MSU LIBRARIES __ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. THE RELATION BETWEEN THE PHYSICAL—CHEMIOAL PROPERTIES OF THE ZOOSPORE PLASMA MEMBRANE AND DIFFERENTIATION IN BLASTOCLADIELLA EMERSONII By Kenneth Stanley Leonards A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1980 Vilm! ABSTRACT THE RELATION BETWEEN THE PHYSICAL-CHEMICAL PROPERTIES OF THE ZOOSPORE PLASMA MEMBRANE AND DIFFERENTIATION IN BLASTOCLADIELLA EMERSONII By Kenneth Stanley Leonards Blastocladiella emersonii is an excellent model system for examining the role(s) of membranes in cellular differentiation. The zoospores are highly differentiated cells which can undergo rapid morphological changes in response to their environment. Cations and temperature play important role(s) in these changes, K+ ions inducing and Ca++ ions inhibiting encystment, in a temperature dependent manner. These developmental changes do not seem to involve either protein or RNA synthesis, but do involve extensive membrane alterations. The first section of this dissertation establishes a correlation between the physio- logical parameters involved in encystment and the physical-chemical properties of the zoospore plasma membrane, in vigg. Intact zoospores and zoospore total lipid extracts were spin-labeled and examined with electron spin resonance spectroscopy. Three breaks points in plots of the hyperfine splitting parameter (2T|l), Kenneth Stanley Leonards order parameter (S), and partition parameter (f) vs. temperature were observed. The first and third breaks (TL and TH) were independent of K+, Ca++, or Mg++ ion concentrations, similar to the breaks observed in lipid extract dispersions, and correlated well with the temperature limits of zoospore viability. In contrast, the middle break point (TM) was affected by Ca++ ions, K+ ions reversed the Ca++ ion effect. The cation-induced changes in TM were closely correlated with the temperature dependence and physiological effects of cations on encystment. The second section expanded upon these results to determine which lipid components were responsible for the observed phenomenon. Lipid dispersions made from various combinations of zoospore glyco-, phospho-, and neutral lipid components indicated that the phase transformation (TL to TH) observed were due to the glycolipid, and not the phospholipid fraction. This transformation seems to represent a gel-to-liquid-crystalline phase transition. Ca++ ions increased the 2Tll values of glycolipid, but not the phospholipid dispersions. The inclusion of neutral lipids did not affect this 2T|l increase in glycolipid dispersions, but was eliminated at a 5 to l phospholipid/glycolipid ratio. K+ ions neither prevented Kenneth Stanley Leonards or reversed the Ca++ ion-induced 2T|l increase in glyco- lipid dispersions. The third section described a procedure for the isolation of zoospore plasma membranes, by rupturing the plasma membrane. Analysis of the lipids present indicated that the plasma membrane was composed of diglucosyldigly- cerides (47% of total lipid) and neutral lipids, rather than phospholipids. No triglycerides were detected. The preponderance of glycolipids in the plasma membrane united the first two sections above. The fourth section details the properties of the middle break point, T in isolated plasma membranes. M3 TM was determined to be the result of a lipid/lipid rather than lipid/protein interaction. Ca++ ions affected this interaction in the same manner as observed in 311g. K+ ion addition had no affect on TM, in the absence of ATP. However, after including ATP, K+ ions did reverse the Ca++ ion effect. ATP was found to generate an "energized membrane" state which K+ ions could depolarize. We suggest that the K+ ion induced reversal of the Ca++ ion effect, and therefore the changes in the lipid/lipid interaction responsible for TM, is a consequence of the K+ ion induced depolarization of the membrane potential. "When I examine myself and my methods of thought, I come to the conclusion that the gift of fantasy has meant more to me than my talent for absorbing positive knowledge." A. Einstein ii ACKNOWLEDGEMENTS I wish to express my appreciation to the members of my committee, Dr. E. C. Cantino, who by his discovery and extensive studies of g. emersonii laid the foundation for my studies, and Dr. A. Haug, in whose laboratory I learned the theoretical and experimental principles of membrane biology. I am also indebted to Dr. K. Poff and Dr. D. Delmer for their advice (scientific and otherwise) during the course of my studies. I especially wish to thank my scientific colleagues, D. Fontana, J. Jen, S. Briggs, and R. Allison who helped make my research fun as well as instructive. I also wish to thank the Plant Research Laboratory and Dr. A. Lang for their financial support during these studies. In addition I wish to thank Royce, my wife, with- out whose help this dissertation would never have been completed. iii LIST OF TABLES LIST OF FIGURES GENERAL INTRODUCTION SECTION I. Effects of Cations on the Plasma Mem- TABLE OF CONTENTS brane of Blastocladiella emersonii Zoospores. An ESR Study II. Electron Spin Resonance Study of the Isolated Lipid Components from Blastocladiella emersonii Zoospores III. Isolation and Characterization of the Plasma Membrane of Blastocladiella emersonii Zoospores IV. Potassium and Calcium Induced Altera- tions in the Lipid Interactions of Isolated Plasma Membranes from Blastocladiella emersonii. for an ATP Requirement GENERAL CONCLUSIONS LIST OF REFERENCES iv Evidence Page vi ll 49 9O 139 178 186 SECTION I. II. LIST OF TABLES Ratio of the heights of the low field peaks (HA/HE) observed in ESR spectra and measured at different temperatures Thermal transformation points of mem- branes of B. emersonii as a function of ion concentration SECTION II I. II. III. Fatty acyl composition of total lipids, phospholipids and glycolipids of B. emersonii zoospores . . . Effect of lipid composition and calcium ions on the hyperfine splitting para- meter (2T ) obtained for aqueous dispersion labeled with 5- NS and measured at 230 C . . Effect of calcium and potassium ion addition on the hyperfine splitting parameter (2T ) values obtained for glycolipid dispersions labeled with 5- -NS, and measured at 2&0 C . SECTION III I. II. III. IV. Comparative quantities of lipid classes in plasma membrane preparations Comparative quantities of lipid compo- nents in plasma membrane preparations Fatty acyl composition of total and plasma membrane lipids extracted from B. emersonii zoospores Comparative composition of glycolipid sugars from plasma membrane preparations and whole cells Page 21 38 61 69 71 111 11a 121 122 LIST OF FIGURES INTRODUCTION 1. SECTION I 1. Schematic drawing of the zoospore of B. emersonii illustrating the ultrastruc- tural organization of the cell Representative ESR spectra of 5- nitroxy- stearate labeled B. emersonii zoospores recorded at different temperatures Plots of 2T I and S vs. temperature for B. emersoni zoospores labeled with 5- Hitroxystearate showing the effects of ions on the position of the break points . . . . . .. . . . 2a. Zoospores in the presence of 50 mM' KCl . . . . . . . . 2b. Zoospores harvested in the presence Of 1 RIM caCl2 . o o o o 0 2c. Zoospores harvested in the presence of 10 mM CaCl2 . . . . 2a. Reversibility of the Ca + effect by K+ ions . . . . . Plot of f x 100 vs. temperature for B. emersonii zoospores labeled with TEMPO (pH 6. 6 - 6. 7) . . . . . . . . Plot of 2TL| vs. temperature for aqueous dispersion of B. emersonii zoospore lipid extract labeled with 5- nitroxy- stearate . . . . . . vi Page 19 2H 2H 26 28 3O 33 36 SECTION II 1. Plot of 2T|i vs. temperature for phospho- lipid and g ycolipid dispersions labeled with 5- NS . . . . . . Plot of 2Téivs temperature for mixed lipid disp sions labeled with 5- NS Plot of ZTél vs. total amount (ul) of CaCl adde to glycolipid dispersions labeIed with 5- NS . . . Representative ESR spectra of 5-NS labeled zoospores recorded at different” temperatures Plot of HMIHA + HB ) x 100 vs. temperature of glycolipid/neutral lipid dispersions (1:3 wt. /wt. ) labeled with 5- -NS, where HA and 3H correspond to the peak heights of the low field peaks . . . . SECTION III 1. Flow diagram of the procedure for isolating plasma membranes from zoospores of B. emersonii Phase-contrast micrographs of B. emersonii zoospores demonstrating the selective rupturing of the plasma membranes during the isolation procedure x 660 Electron micrographsof the isolated plasma membranes from zoospores of B. emersonii Representative labeling patterns on a thin-layer chromatogram for the total lipid extracted from isolated plasma membrane preparations . . . . Thin-layer chromatogram of the total lipid extracted from isolated zoospore plasma membranes (B) and the total lipid extracted from whole cells (for comparison) (A) demonstrating the absence of trigly- cerides in the plasma membrane preparation vii Page 63 66 72 75 77 97 . 