Edward D. Korn

Single bilayer liposomes prepared without sonication

Abstract Single bilayer liposomes, indistinguishable from those obtained by sonication, can be prepared by injecting an thanolic solution of phospholipid into water. The dilute suspension is easily concentrated by ultrafiltration and the mild conditions allow neither degradation nor oxidation of the phospholipid.

Inhibition of actin polymerization by latrunculin A.

Latrunculin A, a toxin purified from the red sea sponge Latrunculia magnifica, was found previously to induce striking reversible changes in the morphology of mammalian cells in culture and to disrupt the organization of their microfilaments. We now provide evidence that latrunculin A affects the polymerization of pureactin in vitro in a manner consistent with the formation of a 1:1 molar complex between latrunculin A and G‐actin. The equilibrium dissociation constant (K d) for the reaction in vitro is about 0.2 μM whereas the effects of the drug on cultured cells are detectable at concentrations in the medium of 0.1–1 μM.

Structure of Biological Membranes

The combined x-ray diffraction and electron microscopic examination of myelin has provided reasonable, but not conclusive, support for its structure as a basically bimolecular leaflet of phospholipid that is partially interspersed with protein. But there is very little basis for extending this concept to biological membranes in general. There is no adequate experimental support for the specific orientation of phospholipids as proposed in the unit membrane theory or for the proposed polar nature of protein-lipid bonds, even in myelin. Membranes differ widely in chemical composition, metabolism, function, enzymatic composition, and even in their electron microscopic image. The only similarity is their general resemblance in electron micrographs, but, until more is known about the chemistry of electron microscopy, this evidence cannot be interpreted with confidence. One positive conclusion to which I have come is that much more chemical evidence must, and can, be obtained. Techniques for the isolation of membranes are improving and protein and lipid chemistry are now highly refined arts. Quantitative analysis of many different membranes is possible and the data can be related in some instances, notably bacterial plasma membranes, to calculations of surface area. Chemical and physical changes induced in membranes of widely different lipid composition by the preparatory procedures of electron microscopy can be determined directly and correlated with the electron microscopic image. Model systems can be assembled whose compositions closely resemble those of biological membranes. Membranes can be disassociated into subunits whose properties can be studied. In particular, x-ray diffraction analysis and electron microscopy by negative staining of reaggregates of lipoproteins isolated from membranes would be very informative. Perhaps most important, the problem of membrane structure must be considered in relation to the problems of membrane function and membrane biosynthesis.

Ultrastructural characterization of F-actin isolated from Acanthamoeba castellanii and identification of cytoplasmic filaments as F-actin by reaction with rabbit heavy meromyosin

Abstract The structures of purified ameba F-actin and cytoplasmic filaments of Acanthamoeba castellanii have been studied by electron microscopy. In negatively stained preparations, purified ameba F-actin is a beaded filament about 60Ain diameter and up to 3 μ long, with a double helical substructure similar to muscle F-actin. When reacted with rabbit muscle heavy meromyosin, ameba F-actin forms a complex consisting of a periodic linear array of ‘arrowheads’ with a spacing of 370Athat is indistinguishable from the complex between rabbit muscle F-actin and heavy meromyosin. The long thin 80Afilaments that are seen in electron micrographs of thin sections of untreated Acanthamoeba are still present after extraction of the amebas with glycerol. After treatment of the glycerinated amebas with the heavy meromyosin, distinct arrowhead complexes form along the filaments. This reaction specifically identifies the filaments as actin, first, because it can be blocked by Mg-pyrophosphate or Mg-ATP, compounds which are known to dissociate actin and heavy meromyosin and second, because the reaction does not occur with the non-specific mixture of proteins found in goat serum. The identification of the cytoplasmic filaments as F-actin strongly suggests that the filaments participate in the movement of the ameba.

