engleski

Entrapment of actin by a Dictyostelium talin fragment visualizes actin flow in chemotaxis, endocytosis and cytokinesis

Actin distribution in a living cell reflects its momentary state resulting from the spatio-temporal patterns of actin polymerization, F-actin translocation, and disassembly. There is ample evidence for the polymerization of actin at the leading edge of a motile cell, (1) and for a retrograde flow of actin associated with cell motility.(2) Directed actin flows are also intrinsic to cytokinesis, and they are essential for cell-surface capping.(3) Direct and indirect methods have been used to probe for actin flows. Evidence for a retrograde flow in cell migration and cytokinesis has been provided by the translocation of crosslinked proteins on the cell surface. A retrograde flow of actin in lamellipodia has been directly shown by the injection of fluorescent actin into fibroblasts. Fluorescent phalloidin has been introduced into cells to visualize specifically the flow of filamentous actin. An optimal probe for monitoring actin flow should bind actin filaments long enough to follow their translocation. It should not, however, prevent remodelling of the actin system during changes in the direction of flow or after the flow has ceased. Here we introduce a fusion of GFP to an actin-binding domain of talin as a fluorescent probe to monitor transient actin flows or flows that rapidly change in direction. Chemotaxis, phagocytosis, and cytokinesis are fast processes in Dictyostelium discoiedum. Re-orientation in a gradient of chemoattractant is recognizable within less than 10 seconds after the onset of stimulation, engulfment of a large particle like a yeast cell takes about 1 minute, and cytokinesis from the commencement of cleavage furrow ingression to a complete separation of the daughter cells requires about 2 minutes. A C-terminal 63 kDa fragment of talin A from Dictyostelium forms a slowly dissociating complex with F-actin in vitro, as established by surface plasmon resonance.(4) This talin fragment (TalC63) has been tagged with GFP and used as a trap for actin filaments in chemotactic cell movement, endocytosis, and mitotic cell division. TalC63 efficiently sequesters actin filaments in vivo. Its translocation reflects the direction and efficiency of an actin flow directed from the front to the tail of a motile Dictyostelium cell (Fig. 1A). Upon chemotactic stimulation, this actin flow is rapidly re-directed in accord with the re-programming of cell polarity in response to changing gradients of cyclic AMP. In endocytosis, the fluorescent complexes are translocated to the base of a phagocytic or macropinocytic cup (Fig. 1B). During mitosis, the complexes of F-actin with TalC63 accumulate within the midzone of anaphase cells. If TalC63 is strongly expressed, the entire cleavage furrow is filled out by sequestered actin filaments, and cytokinesis is severely impaired (Fig. 1C). The presented data indicate that actin cycles comprised of polymerization, transport of actin filaments and their depolymerization, are interrupted at the depolymerization step by the sequestering activity of an actin-binding talin fragment. These data have two implications. First, if the turnover or translocation rates of actin filaments at the sites of polymerization are high, only proteins that bind with high on and off rate constants will mirror the momentary F-actin distribution. On the other hand, only proteins that persist long enough in an actin-bound state can be carried with an actin flow to the end-points of this flow, similar as the actin-binding talin fragment does. It appears therefore that the individual kinetics of actin-binding proteins is crucial for their localization and thus for their function in living cells where the actin network is continuously restructured. Further, if F-actin is effectively translocated to the cleavage furrow, a mechanism of actin disassembly must exist in the midzone of the dividing cell where the cleavage furrow is being formed. The same is true for the tail of a migrating cell and for the base of an endocytic cup. The disassembly of actin at the end points of flow is crucial for recycling the building blocks of actin filaments that are required at sites of active polymerization, and thus for maintaining the actin dynamics. Any impairment of this disassembly will result in the accumulation of filamentous actin at the target sites of flow. The unknown mechanism of actin disassembly may be elucidated by the phenotypic analysis of cytokinesis mutants. Based on the results reported here, mutants deficient in actin disassembly are predicted to exhibit phenotypes similar to TalC63-expressing cells: to accumulate extraordinarily high amounts of F-actin in the cleavage furrow and, in extreme cases, to form bulky, actin-rich midbodies. Similar midbodies separated by constrictions from the daughter cells have been observed in knock-out mutants lacking racE, suggesting a role of this GTPase in actin disassembly.(5) Other candidate constituents of the actin shredding machinery are actin-binding proteins that are enriched in the cleavage furrow, such as cortexillins I and II.(6)

dictyostelium; talin A; actin flow

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