mDia1

Neurotransmission at synapses is mediated by the fusion and subsequent endocytosis of synaptic vesicle membranes. Actin has been suggested to be required for presynaptic endocytosis but the mechanisms that control actin polymerization and its mode of action within presynaptic nerve terminals remain poorly understood. We combine optical recordings of presynaptic membrane dynamics and ultrastructural analysis with genetic and pharmacological manipulations to demonstrate that presynaptic endocytosis is controlled by actin regulatory diaphanous-related formins mDia1/3 and Rho family GTPase signaling in mouse hippocampal neurons...

mDia formin proteins regulate the dynamics and organization of the cytoskeleton through their linear actin nucleation and polymerization activities. We previously showed that mDia1 deficiency leads to aberrant innate immune activation and induces myelodysplasia in a mouse model, and mDia2 regulates enucleation and cytokinesis of erythroblasts and the engraftment of hematopoietic stem and progenitor cells (HSPCs). However, whether and how mDia formins interplay and regulate hematopoiesis under physiological and stress conditions remains unknown...

The diaphanous-related-formin, Diaphanous 1 (DIAPH1), is required for the assembly of Filamentous (F)-actin structures. DIAPH1 is an intracellular effector of the receptor for advanced glycation end products (RAGE) and contributes to RAGE signaling and effects such as increased cell migration upon RAGE stimulation. Mutations in DIAPH1, including those in the basic "RRKR" motif of its autoregulatory domain, diaphanous autoinhibitory domain (DAD), are implicated in hearing loss, macrothrombocytopenia and cardiovascular diseases...

Keratin 7 (Krt7) is a member of the keratin family and is primarily involved in cytoskeleton composition. It has been shown that Krt7 is able to influence its own remodeling and interactions with other signaling molecules via phosphorylation at specific sites unique to Krt7. However, its molecular mechanism in acute lung injury (ALI) remains unclear. In this study, differential proteomics was used to analyze lung samples from the receptor for advanced glycation end products (RAGE)-deficient and (wild-type)WT-septic mice...

Epithelial-mesenchymal transition (EMT) is a key cellular transformation for many physiological and pathological processes ranging from cancer over wound healing to embryogenesis. Changes in cell migration, cell morphology and cellular contractility were identified as hallmarks of EMT. These cellular properties are known to be tightly regulated by the actin cytoskeleton. EMT-induced changes of actin-cytoskeletal regulation were demonstrated by previous reports of changes of actin cortex mechanics in conjunction with modifications of cortex-associated f-actin and myosin...

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape...

Cell processes require precise regulation of actin polymerization at filament plus ends to execute normal functions. The detailed mechanisms used to control filament assembly at plus ends in the presence of diverse and often opposing regulators is not clear. Here we explore and identify residues important for the plus-end related activities of IQGAP1. In multi-wavelength TIRF assays, we directly visualize dimers of IQGAP1, mDia1, and CP on filament ends alone and as a multicomponent end binding complex. IQGAP1 promotes the turnover of end-binding proteins reducing the dwell times of CP, mDia1, or mDia1-CP 'decision complexes' by 8-18-fold...

Living cells assemble their actin networks by regulating reactions at the barbed end of actin filaments. Formins accelerate elongation, capping protein (CP) arrests growth and twinfilin promotes depolymerization at barbed ends. How cells integrate these disparate activities within a shared cytoplasm to produce diverse actin networks, each with distinct morphologies and finely tuned assembly kinetics, is unclear. We used microfluidics-assisted TIRF microscopy to investigate how formin mDia1, CP and twinfilin influence the elongation of actin filament barbed ends...

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified a-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins and there is an underlying concern that these proteins perform differently with actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human β- or γ-actin (i.e., cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin...

Deviations from mirror symmetry in the development of bilateral organisms are common but the mechanisms of initial symmetry breaking are insufficiently understood. The actin cytoskeleton of individual cells self-organises in a chiral manner, but the molecular players involved remain essentially unidentified and the relationship between chirality of an individual cell and cell collectives is unclear. Here, we analysed self-organisation of the chiral actin cytoskeleton in individual cells on circular or elliptical patterns, and collective cell alignment in confined microcultures...

