Margareta Wallin

Effect of S-100 proteins and calmodulin on Ca2+-induced disassembly of brain microtubule proteins in vitro

The brain-specific S-100 protein, discovered in 1965 [l] is a mixture of two very similar proteins, the S-100a and S-100b protein. These proteins are dimers of highly homologous subunits: S-1OOa (a/I) and S-100b (/3/3) [2,3]. Both proteins are small (M, 20 000) very acidic and water soluble. It is assumed, that they are mainly located in the cytosol of glial cells [4] but they have also been found bound to membranes [5,6]. The biological activity of the S-100 proteins remains unknown. However, these proteins share two typical amino acid sequences in their primary structure, associated with the calcium-binding domain [7] which indicates that they also belong to the calcium-binding protein family, such as among others, calmodulin, troponin C and parvalbumin. Therefore, it has been proposed that a calciumsensitizing factor or factors should regulate the microtubule disassembly in vivo [ 10,111. Calmodulin has often been suggested [9,12,13] to have this role as it potentiates the disassembly effect of Ca2+ [ 121 and is found to be localized at the ends of the mitotic spindle [ 131. We now report the effect of S100 proteins on the Ca2+-induced disassembly of microtubule proteins in comparison with the effect of calmodulin. We found that S-100 protein induced disassembly of microtubules with a higher efficiency than calmodulin at mM Ca2 + levels.

Proteolysis of tubulin and microtubule-associated proteins 1 and 2 by calpain I and II. Difference in sensitivity of assembled and disassembled microtubules

Calpain I and II (EC 3.4.22.17) are Ca2+-activated neutral thiol-proteases. Isolated brain tubulin and microtubule-associated proteins were found to be good substrates for proteolytic degradation by brain calpain I and II. The assembly of microtubules was totally inhibited when the calpains were allowed to act on microtubule proteins initially, and a complete disassembly was found after addition of calpain I to assembled microtubules. The high-molecular weight microtubule-associated proteins were degraded within a few minutes following incubation with calpain as shown by SDS-polyacrylamide gel electrophoresis and electron microscopy. When calpain was added to pre-formed microtubules, either in the presence or in the absence of microtubule-associated proteins, the proteolysis was significantly reduced. When tubulin was pre-assembled by taxol, the formation of proteolytic fragments was decreased indicating that assembly alters the availability of tubulin sites for proteolytic cleavage by calpain. Digested tubulin spontaneously formed aberrant polymers. No considerable change of apparent net charge was seen, thus indicating that calpain cleaves off fragments containing neutral amino acid residues and/or that the fragments of tubulin remain associated as an entity with the same charge as native tubulin. The results suggest that the calpains act as irreversible microtubule regulators.

Rapid color change in fish and amphibians – function, regulation, and emerging applications

Physiological color change is important for background matching, thermoregulation as well as signaling and is in vertebrates mediated by synchronous intracellular transport of pigmented organelles in chromatophores. We describe functions of and animal situations where color change occurs. A summary of endogenous and external factors that regulate this color change in fish and amphibians is provided, with special emphasis on extracellular stimuli. We describe not only color change in skin, but also highlight studies on color change that occurs using chromatophores in other areas such as iris and on the inside of the body. In addition, we discuss the growing field that applies melanophores and skin color in toxicology and as biosensors, and point out research areas with future potential.

Evidence for Several Roles of Dynein in Pigment Transport in Melanophores

Melanophores are specialized cells that transport pigment granules to and from the cell center, giving animals the ability to change skin color. A kinesin-related plus-end motor has previously been shown to be responsible for pigment granule dispersion [V.I. Rodionov, F.K. Gyoeva, and V.I. Gelfand. Proc. Natl. Acad. Sci. USA. 1991, 88:4956-4960]. Here, we have microinjected a dynein antibody (70.1) into cultured cod (Gadus morhua) melanophores and used the dynein inhibitor vanadate on permeabilized melanophores in skin pieces, to examine the role of the microtubule minus-end motor dynein in these cells. Both pigment granule aggregation and maintenance of the spherical central pigment mass (CPM) were inhibited by the antibody and by vanadate. Vanadate or antibody treatment of cells with aggregated pigment did not induce pigment dispersion. However, when the antibody-injected cells were induced to disperse pigment, the pigment moved farther to the cell periphery, which resulted in a depletion of pigment in the cell center. Similar superdispersion of previously uniformly distributed pigment was also seen when the antibody was injected in melanophores with dispersed pigment. Our results demonstrate that both pigment aggregation and maintenance of the CPM are dynein-dependent processes. Our data further show that dynein is involved in the homogeneous distribution of dispersed pigment. These results suggest that both dynein and kinesin are active in keeping pigment granules dispersed throughout the cytoplasm, transporting pigment granules in opposite directions. The possibility that dynein is continuously active during both aggregation and dispersion, while kinesin might be the target for regulation, is discussed.