103 , 108 , 112 116 SECTION III, cont. 6. Representative labeling patterns on thin-layer chromatograms for the lipids extracted from the isolated plasma membrane preparations of B. emersonii zoospores SDS-gel electrophoresis recording of absorbance vs. migration distance (cm) of plasma membrane proteins stained with Coomassie brilliant blue SECTION IV 1. Plots of 2T vs. temperature for zoo- spore plasm membrane preparations and aqueous dispersions of the plasma mem- brane lipids spin-labeled with 5-nitroxy- stearate (5-NS) and measured in the presence of CaCl2 Plot of 2T vs. temperature for aqueous dispersion of the plasma membrane lipids spin-labeled with 5-nitroxystearate (5-NS) and measured in the presence of CaCl + EDTA demonstrating the reversal of t e Ca++ effect by EDTA . . Plots of 2T. vs. temperature for zoospore plasma membr ne preparations spin-labeled with 5-nitroxystearate (5-NS) indicating th$+inability of K ions to reverse the Ca ion effect in the isolated membranes Plots of 2TL; vs. temperature for zoospore plasma memb ne preparations spin-labeled with 5-nitroxystearate (S-NS) in the presence of ATP . . . . . . . Plot of 2TLL vs. temperature for zoospore plasma mem ane preparation spin-labeled with 5-nitroxystearate (5-NS) in the presence of AMP-PNP . . . viii Page . 118 . 123 . 152 . 155 . 158 . 162 . 165 ENERAL INTRODUCTION The role(s) of the plasma membrane in development and differentiation is presently an area of intense interest in biology. Unfortunately, most of the existing body of knowledge on plasma membranes is derived either from studies on prokaryotes, erythrocytes, and model membranes, where the difficulties associated . with eukaryotic systems are avoided, or from studies on Saccharomyces cerevisiae (Duran et a1” 1975; Santos et al., 1978) and Neurospora crassa (Scarborough, 1975), where a cell wall must first be removed, either enzyma- tically or by use of cell surface mutants. A desirable alternative would be to find an organism which 1) exhibits the differentiation and development characteristic of an eukaryotic organism, and 2) is structurally organized so as to minimize possible contamination from cytoplasmic organelles, and potential artifacts related to cell wall removal. Zoospores of the chytridiomycete Blastocladiella emersonii fulfill both of these criteria, and are an excellent model system for examining the role(s) of the plasma membrane in cellular differentiation. There are three properties of the zoospore in particular which strongly recommend its use for membrane studies. They are: l.) the ultrastructural organization of the zoo- spore, 2.) the initial changes during zoospore encystment, which do not require either RNA or protein synthesis, but do involve plasma membrane alterations, and 3.) the ability to regulate the encystment process by manipula- ting the ionic and thermal environment of the zoospore. In addition, B. emersonii is one of the most thoroughly investigated of the lower fungi, both physiologically and ultrastructurally. There is, therefore, an extensive body of literature available to provide a foundation for molecular studies. Each of these points is discussed in detail below. 1. The ultrastructural organization of the zoospore Zoospores of B. emersonii are highly differentiated cells, about 7 x 9 uM in size, which are characterized by the lack of a cell wall and an extensive spatial segregation of their internal components. A schematic diagram of a zoospore is shown in Figure l (for detailed reviews see Truesdell & Cantino, 1971; Cantino & Mills, 1976). The cell does not possess an endoplasmic reticulum. Instead, the ribosomes are clustered within a single compartment, the nuclear cap, which is delimited by two parallel unit membranes (Lovett, 1963). The nu- clear cap overlays a significant portion of the nucleus, Figure 1 Schematic drawing of the zoospore of B. emersonii illustrating the ultrastructural organization of the cell. NCOM = nuclear cap outer membrane; NCIM = nuclear cap inner membrane; V = vacuole; M = mitochon- drion; SB = SB matrix of lipid sac; BM = backing membrane; L = lipid globule; G = gamma particle; NC = nuclear cap; MT = member of microtubule triplet; NMP = nuclear membrane pore; N = nucleus; NIM = nuclear inner membrane; NOM = nuclear outer membrane; C = centriole; R rootlet; K = kinetosome; F flagellum; P 'prop' connecting K to plasma membrane of spore. (Drawing taken from Myers and Cantino - see general references) and its membranes are continuous with those enclosing the nucleus proper. Directly adjacent to the nucleus is the zoospore's single large mitochondrion. Appressed against the mitochondrion, and opposite the nucleus, is the side body complex composed of lipid globules (stored energy source) and the side body matrix. The mitochon- drion, lipid globules and side body matrix are enclosed by the backing membrane which is attached to the nuclear cap membrane at numerous points (Myers & Cantino, 197A). The zoospore therefore can be considered to be comprised of an "outer membrane bag", the plasma membrane, which is directly exposed to the cell's exterior environment, and an "inner membrane bag", which encloses almost all of the cell's cytoplasmic organelles. The only major cytoplasmic organelle separate from this inner membrane system is the y-particle, which also contains RNA, and which is intensely stained by neutral red (Myers & Cantino, 19714). The association of almost all of the major cyto- plasmic organelles within such a contiguous membrane structure suggests that it might be possible to separate these components from the plasma membrane as a single unit. In addition, the absence of an endoplasmic reticulum eliminates the most intractable of all plasma membrane preparation contaminants. Moreover, the nuclear cap and lipid globules appear as a large bright crescent and a series of small bright bodies, respectively, with phase contrast microscopy. When the membranes enclosing these organelles are ruptured, the bright crescent disappears, and the lipid globules dissociate from the other organelles. The appearance and location of the nuclear cap and lipid bodies therefore provide an easy method for verifying the integrity of the entire membrane complex during isolation of the plasma membrane. 2. Initial changes during zoospore encystment When zoospores are induced to encyst they proceed irreversibly through a sequence of developmental changes which results in the formation of a chitinous cell wall and the breakdown of the cell's internal organization. Ultrastructurally, the zoospore changes shape, becoming spherical with a concomitant “3% decrease in volume (Truesdell & Cantino,l97l). The cell also retracts its flagellum (Cantino et al., 1968), and experiences a fragmentation of its internal membrane system, including the backing membrane/nuclear cap membrane complex (Truesdell & Cantino, 1971). However, the first detectable changes during encystment involve alterations of the plasma membrane, specifically changes in the cell surface monitored by FITC-Conconavalin A (Jen & Haug, 1979), the induction of cell adhesiveness (Cantino et al., 1968), and the fusion of vesicles derived from the y-particles with the plasma membrane (Truesdell & Cantino, 1970, 1972; Myers & Cantino, 197A; Cantino & Mills, 1976). Biochemically a number of events occur during encystment, but the most relavent to the present study are the changes observed in the cell's lipid composition. The percentage of free sterols drops, and a previously non-detectable monoglyceride appears. Moreover, there is a pronounced change in the zoospore's glycolipid fraction, with the single major glycolipid component, the diglycosyldiglycerides, decreasing by 55% during encystment (Mills et al., 197A). Physiologically membrane fusion processes are of crucial importance in zoospore encystment, since it has been demonstrated that the last enzyme of the biosyn- thetic pathway for chitin formation, chitin synthetase (EC 2.N.l.16), is located in the y-particles (Myers & Cantino, 197A). Deposition of a cell wall has been correlated to the fusion of y-particle derived vesicles with the plasma membrane (Cantino & Myers, 1973; Myers & Cantino, 1974). In addition chemical analysis of the y-particle indicates that a high percentage of the lipids present are glycolipids, especially diglycosyl- diglycerides (Mills & Cantino, 1978). It is also significant that the initial changes which occur during encystment do not require either protein or RNA synthesis (Lovett, 1968, 1975; 8011 & Sonneborn, 1971 a, b; Silverman et al., 197A). As a consequence, the molecular events associated with zoo- spore encystment must involve a re-organization of pre-existing components. The role(s) of the plasma membrane's physical-chemical properties in these events can therefore be examined independently. 