Joann J. Otto

Actin-bundling proteins

Recent studies have greatly expanded our understanding of actin-bundling proteins. A new group of actin-bundling proteins, the fascins, has been recognized. An actin-bundling protein inhibits actin depolymerization even under conditions in which it cannot produce a gel, which suggests that bundling proteins may affect actin filament dynamics. A villin-like protein is present in Dictyostelium, shedding doubt on current ideas on the evolution of villin. Domain mapping continues to be a major thrust of research into most groups of bundling proteins.

Changes in the F‐actin cytoskeleton during neurosensory bristle development in Drosophila: The role of singed and forked proteins

Drosophila neurosensory bristle development provides an excellent model system to study the role of the actin-based cytoskeleton in polarized cell growth. We used confocal fluorescence microscopy of isolated thoracic tissue to characterize changes in F-actin that occurred during macrochaete development in wild type flies and mutants that have aberrant bristle morphology. At the earliest stages in wild type bristle development, cortical patches of F-actin were present, but no bundles were observed. Actin bundles began to form at 31% of pupal development and became more prominent as development progressed. The F-actin patches gradually disappeared and were no longer present by 38% of pupal development. The distribution of F-actin in singed3 mutant macrochaetae was indistinguishable from wild type bristles until 35% of development when the actin bundles began to splay and appear ribbon-like. In forked36a bristles, the mutant phenotype was evident at earlier stages of development than the singed3 mutant. Wild type tissue stained with antibodies against the forked protein demonstrated that the forked protein colocalized with F-actin structures found in early and late stage developing macrochaetae. Antibodies against the singed protein showed it appeared to localize with F-actin structures only at later stages in development. These data suggested that the forked gene product was required for the initiation of fiber bundle formation and the singed gene product was required for the maintenance of fiber bundle morphology during bristle development. Similar analyses of singed3/forked36a double mutants provided additional genetic evidence that the forked gene product was required before the singed gene product. Further, the analyses suggested that at least one additional crosslinking protein was present in these bundles.

Snoods: A periodic network containing cytokeratin in the cortex of starfish oocytes

An extensive fibrous cytoskeletal component in the cortical cytoplasm of oocytes of the starfish Pisaster ochraceus reproducibly stains with anticytokeratin antibody and hence contains cytokeratin. The large-meshed network resembles a snood (hair net). Snood fibers form loops and branches throughout the cortex of a premeiotic oocyte, except at the animal pole where they emanate from a nonstaining zone surrounding the centrosomes. By immunofluorescence microscopy of isolated cortices and electron microscopy of isolated cortices and intact oocytes, snood fibers exhibit complex striations with a periodicity of approximately 0.75 micron. Snoods are not colocalized with the cortical arrays of microtubules and are unaffected by drugs that disrupt microtubules or microfilaments. Stimulation of oocyte maturation by 1-methyladenine causes snoods to disappear, presumably by disassembly, about halfway to the time of germinal vesicle breakdown. They do not reappear during meiosis, fertilization, or development to the two-cell stage, and their functional importance, if any, during oogenesis or development remains to be elucidated.

Vinculin and talin: kinetics of entry and exit from the cytoskeletal pool.

Vinculin and talin, two major components of focal contacts, exist in cytosolic and cytoskeletal pools. The kinetics of entry and exit of the two proteins between the two pools were investigated in normal and transformed cells. In cultured chick embryo fibroblasts, a fraction (2-5%) of the newly synthesized vinculin and talin reached maximal levels in the cytoskeleton in 30-45 min. Both proteins had 2-3 times shorter half-lives in the cytoskeletal pool (t1/2 = 6-7 h) than in the cytosolic pool (t1/2 = 14-15 h), which suggests that the incorporation of cytosolic vinculin and talin into the cytoskeleton does not involve a simple equilibrium between the two pools. However, after disruption of cell-to-substrate adhesion by trypsinization, an equilibrium in the incorporation between the two pools was transiently established, resulting in the use of the preexisting cytosolic pools of the two proteins during re-establishment of cell-to-matrix contacts. Viral transformation did not cause a significant change in the incorporation rates into the cytoskeleton. However, it decreased the half-lives of both proteins in the cytoskeletal pool (t1/2 = approximately 4 h) and in the cytosolic pool (t1/2 = 9-10 h). The increased turn-over rates of vinculin and talin in the cytoskeletal pool in transformed cells may contribute to the enhanced motility of transformed cells.

Profilin functions in cytokinesis, nuclear positioning, and stomatogenesis in Tetrahymena thermophila.

Abstract Expression of the actin-binding protein profilin was disrupted in the ciliate Tetrahymena thermophila by an antisense ribosome method. In cells with the antisense disruption no profilin protein was detected. Cultures of cells with the antisense disruption could be maintained, indicating that profilin was not essential for cytokinesis or vegetative growth. Disruption of the expression of profilin resulted in many cells that were large and abnormally shaped. Formation of multiple micronuclei, which divide mitotically, was observed in cells with a single macronucleus, indicating a defect in early cytokinesis. Some cells with the antisense disruption contained multiple macronuclei, which in Tetrahymena may indicate a function late in cytokinesis. The lack of profilin also affected cytokinesis in the cells that could divide. Normal-sized and normal-shaped cells with the antisense disruption took significantly longer to divide than control cell types. The profilin disruption revealed two new processes in which profilin functions. In cells lacking profilin, micronuclei were not positioned at their normal site on the surface of the macronucleus and phagocytosis was defective. The defect in phagocytosis appeared to be due to disruption of the formation of oral apparatuses (stomatogenesis) and a possible failure in the internalization of phagocytic vacuoles.

