• BMP pathways
• Blood vessels, Zebrafish
• HUVEC
• Rho GTPases
• Tartrazine

• Zebrafish/embryology
• Animals
• Humans
• Human Umbilical Vein Endothelial Cells/drug effects
• Tartrazine/pharmacology
• Tartrazine/toxicity
• Larva/drug effects
• Food Coloring Agents/pharmacology
• Cell Proliferation/drug effects
• Cell Movement/drug effects
• Animals, Genetically Modified
• Neovascularization, Physiologic/drug effects
• Endothelial Cells/drug effects
• Endothelial Cells/metabolism

• CMP8.24 and CMP9.10 Wallonie-Bruxelles International
• Maître de Recherche Fonds De La Recherche Scientifique - FNRS
• Maître de Recherche Fonds De La Recherche Scientifique - FNRS
• Maître de Recherche Fonds De La Recherche Scientifique - FNRS
• Maître de Recherche Fonds De La Recherche Scientifique - FNRS

• Actin
• Angiogenesis
• Endothelium
• RhoGTPase

• Animals
• Actins
• Endothelial Cells
• Actin Cytoskeleton
• Cell Movement
• Morphogenesis
• Mammals

• BT/RLF/Re-entry/51/2019 Department of Biotechnology, Government of India

The restoration of an intact epidermal barrier after wound injury is the culmination of a highly complex and exquisitely regulated physiological process involving multiple cells and tissues, overlapping dynamic events and protein synthesis and regulation. Central to this process is the cytoskeleton, a system of intracellular proteins that are instrumental in regulating important processes involved in wound repair including chemotaxis, cytokinesis, proliferation, migration, and phagocytosis. One highly conserved family of cytoskeletal proteins that are emerging as major regulators of actin and microtubule nucleation, polymerization, and stabilization are the formins. The formin family includes 15 different proteins categorized into seven subfamilies based on three formin homology domains (FH1, FH2, and FH3). The formins themselves are regulated in different ways including autoinhibition, activation, and localization by a range of proteins, including Rho GTPases. Herein, we describe the roles and effects of the formin family of cytoskeletal proteins on the fundamental process of wound healing and highlight recent advances relating to their important functions, mechanisms, and regulation at the molecular and cellular levels.

• Actins/metabolism
• Cytoskeletal Proteins/metabolism
• Formins
• Microfilament Proteins/metabolism
• Protein Structure, Tertiary
• Wound Healing
• rho GTP-Binding Proteins/metabolism

• FASCIN
• actin
• formin
• meiosis
• oocyte

Forward genetic screens have shown the consequences of deleterious mutations; however, they are best suited for model organisms with fast reproductive rates and large broods. Furthermore, investigators must faithfully identify changes in phenotype, even if subtle, to realize the full benefit of the screen. Reverse genetic approaches also probe genotype to phenotype relationships, except that the genetic targets are predefined. Until recently, reverse genetic approaches relied on non-genomic gene silencing or the relatively inefficient, homology-dependent gene targeting for loss-of-function generation. Fortunately, the flexibility and simplicity of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has revolutionized reverse genetics, allowing for the precise mutagenesis of virtually any gene in any organism at will. The successful integration of insertions/deletions (INDELs) and nonsense mutations that would, at face value, produce the expected loss-of-function phenotype, have been shown to have little to no effect, even if other methods of gene silencing demonstrate robust loss-of-function consequences. The disjunction between outcomes has raised important questions about our understanding of genotype to phenotype and highlights the capacity for compensation in the central dogma. This review describes recent studies in which genomic compensation appears to be at play, discusses the possible compensation mechanisms, and considers elements important for robust gene loss-of-function studies.

• CRISPR/Cas
• genetic compensation
• genetic plasticity
• morpholino
• nonsense-mediated mRNA decay
• reverse genetics
• transcription activator-like effector nucleases (TALENs)
• zebrafish

• Animals
• CRISPR-Cas Systems
• Codon, Nonsense
• Gene Editing/methods
• Genetic Association Studies
• Genetic Techniques
• Genomics
• Genotype
• INDEL Mutation
• Mutagenesis
• Mutation
• Nonsense Mediated mRNA Decay/genetics
• Phenotype
• Transcription Activator-Like Effector Nucleases/genetics
• Zebrafish

