Distinct actin oligomers modulate differently the activity of actin nucleators

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Actin polymerization into filaments is crucial for cell structure and function.
• The initial formation of actin nuclei is unstable and difficult to study.
• Understanding actin nucleation factors is key to controlling actin dynamics.

Purpose of the Study:

• To investigate the effects of chemically crosslinked actin oligomers on actin polymerization and nucleation.
• To analyze the differential impact of various actin dimer orientations on actin dynamics.
• To compare the effects of these oligomers with known actin nucleation factors.

Main Methods:

• Chemical crosslinking of actin monomers to form stable oligomers (dimers, trimers).
• Gel filtration for isolation of crosslinked actin oligomers.
• Pyrene assay and total internal reflection fluorescence microscopy to monitor polymerization.
• Native gel electrophoresis to study protein interactions.
• Analysis of actin nucleation activity with gelsolin, mDia formin, and Arp2/3 complex.

Main Results:

• Upper dimer (UD), trimer, and tetramer increasingly stimulated actin polymerization.
• Lower dimer (LD) showed biphasic effect: stimulation at low, inhibition at high concentrations.
• UD and trimer promoted actin repolymerization from thymosin β4 and profilin complexes.
• LD promoted repolymerization from profilin:actin but not actin:Tβ4 complexes.
• UD and trimer enhanced gelsolin and G1-3 nucleating activity, but not mDia or Arp2/3.
• LD stimulated Arp2/3 complex nucleation but inhibited mDia and gelsolin activity.

Conclusions:

• Chemically crosslinked actin oligomers provide stable models for studying nucleation.
• Actin dimer orientation critically influences interactions with nucleation factors.
• LD and UD/trimer exhibit distinct regulatory mechanisms on different actin nucleators.
• These findings offer insights into the precise control of actin dynamics by oligomeric states.