Regulation of the formin INF2 by actin monomers and calcium-calmodulin

In response to increased intracellular calcium, the formin INF2 polymerizes 20-30% of the total cellular actin pool within 30 sec, suggesting robust regulation. INF2 regulation requires an auto-inhibitory interaction between the N-terminal Diaphanous Inhibitory Domain (DID) and the C-terminal Diaphanous Auto-regulatory Domain (DAD). DID mutations are dominantly linked to two human diseases and constitutively activate INF2. However, DAD binding to actin monomers competes with DID binding, disrupting regulation. Here, we use a novel cell-free assay for detailed investigation of INF2 regulation. Contrary to our previous findings, INF2 inhibition does not require CAP proteins but does require actin ‘buffering’ by monomer-binding proteins such as profilin or thymosin. INF2 is activated by calcium-bound calmodulin (CALM) through CALM binding to the N-terminus. In addition, the N-terminus plays an important role in INF2 regulation beyond CALM binding. These findin..., Plasmids and siRNA oligonucleotides
The GFP-F-tractin and mApple-F-tractin plasmids were a gift from A. Pasapera (NIH, Bethesda, MD). The ER-tagRFP (modified from the GFP-N1 backbone) was a gift from E. Snapp (Albert Einstein College of Medicine, New York, NY); and contains a prolactin signal sequence at the 5’ end of the fluorescent protein as well as a KDEL sequence at the 3’ end. The GFP-fusion constructs of human INF2-CAAX (UniProt Q27J81-1) and INF2-nonCAAX (UniProt Q27J81-2) constructs used in this study have been describe previously (Chakrabarti et al., 2018; A et al., 2019), and are in a modified eGFP-C1 vector (Clontech) containing a Strep-tag II (IBA Life Sciences) and an HRV3C cleavage site between the multiple cloning site and the start codon of INF2. INF2 mutations (R218Q; W11L14L18 to A; DNT; NM; LM; R9A, R9K10K15K17 to A; A13T; and E16A) were introduced into the GFP-INF2-CAAX construct using site-directed mutagenesis. For bacterial expression, human INF2 FH1-FH2-C (FFC, a..., # Data from: Regulation of the formin INF2 by actin monomers and calcium-calmodulin

Dataset DOI: [10.5061/dryad.mkkwh71dn](https://doi.org/10.5061/dryad.mkkwh71dn)

## Description of the data and file structure

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You can find the original, unprocessed, and uncropped images and videos corresponding to the figures in this dataset. Detailed figure numbers and their associated file names are listed below.

### Videos for Figure 1C:

Ionomycin induced actin filament assembly: 2021-12-02_12.32.19_U2OS Ionomycin induced CIA Histamine induced actin filament assembly: 2021-12-02_12.32.19_U2OS Ionomycin induced CIA

### Images for Figure 2C:

U2OS INF2 KO cells without stimulation: 2025-02-06_14.42.56_INF2 KO, no
U2OS INF2 KO cells with histamine stimulation: 2025-02-06_14.56.10_INF2 KO, Histamine
U2OS GFP-INF2-CAAX cells without stimulation: 2025-02-06_15.03.50_CAAX, no
U2OS GFP-INF2-CAAX cells with histamine stimulation: 2025-02-06_15.12.20_CAAX, Histamine

### Videos for Figure 4A:
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