Vivianne T. Nachmias

Small actin-binding proteins: the β-thymosin family

Thymosin β4 is a major actin monomer binding protein present at high concentration in many vertebrate cells and cell lines. The interactions of actin with thymosin β4, actobindin and profilin are compared. Nine β-thymosins have been identified; six have been shown to bind to actin. Regulation of the synthesis of thymosin β10 and thymosin β4 has been found in brain development and after stimulation of several cell types, respectively. The extracellular effects of thymosin β4 still need clarification.

AMINE OXIDASE AND 5‐HYDROXYTRYPTAMINE IN DEVELOPING RAT BRAIN

I N THE early development of mammalian brains there is a relatively short period of time during which the activities of several enzymes, though not all, increase from low levels to the higher levels characteristic of the adult. In the guinea pig this period, at least for frontal cerebral cortex, occupies the last trimester of gestation and is heralded by certain well defined cytological signs of differentiation, including the appearance of Nissl bodies and of cell processes (see FLEXNER, 1955, for review). In the rat, the corresponding period, preceded by similar cytological events, is from about the tenth to the twentieth days of postnatal life (SUGITA, 1918). In view of the importance of monoamine oxidase for the metabolism of certain amines in brain (DAVISON, 1958; SJOERDSMA ct ul., 1955) it was of interest to study the development of the enzyme’s activity and to determine whether or not it is present in newborn rat brain. It has been suggested that one of its substrates, 5-hydroxytryptamine (serotonin) may be active at synapses (BRODIE and SHORE, 1957; UDENFRIEND et al., 1957). Therefore, it appeared important to determine its concentration in the developing brain in relation to the development of amine oxidase.

Actin and the elongation of plant cells

SummaryWe have investigated in parallel the effects of different types of inhibitors on elongation of oat coleoptile cells in IAA and on the integrity of the longitudinally oriented actin-containing microfilaments present in control cells as detected by rhodamine phalloidin (RP) staining. Where growth was 50% inhibited by cytochalasin D (CD), we observed extensive to complete breakdown of the microfilaments (MFs) with the appearance of new RP staining in a few nuclei and markedly along the cross walls. When the CD-treated coleoptiles were held at 4°C the nuclei were uniformly strongly stained and cross wall staining was not seen, suggesting that translocation to the nuclei may be an intermediate step in final disposition of the actin. The divalent ions calcium and magnesium both inhibited growth in a dose dependent way, with calcium giving 50% inhibition at 65 mM and magnesium at 25 mM. KCl was not inhibitory and did not reverse the inhibition by divalent ions even at 250 mM. At 50% inhibition by either ion, the long MFs in many cells were replaced either by short fragmented MFs and small brightly staining granules (calcium) or by short usually twisted MFs and large, less intensely staining masses (magnesium). Iodoacetate at 2mM inhibited growth almost completely and resulted in short, fragmented, twisted or curled MFs in most of the cells. Abscisic acid also caused replacement of some MFs with faintly fluorescent bodies somewhat larger than those in CaCl2; occasionally granules similar to those in CaCl2 were also seen. Only mannitol and galactose, which inhibit growth by their osmotic effect, did not cause breakup of the MFs; indeed the MFs in mannitol appeared if anything wider and thicker. The results show that under the influence of three types of growth inhibitors the actin-containing MFs in the cells are broken down to different extents resulting in new structures. The results support the idea that the integrity of the MF bundles is linked, perhaps causally, to the elongation of theAvena cells.

Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-β4

In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of developing muscle cells.

Cap Z, a calcium insensitive capping protein in resting and activated platelets

Capping of the barbed‐ends of actin filaments is an important mechanism for control of the cytoskeleton. In platelets, a valuable model system, it has been thought that gelsolin was the major capping protein. We now report that platelets contain 2 μM Cap Z, a calcium insensitive heterodimeric capping protein; two major and additional minor isoforms of both α and β subunits are present. In lysates from resting platelets 75–80% of the Cap Z sediments with the high speed pellet, but if the platelets are activated with thrombin for 10 s, about 15% of the Cap Z leaves the pellet fraction and is found in the high speed supernatant where it is not bound to actin. This translocation of Cap Z to the supernatant is also observed when resting platelets are lysed into buffer containing 50–100 μM GTPγS and 10 mM EGTA. Our results suggest that release of Cap Z from some actin filaments could generate free filament barbed‐ends. An increase in free barbed‐ends has been reported in platelet lysates prepared shortly after thrombin activation.