Active stresses are generated and transmitted throughout diverse F-actin architectures within the cell cytoskeleton, and drive essential behaviors of the cell, from cell division to migration. However, while the impact of F-actin architecture on the transmission of stress is well studied, the role of architecture on the ab initio generation of stresses remains less understood. Here, we assemble F-actin networks in vitro, whose architectures are varied from branched to bundled through F-actin nucleation via Arp2/3 and the formin mDia1...

The fine regulation of actin polymerization is essential to control cell motility, architecture and to perform essential cellular functions. Formins are key regulators of actin filament assembly, known to processively elongate filament barbed ends and increase their polymerization rate. Different models have been extrapolated to describe the molecular mechanism governing the processive motion of formin FH2 domains at polymerizing barbed ends. Using negative stain electron microscopy, we directly identified for the first time two conformations of the mDia1 formin FH2 domains in interaction with the barbed ends of actin...

As sentinels of our immune system dendritic cells (DCs) rely on efficient cell migration for patrolling peripheral tissues and delivering sampled antigens to secondary lymphoid organs for the activation of T-cells. Dynamic actin polymerization is key to their macropinocytic and migratory properties. Both major actin nucleation machineries, formins and the Arp2/3 complex, are critical for different aspects of DC functionality, by driving the generation of linear and branched actin filaments, respectively. However, the importance of a third group of actin nucleators, the Ena/VASP family, has not been addressed yet...

The DIAPH1 gene fulfills critical immune and neurodevelopmental roles. It encodes the mammalian Diaphanous-related formin (mDia1) protein, which acts downstream of Rho GTPases to promote F-actin polymerization and stabilize microtubules. During mitosis, this protein is expressed in human neuronal precursor cells and considerably affects spindle formation and cell division. In humans, dominant gain-of-function DIAPH1 variants cause sensorineural deafness and macrothrombocytopenia (DFNA1), while homozygous DIAPH1 loss leads to seizures, cortical blindness, and microcephaly syndrome (SCBMS)...

The great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes, including cell migration, endocytosis and cell division. While it is known that this versatility is the result of the many actin-remodeling activities of actin-binding proteins, such as Arp2/3 and Cofilin, recent work also implicates post-translational acetylation or arginylation of the actin N-terminus itself as an equally important regulatory mechanism. However, the molecular mechanisms by which acetylation and arginylation alter the properties of actin are not well understood...

The small GTPase RhoA controls many important cellular processes through its ability to activate multiple downstream effector pathways. Most RhoA effectors contain a Rho-binding domain (RBD), and interaction between active RhoA and the RBD typically induces a conformational change in effectors that stimulates their recruitment or activity. Isolated GTPase binding domains fused to GST have been widely used in so-called pulldown assays to measure the activation state of other GTPases in cell lysates. Similarly, GST fusions containing the RBD of the RhoA effector Rhotekin have been widely adopted as a standardized tool for the measurement of RhoA activation...

SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested...

Myelodysplastic syndromes (MDS) are age-related myeloid neoplasms with increased risk of progression to acute myeloid leukemia (AML). The mechanisms of transformation of MDS to AML are poorly understood, especially in relation to the aging microenvironment. We previously established an mDia1/miR-146a double knockout (DKO) mouse model phenocopying MDS. These mice develop age-related pancytopenia with oversecretion of proinflammatory cytokines. Here, we found that most of the DKO mice underwent leukemic transformation at 12-14 months of age...

The small GTPase protein RhoA has two effectors, ROCK (Rho-associated protein kinase 1) and mDIA1 (protein diaphanous homolog 1), which cooperate reciprocally. However, temporal regulation of RhoA and its effectors in obesity-induced kidney damage remains unclear. Here, we investigated the role of RhoA activation in the proximal tubules at the early and late stages of obesity-induced kidney damage. In mice, a three-week high-fat-diet induced proximal tubule hypertrophy and damage without increased albuminuria, and RhoA/mDIA1 activation without ROCK activation...

Glioblastoma (GBM) is a progressive and lethal brain cancer. Malignant control of actin and microtubule cytoskeletal mechanics facilitates two major GBM therapeutic resistance strategies-diffuse invasion and tumor microtube network formation. Actin and microtubule reorganization is controlled by Rho-GTPases, which exert their effects through downstream effector protein activation, including Rho-associated kinases (ROCK) 1 and 2 and mammalian diaphanous-related (mDia) formins (mDia1, 2, and 3). Precise spatial and temporal balancing of the activity between these effectors dictates cell shape, adhesion turnover, and motility...