Chapter 6 New Insights into Melanosome Transport in Vertebrate Pigment Cells

Pigment cells of lower vertebrates provide an excellent model to study organelle transport as they specialize in the translocation of pigment granules in response to defined chemical cues. This review will focus on the well-studied melanophore/melanocyte systems in fish, amphibians, and mammals. We will describe the roles of melanin, melanophores, and melanocytes in animals, current views on how the three motor proteins dynein, kinesin, and myosin-V are involved in melanosome transport along microtubules and actin filaments, and how signal transduction pathways regulate the activities of the motors to achieve aggregation and dispersion of melanosomes. We will also describe how melanosomes are transferred to surrounding skin cells in amphibians and mammals. Comparative studies have revealed that the ability of physiological color change is lost during evolution while the importance of morphological color change, mainly via transfer of pigment to surrounding skin cells, increases. In humans, pigment mainly has a role in protection against ultraviolet radiation, but also perhaps in the immune system.

Diethylstilbestrol induces metaphase arrest and inhibits microtubule assembly

Diethylstilbestrol produced a dose-dependent increase in the mitotic index of the human prostatic tumour cell line DU 145. This is the result of metaphase arrest which may be induced by the action of diethylstilbestrol on spindle microtubules. Evidence is presented to show that diethylstilbestrol affects microtubules. Diethylstilbestrol completely inhibited the assembly of isolated brain microtubules although only partial disassembly could be induced. In contrast to other microtubule poisons, the inhibitory effect of diethylstilbestrol on taxol-induced self-assembly could be reversed by the addition of GTP.

Interaction of estramustine phosphate with microtubule-associated proteins

We have reported [(1984) Cancer Res., in press] that estramustine phosphate inhibits microtubule assembly and disassembled preformed microtubules. We now present evidence that estramustine phosphate inhibits microtubule assembly by binding to the microtubule‐associated proteins. We have found that: (1) additional microtubule‐associated proteins relieved the inhibition of assembly by estramustine phosphate; (2) 3H‐labelled estramustine phosphate bound predominantly to the microtubule‐associated proteins; and (3) the content of the microtubule‐associated proteins was reduced in taxol reversed estramustine phosphate‐inhibited microtubules.

Effects of potential aneuploidy inducing agents on microtubule assembly in vitro.

The present study was carried out with the 10 known or suspected spindle poisons of the Commission of the European Communities program to study aneuploidy induction. We have investigated these substances on the assembly of isolated bovine microtubules at 10, 100 and 1000 microM and studied morphology by electron microscopy. The substances could be grouped into two categories, strong and weak inhibitors. Colchicine, vinblastine and thimerosal were strong inhibitors; cadmium chloride, thiabendazole, chloral hydrate, hydroquinone, diazepam and econazole were weak inhibitors, the latter three causing aberrant forms visible on electron microscopy. Pyrimethamine did not inhibit the assembly of microtubules, but produced aberrant forms.

Cold-Stable and Cold-Adapted Microtubules

Most mammalian microtubules disassemble at low temperature, but some are cold stable. This probably has little to do with a need for cold-stable microtubules, but reflects that certain populations of microtubules must be stabilized for specific functions. There are several routes by which to achieve cold stability. Factors that interact with microtubules, such as microtubule-associated proteins, STOPs (stable tubule only polypeptides), histones, and possibly capping factors, are involved. Specific tubulin isotypes and posttranslational modifications might also be of importance. More permanent stable microtubules can be achieved by bundling factors, associations to membranes, as well as by assembly of microtubule doublets and triplets. This is, however, not the explanation for cold adaptation of microtubules from poikilothermic animals, that is, animals that must have all their microtubules adapted to low temperatures. All evidence so far suggests that cold adaptation is intrinsic to the tubulins, but it is unknown whether it depends on different amino acid sequences or posttranslational modifications.

Effect of estramustine phosphate on the assembly of trypsin-treated microtubules and microtubules reconstituted from purified tubulin with either tau, MAP2, or the tubulin-binding fragment of MAP2.

Estramustine phosphate, an estradiol nitrogen-mustard derivative is a microtubule-associated protein (MAP)-binding microtubule inhibitor, used in the therapy of prostatic carcinoma. It was found to inhibit assembly and to induce disassembly of microtubules reconstituted from phosphocellulose-purified tubulin with either tau, microtubule-associated protein 2, or chymotrypsin-digested microtubule-associated protein 2. Estramustine phosphate also inhibited assembly of trypsin-treated microtubules, completely depleted of high-molecular-weight microtubule-associated proteins, but with their microtubule-binding fragment present. In all cases estramustine phosphate induced disassembly to about 50%, at a concentration of approximately 100 microM, at similar protein concentrations. However, estramustine phosphate did not affect dimethyl sulfoxide-induced assembly of phosphocellulose-purified tubulin. Estramustine phosphate is a reversible inhibitor, as the nonionic detergent Triton X-100 was found to counteract the inhibition in a concentration-dependent manner. The reversibility was nondisruptive, as Triton X-100 itself did not affect microtubule assembly, microtubule protein composition, or morphology. This new reversible MAPs-dependent inhibitor estramustine phosphate affects the tubulin assembly, induced by tau, as well as by the small tubulin-binding part of MAP2 with the same concentration dependency. This indicates that tau and the tubulin-binding part of MAP2, in addition to their assembly promoting functions also have binding site(s) for estramustine phosphate in common.