The Cytoskeleton of the Blood Platelet: A Dynamic Structure

Publisher Summary This chapter reviews the problem of how the cytoskeleton can determine the shape of the resting platelet and how it may account for shape change, and specific new ideas on secretion and on the possible roles of talin and vinculin. The chapter presents some new data that resolve much of the controversy on the state of the microfilaments in resting platelets. New findings on the role of the microtubule coil are also presented. What makes platelets especially interesting for the study of dynamic changes in the cytoskeleton is that they display remarkably rapid and profound changes in the cytoskeleton correlated with equally striking changes in cell shape and cell–cell or cell–substrate interaction. Platelets circulate in a resting state. They appear as flattened disks only 3μm in diameter, and 0.75μm wide. Within a few seconds after addition of agonists, this alters to a remarkable indented sphere with many filopodia. On surfaces, they first extend filopodia and then spread out in 20–30 minutes into greatly enlarged plate-like structures. After agonists, they also develop the ability to bind fibrinogen and to aggregate and secrete the contents of their storage granules.

Actin mediated calcium dependency of actomyosin in a myxomycete

Abstract A fraction obtained from Physarum polycephalum by differential centrifugation displays magnesium adenosine triphosphatase activity; at low ionic strength (0.07 M KCl) the rate at which ATP ∗ is split in 0.1 mM CaCl 2 is from 1.5 to 6.6 times the rate in 1 mM EGTA ∗ . Both actin and myosin are present in this fraction. On SDS gels several polypeptide bands are present in the range of 39,000 daltons to 14,000 daltons as well as those of actin and myosin. The addition of desensitized rabbit muscle actin to the fraction increased the rate of ATP splitting in EGTA, thereby decreasing the EGTA inhibition 30–50%. We conclude that actomyosin regulation by calcium in this acellular slime mould is, at least in large part, mediated through actin.

Rapid effects ofphorbol ester on platelet shape change, cytoskeleton and calcium transient

Platelets are rapidly activated by several agonists. When phorbol 12‐myristate 13‐acetate (PMA) was added to washed platelet suspensions 10 s prior to either thrombin or ADP, it caused a dose‐dependent inhibition of shape change correlated with decreased myosin association with the cytoskeleton and with inhibition of the calcium transient measured in fura‐2‐loaded platelets. PMA added 5–10 s after agonists did not reverse shape change or the association of myosin with the cytoskeleton, but markedly increased the rate at which the calcium signal returned to the baseline. The analogue, 4α‐phorbol didecanoate did not cause these effects. Our results suggest that one effect of C‐kinase activation is to provide negative feedback in sequential responses.

Development of polarity in human erythroleukemia cells: Roles of membrane ruffling and the centrosome

Cultured human erythroleukemia (HEL) cells were used to study the genesis of polarity in single cells. HEL cells grow in suspension in culture medium, but attach and spread on fibronectin when treated with 10 nM phorbol myristate acetate. If the spread cells are treated with dibutyryl cyclic adenosine monophosphate, about 50% of the cells polarize and form very striking elongated processes. Time-lapse video microscopy showed that elongation develops in these cells because the anterior pole of the cell, which bears a small ruffled membrane, moves slowly (approximately 0.16 microgram/min) forward on the substratum elongating the posterior pole or tail behind it. Using indirect immunofluorescence we found that elongation of the tail correlates with the development of long microtubule bundles emanating from the centrosome, which is located posterior to the nucleus on the trailing side of the cell. Incubation with nocodazole, which inhibited development of the long microtubules and the elongation, resulted in a centrosome positioned over the nucleus in 45% of the cells and extension of the membrane ruffling to many points around the cells periphery. Unexpectedly, time-lapse video microscopy demonstrated that the treated cultures also contained some smaller cells with very marked anterior ruffles and short tails. These cells moved rapidly about the culture dish (maximum 0.8 microgram/min; average 0.5 microgram/min). In these fast moving cells the centrosome was also located posterior to the nucleus. Several recent reports have stressed the importance of relocation of the centrosome to an anterior position in cells developing polarity after experimental wounding. Our results show that both striking polarization and rapid motility can occur without such a relocation. The polarity induced in the HEL cells correlates most clearly with the limitation of membrane ruffling to one region; this limitation is removed by microtubule disassembly. We therefore propose that localized ruffling is the critical first step in polarized motility generally, and that centrosomal position is related to other factors.

Physarum myosin light chain one: A potential regulatory factor in cytoplasmic streaming

SummaryPhysarum myosin is composed of a heavy chain of about 225,000 daltons and two small polypeptides of 17,700 and 16,100 daltons, called light chain one (LC 1) and two (LC 2). Light chain one is shown to belong to the general class of regulating light chains by two independent criteria. After denaturation, purification and renaturation of thePhysarum light chains only LC 1 will combine with scallop myofibrils in which one myosin regulatory light chain has been removed. This LC 1 can restore inhibition of the ATPase activity of the myofibrils at 10−8 M Ca++ just as well as light chains from rabbit skeletal myosin. Secondly, this LC 1 is the only component of the myosin that is significantly phosphorylated by an endogenous kinase present in crude actomyosin. An active phosphatase is also present. Preliminary results could not detect calcium sensitivity for either kinase or phosphatase, nevertheless the importance of phosphorylation in affecting activity of biological systems suggests that LC 1 may serve some regulating function for plasmodial actomyosin.