3. Environmental control of zoospore encystment Cations and temperature play an important role(s) in the regulation of the encystment process. Potassium ion addition has been shown to induce zoospore encystment. This induction process was found to be temperature dependent, with a dramatic change in the kinetics of zoospore encyst- ment occurring between 10 and 15°C. At 15°C or above zoo- spores encysted within minutes, whereas at 100C or lower it took hours, it it occurred at all (8011 & Sonneborn, 1969). 2+ 2+ In contrast, Ca ions (but not Mg ions) can pre- vent encystment (Cantino, et al., 1968; 8011 & Sonneborn, 1969, 1972). In the presence of millimolar Ca2+ ion concentrations (K+ ions not added) zoospores did not encyst at their growth temperature, i.e., 20°C (Soll & Sonneborn, 1972; Suberkropp & Cantino, 1972). However, zoospores did encyst if the temperature was raised to 270C, even in the presence of much higher Ca2+ ion concentrations (Soll & Sonneborn, 1972). These results suggest an upper limit 2+ for the Ca ion effect, which can be overcome at higher temperatures. In addition to the effects of increased tempera- ture, the effect of Ca++ ions on zoospore encystment can be reversed by the addition of K+ ions to the cell suspension. Moreover, zoospores preloaded with “SCa++ have been reported to release the 145Ca++ ions immediately upon the inclusion of K+ ions (Soll & Sonneborn, 1972). Temperature limits for zoospore viability have also been described. At UOC or less zoospore respiration goes to zero, and at O-lOC the cells lyse rather than encyst (Cantino et al., 1969; Truesdell & Cantino, 1972; Suberkropp & Cantino,l972). At 37°C zoospores will encyst and develop normally, but no development occurs at 39°C (Soll & Sonneborn, 1969). Together these results suggest a relationship between temperature, calcium ions and potassium ions on zoospore differentiation. Since the initial changes during zoospore encystment involve the plasma membrane, and temperature and cations have been shown to be the two main factors altering the physical-chemical properties of membranes, it is reasonable to hypothesize that the dynamic properties of the plasma membrane are involved in the regulation of zoospore differentiation. This dissertation is divided into four sections. The purpose of the first section is to establish a corre- lation between the physiological effects of temperature and cations on zoospore differentiation and their effects 10 on the physical-chemical properties of the zoospore plasma membrane 22 2332; The second section expands upon the first and examines the roles of the various lipid fractions of the zoospore in these phenomenon. The third section describes a procedure for the isolation of the zoospore plasma membranes, and their analysis in order to clarify any relationship between the first two sections. The fourth section specifically examines one aspect of the first section, the middle break point, T to determine if it is the result of a M’ lipid/lipid or lipid/protein interaction and to ascer- tain the effects of Ca++ and K+ ions on this interaction. The analytical method employed for the bulk of these studies is electron spin resonance spectroscopy (ESR). This method was chosen because of its extreme sensitivity to subtle changes in the physical-chemical properties of a membrane, and its ability to determine the dynamic properties of such a membrane. SECTION I EFFECTS OF CATIONS ON THE PLASMA MEMBRANE OF BLASTOCLADIELLA EMERSONII ZOOSPORES. AN ESR STUDY. K. S. Leonards and A. Haug MSU-DOE Plant Research Laboratory Michigan State University East Lansing, Michigan A882u Key Words: Membranes, Cations, Differentiation, Blastocladiella emersonii 11 12 Summary The physical properties of the plasma membrane of the aquatic Chytridiomycete Blastocladiella emersonii were investigated, in particular the effects of cations on membrane structure. Intact zoospores and lipid extracts were labelled with the spin-labels 5-nitroxystearate (5-NS), 12-nitroxystearate (l2-NS), and 2,2,6,6-tetramethylpiperi- dine-l-oxyl (TEMPO). Electron spin resonance spectroscopy indicated a total of three breaks in plots of the hyperfine splitting parameter, 2Tll’ order parameter, S, and the partition coefficient, f, vs. temperature. The first and third break points (TL and TH) were found to be independent of the external K+, Caf+, or MgI+ ion concentrations. They were similar to the break points found in aqueous disper- sions of lipid extracts and correlate well with the temperature limits for zoospore viability. In contrast, the middle break point (TM) was markedly influenced by the ++ ion concentration. Ca++ ions increased T external Ca M from 12°C (no Ca++ added) to 22°C (10 mM Ca++), i.e., growth temperature. K+ ions reversed this Ca++ effect, downshifting TM from 22°C to 10°C. A comparison of the physico-chemical effects of these ions on the membrane, as revealed by the cation-induced shift in TM is closely correlated with the temperature dependence and physiolo- gical effects of cations on zoospore differentiation. This suggests that cations may modify the physical state of the 13 plasma membrane and be involved in regulating the initial changes during zoospore encystment. Introduction Zoospores of the aquatic chytridiomycete Blastocla- diella emersonii are highly differentiated cells which can undergo rapid morphological changes in response to their environment (1-5). Prior to encystment and germination, the motile zoospore is characterized by the absence of a cell wall and an extensive spatial segregation of its in- ternal components (6,7). When zoospores are induced to encyst they proceed irreversibly through a sequence of developmental changes which results in the formation of a chitinous cell wall and the breakdown of their internal organization (5,7,8). The encystment process occurs rapid- ly and seems to require neither protein nor RNA synthesis (9-14). However, the first detectable changes during encystment do involve alterations of the plasma membrane, specifically changes in cell surface fluorescence proper- ties (15), the induction of cell adhesiveness (6), and the fusion of vesicles derived from the y-particles with the plasma membrane (16,17). Ions and temperature play an important role(s) in the regulation of the encystment process. Physiological studies on B. emersonii have demonstrated that K+ ions can trigger encystment, and that this induction is temperature dependent (2). In contrast, Ca++ ions (but not Mg++) can + prevent encystment (2,3,6). This Ca+ ion effect can be 14 reversed by increasing the temperature, or including K+ ions in the buffer (3). Cations (especially Ca++) and temperature are known to affect the physical properties of model and biomembranes, including many membrane associated functions (18-24). Since the initial changes during encystment involve the plasma membrane, it is reasonable to hypothesize that at least part of the effects of ions on zoospore differentiation involve changes in the physical properties of this membrane. To test this hypothesis we monitored the state of the zoospore plasma membrane, i2_vivg, with ESR spectroscopy, as a function of temperature and cation concentration using the spin-labels 12-nitroxy- stearate (12-NS), 