• Actin
• Cancer
• Cytoskeleton
• Formins
• MicroRNAs
• Signaling
• WASp family

The vascular system is a hierarchically organized network of blood vessels that play crucial roles in embryogenesis, homeostasis and disease. Blood vessels are built by endothelial cells – the cells lining the interior of blood vessels – through a process named vascular morphogenesis. Endothelial cells react to different biomechanical signals in their environment by adjusting their behavior to: (1) invade, proliferate and fuse to form new vessels (angiogenesis); (2) remodel, regress and establish a hierarchy in the network (patterning); and (3) maintain network stability (quiescence). Each step involves the coordination of endothelial cell differentiation, proliferation, polarity, migration, rearrangements and shape changes to ensure network integrity and an efficient barrier between blood and tissues. In this review, we highlighted the relevance and the mechanisms involving endothelial cell migration during different steps of vascular morphogenesis. We further present evidence on how impaired endothelial cell dynamics can contribute to pathology.

• angiogenesis
• cell migration
• cell polarity
• vascular disease
• vascular remodelling

• Calyculin A
• Cytochalasin D
• blebbistatin
• cellular force generation
• endothelial cytoskeleton
• in vitro angiogenesis
• jasplakinolide

Cell therapies to treat critical limb ischaemia have demonstrated only modest results in clinical trials, and this has been partly attributed to poor cell retention following their delivery directly into the ischaemic limb. The aim of this study was to determine whether alginate encapsulation of therapeutic pro-angio/arteriogenic macrophages enhances their retention and ultimately improves limb perfusion. A reproducible GMP-compliant method for generating 300 µm alginate capsules was developed to encapsulate pro-angio/arteriogenic macrophages. Longitudinal analysis revealed no detrimental effect of encapsulation on cell number or viability in vitro, and macrophages retained their pro-angio/arteriogenic phenotype. Intramuscular delivery of encapsulated macrophages into the murine ischaemic hindlimb demonstrated increased cell retention compared with injection of naked cells (P = 0.0001), and that this was associated both enhanced angiogenesis (P = 0.02) and arteriogenesis (P = 0.03), and an overall improvement in limb perfusion (P = 0.0001). Alginate encapsulation of pro-angio/arteriogenic macrophages enhances cell retention and subsequent limb reperfusion in vivo. Encapsulation may therefore represent a means of improving the efficacy of cell-based therapies currently under investigation for the treatment of limb ischaemia.

Blood vessel-promoting immune cells stay longer in the body and help promote blood flow to the feet and toes of mice with critical limb ischemia when the therapeutic cells are packaged inside tiny bubbles of a biocompatible seaweed derivative called alginate. A team led by Bijan Modarai from King’s College London, UK, developed a reliable method for placing artery-stimulating macrophage cells inside alginate capsules measuring 300 micrometres in diameter, about the thickness of a postcard. In culture, the alginate coating had no effect on the macrophage viability; and when injected into the muscles of mice with artery blockages to their hindlimbs, the encapsulated cells were retained longer and offered greater therapeutic benefit than uncoated cells. This encapsulation strategy may improve the efficacy of comparable cell-based therapies for humans with limb ischemia.

• expected haplotype homozygosity
• hard sweep
• multilocus genotype
• positive selection
• soft sweep

To clarify the underlying mechanism of formin-like 3 (FMNL3) in the promotion of colorectal carcinoma (CRC) cell invasion.

The in vitro biological function analyses of FMNL3 were performed by gain- and loss-of function approaches. Changes in the F-actin cytoskeleton were detected by the technologies of phalloidin-TRITC labeling and confocal microscopy. The signaling pathway mediated by FMNL3 was explored by western blot, gelatin zymograph assay, co-immunoprecipitation (co-IP), immunofluorescence co-localization, and glutathione S-transferase (GST) pull-down assay.

The in vitro experimental results showed that FMNL3 significantly promoted the proliferation, invasion, and migration of CRC cells (P < 0.05 and P < 0.01). Moreover, FMNL3 regulated the remodeling of actin-based protrusions such as filopodia and lamellipodia in a RhoC-dependent manner. The western blot and gelatin zymograph assay results indicated that FMNL3 was involved in the RhoC/ focal adhesion kinase (FAK) pathway and acted as an effector of RhoC to activate the downstream signaling of p-FAK as well as p-MAPK and p-AKT. This resulted in the increased expression of matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF), and the subsequent promotion of CRC cell invasion. The results of TAE226, U0126 or Ly294002 treatment confirmed an essential role of FMNL3 in activation of the RhoC/FAK pathway and the subsequent promotion of CRC invasion. Co-IP, co-localization and GST pull-down assays showed the direct interaction of FMNL3 with RhoC in vivo and in vitro.

