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.

Keywords: Actin; Formin; Macrophage; Motility; Podosome.

Fig. 1.

Genetically targeted depletion of FMNL1 in macrophages impacts macrophage tissue density and in vivo migration. (A) Diagram of FMNL1-targeting vector inserted into the FMNL1 allele through homologous recombination. (B) Diagram depicting exon excision through Cre-mediated recombination. Position of oligonucleotides for genotyping (Fmnl1-wtF2, Fmnl1-wtR2, CSD-flpF, CSD-flpR2, CRE-68 and CRE-67) are indicated on the gene alignment. Lane designations (lanes 1–4) refer to the PCR products of genotyping shown in Fig. 1C. β-galactosidase and neomycin cassettes were removed from the targeting vector using flippase-mediated recombination to generate a conditional knockout. FMNL1 floxed mice were bred with LysMcre mice to delete FMNL1 expression through Cre-mediated recombination under the lysozyme 2 promoter (Lyz2). (C) Agarose gel depicts representative PCR reaction products used to determine the genotypes of progeny yielded from crossing fFMNL1(+/−)/LysMcre (+/−) mice. Lane (‘L’) 1 shows a 235 bp product for any wild-type FMNL1 allele. Lane 2 shows either a 2107 bp floxed FMNL1 allele if Cre is absent or a 432 bp allele if Cre is present, with both products present in heterozygotes. Lane 3 shows the normal Lyz2 allele, and lane 4 the insertion of Cre into the Lyz2 allele. (D) Equal amounts of protein from lysates of purified macrophages from the indicated mouse genotypes from LysMcre X fFMNL1 crosses were separated using SDS-PAGE. FMNL1, Dia1, Dia2, FHOD1 and FHOD3 were detected via western blot analysis, using transaldolase as a loading control. A representative example is shown. Macrophages from animals heterozygous for either fFMNL1 or LyzMcre show a partial reduction in FMNL1 expression. Complete FMNL1 loss is seen in animals homozygous for fFMNL1 and LysMcre. (E) Frozen liver sections from WT and FMNL1 KO mice were probed for F4/80 to identify Kupffer cells and quantified as described in Materials and Methods. Data are mean±s.d. from five separate studies, each with n>6 per genotype. Asterisk indicates P<0.05, Student's t-test. (F) Macrophages that migrated into the peritoneal cavity after inducing inflammation were harvested, detected by F4/80 reactivity, and quantified as described in Materials and Methods. Data are mean±s.d. from four separate studies, with 2000 cells analyzed for each genotype. Asterisks between groups indicate P<0.05, Student's t-test. (−/−)/(−/−), fFMNL1(−/−)/LysMcre (−/−); (+/+)/(+/+), fFMNL1(+/+)/LysMcre(+/+); (+/+)/(+/−), fFMNL1(+/+)/LysMcre(+/−); (+/−)/(+/−), fFMNL1(+/−)/LysMcre(+/−).

Fig. 2.

Loss of FMNL1 in macrophages disrupts podosome formation and directed migration but does not affect random motility. (A) Macrophages from the indicated genotypes that migrated through an in vitro barrier in the presence or absence of the formin inhibitor SMIFH2 were quantified using light microscopy after performing differential staining as described in Materials and Methods. Data are mean±s.d. from three separate studies with n=10 (transwell inserts examined) per genotype. Asterisks between groups indicate P<0.05, ANOVA with Dunnet's comparison between groups. Migration is given as a percentage of the untreated WT macrophages. Genotypes of the animals from which macrophages were isolated are indicated on the x axis. (B) The number of macrophages from the indicated genotypes displaying podosomes was quantified as described in Materials and Methods. Data are mean±s.d. from three biological replicates where all podosomes in 100 macrophages were counted. Asterisk indicates P<0.05, Student's t-test. Representative images of each genotype are shown with an enlarged inset to demonstrate that podosomes do form in both genotypes. (C) The average distance traveled and average velocity during random motility by macrophages of the indicated genotypes were imaged using live-cell microscopy for 6 h, followed by ImageJ analysis as described in Materials and Methods. Data are mean±s.d. for three studies tracking n>10 macrophages in each once. (D) Podosome height and width was measured in macrophages from WT (−/−)/(−/−) and FMNL1-depleted (+/+)/(+/+) mice using z-stack analysis after staining with Rhodamine–phalloidin for actin visualization. Data are mean±s.d. for three separate studies where all podosomes in n>5 macrophages per genotype were measured. (+/+)/(+/−), fFMNL1(+/+)/LysMcre(+/−); (+/−)/(+/−), fFMNL1(+/−)/LysMcre(+/−).

Fig. 3.

Macrophage podosomes and migration are dependent on a specific FMNL1 isoform. (A) FMNL1 KO macrophages that had been infected with lentivirus expressing GFP, GFP–FMNL1β, GFP–FMNL1γ or GFP–FMNL1γ ABM were permitted to attach to glass in the presence of serum and stained with Rhodamine–phalloidin for actin visualization. Shown are representative images. Regions of cells containing podosomes, only seen in cells expressing FMNL1γ or FMNL1γ ABM are outlined and expanded. Merged images illustrate colocalization of actin and FMNL1γ. Scale bar: 20 μm. (B) FMNL1 KO macrophages were infected (underlined groups) with lentivirus expressing GFP, GFP–FMNL1β, GFP–FMNL1γ, GFP–FMNL1γ ABM, or mock (dash). In vitro migration assays were performed as described in Materials and Methods, and the number of macrophages migrating through the barrier were quantified using light microscopy and differential staining. The absolute number of cells was normalized to that of WT macrophages (−/−)/(−/−) and are presented as the percent of WT. Data are mean±s.d. from multiple separate experiments with triplicate samples. Asterisks indicate P<0.05 between each group versus WT, ANOVA with Dunnet's comparison. (C) FMNL1 KO macrophages were infected with lentivirus expressing GFP, GFP–FMNL1β or GFP–FMNL1γ and then permitted to attach to glass in the presence of serum and stained with Rhodamine–phalloidin to visualize podosomal actin cores. The mean percentage of macrophages forming podosomes in each group±s.d. is shown, asterisk indicates P<0.05 compared to expression of GFP alone (Student's t-test).