysoin proteins to contract the cell (Fukui, 2002). The filaments disassociate into unlinked filaments, and then are re-linked (a linked fiber is a fiber that is connected to another fiber via a linking protein (Cooper, Hausman, 2004). A collection of cross-linked fibers is also known as a "gel") in a pseudopod to give the protrusion structural stability. As a pseudopod increases in size, the cell can displace more and more of its contents into the pseudopod until what was once a pseudopod is now so large that it is considered part of the cell body, and the cell has displaced itself into a new position, and has therefore moved. This is the process of amoeboid movement; a form of movement which acts via displacement and cellular reformation. An amoeba is a single-celled organism protozoic organism that lives in freshwater environments such as ponds, rivers, or any other freshwater ecosystem. It is known that actin filaments play a significant role in the motility of cells through their de-linking and re-geling at the movement front (Cooper, Hausman, 2004).
    [Show full text]