Origin, Organization, Dynamics, and Function of Actin and Actomyosin Networks at the T Cell Immunological Synapse.

The engagement of a T cell with an antigen-presenting cell (APC) or activating surface results in the formation within the T cell of several distinct actin and actomyosin networks. These networks reside largely within a narrow zone immediately under the T cell's plasma membrane at its site of contact with the APC or activating surface, i.e., at the immunological synapse. Here we review the origin, organization, dynamics, and function of these synapse-associated actin and actomyosin networks. Importantly, recent insights into the nature of these actin-based cytoskeletal structures were made possible in several cases by advances in light microscopy.

Filopodia In Vitro and In Vivo.

Filopodia are dynamic cell surface protrusions used for cell motility, pathogen infection, and tissue development. The molecular mechanisms determining how and where filopodia grow and retract need to integrate mechanical forces and membrane curvature with extracellular signaling and the broader state of the cytoskeleton. The involved actin regulatory machinery nucleates, elongates, and bundles actin filaments separately from the underlying actin cortex. The refined membrane and actin geometry of filopodia, importance of tissue context, high spatiotemporal resolution required, and high degree of redundancy all limit current models. New technologies are improving opportunities for functional insight, with reconstitution of filopodia in vitro from purified components, endogenous genetic modification, inducible perturbation systems, and the study of filopodia in multicellular environments. In this review, we explore recent advances in conceptual models of how filopodia form, the molecules involved in this process, and our latest understanding of filopodia in vitro and in vivo.

F-Actin Cytoskeleton Network Self-Organization Through Competition and Cooperation.

Many fundamental cellular processes such as division, polarization, endocytosis, and motility require the assembly, maintenance, and disassembly of filamentous actin (F-actin) networks at specific locations and times within the cell. The particular function of each network is governed by F-actin organization, size, and density as well as by its dynamics. The distinct characteristics of different F-actin networks are determined through the coordinated actions of specific sets of actin-binding proteins (ABPs). Furthermore, a cell typically assembles and uses multiple F-actin networks simultaneously within a common cytoplasm, so these networks must self-organize from a common pool of shared globular actin (G-actin) monomers and overlapping sets of ABPs. Recent advances in multicolor imaging and analysis of ABPs and their associated F-actin networks in cells, as well as the development of sophisticated in vitro reconstitutions of networks with ensembles of ABPs, have allowed the field to start uncovering the underlying principles by which cells self-organize diverse F-actin networks to execute basic cellular functions.

Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

Cell Motility and Cytokinesis: From Mysteries to Molecular Mechanisms in Five Decades.

This is the story of someone who has been fortunate to work in a field of research where essentially nothing was known at the outset but that blossomed with the discovery of profound insights about two basic biological processes: cell motility and cytokinesis. The field started with no molecules, just a few people, and primitive methods. Over time, technological advances in biophysics, biochemistry, and microscopy allowed the combined efforts of scientists in hundreds of laboratories to explain mysterious processes with molecular mechanisms that can be embodied in mathematical equations and simulated by computers. The success of this field is a tribute to the power of the reductionist strategy for understanding biology.

FMN2 Makes Perinuclear Actin to Protect Nuclei during Confined Migration and Promote Metastasis.

Cell migration in confined 3D tissue microenvironments is critical for both normal physiological functions and dissemination of tumor cells. We discovered a cytoskeletal structure that prevents damage to the nucleus during migration in confined microenvironments. The formin-family actin filament nucleator FMN2 associates with and generates a perinuclear actin/focal adhesion (FA) system that is distinct from previously characterized actin/FA structures. This system controls nuclear shape and positioning in cells migrating on 2D surfaces. In confined 3D microenvironments, FMN2 promotes cell survival by limiting nuclear envelope damage and DNA double-strand breaks. We found that FMN2 is upregulated in human melanomas and showed that disruption of FMN2 in mouse melanoma cells inhibits their extravasation and metastasis to the lung. Our results indicate a critical role for FMN2 in generating a perinuclear actin/FA system that protects the nucleus and DNA from damage to promote cell survival during confined migration and thus promote cancer metastasis.

SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II.

