Formin Homology Proteins (Formins) are a highly conserved family of cytoskeletal regulatory proteins that participate in a diverse range of cellular processes. FMNL2 is a member of the Diaphanous-Related Formin sub-group, and previous reports suggest FMNL2\'s role in filopodia assembly, force generation at lamellipodia, subcellular trafficking, cell-cell junction assembly, and focal adhesion formation. How FMNL2 is recruited to these sites of action is not well understood. To shed light on how FMNL2 activity is partitioned between subcellular locations, we used biotin proximity labeling and proteomic analysis to identify an FMNL2 interactome. The interactome identified known and new FMNL2 interacting proteins with functions related to previously described FMNL2 activities. In addition, our interactome predicts a novel connection between FMNL2 and extracellular vesicle assembly. We show directly that FMNL2 protein is present in exosomes.

Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed.
Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number.
Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells.
Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.

The actin nucleating and polymerizing formin-like 2 (FMNL2) is upregulated in several cancers and has been shown to play important roles in cell migration, invasion, cell-cell adhesion and filopodia formation. Here, using structured illumination microscopy we show that FMNL2 promotes rapid and highly dynamic filopodia formation in epithelial cells while remaining on the tip of the growing filopodia. This filopodia tip localization depends fully on its N-terminal myristoylation. We further show that FMNL2-dependent filopodia formation requires its serine 1072 phosphorylation within the diaphanous-autoregulatory domain (DAD) by protein kinase C (PKC) α. Consistent with this, filopodia formation depends on PKC activity and PKCα localizes to the base of growing filopodia. Thus, a PKCα-FMNL2 signaling module spatiotemporally controls dynamic filopodia formation.

Increasing evidence indicates that angiogenesis plays a pivotal role in tumor progression. Formin-like 2 (FMNL2) is well-known for promoting metastasis; however, the molecular mechanisms by which FMNL2 promotes angiogenesis in colorectal cancer (CRC) remain unclear. Here, we found that FMNL2 promotes angiogenesis and metastasis of CRC in vitro and in vivo. The GDB/FH3 domain of FMNL2 directly interacts with epidermal growth factor-like protein 6 (EGFL6). Formin-like 2 promotes EGFL6 paracrine signaling by exosomes to regulate angiogenesis in CRC. Cytoskeleton associated protein 4 (CKAP4) is a downstream target of EGFL6 and is involved in CRC angiogenesis. Epidermal growth factor-like protein 6 binds to the N-terminus of CKAP4 to promote the migration of HUVECs by activating the ERK/MMP pathway. These findings suggest that FMNL2 promotes the migration of HUVECs and enhances angiogenesis and tumorigenesis in CRC by regulating the EGFL6/CKAP4/ERK axis. Therefore, the EGFL6/CKAP4/ERK axis could be a candidate therapeutic target for CRC treatment.

Alzheimer\'s disease (AD) has been associated with cardiovascular and cerebrovascular risk factors (CVRFs) during middle age and later and is frequently accompanied by cerebrovascular pathology at death. An interaction between CVRFs and genetic variants might explain the pathogenesis. Genome-wide, gene by CVRF interaction analyses for AD, in 6568 patients and 8101 controls identified FMNL2 (p = 6.6 × 10-7). A significant increase in FMNL2 expression was observed in the brains of patients with brain infarcts and AD pathology and was associated with amyloid and phosphorylated tau deposition. FMNL2 was also prominent in astroglia in AD among those with cerebrovascular pathology. Amyloid toxicity in zebrafish increased fmnl2a expression in astroglia with detachment of astroglial end feet from blood vessels. Knockdown of fmnl2a prevented gliovascular remodeling, reduced microglial activity and enhanced amyloidosis. APP/PS1dE9 AD mice also displayed increased Fmnl2 expression and reduced the gliovascular contacts independent of the gliotic response. Based on this work, we propose that FMNL2 regulates pathology-dependent plasticity of the blood-brain-barrier by controlling gliovascular interactions and stimulating the clearance of extracellular aggregates. Therefore, in AD cerebrovascular risk factors promote cerebrovascular pathology which in turn, interacts with FMNL2 altering the normal astroglial-vascular mechanisms underlying the clearance of amyloid and tau increasing their deposition in brain.

