Biomedical subjects

Elizabeth S Harris

Publications and source records attributed to Elizabeth S Harris.

5 recordsLinked to original sources

PubMed2006

Mechanistic differences in actin bundling activity of two mammalian formins, FRL1 and mDia2.

Formin proteins are regulators of actin dynamics, mediating assembly of unbranched actin filaments. These multidomain proteins are defined by the presence of a Formin Homology 2 (FH2) domain. Previous work has shown that FH2 domains bind to filament barbed ends and move processively at the barbed end as the filament elongates. Here we report that two FH2 domains, from mammalian FRL1 and mDia2, also bundle filaments, whereas the FH2 domain from mDia1 cannot under similar conditions. The FH2 domain alone is sufficient for bundling. Bundled filaments made by either FRL1 or mDia2 are in both parallel and anti-parallel orientations. A novel property that might contribute to bundling is the ability of the dimeric FH2 domains from both FRL1 and mDia2 to dissociate and recombine. This property is not observed for mDia1. A difference between FRL1 and mDia2 is that FRL1-mediated bundling is competitive with barbed end binding, whereas mDia2-mediated bundling is not. Mutation of a highly conserved isoleucine residue in the FH2 domain does not inhibit bundling by either FRL1 or mDia2, but inhibits barbed end activities. However, the severity of this mutation varies between formins. For mDia1 and mDia2, the mutation strongly inhibits all effects of barbed end binding, but affects FRL1 much less strongly. Furthermore, our results suggest that the Ile mutation affects processivity. Taken together, our data suggest that the bundling activities of FRL1 and mDia2, while producing phenotypically similar bundles, differ in mechanistic detail.

Actin Cytoskeleton↗

PubMed2006

Control of the assembly of ATP- and ADP-actin by formins and profilin.

Formin proteins nucleate actin filaments, remaining processively associated with the fast-growing barbed ends. Although formins possess common features, the diversity of functions and biochemical activities raised the possibility that formins differ in fundamental ways. Further, a recent study suggested that profilin and ATP hydrolysis are both required for processive elongation mediated by the formin mDia1. We used total internal reflection fluorescence microscopy to observe directly individual actin filament polymerization in the presence of two mammalian formins (mDia1 and mDia2) and two yeast formins (Bni1p and Cdc12p). We show that these diverse formins have the same basic properties: movement is processive in the absence or presence of profilin; profilin accelerates elongation; and actin ATP hydrolysis is not required for processivity. These results suggest that diverse formins are mechanistically similar, but the rates of particular assembly steps vary.

Actins↗

PubMed2006

Biochemical analysis of mammalian formin effects on actin dynamics.

Formins are members of a conserved family of proteins, present in all eukaryotes, that regulate actin dynamics. Mammals have 15 distinct formin genes. From studies to date, surprising variability between these isoforms has been uncovered. All formins examined have several common effects on actin dynamics in that they: (1) accelerate nucleation rate; (2) alter filament barbed end elongation/depolymerization rates; and (3) antagonize capping protein. However, the potency of each effect can vary greatly between formins. In addition, a subset of formins binds tightly to filament sides and bundle filaments. Even isoforms that are closely related phylogenetically can display marked differences in their effects on actin. This chapter discusses several methods for examining formin function in vitro. We also discuss pitfalls associated with these assays. As one example, the effect of profilin on formin function is difficult to interpret by "pyrene-actin" polymerization assays commonly used in the field and requires assays that can distinguish between filament nucleation and filament elongation. The regulatory mechanisms for formins are not clear and certainly vary between isoforms. A subset of formins is regulated by Rho GTPases, and the assays described in this chapter have been used for characterization of this regulation.

Actins↗

PubMed2005

Actin dynamics: growth from dendritic branches.

The dendritic nucleation model was devised to explain the cycle of actin dynamics resulting in actin filament network assembly and disassembly in two contexts--at the leading edge of motile cells and in the actin comet tails of intracellular pathogenic bacteria and viruses. Due to the detailed nature of its biochemical predictions, the model has provided an excellent focus for subsequent experimentation. This review summarizes recent work on actin dynamics in the context of the dendritic nucleation model. One outcome of this research is the possibility that additional proteins, as well as the six proteins included in the original model, might increase the efficiency of dendritic nucleation or modify the resulting actin network. In addition, actin dynamics at the leading edge might be influenced by a second actin filament network, independent of dendritic nucleation.

Actins↗

PubMed2004

The mouse formin, FRLalpha, slows actin filament barbed end elongation, competes with capping protein, accelerates polymerization from monomers, and severs filaments.

Formins are a conserved class of proteins expressed in all eukaryotes, with known roles in generating cellular actin-based structures. The mammalian formin, FRLalpha, is enriched in hematopoietic cells and tissues, but its biochemical properties have not been characterized. We show that a construct composed of the C-terminal half of FRLalpha (FRLalpha-C) is a dimer and has multiple effects on muscle actin, including tight binding to actin filament sides, partial inhibition of barbed end elongation, inhibition of barbed end binding by capping protein, acceleration of polymerization from monomers, and actin filament severing. These multiple activities can be explained by a model in which FRLalpha-C binds filament sides but prefers the topology of sides at the barbed end (end-sides) to those within the filament. This preference allows FRLalpha-C to nucleate new filaments by side stabilization of dimers, processively advance with the elongating barbed end, block interaction between C-terminal tentacles of capping protein and filament end-sides, and sever filaments by preventing subunit re-association as filaments bend. Another formin, mDia1, does not reduce the barbed end elongation rate but does block capping protein, further supporting an end-side binding model for formins. Profilin partially relieves barbed end elongation inhibition by FRLalpha-C. When non-muscle actin is used, FRLalpha-C's effects are largely similar. FRLalpha-C's ability to sever filaments is the first such activity reported for any formin. Because we find that mDia1-C does not sever efficiently, severing may not be a property of all formins.

Actins↗