The nucleoskeleton is essential for nuclear architecture as well as genome integrity and gene expression. In addition to lamins, titin or spectrins, dynamic actin filament polymerization has emerged as a potential intranuclear structural element but its functions are less well explored. Here we found that calcium elevations trigger rapid nuclear actin assembly requiring the nuclear membrane protein SUN2 independently of its function as a component of the LINC complex. Instead, SUN2 colocalized and associated with the formin and actin nucleator INF2 in the nuclear envelope in a calcium-regulated manner. Moreover, SUN2 is required for active RNA polymerase II (RNA Pol II) clustering in response to calcium elevations. Thus, our data uncover a SUN2-formin module linking the nuclear envelope to intranuclear actin assembly to promote signal-dependent spatial reorganization of active RNA Pol II.

Formin tails act as a switch, inhibiting or enhancing processive actin elongation.

Formins are large, multidomain proteins that nucleate new actin filaments and accelerate elongation through a processive interaction with the barbed ends of filaments. Their actin assembly activity is generally attributed to their eponymous formin homology (FH) 1 and 2 domains; however, evidence is mounting that regions outside of the FH1FH2 stretch also tune actin assembly. Here, we explore the underlying contributions of the tail domain, which spans the sequence between the FH2 domain and the C terminus of formins. Tails vary in length from ∼0 to >200 residues and contain a number of recognizable motifs. The most common and well-studied motif is the ∼15-residue-long diaphanous autoregulatory domain. This domain mediates all or nothing regulation of actin assembly through an intramolecular interaction with the diaphanous inhibitory domain in the N-terminal half of the protein. Multiple reports demonstrate that the tail can enhance both nucleation and processivity. In this study, we provide a high-resolution view of the alternative splicing encompassing the tail in the formin homology domain (Fhod) family of formins during development. While four distinct tails are predicted, we found significant levels of only two of these. We characterized the biochemical effects of the different tails. Surprisingly, the two highly expressed Fhod-tails inhibit processive elongation and diminish nucleation, while a third supports activity. These findings demonstrate a new mechanism of modulating actin assembly by formins and support a model in which splice variants are specialized to build distinct actin structures during development.

Methylation and phosphorylation of formin homology domain proteins (Fhod1 and Fhod3) by protein arginine methyltransferase 7 (PRMT7) and Rho Kinase (ROCK1).

Protein post translational modifications (PTMs) can regulate biological processes by altering an amino acid's bulkiness, charge, and hydrogen bonding interactions. Common modifications include phosphorylation, methylation, acetylation and ubiquitylation. Although a primary focus of studying PTMs is understanding the effects of a single amino acid modification, the possibility of additional modifications increases the complexity. For example, substrate recognition motifs for arginine methyltransferases and some serine/threonine kinases overlap, leading to potential enzymatic crosstalk. In this study we have shown that the human family of formin homology domain containing proteins (Fhods) contain a substrate recognition motif specific for human protein arginine methyltransferase 7 (PRMT7). In particular, PRMT7 methylates two arginine residues in the diaphanous autoinhibitory domain (DAD) of the family of Fhod proteins: R1588 and/or R1590 of Fhod3 isoform 4. Additionally, we confirmed that S1589 and S1595 in the DAD domain of Fhod3 can be phosphorylated by Rho/ROCK1 kinase. Significantly, we have determined that if S1589 is phosphorylated then PRMT7 cannot subsequently methylate R1588 or R1590. In contrast, if R1588 or R1590 of Fhod3 is methylated then ROCK1 phosphorylation activity is only slightly affected. Finally, we show that the interaction of the N-terminal DID domain can also inhibit the methylation of the DAD domain. Taken together these results suggest that the family of Fhod proteins, potential in vivo substrates for PRMT7, might be regulated by a combination of methylation and phosphorylation.

The role of SPIRE actin nucleators in cellular transport processes.

