Two isoforms of Drosophila dynamin in wild-type and shibire(ts) neural tissue: different subcellular localization and association mechanisms.

The temperature-sensitive mutations of the shibire (shi) gene in Drosophila cause endocytic arrest, resulting in neurotransmission block and paralysis at high temperatures. However, underlying mechanism for the defects is not yet known. We examined the subcellular distribution of dynamin, a product of the shi gene, by immunoblotting and immunocytochemical assays. Two isoforms of dynamin with apparent M(r) of 92 kD and 94 kD have been detected in wild-type and shi(n) adult neural tissue. The two isoforms were reproducibly associated with different subcellular fractions of head homogenates. The 94kD isoform is fractionated in the low speed (2.000 x g) pellet containing plasma membrane fragments, and the 92kD isoform in the high speed (130,000 x g) pellet. In this procedure, very little dynamin remained in the high speed supernatant fraction. The 94 kD isoform represents the majority (65-75%) of total dynamin and appears to be a peripheral membrane protein. It can be extracted from the low speed membrane pellet by high salt, Na2CO3 (pH 11) or Triton X-100 treatments. Extracted 94kD dynamin from both wild-type and mutant homogenates is able to reassociate with artificial phospholipid vesicles at both permissive and restrictive temperatures. Binding of the 94 kD dynamin to liposomes appears to be pH-dependent, varying most significantly within the physiological pH range, which may be functionally important. The 92 kD isoform cannot be released by high salt or Na2CO3 treatments and only a small fraction is released by Triton X-100, suggesting a different mechanism of association with cell structures. The distribution of the two isoforms is not altered by the presence of stabilized microtubules in homogenates. No apparent degradation or subcellular redistribution of mutant dynamin was detected in two shi(n) alleles after heat shock or block of the dynamin GTPase activity, suggesting that intracellular redistribution or degradation of mutant dynamin are not involved in the endocytosis arrest in these mutants. These observations resemble the effect of endocytosis arrest by GTP-gamma-S in rat brain synaptosomes (Takei et al., 1995), in which dynamin is trapped at the neck of invaginated pits but is absent in the clathrin-coated distal end undergoing internalization. Our finding that endocytosis arrest by shi(n) mutations and GTP-gamma-S do not lead to cumulation of dynamin in the low speed pellet fraction further suggests that the 94 kD isoform remains associated with the plasma membrane during coated vesicle pinch-off and that the two isoforms do not appear to correspond to different functional states of dynamin but are likely to be involved in separate cellular compartments within the membrane cycling pathway (e.g., the plasma membrane, endosomes, and endoplasmic reticulum).

Local deformation of human red blood cells in high frequency electric field.

A method of local and general deformation of single erythrocytes by external forces in high-frequency electric field is described. The method allows the avoidance of any mechanical contact of the cell with electrodes. Under the action of the forces applied human erythrocytes change their shape and produce various membrane structures: long filopodia-like processes, retraction fibers and lamella-like structures. These structures are never formed by erythrocytes under normal conditions, but are typical for fibroblasts, macrophages and epithelium cells. By the method developed the elastic properties of spicules on the membranes of echinocytes were also studied. Deformation of echinocyte in high-frequency electric field leads to the smoothing out of spicules. However, after the electric field is turned off, echinocyte restores its initial forms including the number and localization of all initial spicules on the cell surface.

Reversible large-scale deformations in the membranes of electrically-treated cells: electroinduced bleb formation.

Morphological changes in electrically-treated cells have been investigated by light and scanning electron microscopy. The application of 100-microseconds rectangular pulses of 1.3 kV/cm electric field to different types of cells (FBT, MEF, RAT-1, L-cells) in the physiological medium leads to the formation and growth of spherical and hemispherical protuberances of the cell membrane. The formation of such electroinduced blebs is not associated with the cells' death and is reversible. The electroinduced blebs are mainly formed at those sites of the cell membrane which are subjected to the highest voltage during the electric pulses. Increasing the tonicity of the medium by introducing 20 mM of inulin prevents the bleb formation, indicating the osmotically-dependent nature of the processes involved. When electric pulses are applied to the cells pre-treated with cytochalasin B, the formation of electroinduced blebs occurs independently from cytochalasin-induced ones originally present on such cells. Speculations are presented concerning the nature of the membrane structural changes underlying the electroinduced blebbing and their possible role in some electrically-induced processes.