Non-canonical activity of the podosomal formin FMNL1γ supports immune cell migration

Abstract

Having previously located the formin FMNL1 in macrophage podosomes, we developed an in vivo model to assess the role of FMNL1 in the migration activities of primary macrophages. Deletion of FMNL1 in mice was genetically lethal; however, targeted deletion in macrophages was achieved by employing macrophage-specific Cre. Unchallenged FMNL1-deficient mice exhibited an unexpected reduction in tissue-resident macrophages despite normal blood monocyte numbers. Upon immune stimulus, the absence of FMNL1 resulted in reduced macrophage recruitment in vivo, decreased migration in two-dimensional in vitro culture and a decrease in the number of macrophages exhibiting podosomes. Of the three described isoforms of FMNL1 -α, β and γ -only FMNL1γ rescued macrophage migration when expressed exogenously in depleted macrophages. Surprisingly, mutation of residues in the FH2 domain of FMNL1γ that disrupt barbed-end actin binding did not limit rescue of macrophage migration and podosome numbers. These observations suggest that FMNL1 contributes to macrophage migration activity by stabilizing the lifespan of podosomes without interaction of fast-growing actin termini.

Stress hormone signaling through β‐adrenergic receptors regulates macrophage mechanotype and function

“…Our results show that inhibition of Arp2/3 or formin activity blocks the b-AR-induced increase in phagocytosis, consistent with the role of actin polymerization and branching in generating protrusions required to engulf particles (60)(61)(62). The activity of Arp2/3 and formins is also critical for the motility of macrophages and dendritic cells (63)(64)(65), where force generation is required for the formation of protrusive structures that promote invasion (66,67). Arp2/3 was also recently identified to contribute to the deformation of the nucleus during the migration of cells through narrow gaps (64).…”

The carboxy‐terminus of the formin FMNL1ɣ bundles actin to potentiate adenocarcinoma migration

“…All FMNL1 alternative splice isoforms and mutants were generated from primary human macrophage cDNA as was previously described. 12 The truncation mutant GFP-FMNL1Δ was cloned via PCR using the forward primer 5′-GCACTGAAACCCAGCCAGATCACC-3′ and the reverse primer 5′-CCGGCGGCCGCTATGTGATGATGTCTTCAAT Low-speed co-sedimentation assays were performed using 10 μM actin and 500 nM indicated purified fusion protein as described in section 2, control lane contains buffer only. Data is representative of multiple experiments.…”

“…3,38 Interestingly, we have previously demonstrated that the inhibition of barbed end binding of actin filaments through these FH2 domain mutations does not impede the function or localization of FMNL1ɣ with macrophage podosomal actin, nor its ability to rescue migration in FMNL1-null macrophages. 12 To test whether FH2 domain engagement of barbed ends contributed to the inhibition of actin assembly by FMNL1ɣ, we prepared a fusion protein incorporating the disabling FH2 mutations (Figure 1 and 2F inset). FMNL1ɣ-CT and FMNL1ɣFH2ø-CT both exhibited simiinhibition of actin assembly rates, 0.48 and 0.34 nmol/s, respectively ( Figure 2D-F).…”

“…We have previously demonstrated that FMNL1ɣ is crucial for proper macrophage adhesion and migration. 12,13,39 This led us to question whether this may be true for cancer cells as well. We observed that siRNA depletion of FMNL1 appeared to decrease MDA-MB-231 cell size and deplete cultures of adherent cells ( Figure 4G).…”

“…13 No changes in GTPase levels were detected upon genetic deletion of FMNL1 in murine macrophages. 12 It has also been suggested that FMNL1ɣ may not be subject to GTPase conformational regulation. FMNL1α and FMNL1β include the nonpolar amino acid residues isoleucine and leucine, respectively, at position 1071, while FMNL1ɣ specifies the positively-charged, polar amino acid residue lysine at this position.…”