Looking back at two decades of research on SPIRE actin nucleator proteins, the first decade was clearly dominated by the discovery of SPIRE proteins as founding members of the novel WH2-domain-based actin nucleators, which initiate actin filament assembly through multiple WH2 actin-binding domains. Through complex formation with formins and class 5 myosins, SPIRE proteins coordinate actin filament assembly and myosin motor-dependent force generation. The discovery of SPIRE-regulated cytoplasmic actin filament meshworks in oocytes initiated the next phase of SPIRE research, which has found that SPIRE proteins are integrated in a diverse range of cell biological processes. In addition to regulating vesicle-based actin filament meshworks, SPIRE proteins function in the organisation of actin structures driving the inward movement of pronuclei of the mouse zygote. Localisation at cortical ring structures and the results of knockdown experiments indicate that SPIRE proteins function in the formation of meiotic cleavage sites in mammalian oocytes and the externalisation of von Willebrand factor from endothelial cells. Alternative splicing targets mammalian SPIRE1 towards mitochondria, where it has a role in fission. In this Review, we summarise the past two decades of SPIRE research by addressing the biochemical and cell biological functions of SPIRE proteins in mammalian reproduction, skin pigmentation and wound healing, as well as in mitochondrial dynamics and host-pathogen interactions.

The expression of the formin Fhod3 in mouse tongue striated muscle.

The sarcomere is the contractile unit of striated muscle and is composed of actin and myosin filaments. There is increasing evidence to support that actin assembly mediated by Fhod3, a member of the formin family of proteins, is critical for sarcomere formation and maintenance in cardiac muscle. Fhod3, which is abundantly expressed in the heart, localizes to the center of sarcomeres and contributes to the regulation of the cardiac function, as evidenced by the fact that mutations in Fhod3 cause cardiomyopathy. However, the role of Fhod3 in skeletal muscle, another type of striated muscle, is unclear. We herein show that Fhod3 is expressed in the tongue at both mRNA and protein levels, although in smaller amounts than in the heart. To determine the physiological role of Fhod3 expressed in the tongue, we generated embryos lacking Fhod3 in the tongue. The tongue tissue of the Fhod3-depleted embryos did not show any significant structural defects, suggesting that Fhod3 is dispensable for normal development of the mouse tongue. Unexpectedly, the immunostaining analysis revealed the absence of specific sarcomeric signals for Fhod3 in the wild-type tongue when compared to the Fhod3-depleted tongue as a negative control, despite the use of antibodies that had previously been validated by immunostaining of heart tissues. Taken together, although Fhod3 protein is expressed at a significant level in the tongue, Fhod3 in the tongue does not appear to exhibit the same sarcomeric pattern as observed in the heart, suggesting a different role for Fhod3 in the tongue muscles.Key words: actin, formin, sarcomere, striated muscle.

Distinct roles of the Chlamydia trachomatis effectors TarP and TmeA in the regulation of formin and Arp2/3 during entry.

The obligate intracellular pathogen Chlamydia trachomatis manipulates the host actin cytoskeleton to assemble actin-rich structures that drive pathogen entry. The recent discovery of TmeA, which, like TarP, is an invasion-associated type III effector implicated in actin remodeling, raised questions regarding the nature of their functional interaction. Quantitative live-cell imaging of actin remodeling at invasion sites revealed differences in recruitment and turnover kinetics associated with the TarP and TmeA pathways, with the former accounting for most of the robust actin dynamics at invasion sites. TarP-mediated recruitment of actin nucleators, i.e. formins and the Arp2/3 complex, was crucial for rapid actin kinetics, generating a collaborative positive feedback loop that enhanced their respective actin-nucleating activities within invasion sites. In contrast, the formin Fmn1 was not recruited to invasion sites and did not collaborate with Arp2/3 within the context of TmeA-associated actin recruitment. Although the TarP-Fmn1-Arp2/3 signaling axis is responsible for the majority of actin dynamics, its inhibition had similar effects as the deletion of TmeA on invasion efficiency, consistent with the proposed model that TarP and TmeA act on different stages of the same invasion pathway.

Actin assembly requirements of the formin Fus1 to build the fusion focus.

In formin-family proteins, actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, with reaction rates that vary by at least 20-fold between formins. Each cell expresses distinct formins that assemble one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates in vivo. Fus1 assembles the actin fusion focus, necessary for gamete fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on fusion focus architecture, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in Fus1 FH2 that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional FH2 domain property. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

Formin 3 directs dendritic architecture via microtubule regulation and is required for somatosensory nociceptive behavior.