BACKGROUND: Breast cancer is the leading cause of cancer-related deaths in females worldwide. Formin-like protein 2 (FMNL2) is a member of formin family that governs cytokinesis, cell polarity, morphogenesis and cell division. To our knowledge, the function of FMNL2 in breast cancer proliferation still remains uncovered.
METHODS: Tumor immune estimation resource (TIMER) analysis was used to detect the correlation between FMNL2 and Ki67 in breast cancer tissues. Quantitative real-time transcription polymerase chain reaction (qRT-PCR) and western blotting were performed to analyze the expression in human breast cancer cells. Moreover, RNA interference (RNAi) and plasmids were performed to silence and overexpress FMNL2 and p27. The CCK8, MTT, cell counting, colony formation, and 5-ethynyl-2-deoxyuridine (EdU) incorporation assays were used to detect cell proliferation, respectively. Flow cytometry analysis was used to detect cell cycle distribution. Further, the distribution of p27 was examined using immunofluorescence.
RESULTS: We found that FMNL2 expression was positively associated with Ki67 among collected breast cancer tissues and in TCGA database. Compared to lower proliferative cells MCF7 and T47D, FMNL2 was overexpressed in highly proliferative breast cancer cells MDA-MB-231, BT549 and SUM159, accompanied by reduced levels of p27 and p21, and elevated CyclinD1 and Ki67 expression. FMNL2 silencing significantly inhibited the cell proliferation of MDA-MB-231 and BT549 cells. Meanwhile, FMNL2 overexpression distinctly promoted the cell proliferation of MCF7 cells. Furthermore, FMNL2 suppressed the nuclear levels of p27 and promoted p27 proteasomal degradation in human breast cancer cells. The ubiquitination of p27 was inhibited by FMNL2 silencing in BT549 cells. Besides, p27 silencing markedly elevated Ki67 expression and cell viability, which could be blocked by additionally FMNL2 silencing in MDA-MB-231 and BT549 cells. Furthermore, overexpression of p27WT significantly reversed the increased levels of FMNL2 and Ki67, cell viability and cell cycle progression induced by FMNL2 overexpression in MCF7 cells. More importantly, compared to p27WT group, those effects could be significantly reversed by p27△NLS overexpression.
CONCLUSIONS: These results demonstrated that FMNL2 promoted cell proliferation partially by reducing p27 nuclear localization and p27 protein stability in human breast cancer cells, suggesting the pivotal role of FMNL2 in breast cancer progression.

BACKGROUND: Epithelial collective cell migration requires the intrinsic locomotor activity of cells to be coordinated across populations. This coordination is governed by the presence of cell-cell adhesions as well as the cooperative behaviour of cells within the monolayer.
RESULTS: Here, we report a role for Caveolin-1 (CAV1) in epithelial collective cell migration. CAV1 depletion reduced the migratory behaviour of AML12 liver epithelial cells when grown as monolayers, but not as individual cells. This suggested that CAV1 is a component of the process by which multicellular collectivity regulates epithelial motility. The correlation length for migration velocity was increased by CAV1 RNAi, a possible sign of epithelial jamming. However, CAV1 RNAi reduced migration, even when monolayers were allowed to migrate into unconfined spaces. The migratory defect was ameliorated by simultaneous depletion of the FMNL2 formin, whose cortical recruitment is increased in CAV1 RNAi cells.
CONCLUSIONS: We therefore suggest that CAV1 modulates intraepithelial motility by controlling the cortical availability of FMNL2.
CONCLUSIONS: Although epithelial collective cell migration has been observed in multiple contexts both in vivo and in vitro, the inherent coupling and coordination of activity between cells within the monolayer remain incompletely understood. Our study highlights a role for CAV1 in regulating intraepithelial motility, an effect that involves the formin FMNL2.