Association of actin and myosin in the contractile ring.

The process of cell division culminates in cytokinesis, the actual cleavage of one cell into two. Cytokinesis is powered by the interaction of actin and myosin in the contractile ring, a specialized, transient organelle that forms immediately at the onset of cytokinesis and disappears once cleavage of the cell is complete. Because of its transient nature and small size relative to the entire cell, the contractile ring has proven difficult to study at the biochemical level. Although it is clear that the contraction of actin and myosin occurs in the contractile ring (reviewed in refs. 1 and 2), most details of their organization are still unknown. In addition, little is known about other components of the ring. These components probably include molecules that regulate the interaction of actin and myosin, those that are involved in the specific localization of actin and myosin into the ring, and those that are involved in and regulate the polymerization and depolymerization of actin and myosin in the ring. Furthermore, to have an effective cleavage, the contractile complex of actin and myosin in the contractile ring must be attached by at least one component to the plasma membrane at the cleavage furrow. To address some of these problems, we have been studying the organization of actin and myosin in the contractile ring in preparations of isolated cortices of dividing sea urchin eggs. This paper reviews our results on the morphology and localization of actin and myosin in the contractile ring and our data on the association of actin and myosin with the cortex in the cleavage furrow. To prepare isolated cortices containing contractile rings, sea urchin eggs that are just beginning to undergo cleavage are pipetted onto polylysine-coated coverslips. After a short time of attachment, the eggs are sheared with a stream of buffer (75 mM KCl, 1 mM EGTA, 1 mM MgCl,, 1 mM TAME, and 20 mM Hepes, pH 7.4). The isolated cortices can then be fixed immediately for immunofluorescence staining or they can be treated with reagents that might alter one or more ring components and then fixed and stained with the desired antibodies. Details of this procedure can be found in Schroeder and Otto?

Chapter 6 Immunoblotting

Publisher Summary This chapter presents the technique of immunoblotting with emphasis on blotting of one-dimensional SDS gels onto nitrocellulose. Immunoblotting or Western blotting is a procedure in which a replica of a separating gel is produced by transferring proteins from a gel to a membrane such as nitrocellulose. This replica, or blot, is subsequently incubated or stained with antibodies. Immunoblotting provides information about an antigen, such as the subunit molecular weight of the antigens can be determined from its mobility in a sodium dodecyl sulfate (SDS) gel that is blotted. A blot can also give information about the purity of antibodies. Most types of gels, both one and two dimensional, can be blotted provided they are strong enough to withstand physical manipulation. Gels can be blotted either by submersed (wet) methods or by buffer-soaked filter paper (semidry) methods. Transfer can be accomplished either by passive diffusion or electrophoresis. The gel sandwich consists of the gel adjacent to the nitrocellulose with both enclosed by heavy filter paper. The paper and nitrocellulose are saturated with transfer solution to carry the electrical current. Nitrocellulose and other such membranes have a high affinity for proteins (and usually nucleic acids). The entire paper must be coated with protein, otherwise the primary antibody will bind everywhere nonspecifically.

Chapter 7 Immunoprecipitation Methods

Publisher Summary This chapter highlights immunoprecipitation methods along with variations that may be necessary under certain conditions. These methods are used in several major techniques for research in cell biology, such as identifying the molecular mass of an antigen, characterizing the specificity of antibodies, identifying molecules associated with antigens, and quantifying amounts of antigen with radioimmunoassays. The chapter emphasizes on the first three techniques, each of which uses gel electrophoresis to analyze the results. The basic steps for immunoprecipitation include (1) solubilizing the antigen preparation, (2) clearing the preparation of any insoluble material and molecules that bind, (3) incubating with primary antibody, (4) precipitating the primary antibody with protein A or G attached to a matrix, (5) washing the immunoprecipitate, and (6) resolving the immunoprecipitate on electrophoretic gels. The primary antibody is directed against the antigen. For most kinds of analysis, the use of purified immunoglobulin is required rather than crude serum. If the primary antibody is not in a class bound by protein A or G , a bridge antibody that is directed against the species of the primary antibody and that binds protein A or G is required. A secondary antibody directed against the immunoglobulin of the primary antibody can be coupled to a matrix such as Sepharose or agarose.

Relative changes in F-actin during the first cell cycle: Evidence for two distinct pools of F-actin in the sea urchin egg

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin.

Culturing Hydra of the Same Species but of Different Sizes

Size and morphology are controlled by patterning agents such as morphogenetic and positional information fields. In order to study the nature of these fields it is useful to be able to modify them. Controlling hydra size by varying the amounts of food given to them provides an experimental method for manipulating the sizes and properties of patterning fields, and also for obtaining hydra of particular sizes for experiments.