FMNL3 regulates the RhoC/FAK signaling pathway and RhoC-dependent remodeling of actin-based protrusions to promote CRC invasion.

• Formin-like 3
• Colorectal carcinoma
• Invasion
• RhoC/FAK pathway
• Actin assembly

• Actins/metabolism
• Cell Adhesion/drug effects
• Cell Line, Tumor
• Cell Movement/drug effects
• Colorectal Neoplasms/pathology
• Enzyme Inhibitors/pharmacology
• Focal Adhesion Kinase 2/genetics
• Focal Adhesion Kinase 2/metabolism
• Formins
• HEK293 Cells
• Humans
• Neoplasm Invasiveness/pathology
• Proteins/antagonists & inhibitors
• Proteins/genetics
• Proteins/metabolism
• RNA, Small Interfering/metabolism
• Signal Transduction/drug effects
• rhoC GTP-Binding Protein/metabolism

The mammalian Diaphanous-related (mDia) formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin), as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2) inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf) in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ≥5–10 μM induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10–20 and 50 μM, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.

• SMIFH2
• actin cytoskeleton
• formin
• intramimics
• invasion
• mDia
• xenograft
• zebrafish

• Animals
• Cell Proliferation
• Gene Expression Regulation, Developmental
• Gene Knockout Techniques
• Heart/growth & development
• Heart Ventricles/cytology
• Heart Ventricles/growth & development
• Heart Ventricles/metabolism
• Male
• Mice
• Multigene Family
• Myocardium/cytology
• Myocardium/metabolism
• SOXD Transcription Factors/genetics
• SOXD Transcription Factors/metabolism
• Transcriptome

• R01 AR055209 NIAMS NIH HHS
• American Heart Association
• National Institutes of Health
• National Science Foundation
• RIKEN
• Ministry of Education, Culture, Sports, Science, and Technology
• Japan Science and Technology Agency

• Actin
• Colorectal cancer
• FMNL2
• Formins
• Invasion
• Melanoma
• Metastasis

• Adherens junction
• Cardiovascular disease
• Catenin
• Cytoskeleton
• Endothelial integrity
• Mechanosensing
• PECAM-1
• Permeability
• Vascular stiffness

• Blotting, Western
• Cells, Cultured
• Fetal Proteins/metabolism
• Flow Cytometry
• Formins
• Human Umbilical Vein Endothelial Cells/metabolism
• Human Umbilical Vein Endothelial Cells/microbiology
• Humans
• Listeria monocytogenes
• Nuclear Proteins/metabolism
• Phagocytosis/physiology
• Polymerase Chain Reaction
• Proteins/metabolism

• T32 GM007365 NIGMS NIH HHS
• R01 AI036929 NIAID NIH HHS
• P50 GM107615 NIGMS NIH HHS
• S10 RR027431 NCRR NIH HHS
• R37 AI036929 NIAID NIH HHS
• Howard Hughes Medical Institute
• National Institute of Allergy and Infectious Diseases

• Angiogenesis
• Coculture
• Endothelial cells
• Fibroblasts
• Imaging
• VEGF

• Coculture Techniques/methods
• Epithelial Cells/cytology
• Fibroblasts/cytology
• Fibroblasts/metabolism
• Human Umbilical Vein Endothelial Cells
• Humans
• Neovascularization, Physiologic
• Optical Imaging

• PG/10/014/28224 British Heart Foundation
• PG/11/68/29074 British Heart Foundation

• actin
• cell–cell contacts
• epithelialization
• formins
• lumen formation

• Colorectal carcinoma (CRC)
• Formin-like 3 (FMNL3)
• Metastasis
• Prognosis

FMNL3 localizes broadly over the plasma membrane as discrete puncta, with particular enrichment in filopodia and ruffles and at cell–cell contacts. In addition, a population of FMNL3-containing vesicles of endocytic origin can fuse with the plasma membrane. FMNL3 suppression causes reductions in filopodia and cell–cell adhesion.