I. loss of lipids during preparation of amoebae for electron microscopy

Abstract 1. 1. Amoebae were fixed in glutaraldehyde, potassium permanganate or osmium tetroxide, alone or in combination, and extracted with ethanolic solutions, propylene oxide and embedding solution as in preparation for electron microscopy. The lipid that was extracted by each solvent was measured chemically (ester and phosphorus analyses) and by radioactive analysis using amoebae grown in the presence of [ 3 H]-palmitic acid. The extracted lipids were fractionated by silicic acid chromatography into neutral lipids and phospholipids and the fatty acid composition of each was determined. 2. 2. After fixation in glutaraldehyde, the lipids were unchanged and were almost completely extracted by ethanol during the dehydration procedure. After perman-ganate fixation, all of the neutral lipid and about 25% of the phospholipid were extracted by the fixative and ethanol. Following fixation in osmium tetroxide, most of the neutral lipid and some of the phospholipid were extracted by ethanol and the embedding medium. Osmium tetroxide destroyed the unsaturated fatty acids. 3. 3. It is concluded that autoradiographic studies of lipid metabolism at the electron microscopic level are not possible if the usual methods of fixation and dehydration are employed. Also, structures visualized in the cell after fixation in glutaraldehyde alone probably do not contain lipid at the time of observation.

Regulation of Class I and Class II Myosins by Heavy Chain Phosphorylation

Myosins have been traditionally viewed as mechanochemical, actin-activated MgATPases that convert the energy of ATP hydrolysis into force between actin and myosin filaments exhibited as either movement (isotonic contraction) or tension (isometric contraction). “Conventional” myosins, i.e.myosins that form filaments, consist of a pair of heavy chains (;200 kDa) and two pairs of light chains, the regulatory light chain and the essential light chain. Each heavy chain has an N-terminal, globular head, where the actin-activated ATPase activity resides, and a C-terminal tail, through which the two heavy chains interact to form an a-helical coiled-coil rod. One of each pair of light chains is bound to the neck region of the globular heads (subfragment-1) near the head-tail junction. The tail also mediates the self-association of multiple myosin molecules into bipolar thick filaments. Ca activates muscle actomyosins predominantly in one of two general ways (1, 2): (i) actin-based regulation in which Ca binds to troponin C, a component of the tropomyosin-troponin complex that lies in the groove of the a-helical coiled-coil actin thin filament (skeletal muscle); (ii) myosin light chain-based regulation in which either Ca/calmodulin-dependent myosin light chain kinase phosphorylates a serine in the regulatory light chain (smooth muscle) or Ca binds directly to the regulatory light chain (molluscan muscle). For many years, myosin filaments, as well as actin filaments, were thought to be essential for physiologically meaningful interactions of actin and myosin. The discovery of monomeric Acanthamoeba myosin I (3), however, proved that single-headed, nonfilamentous myosins, i.e. “unconventional” myosins, could exert force between, and translocate along, actin filaments. This expanded the potential physiological roles for myosins beyond those that might be performed by filamentous myosins and initiated a search for other unconventional myosins. We now recognize the existence of a myosin family presently comprising 11 classes defined by the extent of sequence homology within the subfragment-1 (S-1) domain: 10 unconventional myosin classes and 1 conventional (so named because it was the first to be discovered) class. There are several excellent recent reviews of the sequence, structural diversity, and possible biological functions of the multiple members of this extended family (1, 4–6). Most conventional (class II, by the current classification system) nonmuscle myosins are regulated by phosphorylation of their regulatory light chains, similarly to smooth muscle myosin (7). All unconventional myosins contain at least one light chain which, with the notable exception of the amoeba myosins, appears to be calmodulin (4, 8). In some cases, calmodulin has been shown to be associated with the purified myosin, but in many cases a calmodulin light chain is inferred from the heavy chain sequence, which can contain from one to six calmodulin-binding IQ motifs in the light chain-binding region (4, 8). The IQ motifs and the regulation of unconventional myosins by Ca interacting with calmodulin, which is reminiscent even if different in detail (e.g. in some cases Ca causes calmodulin to dissociate from the heavy chain with loss of actomyosin activities) of the regulation of molluscan myosins, were reviewed recently (4, 8, 9) and will not be discussed here. Early in the study of the Acanthamoeba myosins it became clear that they were regulated exclusively by a heretofore unknown mechanism: heavy chain phosphorylation (10). The phosphorylation occurs in the S-1 domain of the unconventional, class I myosins, as one might expect for a covalent modification that regulates MgATPase activity, but, and quite unexpectedly, near or at the end of the C-terminal tail of the conventional, class II myosin (6). The latter modification is quite remote from the regulated ATPase site in the S-1 domain and separated from it by a relatively rigid a-helical coiled-coil, thus posing some very interesting questions about the mechanism of signal transduction between the phosphorylation site and the catalytic site. Thus far, only Acanthamoeba, Dictyostelium, and Physarummyosins have been shown definitively to be regulated by heavy chain phosphorylation. However, there are numerous examples of heavy chain phosphorylation of vertebrate nonmuscle class II myosins, in vivo as well as in vitro, with as yet no known substantial biochemical or physiological consequences (6, 11). Heavy chain phosphorylation of vertebrate nonmuscle class II myosins deserves more attention than it has received. Possibly this review will stimulate interest in those systems. In addition to novel mechanisms for regulation of actomyosin MgATPase activity, an emerging feature from the study of unconventional myosins is the probable role of their nonfilamentous tails in regulating function. Tail domains may determine the intracellular localization of the myosin and the physiological task for which the mechanochemical activity of its S-1 domain is used. This aspect of unconventional myosins will be briefly addressed.

