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The actin microfilament network is important in maintaining cell shape and function in eukaryotic cells. It has a multitude of roles in cellular processes such as cell adhesion, motility, cellular signalling, intracellular trafficking and cytokinesis. Alterations in the organisation of the cytoskeleton and changes in cellular morphology, motility and adhesiveness are characteristic features of transformed cancer cells. For this reason cytoskeletal microfilaments have become promising targets for chemotherapy. In contrast to the microtubules, which have been targeted successfully with anti-tumour drugs such as Taxol-like compounds and the Vinca alkaloids, very few actin targeting drugs have been characterised. To date, no actin targeting drugs have been used in clinical trials due to their severe cytotoxicity. One reason for this cytotoxicity is that drugs such as the cytochalasins and latrunculins disrupt actin microfilaments in both non-tumour and tumour cells. To circumvent this problem, actin filament populations need to be targeted more specifically. Not all actin filaments are the same and there is growing evidence that within a cell there are different populations of actin filaments which are spatially organised into distinct cellular compartments each with a unique function. The structure and function of the actin cytoskeleton is primarily regulated by the associated actin binding proteins. Tropomyosin is an intrinsic component of most actin filaments and over 40 isoforms have been identified in non-muscle cells. Tm isoforms are spatially segregated and current evidence suggests that they can specify the functional capacity of the actin microfilaments. Therefore the composition of these functionally distinct actin filaments may be important in determining their stability and function within the cell. If actin filament populations can be discriminated and targeted based on their tropomyosin composition then this becomes a powerful approach for anticancer therapy.

Microfilaments have been reported to be polarized in a number of cell types based both on function and isoform composition. There is evidence that microfilaments are involved in the movement of vesicles and the polarized delivery of proteins to specialized membrane domains. We have investigated the composition of actin microfilaments in gastrointestinal epithelial cells and their role in the delivery of the cystic fibrosis transmembrane conductance regulator (CFTR) into the apical membrane using cultured T84 cells as a model. We identified a specific population of microfilaments containing the tropomyosin (Tm) isoforms Tm5a and/or Tm5b, which are polarized in T84 cell monolayers. Polarization of this microfilament population occurs very rapidly in response to cell-cell and cell-substratum contact and is not inhibited by jasplakinolide, suggesting this involves the movement of intact filaments. Colocalization of Tm5a and/or Tm5b and CFTR was observed in long-term cultures. A reduction in Tm5a and Tm5b expression, induced using antisense oligonucleotides, resulted in an increase in both CFTR surface expression and chloride efflux in response to cAMP stimulation. We conclude that Tm isoforms Tm5a and/or Tm5b mark an apical population of microfilaments that can regulate the insertion and/or retention of CFTR into the plasma membrane.

Caldesmon inhibits actomyosin ATPase and filament sliding in vitro, and therefore may play a role in modulating smooth and non-muscle motile activities. A bacterially expressed caldesmon fragment, 606C, which consists of the C-terminal 150 amino acids of the intact molecule, possesses the same inhibitory properties as full-length caldesmon and was used in our structural studies to examine caldesmon function. Three-dimensional image reconstruction was carried out from electron micrographs of negatively stained, reconstituted thin filaments consisting of actin and smooth muscle tropomyosin both with and without added 606C. Helically arranged actin monomers and tropomyosin strands were observed in both cases. In the absence of 606C, tropomyosin adopted a position on the inner edge of the outer domain of actin monomers, with an apparent connection to sub-domain 1 of actin. In 606C-containing filaments that inhibited acto-HMM ATPase activity, tropomyosin was found in a different position, in association with the inner domain of actin, away from the majority of strong myosin binding sites. The effect of caldesmon on tropomyosin position therefore differs from that of troponin on skeletal muscle filaments, implying that caldesmon and troponin act by different structural mechanisms.

Although muscle contraction is known to result from movement of the myosin heads on the thick filaments while attached to the thin filaments, the myosin head movement coupled with ATP hydrolysis still remains to be investigated. Using a gas environmental (hydration) chamber, in which biological specimens can be kept in wet state, we succeeded in recording images of living muscle thick filaments with gold position markers attached to the myosin heads. The position of individual myosin heads did not change appreciably with time in the absence of ATP, indicating stability of the myosin head mean position. On application of ATP, the position of individual myosin heads was found to move by approximately 20 nm along the filament axis, whereas no appreciable movement of the filaments was detected. The ATP-induced myosin head movement was not observed in filaments in which ATPase activity of the myosin heads was eliminated. Application of ADP produced no appreciable myosin head movement. These results show that the ATP-induced myosin head movement takes place in the absence of the thin filaments. Because ATP reacts rapidly with the myosin head (M) to form the complex (M. ADP.Pi) with an average lifetime of >10 s, the observed myosin head movement may be mostly associated with reaction, M + ATP --> M.ADP. Pi. This work will open a new research field to study dynamic structural changes of individual biomolecules, which are kept in a living state in an electron microscope.

We have investigated the effect of methanol at concentrations below 15% (v/v) on acto-heavy meromyosin (HMM)- and tropomyosin (Tm)-troponin (Tn)-acto-heavy meromyosin-ATPase activities. Methanol slightly enhanced ATPase activity of acto-HMM alone. It had no apparent effect on normalized Tm-Tn-acto-HMM-ATPase activity in the absence of Ca2+. In the presence of Ca2+, however, methanol markedly inhibited normalized Tm-Tn-acto-HMM ATPase activity. These results show that methanol affects the troponin-tropomyosin regulation of acto-HMM ATPase: methanol suppresses the Ca(2+)-sensitivity of the regulatory system and traps it in an "off-state."

We compared the mechanisms by which caldesmon inhibits actin and actin-tropomyosin activation of myosin subfragment 1 (S1) MgATPase activity. Caldesmon always inhibited actin activation by displacing S1.ADP.Pi from actin and inhibition required at least 0.7 caldesmon molecules bond per actin for 90% inhibition. Caldesmon inhibited actin-tropomyosin without any displacement of S1.ADP.Pi; thus it inhibits a rate-limiting step. Inhibition is highly cooperative, requiring no more than one caldesmon bound per 10 actins for 90% inhibition of activation by actin and smooth muscle tropomyosin. The degree of cooperativity is defined by the tropomyosin since inhibition by skeletal tropomyosin requires up to one caldesmon bound per 4 actins for 90% inhibition under identical conditions. Both noncooperative inhibition of actin and cooperative, tropomyosin-dependent, inhibition are manifested by a fragment of caldesmon containing only the C-terminal 99 amino acids (658C), although this fragment does not itself bind to tropomyosin. The functional properties of 658C are very similar to striated muscle troponin I, consequently we propose a similar mechanism for tropomyosin-dependent regulation by caldesmon. Caldesmon binding switches actin-tropomyosin to the "off" or "weak" state and Ca2+/calmodulin binding to caldesmon blocks this switch and thus reactivates the actin filament.