Dendrite shape impacts functional connectivity and is mediated by organization and dynamics of cytoskeletal fibers. Identifying the molecular factors that regulate dendritic cytoskeletal architecture is therefore important in understanding the mechanistic links between cytoskeletal organization and neuronal function. We identified Formin 3 (Form3) as an essential regulator of cytoskeletal architecture in nociceptive sensory neurons in Drosophila larvae. Time course analyses reveal that Form3 is cell-autonomously required to promote dendritic arbor complexity. We show that form3 is required for the maintenance of a population of stable dendritic microtubules (MTs), and mutants exhibit defects in the localization of dendritic mitochondria, satellite Golgi, and the TRPA channel Painless. Form3 directly interacts with MTs via FH1-FH2 domains. Mutations in human inverted formin 2 (INF2; ortholog of form3) have been causally linked to Charcot-Marie-Tooth (CMT) disease. CMT sensory neuropathies lead to impaired peripheral sensitivity. Defects in form3 function in nociceptive neurons result in severe impairment of noxious heat-evoked behaviors. Expression of the INF2 FH1-FH2 domains partially recovers form3 defects in MTs and nocifensive behavior, suggesting conserved functions, thereby providing putative mechanistic insights into potential etiologies of CMT sensory neuropathies.

Transmembrane formins as active cargoes of membrane trafficking.

Formins are a large, evolutionarily old family of cytoskeletal regulators whose roles include actin capping and nucleation, as well as modulation of microtubule dynamics. The plant class I formin clade is characterized by a unique domain organization, as most of its members are transmembrane proteins with possible cell wall-binding motifs exposed to the extracytoplasmic space-a structure that appears to be a synapomorphy of the plant kingdom. While such transmembrane formins are traditionally considered mainly as plasmalemma-localized proteins contributing to the organization of the cell cortex, we review, from a cell biology perspective, the growing evidence that they can also, at least temporarily, reside (and in some cases also function) in endomembranes including secretory and endocytotic pathway compartments, the endoplasmic reticulum, the nuclear envelope, and the tonoplast. Based on this evidence, we propose that class I formins may thus serve as 'active cargoes' of membrane trafficking-membrane-embedded proteins that modulate the fate of endo- or exocytotic compartments while being transported by them.

CYK-1/Formin activation in cortical RhoA signaling centers promotes organismal left-right symmetry breaking.

Proper left-right symmetry breaking is essential for animal development, and in many cases, this process is actomyosin-dependent. In Caenorhabditis elegans embryos active torque generation in the actomyosin layer promotes left-right symmetry breaking by driving chiral counterrotating cortical flows. While both Formins and Myosins have been implicated in left-right symmetry breaking and both can rotate actin filaments in vitro, it remains unclear whether active torques in the actomyosin cortex are generated by Formins, Myosins, or both. We combined the strength of C. elegans genetics with quantitative imaging and thin film, chiral active fluid theory to show that, while Non-Muscle Myosin II activity drives cortical actomyosin flows, it is permissive for chiral counterrotation and dispensable for chiral symmetry breaking of cortical flows. Instead, we find that CYK-1/Formin activation in RhoA foci is instructive for chiral counterrotation and promotes in-plane, active torque generation in the actomyosin cortex. Notably, we observe that artificially generated large active RhoA patches undergo rotations with consistent handedness in a CYK-1/Formin-dependent manner. Altogether, we conclude that CYK-1/Formin-dependent active torque generation facilitates chiral symmetry breaking of actomyosin flows and drives organismal left-right symmetry breaking in the nematode worm.

Spatial focalization of pheromone/MAPK signaling triggers commitment to cell-cell fusion.

Cell fusion is universal in eukaryotes for fertilization and development, but what signals this process is unknown. Here, we show in Schizosaccharomyces pombe that fusion does not require a dedicated signal but is triggered by spatial focalization of the same pheromone-GPCR (G-protein-coupled receptor)-MAPK signaling cascade that drives earlier mating events. Autocrine cells expressing the receptor for their own pheromone trigger fusion attempts independently of cell-cell contact by concentrating pheromone release at the fusion focus, a dynamic actin aster underlying the secretion of cell wall hydrolases. Pheromone receptor and MAPK cascade are similarly enriched at the fusion focus, concomitant with fusion commitment in wild-type mating pairs. This focalization promotes cell fusion by immobilizing the fusion focus, thus driving local cell wall dissolution. We propose that fusion commitment is imposed by a local increase in MAPK concentration at the fusion focus, driven by a positive feedback between fusion focus formation and focalization of pheromone release and perception.