5-nitroxystearate (5-NS), and 2,2,6,6- tetramethylpiperidine-l-oxyl (TEMPO). The information presented in this paper indicates that cations do affect the dynamic state of the plasma membrane, in vivo, in a way which correlates very well with the previously reported effects of ions on zoospore differentiation. Materials and Methods Organism,gMedium and Growth Blastocladiella emersonii was kindly supplied by Dr. E. C. Cantino (Department of Botany and Plant Pathology, Michigan State University). The organism was routinely grown on PYG agar at 22°C as previously described (25). Zoospores were harvested from first generation plants by flooding each plate with a buffered (5 mM Morpholinopro- pane sulfonic acid (MOPS), final pH 6.6 - 6.8) solution 15 containing the particular Ca++ or Mg++ ion concentration being tested (K+ ions, final concentration 50 mM, were added after resuspension of the pellet to avoid encystment during harvesting). The zoospore suspension was then collected, filtered to remove germlings and plants, and gently pelleted by centrifugation (1000 x g for 5 min at room temperature). Spin-Labeling Procedure Pellets were resuspended, 0.5 m1 samples removed (A.O to 5.0 x 109 cells) and labelled with an ethanolic solution of 12-NS, 5-NS, or TEMPO (conc. approx. 1 molecule spin-label/6000 molecules of lipid). Just prior to trans- fer of the sample into the ESR cuvette, K3Fe(CN)6 was added (final cone. 1 mM) to prevent disappearance of the spin-label. Control experiments (not shown) indicated that neither the concentration of ethanol (approx. 1%) nor K3Fe(CN)6 used altered the ESR spectra recorded. The ethanol concentration used was also shown not to affect cell viability. In some experiments ethylene glycol (final conc. 33%) was added to the sample Just before transfer into the ESR cuvette to depress the freezing point. Control experiments demonstrated that though there was an increase in the hyperfine splitting parameter (ZTII) values obtained, there was less than a one degree shift in the temperature at which break points were found in plots of 2T|| vs.temperature. l6 Lipid Extraction and Vesicle Preparation Lipids were isolated from zoospores as previously reported (26). Aqueous dispersions were prepared from the total lipid extract by drying an aliquot of the mixture (30-40 mg dry wt.) first under N , and then under vacuum. 2 The sample was resuspended on a vortex stirrer into 0.5 ml buffer (pH 6.7) with a glass bead. The suspension was then sonicated (125 watt sonic water bath), spin-label was added, and the suspension was again sonicated. The concen- tration of spin-label was less than 0.2% wt./wt. Spin-Label Measurements and Analysis ESR measurements were made with a Varian X-band spec- trometer (Model E-112). The temperature was regulated with a Varian variable temperature controller and monitored inside the cuvette with a calibrated thermocouple which was connected to a digital readout meter (Omega model 250). All spectra were recorded at power and modulation amplitude settings which were previously determined to be below those causing saturation or line width broadening. The preferen- tial incorporation of the spin-label into the plasma membrane was ascertained by ascorbate reduction of the spin-probe (27), and protection of the spin-label from reduction using K Fe(CN)6 or Na3Fe(CN)6 (28). 3 The spectra were analyzed with a Varian 620/L-100 computer. The order parameter (S) was calculated accord- ing to method number two of Jost and Griffith (29). The l7 partition parameter (f) was determined according to the method of Shimshick and McConnell (30). The experimental plots (hyperfine splitting parameter 2Tll, S, or f vs. temperature) were analyzed using two methods. The first method was in terms of linear components by fitting regression lines to appropriate sections using the method of least squares. This method of analysis has been frequently used in the interpretation of results obtained from ESR experiments,and the temperatures at which break points are observed (indicated by the intersections of straight lines) are in good agreement with those obtained by other physical-chemical techniques (22,30,31). This method also included the determination of the correlation coefficient, r, a measure of the goodness of fit, which indicated whether the initial assumption of a stright line was valid. An r value of 1.0 indicates a perfect fit between the data points and the calculated line. In all cases r >0.96 with most lines having r 10.98. The second method of analysis was in terms of an iterative least squares program (Brunder, Coughlin, and McGroarty, unpublished) using normalized B-splines develOped by Dierckx (32). This method involved the simultaneous analysis of ZTII’ S, or f over the entire temperature range, and determined the temperatures at which break points occurred. Both methods of analysis gave the same results. The spectra obtained with l2-NS, however, could not be 18 analyzed in this fashion due to the absence of a high field trough even at 000 which made measurements of 2Tll impossible. Use of this spin-label was therefore discontinued. Results Representative spectra of 5-NS labeled zoospores recorded at different temperatures are shown in Figure l. The spectra obtained, especially at higher temperatures, are similar to those observed for spin-labeled sarco- plasmic reticulum, spinach chloroplasts, yeast cells (18), erythrocytes (33), and B. 39;; membranes (3A). These previously published spectra were interpreted as indica- ting the presence of at least two superimposed spectral components, one corresponding to a relatively immobilized spin-label population, and another to a relatively mobile population, here associated with peaks A and B. To gain further insight into the spectral shape changes observed, the heights of the two low field peaks A and B, were measured at different temperatures. The ratio of the two peak heights.(HA/HB) was found to change as a function of temperature (Table I). This change is similar to that recently observed for spin-labeled B. 39;; membranes, where changes in the (HA/HE) ratio were measured as a function of divalent cation concentration (34). In B. emersonii zoospores the spectral contribution of peak B increased with increasing temperature. However, there 19 Figure 1 Representative ESR spectra of 5-nitroxy- stearate labelled B. emersonii zoospores recorded at different temperatures. The ratio of the two low field peak heights (HA/HE) changed reversibly as a function of temperature. ——————, 2°C; °°°°°°° , 900; ------- , 36°C. - “F‘d ’ 1’ ' ’I o' I o I °. ( " \\ { ‘\‘ .‘o. “‘ ......'.I ‘ ...o° - - ~~ . ‘ ‘-- - OOOOOQOQOOOIOOQOQO. ‘~~ ‘ ~ ‘ ‘ I ‘ ‘ 00...... ~ ' 0"“ ) ....I ’ .0 ' -- ..0 -fl — - ‘-_,..p m - e . \\ : \ o \ .' ."x I a I 10 GAUSS “ow-no.3.- -"‘" 21 Table 1. Ratio of the heights of the low field peaks (HA/H3) observed in ESR spectra and measured at different temperatures. B. emersonii zoospores spin-labeled with 5-nitroxystearate (5-NS). Values are averages of four experi- ments. Temperature 0C 0° 5° 10° 15° 20° 25° Ratio HA/HB 3.54 2.38 1.62 1.24 0.97 0.84 S. D. £0.22 £0.18 i0.13 £0.07 $0.048 £0.059 22 was little, if any, Change in its position. In contrast, the spectral contribution of peak A changed both in rela- tive peak height and position as a function of temperature (Figure 1). Addition of ascorbate to reduce the spin-label resulted in the simultaneous and total disappearance of both low field peaks (data not shown). The effects of ion addition on the plasma membrane of intact zoospores labelled with 5-NS were evaluated by measuring the hyperfine splitting parameter, 2T||, and where possible, the order parameter, S. The hyperfine splitting value, 2TII’ is highly sensitive to Changes in the molecular environment of the spin-label (35), and is related to the freedom of motion of the spin-label in the membrane, lower 2Tll values being associated with a greater freedom of motion of the probe (36,37). The order para- meter, S, is dependent on 2TI as well as 2T||° Various models have been proposed fo; the interpretation of the order parameter at the molecular level, including models based upon molecular motion (29), and others based upon molecular orientation (38). In either case a large value for S (up to 1.0) indicates a high degree of order and a small value for S (down