II. Synthesis of bis(methyl 9,10-dihydroxystearate)osmate from methyl oleate and osmium tetroxide under conditions used for fixation of biological material

Abstract 1. 1. Methyl oleate was reacted with osmium tetroxide in water at 0°, conditions very similar to those used for fixation of biological material with osmium tetroxide. 2. 2.The major reaction product (the only one containing the fatty acid) was identified as bis(methyl 9,10-dihydroxystearate)osmate. 3. 3.Methods are described for the isolation of this compound and for its characterization by thin-layer and gas-liquid chromatography.

III. Modification of oleic acid during fixation of amoebae by osmium tetroxide

Abstract 1. 1. Amoebae (Acanthamoeba sp.) were fixed with 1% osmium tetroxide exactly as for electron microscopy. The fixed cells were extracted with ethanol and the lipids in the extract and residue were separately trans-esterified in 0.5 N sodium methoxide. The fatty acid methyl esters were then separated and identified by gas-liquid and thin-layer chromatography. 2. 2. All of the unsaturated fatty acids, that normally account for about 85% of the total fatty acids, were destroyed by osmium tetroxide. At least 40% of the oleic acid (the major fatty acid of Acanthamoeba) was recovered as bis(methyl 9,10-dihydroxystearate)osmate. No methyl 9,10-dihydroxystearate was found. 3. 3. Thus, it seems most likely that during fixation of biological material with osmium tetroxide unsaturated fatty acids are converted to stable glycol osmates.

The sterols of Trypanosoma cruzi and Crithidia fasciculata

Abstract 1. 1. Six sterols have been detected in Trypanosoma cruzi cultured on serum-free medium. Four of these are ergosterol, 22,23-dihydroergosterol, 7-dehydroporiferasterol and 7-dehydroclionasterol (or their C24 isomers from which they cannot be distinguished with the small quantities available). The remaining two sterols may be 24-methylene-7-dehydrocholesterol and 24-ethylidene-7-dehydrocholesterol but this has not been proven. 2. 2. In addition to these six sterols, cholesterol is also a major component of T. cruzi cultured in the presence of serum. 3. 3. Ergosterol (or its C24 isomer) was the only sterol detected in Crithidia fasciculata cultured in serum-free medium. When grown in the presence of serum the cells also contained cholesterol and 22,23-dihydroergosterol (or its C24 isomer).

Ameba actin: The presence of 3-methylhistidine

Abstract Actin has been isolated from the ameba, Acanthamoeba castellanii (Neff) by procedures similar to those used previously for the isolation of actin from slime mold and skeletal muscle. The three actins are very similar in their amino acid composition, and ameba actin, like skeletal muscle actin, contains one mole of 3-methylhistidine per mole of protein. Ameba actin resembles the other two actins in its molecular weight, its precipitation by added myosin at low ionic strength, its increase in viscosity upon the addition of myosin at high ionic strength which is reversed by ATP, and its ability to undergo a reversible G-F transition.