Addendum: CYTOPLASMIC FILAMENTS OF AMOEBA PROTEUS: I. The Role of Filaments in Consistency Changes and Movement

Although cytoplasmic streaming is a basic property of cells, the mechanism of the movement of subcellular particles is not clearly understood. Since cytoplasm isolated from plant cells and amoebas exhibited independent motility (Jarosch, 1956; Allen et al., 1960; Thompson and Wolpert, 1963), the motive force must be generated in the cytoplasm. The structures thought most likely to produce movement are the two types of cytoplasmic filaments: (a) solid filaments, which occur in several sizes, and (b) hollow microtubules. Thin filaments, about 75 Å in diameter, and thicker filaments, 150 Å in diameter and up to 0.5 µm long, were discovered in electron micrographs of thin sections of giant amoebas whose plasma membranes were torn before fixation (Nachmias, 1964) or extracted with glycerol (Schäfer-Danneel, 1967). Glycerinated amoebas contracted slightly upon adding ATP (Schäfer-Danneel, 1967).

We did our work in the spring of 1968 and showed our movies and electron micrographs at the 1968 ASCB meeting. Unknown to us at that time, Sadshi Hatano and Fumio Oosawa (1966) had isolated actin from Physarum polycephalum. Subsequently, Weihing and Korn (1969) isolated actin from Acanthamoeba, and Hatano, Kondo, and Miki-Noumura (1969) isolated actin from sea urchin eggs.

We worked on the carnivorous giant amoeba called Amoeba proteus, which moves rapidly by vigorous cytoplasmic streaming (Video 1). Inspired by the success of Hugh Huxley using electron microscopy to understand the contractile apparatus of striated muscle (Huxley, 1957), we initially tried a purely anatomical approach, which failed. Fortunately, we learned about work by Thompson and Wolpert (1963), who reported that a crude cytoplasmic extract from Amoeba proteus exhibited vigorous movements when ATP or ADP was added and it was warmed from 4° to 20°C. We did not know that others had tried but failed to confirm these findings.

We grew large-scale cultures of Amoeba proteus in a dilute buffer with living Tetrahymena pyriformis as food. We modified the method of Thompson and Wolpert (1963) to prepare the motile fraction of cytoplasm. We cooled amoebas to 4°C for 12–24 h and concentrated them by low-speed centrifugation. 5 cc of cell slurry were centrifuged at 4°C for 10 min at 18,000 rpm in a Beckman Type SW 39L rotor. The centrifugal force of 35,000 g at the tip of the tube fragmented the cells into four distinct layers. The second layer (about 1–2 ml) consisted of membrane-bounded bags of cytoplasm. We mixed this layer with an equal volume of glass-distilled water or Tris-maleate buffer, pH 7.0 (10 or 20 mM), and lysed the cells with five gentle strokes of a Teflon-glass homogenizer. We removed large fragments of plasma membrane by centrifugation at 1,000 g for 5 min. The supernatant was called Extract 1.

The original paper did not include the conditions used to make the movies, as no way existed in 1968 to share the movies with the publication. We used a Zeiss phase-contrast microscope to make movies on 16-mm film at 30 frames per second. In 2015, I digitized a sample of these movies. The movies are played back in these videos at the same rate. Thus, all sequences are in real time. Our JCB paper did not list the optics we used, but I am confident that most of the movies were made with a 100x objective. Magnifications are approximate, based on the sizes of membrane-bound particles in the movies and electron micrographs in our paper.

Modified from the abstract to the paper: At 0°C, this extract was nonmotile and similar in structure to amoeba cytoplasm, consisting of groundplasm, vesicles, mitochondria, and a few short filaments 160 Å in diameter. The extract underwent striking ATP-stimulated streaming when warmed to 22°C. We observed two phases of movement. During the first phase, the apparent viscosity usually increased, and numerous 50–70 Å filaments appeared in samples of the extract prepared for electron microscopy, suggesting that the increase in viscosity was caused, at least in part, by the formation of these thin filaments. During this initial phase of ATP-stimulated movement, these thin filaments were not detected by phase-contrast or polarization microscopy, but later, in the second phase of movement, 70 Å filaments aggregated to form birefringent microscopic fibrils. Centrifugation of Extract 1 at 10,000 g for 10 minutes produced Extract 2 consisting of pure groundplasm with no 160 Å filaments or membranous organelles. Extract 2 exhibited little or no ATP-stimulated movement, but 50–70 Å filaments formed and aggregated into birefringent fibrils. This observation and the structural relationship of the 70 Å and the 160 Å filaments in the motile extract suggested that both types of filaments may be required for movement. These two types of filaments, 50–70 Å and 160 Å, are also present in the cytoplasm of intact amoebas. Fixed cells could not be used to study the distribution of these filaments during natural amoeboid movement because of difficulties in preserving the normal structure of the amoeba during preparation for electron microscopy.

A subsequent paper identified the 70 Å filaments as actin filaments by decoration with myosin heads (Pollard and Korn, 1971).