erythro-9-[3-(2-Hydroxynonyl)]adenine is an effective inhibitor of cell motility and actin assembly.

Abstract

erythro-9-[3-(2-Hydroxynonyl)Jadenine (EHNA) has been reported previously to be an agent that arrests sperm motility by inhibiting the axonemal dynein ATPase activity and has been used to probe the involvement of putative cytoplasmic dyneins in mitosis and intracellular organelle transport. We report here that EHNA profoundly and reversibly affects several actin-dependent processes, both in vivo and in vitro. It induces dramatic changes in actin organization in cultured cells, inhibits cell translocation, blocks actin-dependent cytoplasmic streaming, interferes with actin-dependent gelation of cytoplasmic extracts, and inhibits actin assembly. Just as the cytochalasins, -EHNA appears to be a highly effective inhibitor of actin-based motility, whose effects in complex biological systems should be interpreted with caution.Analysis of the function of cytoskeletal components greatly profits from the use of specific inhibitors. Prime examples include colchicine, which depolymerizes microtubules and inhibits their assembly, and the cytochalasins, which decrease the rate of actin assembly and interfere with actin network formation. Such inhibitors, of which colchicine and cytochalasin are just two of the most widely known, have become invaluable tools in research on cell motility. Recently, an inhibitor of dynein ATPase activity, erythro-9-[3-(2-hydroxynonyl)]adenine (EHNA), became available, which, unlike vanadate, another potent dynein inhibitor (1), can easily cross the cell membrane and thus can be applied to living cells (2). Its potential usefulness in the search for "cytoplasmic" dyneins involved in motility phenomena, such as intracellular transport, was immediately realized. Thus, in addition to its use in the study of the properties of ciliary and flagellar dynein (2, 3), EHNA has been employed as a probe for the possible involvement of dynein-like ATPases in chromosome movement in mitosis (4), particle transport in chromatophores (5, 6), and organelle movements along axons (7,8). Inhibition of these forms of motility by EHNA has been taken as an indication for the involvement of a cytoplasmic, possibly microtubule-associated, dynein ATPase activity.In the course of studies on intracellular organelle movements using EHNA, we noted effects of this compound apparently unrelated to a possible action on dynein-like molecules. Here we report that EHNA interferes with the functional organization of actin in vivo and in vitro. It profoundly affects actin filament organization in cultured cells, reversibly interferes with actin-dependent forms of motility, and inhibits gel formation of cytoplasmic extracts and actin assembly in vitro. Next to the cytochalasins, it is probably one of the most effective inhibitors of actin-based motility.MATERIALS AND METHODS Cells. African green monkey kidney cells (strain BSC-1) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (GIBCO). For experiments, cells were seeded on coverslips and were used in the exponential growth phase. M...

The Rap1–cofilin-1 pathway coordinates actin reorganization and MTOC polarization at the B cell immune synapse

“…5A-D). Although EHNA concentrations 25-200 times greater than we used have profound effects on actin organization (Schliwa et al, 1984), in our experiments F-actin accumulation at the bead contact site occurred normally in B cells treated with these dynein inhibitors (Fig. 5C).…”

KIF5B and KIF3A/KIF3B kinesins drive MT1-MMP surface exposure, CD44 shedding, and extracellular matrix degradation in primary macrophages

“…on May 10, 2018. by guest www.bloodjournal.org From results were gained by inhibition of dynein using the chemical inhibitor erythro-9-[3-(2-hydroxynonyl)] adenine (EHNA; supplemental Figure 2 and supplemental Table 1). 30 In all cases, the integrity of the microtubule system was not compromised ( Figure 2M and not shown). Combined, these results indicate that retrograde movement of MT1-MMP-mCherry vesicles along microtubules depends on cytoplasmic dynein.…”

cGMP-stimulated cyclic nucleotide phosphodiesterase regulates the basal calcium current in human atrial myocytes.

“…3), which suggests that EHNA does not directly interact with the Ca channels, in their phosphorylated or nonphosphorylated states. In addition to inhibition of adenosine deaminase and PDE2, EHNA has been found to be an inhibitor of dynein ATPase activity, actin assembly, and cell motility (38,39). Although we did not address these questions specifically, it is hard to anticipate how these alternative mechanisms could account for the observed effects of EHNA on I Ca .…”