FMNL3 is a vertebrate-specific formin protein previously shown to play a role in angiogenesis and cell migration. Here we define the cellular localization of endogenous FMNL3, the dynamics of GFP-tagged FMNL3 during cell migration, and the effects of FMNL3 suppression in mammalian culture cells. The majority of FMNL3 localizes in a punctate pattern, with >95% of these puncta being indistinguishable from the plasma membrane by fluorescence microscopy. A small number of dynamic cytoplasmic FMNL3 patches also exist, which enrich near cell–cell contact sites and fuse with the plasma membrane at these sites. These cytoplasmic puncta appear to be part of larger membranes of endocytic origin. On the plasma membrane, FMNL3 enriches particularly in filopodia and membrane ruffles and at nascent cell–cell adhesions. FMNL3-containing filopodia occur both at the cell–substratum interface and at cell–cell contacts, with the latter being 10-fold more stable. FMNL3 suppression by siRNA has two major effects: decrease in filopodia and compromised cell–cell adhesion in cells migrating as a sheet. Overall our results suggest that FMNL3 functions in assembly of actin-based protrusions that are specialized for cell–cell adhesion.

Formin proteins were recognized as effectors of Rho GTPases some 15 years ago. They contribute to different cellular actin cytoskeleton structures by their ability to polymerize straight actin filaments at the barbed end. While not all formins necessarily interact with Rho GTPases, a subgroup of mammalian formins, termed Diaphanous-related formins or DRFs, were shown to be activated by small GTPases of the Rho superfamily. DRFs are autoinhibited in the resting state by an N- to C-terminal interaction that renders the central actin polymerization domain inactive. Upon the interaction with a GTP-bound Rho, Rac, or Cdc42 GTPase, the C-terminal autoregulation domain is displaced from its N-terminal recognition site and the formin becomes active to polymerize actin filaments. In this review we discuss the current knowledge on the structure, activation, and function of formin-GTPase interactions for the mammalian formin families Dia, Daam, FMNL, and FHOD. We describe both direct and indirect interactions of formins with GTPases, which lead to formin activation and cytoskeletal rearrangements. The multifaceted function of formins as effector proteins of Rho GTPases thus reflects the diversity of the actin cytoskeleton in cells.

• Cdc42
• Daam1
• Daam2
• FHOD1
• FHOD3
• FMNL1
• FMNL2
• FMNL3
• ROCK
• Rac
• Rho
• filopodia
• formin
• lamellipodium
• mDia1
• mDia2
• mDia3
• stress fiber

• Actin cytoskeleton
• DRF
• Formin

• Actin Cytoskeleton/metabolism
• Animals
• Cardiomyopathies/metabolism
• Cell Differentiation
• Cell Movement
• Embryonic Development
• Humans
• Microfilament Proteins/chemistry
• Microfilament Proteins/metabolism
• Neoplasms/metabolism
• Protein Conformation

• PG/11/123/29317 British Heart Foundation

The ARP2/3 complex controls junction-associated intermittent lamellipodia (JAIL), which trigger VE-cadherin adhesion and dynamics. JAIL formation maintains paraendothelial barrier function under physiological conditions and depends on the local VE-cadherin concentration.

Maintenance and remodeling of endothelial cell junctions critically depend on the VE-cadherin/catenin complex and its interaction with the actin filament cytoskeleton. Here we demonstrate that local lack of vascular endothelial (VE)-cadherin at established cell junctions causes actin-driven and actin-related protein 2/3 complex (ARP2/3)–controlled lamellipodia to appear intermittently at those sites. Lamellipodia overlap the VE-cadherin–free adjacent plasma membranes and facilitate formation of new VE-cadherin adhesion sites, which quickly move into the junctions, driving VE-cadherin dynamics and remodeling. Inhibition of the ARP2/3 complex by expression of the N-WASP (V)CA domain or application of two ARP2/3 inhibitors, CK-548 and CK-666, blocks VE-cadherin dynamics and causes intercellular gaps. Furthermore, expression of carboxy-terminal–truncated VE-cadherin increases the number of ARP2/3-controlled lamellipodia, whereas overexpression of wild-type VE-cadherin largely blocks it and decreases cell motility. The data demonstrate a functional interrelationship between VE-cadherin–mediated cell adhesion and actin-driven, ARP2/3-controlled formation of new VE-cadherin adhesion sites via intermittently appearing lamellipodia at established cell junctions. This coordinated mechanism controls VE-cadherin dynamics and cell motility and maintains monolayer integrity, thus potentially being relevant in disease and angiogenesis.