to 0) indicates a low degree of order. Plots of these two parameters vs. temperature have been used extensively to determine changes in the physical properties of both model and natural biomembranes (22,24, 31). To eliminate possible artifacts due to overlap of 23 the two low field peaks 2T and S were only measured at temperatures where the twolJow field peaks of the spectrum were clearly resolvable (up to 26-28OC). By restricting the measurement of 2Tll to this temperature range and observing the spectra obtained at low temperatures, where the spectral contribution of peak B was minimized, it was possible to attribute changes in 2Tll to changes in peak A. Figure 2 illustrates the effects of various ions and ion concentrations on 2Tll and S as a function of temperature. In all cases two break points were observed. Plots of 2Tl| and S exhibited the same break point temperatures. The results obtained in the presence of buffer only, buffer + 10 mM MgClz, or buffer + 50 mM KCl were very similar . (Figure 2a). The first break point occurred at 3-4°C in buffer + 50 mM KCl, or at 4-6OC in buffer only or buffer + 10 mM MgC12. The second break point varied only slightly, occurring at 8-9°C in buffer + 50 mM KCl, at 10-12°C in buffer only, or at 11-13OC in buffer + 10 mM MgC12. Ca++ ions did not significantly alter the temperature at which the first break occurred (5-700), but had a marked affect on the temperature at which the second break point, TM, was observed (Plots 2b,c). In the presence of 1 mM Ca++ this break point was shifted from 10-12OC to 17-18OC. Increasing the Ca++ ion concentration to 5 mM Ca++ shifted TM to 19-20°C, and 10 mM Ca++ further shifted the break + point to 20-22OC. 20 mM Ca+ did not further change the Figure 2 24 Plots of 2T and S vs. temperature for B. emersonii zoospores labelled with 5- nitroxystearate showing the effects of ions on the position of the break points. Two breaks for 2Tll were found in all cases. 8 was calculated where possible. Arrows indi- cate break points. Note the agreement between the break points determined with 2Tll and those obtained with S (pH 6.6 to 6.7). 2a. Zoospores in the presence of 50 mM KCl. Break points were very similar to those determined in the presence of buffer only or 10 mM MgC12. 2TI' ,O-O; S,D-—[:]. 25 14 U I 1' t AGv F n b P 15 20 10 TEMPERATURE °C 64- m «w olllo 721$ __.—.N 52 2b. 26 Zoospores harvested in the presence of increase in the temperature at which the second break occurred. 27 65- 60-\\ ole 738V =._. n.o 5 N 45L TEMPERATURE °C 2c. 28 Zoospores harvested in the presence Of 10 mM CaClz. Break points were the same as those obtained in the presence of 20 mM CaCl2. 2Tl |,O-O; S,D"'D . 29 - P - 25 15 20 TEMPERATURE °C 10 62- . 8 5 4 5 o|.o ASBGU =._. 0 5 N . 4e- 2d. 30 Reversibility of the Ca++ effect by K+ ions. Zoospores were harvested as in experiments represented in Figure 2c. After spin-labeling, but before transfer to the ESR cuvette, KCl was added (final conc. 50 mM KCl). 2T ,O—O; S,D-D . 2 T” (OM15!) o—o 9 8 l l l w» l 11 \l O L 0 5 1O 15 20 TEMPERATURE °C 25 0—0 Sx 100 32 temperature at which the second break point occurred, suggesting a possible saturation effect. The Ca++ ion effect observed could be reversed by K+ ions (Figure 2d). When KCl was added (50 mM final conc.) to samples already in the presence of 10 mM Ca++, the second break point TM was shifted back to lO-llOC. To ascertain whether any break points occurred below 0°C, 5-NS labeled zoospores were studied in the presence of ethylene glycol. No additional break points were detected between -l2°C and 0°C, nor were the positions of the break points above 0°C significantly altered (data not shown). To determine whether there were any break points at higher temperatures (>25°C) which could not be observed with 5—NS, we used the spin-label TEMPO. TEMPO is a mole- cule which has been shown to be soluble in water and in fluid, liquid-crystalline membranes, but not in bilayers in the gel state(29,30,39). The spectral parameter, f, is related to the fraction of the membrane which is accessible to the spin probe. Since f is determined by the partition- ing of the spin-label between the aqueous phase and the fluid membrane, TEMPO not only allowed us to measure the physical behavior of the zoospore membrane at higher temperatures, but also provided a different method of verifying the results obtained with 5-NS. Figure 3 illustrates the data obtained for zoospores labelled with TEMPO in the presence of different Ca++ ion Figure 3 33 Plot of f x 100 vs. temperature for B.emersonii zoospores labelled with TEMPO (pH 6.6 to 6.7). f was calculated as indicated since the line widths for both H and P were found to be equal. Zoospores harvested in buffer only,;)-——(3; 1 mM CaC12,A—-A; 10 mM CaCl2,D-—D Plots of 10 mM MgCl2 were the same as buffer only. n—l °C TEMPERATURE 35 concentrations as a function of temperature. In these experiments it was not possible to determine the absolute amount of membrane lipid in the liquid-crystalline state, due to the variable number of cells present. However, changes in the partitioning of the spin-label measured in the same sample as a function of temperature do reflect changes in the relative fraction of the zoospore plasma membrane which was accessible to the spin-label. In all cases, an upper break point, TH, was observed between 32 and 34°C which was independent of Ca++ ion concentrations. In contrast to this upper break point, another break occurred at lower temperatures which was influenced by the Ca++ ion concentration.' For samples in the presence of 10 mM CaCl2 this break occurred at 22-23°C. Reducing ++ ion concentration to 1 mM CaCl2 shifted this break point down to 17-18°C. Zoospores in the presence of buffer the Ca only, or buffer + 10 mM MgCl2 were characterized by a shallow break which occurred at 10-1200. Thus, the results derived from the TEMPO experiments confirm those obtained with 5-NS for the Ca++ ion dependent break point, TM These results are summarized in Table II. Breaks were not detected at 4-600 using the spin-label TEMPO. However, no firm conclusions can be drawn since it was not possible to determine f accurately below 0°C. To examine the role(s) of lipids in these observed phenomena, zoospore lipids were isolated, aqueous Figure 4 36 Plot of 2T vs. temperature for aqueous dis- l l persions of B. emersonii zoospore lipid extract labelled with 5-nitroxystearate. 0, sample run in the presence of buffered ethylene glycol (final conc. 33%). 0, sample run in the presence of buffer only. Note: the ethylene glycol did affect the absolute value of 2TII observed and the data presented were normalized to those obtained with buffer only. Break points were shifted less than one degree in the presence of ethylene glycol. 37 1 30 L 10 TEMPERATURE 4O 20 -1O 70‘- (ssnv o) "13 40 °C 38 Table II. Thermal transformation points of membranes of B. emersonii as a function of ion concentration Break point Spin-label Lowgr Midd e Highgr TL ( C) TM ( C) TH ( C) 5-NS Buffer only* 4-6 10-12 + 10 mM MgCl2 4-6 ll-l3 + 50 mM KCl 3-4 8- 9 + 1 mM CaCl2 5-7 17-18 + 10 mM CaCl2 5-7 19-20 + 20 mM CaCl2 5-7 20-22 + 10 mM CaC12_+ 2-3 10-11 50 mM KCl Tempo Buffer only 10-12 32-34 + 1 mM CaCl2 17-18 33-34 + 10 mM CaCl2 22-23 33-34 + 10 mM MgCl2 10-12 33-34 5-NS labeled lipid extract 2-4 30-32 Buffer only *Buffer: 5 mM Mops, pH 6.7 39 dispersions were labelled with 5-NS, and the temperature dependence of the hyperfine splitting parameter, 2Tll, was determined over the temperature range -l2°C to 42°C. A plot of 2T1] as a function of temperature is shown in Figure 4. Two break points were observed, one at 2-4°C and the other at 30-3200. These values are very similar to the lower and upper break points, T L in whole cells with 5-NS and TEMPO respectively (Table and TH, observed II). Ca++ ion addition did not affect the position of these break points. However, it did increase the 2Tll values obtained (by 5-7 gauss) over the entire temperature range. In addition, no breaks were detected between these ++ ' two, in the presence or absence of Ca ions. Since the HA/HB ratios were significantly different from those obtained with zoospores labelled with 5-NS, there are probably differences in the relative amounts and organi- zation of the extracted lipids as compared to the plasma membrane of the zoospore. Such