The activities of the C-terminal regions of the formin protein disheveled-associated activator of morphogenesis (DAAM) in actin dynamics

Abstract

Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N- and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. Here, to better understand the biological activities of DAAM, we studied the role of DAD-CT regions of DAAM in its interaction with actin with biochemical and genetic approaches. We found that the DAD-CT region binds actin and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, we also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. We also demonstrate that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, our results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context.

The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics

“…Polymer growth ( Figure 7E , green portion) was observed from preformed F-actin seeds ( Figure 7E , magenta portion) both in the absence and presence of Fli-I. In control samples, profilin:G-actin assembled at the barbed ends of preformed F-actin actin seeds at a rate of v PA = 3.14 ± 0.58 subunit × s –1 ( n = 37) that is consistent with the slightly reduced association rate constant of profilin:G-actin to the barbed ends as compared to free G-actin (k + = 7.86 ± 1.45 subunit × s –1 ; Barko et al, 2010 ; Toth et al, 2016 ; Vig et al, 2017 ; Figure 7F ). In the presence of Fli-I GST-GH16 (10 nM) or GST-GH13 (10 nM) the number of elongating barbed ends, as well as the rate of profilin:G-actin association to F-actin seeds was severely reduced [v Fli–I GST–GH16 = 0.45 ± 0.14 subunit × s –1 ( n = 62, p ≤ 0.0001), v Fli–I GST–GH13 = 0.46 ± 0.23 subunit × s –1 ( n = 57)] ( Figures 7E,F ).…”

“…An N-terminally truncated, constitutively active DAAM construct comprising the formin homology (FH) domains, FH1 and FH2 and the C-terminal DAD-CT regions (FH1FH2-DAD-CT), as well as the isolated DAAM FH1FH2 domains, were studied in pyrenyl polymerization experiments ( Figure 9A ; Matusek et al, 2008 ; Barko et al, 2010 ; Vig et al, 2017 ). Our previous work showed that the FH1FH2-DAD-CT of DAAM is more potent in promoting actin assembly as compared to FH1FH2 due to the presence of the DAD-CT region ( Vig et al, 2017 ; Figures 9B,C ). We found that while Fli-I GST-GH46 does not influence FH1FH2-mediated actin polymerization, it inhibits the DAAM FH1FH2-DAD-CT-catalyzed actin assembly in a concentration-dependent fashion ( Figures 9B,C ).…”

Myosin XVI in the Nervous System

“…Profilin is an actin-binding protein that also binds proline-rich ligands, such as formins, VASP and the WAVE family proteins [ 50 , 80 , 91 , 92 ]. The protein has four isoforms (PFN1-4), each is encoded by a distinct gene ( PFN1-4 ).…”

Microtubule organization in presynaptic boutons relies on the formin DAAM

“…We performed a series of rescue experiments with mutant versions of the full-length protein (FLDAAM), in which we impaired actin interaction alone or actin and MT interactions together. We used FLDAAM-I732A for the former (Gombos et al, 2015;Vig et al, 2017), mutating a conserved isoleucine residue within the FH2 domain crucial for actin interaction, while for the latter we created an FLDAAM-R876A-K881A double mutant, which is equivalent to the K989/994A mutation of mouse Dia1 that was shown to compromise both actin and MT regulation (Daou et al, 2014). Comparably to WT UAS-FLDAAM, when driven with D42-Gal4 the UAS-FLDAAM I732A mutant transgene could almost completely rescue the bouton number phenotype of DAAM Ex68 (Fig.…”