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

Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Flightless-I by bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as genetic approaches.

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

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.

The formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones.

Directed axonal growth depends on correct coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in actin-microtubule crosstalk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. Here, we identified the formin Dishevelled-associated activator of morphogenesis (DAAM) as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in primary neurons.

Biochemical Activities of the Wiskott-Aldrich Syndrome Homology Region 2 Domains of Sarcomere Length Short (SALS) Protein.

Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich syndrome protein homology 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcomeric actin filaments. Here, we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2).

Purification of tropomyosin Br-3 and 5NM1 and characterization of their interactions with actin.

Tropomyosins were first identified in neuronal systems in 1973. Although numerous isoforms were found and described since then, many aspects of their function and interactions remained unknown. Tropomyosin isoforms show different sorting pattern in neurogenesis.

The effect of toxins on inorganic phosphate release during actin polymerization.

During the polymerization of actin, hydrolysis of bound ATP occurs in two consecutive steps: chemical cleavage of the high-energy nucleotide and slow release of the γ-phosphate. In this study the effect of phalloidin and jasplakinolide on the kinetics of P(i) release was monitored during the formation of actin filaments. An enzyme-linked assay based spectrophotometric technique was used to follow the liberation of inorganic phosphate.

EFFECT OF PHALLOIDIN ON FILAMENTS POLYMERIZED FROM HEART MUSCLE ADP-ACTIN MONOMERS.

The effect of phalloidin on filaments polymerized from ADP-actin monomers of the heart muscle was investigated with differential scanning calorimetry. Heart muscle contains alpha-skeletal and alpha-cardiac actin isoforms. In the absence of phalloidin the melting temperature was 55 degrees C for the alpha-cardiac actin isoform and 58 degrees C for the alpha-skeletal one when the filaments were generated from ADP-actin monomers.

Effect of tropomyosin on formin-bound actin filaments.

Formins are conservative proteins with important roles in the regulation of the microfilament system in eukaryotic cells. Previous studies showed that the binding of formins to actin made the structure of actin filaments more flexible. Here, the effects of tropomyosin on formin-induced changes in actin filaments were investigated using fluorescence spectroscopic methods.

The effect of jasplakinolide on the thermodynamic properties of ADP.BeF(x) bound actin filaments.

The effect of BeF(x) and a natural toxin (jasplakinolide) was examined on the thermal stability of actin filaments by using differential scanning calorimetry. The phosphate analogue beryllium fluoride shifted the melting temperature of actin filaments (67.4 degrees C) to 83.