differences would explain the absence of the middle, ion dependent break point, TM. Discussion The data presented in this paper support two conclusions. (1) The temperature limits of zoospore viability are related to the phase transformation tem- peratures of the lipid matrix. If T and TH represent L the onset and completion of a gel-to-liquid-crystalline phase transition, respectively, zoospore viability is 40 related to a mixed lipid state. (2) Specific ions markedly affect the physical properties of the plasma membrane, which in turn are related to the physiological effects of these ions on zoospore differentiation. The hyperfine splitting parameter (2Tll) values obtained for 5-NS labelled zoospores indicate that the plasma membrane is much more fluid at its growth tempera- ture than the plasma membrane of organisms such as T. acidophila (22), or B. coli (31). Initial experiments utilizing 12-NS also suggest a very fluid microenvironment. This observation is consistent with preliminary findings in our laboratory showing that the isolated plasma mem- brane contains a large percentage of unsaturated fatty acyl chains, including arachidonic acid (20:4). Similar levels of unsaturated lipids have also been reported in B. emersonii zoospore lipid extracts (26). Analysis of the data obtained from spin-labelled zoospores indicated three break points in plots of spectral parameters (2Tll, S, f) vs. temperature (Table II). Of these three, the lower and upper transition points, T L and T , were found to be independent of ion concentration. H They were also very similar to the two break points found in 5-NS labelled aqueous dispersions of whole cell lipid extracts. Both intact cells and lipid extracts were char- acterized by the same transformation temperature range AT of about 28°C, where AT = TH - TL. In lipid extracts 41 TL and TH were shifted to lower temperatures by approxi- mately three degrees as compared to the break points determined in spin-labeled zoospores. Such a downshift of TL and TH probably reflects the absence of proteins in the lipid extract dispersions, or organizational differences in the two membranes. Break points observed with ESR spectroscopy in multicomponent systems have been correlated with lipid phase transitions, lateral phase separations, lipid clusters or lipid/protein interactions (30,39,40-43). In our experiments both protein-free lipid extracts and intact zoospores had similar break points (Table II). In addition, the partition parameter, f, only increased slightly above TH (Figure 3). This has previously been interpreted to indicate the presence of a fluid phase membrane (29,30,39). The temperatures, TL and TH, may therefore represent the onset and end of a gel-to-liquid- crystalline phase transformation of the lipid environment associated with spectral component A. The lower and upper phase transformation tempera- tures, TL and TH, correlate well with the temperature range of zoospore viability. Previous studies indicated a lower temperature viability limit of l-4°C (5), and an upper growth temperature limit of 37-3900. We confirmed these results finding temperature limits of l-3°C and 34- 36°C (data not shown). These observations suggest that the 42 temperature range over which the zoospore remains viable is correlated with that where plasma membrane lipids exist in a mixed state. The necessity of a mixed lipid state for cell viability is in accordance with findings for B, 92;; outer membranes (31). In contrast to the lower and upper break points, T and T L H’ point occurred (TM) was markedly influenced by the ionic the temperature at which the middle break environment (Figures 2 and 3, Table II). The reference value determined for TM in buffer alone was about 12°C. Addition of either K+ ions or Mg++ ions changed this value only slightly. However, increasing the external Ca++ ion concentration progressively shifted the TM value in both 5-NS and TEMPO labelled cells up to about 22°C, i.e., around the growth temperature, where this effect saturated. These results suggest that the shift of TM upwards was Ca++ ion selective rather than an electrostatic divalent cation effect. Furthermore, ionic strength could not account for this shift since the concentrations of K+ ions employed were much greater than those of Ca++ ions. A comparison of these ionic effects on TM with the temperature dependence and physiological effects of the same ions on zoospore differentiation indicated a high degree of similarity. A dramatic change in zoospore encystment kinetics was found to occur between 10°C and 15°C in the presence of K+ ions (2). At 15°C or above 43 zoospores encysted within minutes, whereas at 10°C or lower it took hours if it occurred at all. In the presence of millimolar Ca++ion concentrations (K+ ions not added), zoospores did not encyst at their growth temperature, i.e., 20°C (3,4). However, zoospores did encyst if the temperature was raised to 27°C, even in the presence of much higher Ca++ ion concentrations (3). These results indicate that there is an upper limit for the Ca++ ion effect, which lies between 20 and 27°C. This is consistent with our experiments which demonstrated an ++ upper limit for TM at about 22°C as a function of Ca ion concentration. In addition, Ca++ ions were found to be more effective than Mg++ ions in preventing encystment, indicating that the inhibition was relatively Ca++ selec- tive (5). Similar effects of these ions on TM are shown in Table II. Physiological experiments indicate that K+ ions can reverse the effect of Ca++ ions on zoospore encystment (3). This implies that a similar effect should be ob- served physico-chemically for TM, if the two ion effects are indeed related. The results presented in Figure 2 (c,d) demonstrate that K+ ions do reverse the effects of Ca++ ions on the plasma membrane. This hypothesis is °°Ca++ ions were immediately in accord with findings that released from preloaded zoospores upon K+ addition (3). The molecular nature of the phenomena responsible 44 for the middle break point and the ion-induced shifts in TM remains unknown. Since both TEMPO and 5-NS were sensi- tive to these shifts in T the membrane lipids are M, certainly involved. In addition, ESR experiments with the isolated zoospore plasma membrane, spin-labeled with 5-NS, also show a distinct middle break point (TM) at 23-25°C 1 2 temperature, measured over the temperature range 0-40°C in the presence of 10 mM CaCl in plots of 2Tll vs. (Leonards and Haug, unpublished). It is noteworthy, however, that irrespective of the position of TM, the 2T|| and S values observed remained relatively unchanged as determined by the 5-NS spin probe (Figure 2). In model systems addition of Ca++ to negatively charged phos- pholipids resulted in an increase in 2Tll and S as well as a shift in the transition point (18,40,44). This suggests that the actual fluidity of the bulk phase lipids in the zoospore membrane may not be affected by Ca++ ions. Instead, the Ca++ and K+ ions may be acting more specifi- cally at the cell surface, perhaps by altering lipid/ protein interactions, lipid head-group orientations, or the clustering of membrane components. These interactions may be similar to the surface antagonism of Na+ and Mg++ observed in chloroplast grana thylakoid membranes (45). Such interactions would be consistent with the observation that addition of K+ ions alters the fluorescence properties of the cell surface within 15 seconds (15). These 45 possibilities are now being investigated. Acknowledgements This study was funded by United States DOE contract EY-76-C-02-l338. We are grateful to Dr. E. C. Cantino for providing cultures of B. emersonii and to Donna Fontana and Dr. K. Poff for helpful discussions. 10. 11. 12. 13. 14. 15. 16. References Silverman, P. M. and Epstein, P. (l biology (Schlessinger, D., ed.) for Microbiology. $5) in Micro- O 4 4-489, Am. Soc. Soll, D. R. and Sonneborn, D. R. (1969) Develop. Biol. 20, 218—235. Soll, D. R. and Sonneborn, D. R. (1972) J. Cell. Sci. 10, 315-333. Suberkropp, K. F. and Cantino, E. C. (1972) Trans. Br. Mycol. Soc. 59(3), 463-475. Truesdell, L. C. and Cantino, E. C. (1971) in Current Topics in Developmental Biology (Moscona, A. A. and Monroy, A. eds.) Vol. 6, 1-44, Academic Press, Inc., New York. Cantino, E. C., Turesdell, L. C. and Shaw, D. S. (1968) J. EllshiMitChell Sci. SOC. 84(1), 125-146. Cantino, E. C. and Mills, G. L. (1976) in The Fungal Spore, Form and Function (Weber, D. J. and Hess, W. M., eds.),501-557, J. Wiley and Sons, Inc., New York. 8011, D. R., Bromberg, R., and Sonneborn, D. R. (1969) Develop. Biol. 20, 183-217. Leaver, C. J. and Lovett, J. S. (1974) Cell Differ. 