Increasing evidences demonstrate that circular RNAs (circRNAs) are extensively implicated in various cancers including colorectal cancer (CRC). In the present study, we found that circRNA HIPK3 (circPIK3) was upregulated in CRC. We identified that circHIPK3 was closely related with unfavorable clinicopathological features in patients with CRC. Functional transwell assay and proliferation assay indicated that circHIPK3 served as an oncogene and promoted CRC cells migration, invasion and proliferation. Meanwhile, we found that formin like 2 (FMNL2) was a key downstream molecule in circHIPK3-induced metastasis and proliferation in CRC cells. We further verified that circHIPK3 was mainly located at cytoplasm through an immunofluorescence assay. An online bioinformatics screening and a GEO datasets analysis showed that microRNA 1207-5p (miR-1207-5p) was downregulated in CRC. Also, we found that miR-1207-5p shared a similar miR-1207-5p response elements (MREs-1207-5p). Meanwhile, we showed that miR-1207-5p suppressed CRC cells migration, invasion and proliferation via directly targeting of FMNL2. Even further, via a constructed luciferase assay, we indicated that circHIPK3 was another target of miR-1207-5p. Functionally, we proved that circHIPK3 enhanced FMNL2 mediated promotion of migration, invasion and proliferation by sponging of miR-1207-5p in CRC cells. In summary, the outcomes of this study illustrated that circHIPK3 promoted CRC cells migration, invasion and proliferation modulating of FMNL2 by sponging of miR-1207-5p. Our findings indicated that circHIPK3/miR-1207-5p/FMNL2 axis might be a new strategy in molecular treatment of CRC.

T-complex 11 like 2 (TCP11L2) is a protein containing a serine-rich region in its N-terminal region. However, the function of TCP11L2 is unclear. Here, we showed that TCP11L2 expression gradually increased during muscle-derived satellite cell (MDSC) differentiation in vitro, reaching a peak on Day 3, which is the migration and fusion stage of MDSCs. Using CRISPR/dCas9 gene-editing technology to elevate or repress the expression of TCP11L2, we also showed that TCP11L2 promoted MDSC differentiation. Moreover, wound-healing assays showed that TCP11L2 promoted the migration of MDSCs during differentiation. Additionally, immunofluorescence analyses showed that TCP11L2 was mainly distributed around the microfilament and microtubules. Furthermore, the expression of TCP11L2 affected the expression of actin-related protein 2/3 (ARP2/3) complex. Co-immunoprecipitation assays and immunofluorescence analysis showed that TCP11L2 interacted with formin-like 2 (FMNL2). This protein promoted migration of bovine MDSCs by affecting the expression of ARP2/3. Finally, the activities of TCP11L2 during MDSC differentiation and migration were blocked when FMNL2 was inhibited. Taken together, our data established that TCP11L2 interacted with FMNL2 to promote MDSC migration and differentiation.

EMT (Epithelial-Mesenchymal Transition) is one of the factors in the pathogenesis of adenomyosis. FMNL2 induced invasion of cancer cell through promoting EMT, but it is unclear the role of FMNL2 in the adenomyosis. By IHC staining, we found the expression level of FMNL2 was significantly higher in the ectopic endometrial stromal cells from women with adenomyosis when compared with normal endometrial stromal cells. Knockdown of FMNL2 inhibited the invasion and migration of ectopic endometrial stromal cells and promoted the protein levels of E-cadherin and Vimentin. Meanwhile, inhibition of FMNL2 could induce the cell membrane localization of E-cadherin. Our findings reveal that the aberrant activation of FMNL2 promotes the pathogenesis of adenomyosis through inducing the EMT process. On the contrary, inhibition of FMNL2 promotes the transition of ectopic endometrial stromal cells to epithelial cells in adenomyosis through a MET-like process.