TAXOL (Fig. 1) was isolated from the plant Taxus brevifolia (western yew) by Wani et al., who reported that the molecule has antitumour activity in several experimental systems1. In our laboratory we have found that taxol, a low molecular weight neutral compound, completely inhibits division of exponentially growing HeLa cells at low concentrations of drug (0.25 µM) that have no significant effects on DNA, RNA or protein synthesis during a 4-h incubation with the cells. HeLa cells incubated with taxol for 20 h are blocked in late G2 and/or M (ref. 2). We report here that taxol acts as a promoter of calf brain microtubule assembly in vitro, in contrast to plant products such as colchicine and podophyllotoxin, which inhibit assembly. Taxol decreases the lag time for microtubule assembly and shifts the equilibrium for assembly in favour of the microtubule, thereby decreasing the critical concentration of tubulin required for assembly. Microtubules polymerised in the presence of taxol are resistant to depolymerisation by cold (4 °C) and CaCl2 (4 mM).

Promotion of microtubule assembly in vitro by taxol

The polymerization dynamics of microtubules are central to their biological functions. Polymerization dynamics allow microtubules to adopt spatial arrangements that can change rapidly in response to cellular needs and, in some cases, to perform mechanical work. Microtubules utilize the energy of GTP hydrolysis to fuel a unique polymerization mechanism termed dynamic instability. In this review, we first describe progress toward understanding the mechanism of dynamic instability of pure tubulin and then discuss the function and regulation of microtubule dynamic instability in living cells.

/pdf/microtubule-polymerization-dynamics-27938flntt.pdf

Microtubule polymerization dynamics

Integrin-mediated cell adhesions provide dynamic, bidirectional links between the extracellular matrix and the cytoskeleton Besides having central roles in cell migration and morphogenesis, focal adhesions and related structures convey information across the cell membrane, to regulate extracellular-matrix assembly, cell proliferation, differentiation, and death This review describes integrin functions, mechanosensors, molecular switches and signal-transduction pathways activated and integrated by adhesion, with a unifying theme being the importance of local physical forces

Transmembrane crosstalk between the extracellular matrix--cytoskeleton crosstalk.

Focal adhesions are sites of tight adhesion to the underlying extracellular matrix developed by cells in culture. They provided a structural link between the actin cytoskeleton and the extracellular matrix and are regions of signal transduction that relate to growth control. The assembly of focal adhesions is regulated by the GTP-binding protein Rho. Rho stimulates contractility which, in cells that are tightly adherent to the substrate, generates isometric tension. In turn, this leads to the bundling of actin filaments and the aggregation of integrins (extracellular matrix receptors) in the plane of the membrane. The aggregation of integrins activates the focal adhesion kinase and leads to the assembly of a multicomponent signaling complex.