3(3), 165-192. Lovett, J. S. (1968) J. Bact. 96(4), 962—969. Lovett, J. S. (1975) Bacteriol. Rev. 39(4), 345-404. Silverman, P. M., Moo-On Huh, M., and Sun, L. (1974) Develop. Biol.40, 59-70. Soll, D. R. and Sonneborn, D. R. (1971) Proc. Nat. Acad. Sci., 68(2), 459-463. Soll, D. R. and Sonneborn, D. R. (1971) J. Cell Sci., 9, 679-699. Jen, C. J. and Haug, A. J. (1979) J. Gen. Microbiol., in press. Myers, R. B. and Cantino, E. C. (1974) The Gamma 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 47 Particle, Monographs in Developmental Biology (Wolsky, A., ed.), Vol. 8, S. Karger, Basel, Switzerland. Truesdell, L. C. and Cantino, E. C. (1970) Arch. Mikrobiol. 70, 378-392. Ohnishi, s. (1975) Adv. Biophys. 8, 35-82. Ueda, J. J., Ito, T., Okada, T. S., and Ohnishi, S. (1976) J. Cell Biol. 71, 670-674. Newton, C., Pangborn, W., Nir, S., and Papahad- Jopoulos, D. (1978) Biochim. Biophys. Acta 506, 281-287. Knudsen, K. and Horwitz, A. F. (1977) Develop. Biol. 58, 328-338. Weller, H. and Haug, A. (1977) J. Gen. Microbiol. 99, 379-382. Machida, K. and Ohnishi, S. (1978) Biochim. Biophys. Acta 507, 156-164. - Leterrier, F., Breton, J., Daveloose, D., Viret, J., LeSaux, F., and Pollet, S. (1978) Biochim. Biophys. Acta 507, 525-530. Cantino, E. C. and Hyatt, M. T. (1953) Antonie van Leeuwenhoek J. MicrObiol. Serol. 19, 25-70. Mills, G. L. and Cantino, E. C. (1974) J. Bacteriol. 118(1), 192-201. Schreier-Muccielo, S., Marsh, D., and Smith, I.C.P. (1976) Arch. Biochem. Biophys. 172, 1-11. Kaplan, J., Canonico, P. G., and Caspary, W. J. (1973) Proc. Natl. Acad. Sci. 70(1), 66-70. Griffith, 0. H. and Jost, P. C. (1976) in Spin Labeling: Theory and Applications (Berliner, L. J., ed.), p. 483, Academic Press, Inc., New York. Shimshick, E. J. and McConnell, H. M. (1973) Biochem- istry 12(12), 2351-2360. Janoff, A., Haug, A., and McGroarty, E. J. (1979) Biochim. Biophys. Acta 555, 56-66. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 48 Dierckyx, P. (1975) J. Comp. Appl. Math 1(3), 165-184. Shiga, T., Suda, T., and Maeda, N. (1977) Biochim. Biophys. Acta 466, 231-244. Takenchi, Y., Ohnishi, S., Ishinaga, M., and Kito, M. (1978) Biochim. Biophys. Acta 506, 54-63. Gaffney, B. J. (1974) in Methods in Enzymology (Flei- sher, S. and Packer, L., eds.), Vol. 32B, Academic Press, Inc., New York. Hubbell, W. L. and McConnell, H. M. (1969) Proc. Natl. Acad. Sci. U. S. 64, 20-27. Schroit, A. J., Rottem, S., and Gallily, R. (1976) Biochim. Biophys. Acta 426, 499-512. Schreier, S., Polnaszek, C. F., and Smith, I. C. P. (1978) Biochim. Biophys. Acta 515, 375-436. McConnell, H. M. (1976) in Spin Labeling: Theory and Applications (Berliner, L. J., Ed.), Academic Press, Inc,, New York. Lee, Aé G. (1975) Prog. Biophys. Molec. Biol. 29(1), 3-5 . Lee, A. G. (1977) Biochim. Biophys. Acta 472, 237-281. Lee, A. G. (1977) Biochim. BiOphys. Acta 472, 285-344. Lee, A. G., Birdsall, N. J. M., Metcalfe, J. C., Toon, P. A., and Warren, G. B. (1974) Biochemistry 13(18), 3699-3705. Ito, T. and Ohnishi, S. (1974) Biochim. Biophys. Acta 352’ 29-370 Gross, E. L. and Prasher, S. H. (1974) Arch. Biochem. Biophys. 164, 460-468. SECTION II ELECTRON SPIN RESONANCE STUDY OF THE ISOLATED LIPID COMPONENTS FROM BLASTOCLADIELLA EMERSONII ZOOSPORES K. S. Leonards and A. Haug MSU-DOE Plant Research Laboratory Michigan State University East Lansing, Michigan 48824 Key Words: Lipid Dispersions, ESR, Glycolipids, Cation Effects, Phase Transitions, Blastocladiella emersonii, Zoospores 49 50 Abbreviations: MOPS, Morpholinopropane sulfonic acid; TLC, Thin-layer chromatography; GLC, Gas-liquid chromatograth; 5-NS or 5-nitroxystearate, {2-(3-Carboxypropyl)4, 4-dimethyl-2-tridecy1-3-oxazo- lidinyloxyl} 51 SUMMARY The physical-chemical properties of lipid compo- nents isolated from zoospores of the aquatic phycomycete Blastocladiella emersonii were investigated with electron spin resonance (ESR) spectroscopy using the spin label 5-nitroxystearate. Lipid dispersions were made from zoospore phospholipids and glycolipids, both singly and in combination with each other and with isolated neutral lipid components. Plots of the hyperfine splitting parameter (2Tll) vs. temperature indicate that it is the zoospore glyco- lipids rather than the phospholipids which are responsible for the phase transformations previously observed in aqueous dispersions of the total lipids extracted from zoospores and in zoospores 1E XlXQ- The discontinuities observed in the glycolipid dispersions seem to represent the onset and completion of a gel-to-liquid-crystalline phase transition. Over the temperature range tested, Ca2+ increased the rigidity of the glycolipid dispersions, the major component of which is probably a diglucosyldi- glyceride, but had no effect on the phospholipid dispersions. This increase in 2T|l was not affected by inclusion of neutral lipids into the glycolipid disper- sion but was eliminated at high (5 to l wt./wt.) phospholipid to glycolipid ratios. The Ca2+ effect was relatively independent of both the absolute rigidity 52 of the dispersion and its phase (gel or liquid-crystal- line), suggesting an interaction with the glycolipid head group rather than the hydrocarbon core. The Ca2+ ion-induced increase in 2T11 was neither prevented nor reversed by the presence of K+ ions. The presence of two spin label populations co-existing in a dynamic equilibrium was found in glycolipid/neutral lipid dispersions. Plots of the percentage ((HA/(HA + HB)} x 100) of the spin label population, as measured by the peak height of the low field peaks, corresponding to the mOre immobilized component (HA) vs. temperature indicated two break points. The temperatures at which these break points occurred are similar to those obtained for the glycolipid dispersions, and match the break points (TL and TH) found in ESR experiments using zoospores $2 gizg. The importance of the glycolipids in the develop- ment of this organism is discussed. 53 INTRODUCTION The Chytridiomycete Blastocladiella emersonii is an excellent modelsystem for studying the role(s) of membranes in cellular differentiation. The zoospores of this organism are highly differentiated cells which can be triggered to undergo rapid and irreversible mor- phological changes (1-4). These developmental changes do not seem to require either protein or RNA synthesis (5,6), but do involve extensive membrane alterations. The membrane changes include the onset of cell adhesive- ness (7), the fusion of cytoplasmic vesicles with the plasma membrane (8,9), and the appearance of a chitinous cell wall (4-6). In a previous study, we established a correlation between cation and temperature induced changes in the physical-chemical properties of the zoospore plasma membrane, BB KEYS: observed with ESR spectroscopy, and the effects of temperature and ions on zoospore differ- entiation and viability (10,11). The ESR spectra pre- viously obtained for spin labeled zoospores were also interpreted as indicating the presence of two co-existing spin label populations which were in dynamic equilibrium with each other. Of these two pOpulations, it was the one characterized as the more rigid which seemed to determine the temperature limits of zoospore viability and be involved in the regulation of encystment. 54 Experiments with aqueous dispersions of the total lipids (phospho-, glyco-, and neutral lipids) extracted from zoospores indicated that of three "break points" observed 12 vivo, with ESR (TL, T , and TH), at least M the lower (TL) and upper (TH) were due to bulk changes in the properties of the zoospore lipids. The purpose of this report is to expand upon these earlier results, especially, 1.) to determine which lipid components give rise to the phase transformations obser- ved in the total lipid extract preparations, 2.) to indicate some general properties concerning the inter- actions of these components with other lipids and ions, and 3;) to suggest how these properties may affect zoospore differentiation. Part of the results obtained in this study were presented previously (12). MATERIALS AND METHODS Organism, Medium and Growth Cultures of Blastocladiella emersonii were routinely grown on PYG agar at 22°C as previously des- cribed (13). Zoospores were harvested from first gener- ation plants with a 5 mM Morpholinopropane sulfonic acid (MOPS) + 10 mM Ca012 solution (pH 6.7). The resulting zoospore suspension was then collected, filtered to remove germlings and plants, and pelleted by centrifugation (2000 x g for 5 minutes at room temperature). 