INF2 is an endoplasmic reticulum-associated formin protein

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Formins are proteins that regulate actin dynamics, primarily by promoting filament assembly.
• INF2 is a unique formin with the distinct ability to accelerate actin filament depolymerization, dependent on its WASP homology 2 (WH2) motif.

Purpose of the Study:

• To investigate the localization and regulation of the INF2 protein.
• To elucidate the role of post-translational modifications and domain interactions in INF2 function.
• To determine the association of actin-assembly factors with the endoplasmic reticulum (ER).

Main Methods:

• Immunofluorescence microscopy to determine INF2 localization in Swiss 3T3 cells.
• Expression of GFP-fusion constructs of INF2 to confirm localization.
• Biochemical assays to study the interaction between INF2's diaphanous inhibitory domain (DID) and diaphanous autoregulatory domain (DAD)/WH2 motif.
• Site-directed mutagenesis to disrupt DID-DAD interaction and depolymerization activity.

Main Results:

• INF2 is peripherally bound to the cytoplasmic face of the ER, requiring C-terminal farnesylation and ionic interactions for association.
• The WH2 motif of INF2 functions as a DAD, interacting with the DID domain.
• While the DID-DAD interaction does not inhibit actin nucleation, it significantly inhibits depolymerization activity.
• Mutations disrupting the DID-DAD interaction abolish depolymerization activity and cause ER collapse with actin accumulation.

Conclusions:

• INF2 is the first identified actin-assembly factor to associate with the ER.
• Post-translational farnesylation and specific domain interactions regulate INF2's localization and unique depolymerization activity.
• Disruption of INF2's autoregulation leads to cellular defects, including ER morphology changes and actin redistribution.