Fluctuations of formin binding in the generation of membrane patterns.

Circular actin waves that propagate on the substrate-attached membrane of Dictyostelium cells separate two distinct membrane domains from each other: an inner territory rich in phosphatidyl-(3,4,5) trisphosphate (PIP3) and an external area decorated with the PIP3-degrading 3-phosphatase PTEN. During wave propagation, the inner territory increases at the expense of the external area. Beyond a size limit, the inner territory becomes unstable, breaking into an inner and an external domain. The sharp boundary between these domains is demarcated by the insertion of an actin wave. During the conversion of inner territory to external area, the state of the membrane fluctuates, as visualized by dynamic landscapes of formin B binding. Here we analyze the formin B fluctuations in relation to three markers of the membrane state: activated Ras, PIP3, and PTEN.

Single particle cryo-EM analysis of Rickettsia conorii Sca2 reveals a formin-like core.

Spotted fever group Rickettsia undergo actin-based motility inside infected eukaryotic cells using Sca2 (surface cell antigen 2): an âˆ¼ 1800 amino-acid monomeric autotransporter protein that is surface-attached to the bacterium and responsible for the assembly of long unbranched actin tails. Sca2 is the only known functional mimic of eukaryotic formins, yet it shares no sequence similarities to the latter. Using structural and biochemical approaches we have previously shown that Sca2 uses a novel actin assembly mechanism. The first âˆ¼ 400 amino acids fold into helix-loop-helix repeats that form a crescent shape reminiscent of a formin FH2 monomer. Additionally, the N- and C- terminal halves of Sca2 display intramolecular interaction in an end-to-end manner and cooperate for actin assembly, mimicking a formin FH2 dimer. Towards a better structural understanding of this mechanism, we performed single-particle cryo-electron microscopy analysis of Sca2. While high-resolution structural details remain elusive, our model confirms the presence of a formin-like core: Sca2 indeed forms a doughnut shape, similar in diameter to a formin FH2 dimer and can accommodate two actin subunits. Extra electron density, thought to be contributed by the C-terminal repeat domain (CRD), covering one side is also observed. This structural analysis allows us to propose an updated model where nucleation proceeds by encircling two actin subunits, and elongation proceeds either by a formin-like mechanism that necessitates conformational changes in the observed Sca2 model, or via an insertional mechanism akin to that observed in the ParMRC system.

Cooperative assembly of filopodia by the formin FMNL2 and I-BAR domain protein IRTKS.

Filopodia are long finger-like actin-based structures that project out from the plasma membrane as cells navigate and explore their extracellular environment. The initiation of filopodia formation requires release of tension at the plasma membrane followed by the coordinated assembly of long unbranched actin filaments. Filopodia growth is maintained by a tip complex that promotes actin polymerization and protects the growing barbed ends of the actin fibers from capping proteins. Filopodia growth also depends on additional F-actin bundling proteins to stiffen the actin filaments as well as extension of the membrane sheath projecting from the cell periphery. These activities can be provided by a number of actin-binding and membrane-binding proteins including formins such as formin-like 2 (FMNL2) and FMNL3, and Inverse-Bin-Amphiphysin-Rvs (I-BAR) proteins such as IRTKS and IRSp53, but the specific requirement for these proteins in filopodia assembly is not clear. We report here that IRTKS and IRSp53 are FMNL2-binding proteins. Coexpression of FMNL2 with either I-BAR protein promotes cooperative filopodia assembly. We find IRTKS, but not IRSp53, is required for FMNL2-induced filopodia assembly, and FMNL2 and IRTKS are mutually dependent cofactors in this process. Our results suggest that the primary function for FMNL2 during filopodia assembly is binding to the plasma membrane and that regulation of actin dynamics by its formin homology 2 domain is secondary. From these results, we conclude that FMNL2 initiates filopodia assembly via an unexpected novel mechanism, by bending the plasma membrane to recruit IRTKS and thereby nucleate filopodia assembly.