55 Lipid Isolation and Characterization Lipids were immediately extracted from the zoo- spores, the proteins removed, and the separated lipids fractionated into neutral, glyco-, and phospholipid classes as previously described (14). Both lipid ex- traction and subsequent fractionations were carried out under N . All solvent evaporations were carried out at 2 40°C or less, and samples were stored under N at -20°C. 2 Solvents were spectra grade or redistilled. Individual lipid components were separated by TLC on Silica gel G, Silica gel H, and Aflasil plates (Supelco) using chloroform/methanol/water (65:25:4 v/v/v) and petroleum ether/diethyl ether/acetic acid (80:20:l v/v/v). The separated lipids were detected with iodine vapor, sulfuric acid charring, ninhydrin, Rhodamine 6 G, and FeCl3 in H280“ (for sterols). Zoospore neutral lipids were separated on a preparative scale by TLC on silica gel G plates. Bands were found by blowing iodine vapor (iodine crystals in a Pasteur pipette) over end strips of the TLC plates. The four major spots were marked and the in-between silica gel bands scraped off. The lipids were extracted from the silica gel using chloroform/methanol (1:1 v/v) as solvent, and stored under N2 at -20°C. Comparable experiments with lipids (dipalmitoyl phosphatidylcholine) indicated a recovery rate of 90 to 93%. Lipid components were identified 56 by co-chromatography with standards (Supelco), color reactions to the detection sprays, and comparison to published analyses of B. emersonii zoospore lipids (l4). Fatty Acid and Glycolipid Sugar Analysis A Hewlett-Packard gas chromatograph, Model 402, equipped with a flame ionization detector was used. Peak areas were determined with a Hewlett-Packard inte- grator, Model 3380 A. Fatty acyl methyl esters were separated by GLC chromatography at 170°C on a 2m x 2mm glass column containing 3% SP-2330 on 80 - 100 mesh, Supelcoport. The fatty acyl methyl esters were identified by comparing their retention times to standard mixtures (Supelco). Glycolipid sugars were analyzed on a 3% OV-l column at 190°C according to the method of Yang and Hakamori (15). This method allowed the detection of amino sugars. The sugars were identified by compari- son of their retention times with those obtained for standards. Preparation of Aqueous Dispersions and Spin-Labeling Procedure The spin label 5-nitroxystearate was used for these ESR studies. Aqueous dispersions of the lipid components were made by mixing aliquonts of the desired lipids in chloroform/methanol (2:1 v/v) and drying the mixture first under N2, then under vacuum. The sample was resus- pended on a vortex stirrer into 5 mM MOPS buffer (pH 6.7) 57 with a glass bead. The ratio of buffer to lipid was adjusted to give a final lipid concentration of at least 5.0 mg/ml. The suspension was then sealed in a screw cap test tube under nitrogen and sonicated (125 Watt sonic water bath). An ethanolic solution of the spin-label was then added to the lipid dispersion, the tubes resealed and the dispersion sonicated for another 5 to 10 minutes to incorporate the label. The concentra- tion of the spin-label was less than 0.2% (wt./wt.). Control experiments indicated that the small amounts of ethanol added did not alter the spectra measured. For some experiments ethylene glycol (final conc. 33%) was added to the sample just before transfer into the ESR cuvette to depress the freezing point. Previous experiments had shown that this procedure did not affect the temperature at which break points were found in plots of 2Tl' vs. temperature (11). In experiments to test the effects of Ca2+ ions on membrane rigidity, CaCl2 (final conc. 10 - 20 mM) was added both to samples which had just been tested without calcium, and to freshly prepared samples just before transfer into the ESR cuvette. The effect of calcium was found to be the same in both cases. Since the addition of both K+ and Ca2+ ions could cause aggregation, ions were added after the formation of the dispersion immediately before transfer into the EPR cuvette. Control experiments indicated 58 that the aggregation itself did not affect rigidity. Spin-Label Measurements and Analysis ESR measurements were made with a Varian X-band spectrometer (Model E-ll2). The temperature was regu- lated with a Varian variable temperature controller and monitored inside the cuvette with a calibrated thermo- couple connected to a digital readout meter (Omega model 250). All spectra were recorded at power and modulation amplitude settings which were previously determined to be below those causing saturation or line width broaden- ing. The spectra were analyzed with a Varian 620/L-100 computer. The experimental plots of the hyperfine split- ting parameter (2Tll), and the HA/(HA + HB) ratios vs. temperature, (where HA and HB are the heights of the peaks A and B of Figure 4, respectively), were analyzed in terms of linear components by fitting regression lines to appropriate sections using the method of least squares. The hyperfine splitting parameter 2T|l is indicative of motional properties of the spin probe in the membrane. A high value of 2T11 is associated with restricted motion or increased rigidity and a low °T|l value corresponding to a decrease in rigidity. This empirical method of analysis has been frequently used in the interpretation of results obtained from ESR experiments, and the temperatures at which break points are observed (indicated by the intersections of straight 59 lines) are in good agreement with those obtained by other physical-chemical techniques (16,17,18). RESULTS Lipids - Analytical Studies The percentages of neutral, glyco- and phospho- lipid fractions found in zoospores were 56, 12 and 31%, by weight. This is similar to the results previously reported for B. emersonii zoospores (14). The major phospholipids detected by TLC were phosphatidylcholine and phosphatidylethanolamine. These were previously shown to account for 55 and 22%, respectively, of the zoospore's phospholipids (14). A minor difference found was the absence in our preparations of lysophosphatidyl- choline and phosphatidic acid (lysophosphatidylethanolamine not determined) which had been previously observed (14). Both lysophosphatidylcholine and phosphatidic acid could be detected by TLC if the samples were exposed to air or heated above 40°C for too long. The glycolipid frac- tion was composed of monoglycosyldiglycerides, diglycosyldiglycerides and a small amount of polyglyco- syldiglycerides. These components have previously been shown to comprise 18, 70 and 12%, respectively, of the glycolipid fraction (14). The neutral lipid fraction was resolved into seven components with the solvent system employed. The four major components were 60 triglycerides, sterols, diglycerides and free fatty acids. These four components have previously been re- ported to account for 30, 13, 9 and 8%, respectively, of the total neutral lipid fraction (14). The fatty acid composition of the total, phospho- and glycolipid fractions are shown in Table I. The major fatty acids detected were palmitic (16:0), oleic (18:1), y-linolenic (18:3) and arachidonic (20:4). The phospholipid fraction was significantly enriched for palmitic and arachidonic acids in comparison with the glycolipid fraction. The glycolipid fraction in turn contained more oleic and y-linolenic acids than the phospholipid fraction. Analysis of the glycolipid sugars indicated that the major sugars present were glucose (83%), and mannose (15%). No N-acetylglucosamine was detected. Lipids - ESR SpectrOSCOpy Phase transformations were previously observed with zoospores $2.11XP’ and in the zoospore total lipid extract (10). In multicomponent systems, i.e., bio- membranes, such phase transformations can be correlated to lipid phase transitions, lateral phase separations or lipid clusters. 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