/pdf/focal-adhesions-contractility-and-signaling-ij3wyc7tuo.pdf

Focal adhesions, contractility, and signaling

Ca(2+) regulation of contraction in vertebrate striated muscle is exerted primarily through effects on the thin filament, which regulate strong cross-bridge binding to actin. Structural and biochemical studies suggest that the position of tropomyosin (Tm) and troponin (Tn) on the thin filament determines the interaction of myosin with the binding sites on actin. These binding sites can be characterized as blocked (unable to bind to cross bridges), closed (able to weakly bind cross bridges), or open (able to bind cross bridges so that they subsequently isomerize to become strongly bound and release ATP hydrolysis products). Flexibility of the Tm may allow variability in actin (A) affinity for myosin along the thin filament other than through a single 7 actin:1 tropomyosin:1 troponin (A(7)TmTn) regulatory unit. Tm position on the actin filament is regulated by the occupancy of NH-terminal Ca(2+) binding sites on TnC, conformational changes resulting from Ca(2+) binding, and changes in the interactions among Tn, Tm, and actin and as well as by strong S1 binding to actin. Ca(2+) binding to TnC enhances TnC-TnI interaction, weakens TnI attachment to its binding sites on 1-2 actins of the regulatory unit, increases Tm movement over the actin surface, and exposes myosin-binding sites on actin previously blocked by Tm. Adjacent Tm are coupled in their overlap regions where Tm movement is also controlled by interactions with TnT. TnT also interacts with TnC-TnI in a Ca(2+)-dependent manner. All these interactions may vary with the different protein isoforms. The movement of Tm over the actin surface increases the "open" probability of myosin binding sites on actins so that some are in the open configuration available for myosin binding and cross-bridge isomerization to strong binding, force-producing states. In skeletal muscle, strong binding of cycling cross bridges promotes additional Tm movement. This movement effectively stabilizes Tm in the open position and allows cooperative activation of additional actins in that and possibly neighboring A(7)TmTn regulatory units. The structural and biochemical findings support the physiological observations of steady-state and transient mechanical behavior. Physiological studies suggest the following. 1) Ca(2+) binding to Tn/Tm exposes sites on actin to which myosin can bind. 2) Ca(2+) regulates the strong binding of M.ADP.P(i) to actin, which precedes the production of force (and/or shortening) and release of hydrolysis products. 3) The initial rate of force development depends mostly on the extent of Ca(2+) activation of the thin filament and myosin kinetic properties but depends little on the initial force level. 4) A small number of strongly attached cross bridges within an A(7)TmTn regulatory unit can activate the actins in one unit and perhaps those in neighboring units. This results in additional myosin binding and isomerization to strongly bound states and force production. 5) The rates of the product release steps per se (as indicated by the unloaded shortening velocity) early in shortening are largely independent of the extent of thin filament activation ([Ca(2+)]) beyond a given baseline level. However, with a greater extent of shortening, the rates depend on the activation level. 6) The cooperativity between neighboring regulatory units contributes to the activation by strong cross bridges of steady-state force but does not affect the rate of force development. 7) Strongly attached, cycling cross bridges can delay relaxation in skeletal muscle in a cooperative manner. 8) Strongly attached and cycling cross bridges can enhance Ca(2+) binding to cardiac TnC, but influence skeletal TnC to a lesser extent. 9) Different Tn subunit isoforms can modulate the cross-bridge detachment rate as shown by studies with mutant regulatory proteins in myotubes and in in vitro motility assays. (ABSTRACT TRUNCATED)

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.2.853

Regulation of Contraction in Striated Muscle

Head to tail polymerization of actin.

Equilibrium of the actin-tropomyosin interaction.

ADP-ribosylated actin caps the barbed ends of actin filaments.

The kinetics of actin polymerization were analyzed by taking into account nucleation, elongation, and spontaneous fragmentation of filaments. Polymerization curves measured in the presence of potassium (40 mM) were found to be in good agreement with curves calculated for the assumption that nucleation and elongation but no fragmentation reactions occur. Polymerization curves measured in the presence of calcium (1.8 mM) or magnesium (0.6 mM MgCl2 and 0.5 mM EGTA) could only be stimulated by calculated curves when spontaneous fragmentation was assumed to occur in addition to nucleation and elongation. The experiments reported in this study that even in the absence of ultrasonication or shear forces actin filaments may break spontaneously and that the extent of fragmentation depends strongly on the experimental conditions. Spontaneous fragmentation changes the shape of the polymerization curves significantly. When fragmentation of filaments takes place, a relatively long lag phase of polymerization is observed that is followed by a strongly increasing polymerization rate to reach the final constant value quickly. On the other hand, when filaments are formed exclusively by nucleation, the polymerization curves approach the final constant value slowly after a relatively short initial lag phase.

Fragmentation of actin filaments

ADP-rlbosylation of regulatory proteins is an important mechanism by which bacterial toxins affect the eukaryotic organism.9,27,32 Diphtheria toxin and Pseudomonas aeruginosa exotoxin A modify elongation factor 2 thereby inhibiting protein synthesis.9,26,32 Cholera toxin, Escherichia coli heat-labile enterotoxin and pertussis toxin ADP-ribosylate regulatory G-proteins involved in a variety of transmembranous signaling processes in eukaryotic cells.8,9,28,32 These G-proteins (Gg, Gi, Go, transducin) comprise a family of highly homologous proteins of heterotrimeric structure,25 which function by utilizing a guanine nucleotide binding and GTPase cycle. The toxins ADP-ribosylate the α-subunit of the G-proteins thereby either enhancing (cholera toxin) or inhibiting (pertussis toxin) signal transduction.25

/pdf/adp-ribosylation-of-actin-by-clostridial-toxins-4golz2j60l.pdf

Albrecht Wegner

Papers

Head to tail polymerization of actin.

Equilibrium of the actin-tropomyosin interaction.

ADP-ribosylated actin caps the barbed ends of actin filaments.

Fragmentation of actin filaments

ADP-Ribosylation of Actin by Clostridial Toxins