No abstract

Focal adhesions (FAs) mechanically couple the extracellular matrix (ECM) to the dynamic actin cytoskeleton, via transmembrane integrins and actin-binding proteins. The molecular mechanisms by which protein machineries control force transmission along this molecular axis, i.e. modulating integrin activation and controlling actin polymerization, remain largely unknown. Talin is a major actin-binding protein that controls both the inside-out activation of integrins and actin-filament anchoring and thus plays a major role in the establishment of the actin-ECM mechanical coupling. Talin contains three actinbinding domains (ABDs). The N-terminal head domain contains both the F3 integrin-activating domain and ABD1, while the C-terminal rod contains the actin-anchoring ABD2 and ABD3. Integrin binding is regulated by an intramolecular interaction between the N-terminal head and a Cterminal five-helix-bundle (R9). Whether talin ABDs regulate actin polymerization in a constitutive or regulated manner has not been fully explored. Here, we combine kinetics assays using fluorescence spectroscopy and single actin filament observation in TIRF microscopy, to examine relevant functions of the three ABDs of talin. We find that the N-terminal ABD1 blocks actin filament barbed end elongation while ABD2 and ABD3 do not show any activity. By mutating residues in ABD1, we find that this activity is mediated by a positively charged surface that is partially masked by its intramolecular interaction with R9. Our results also demonstrate that, once this intramolecular interaction is released, integrinbound talin head retains the ability to inhibit actin assembly.Cell adhesion to the extracellular matrix plays a critical role in many physiological functions such as cell migration, invasion or epithelial basement membrane attachment. Among the multiple adhesion structures, focal adhesions (FAs) play a major role (1,2). These multiprotein complexes couple various extracellular matrices to the actin cytoskeleton via the transmembrane heterodimeric αβ integrins and actin-binding proteins (ABPs) (3,4). The control of actin polymerization by ABPs is thought to play an important role to initiate the formation of the actomyosin stress fibers and control their tension by modulating their elongation. We showed previously that vinculin blocks actin filament barbed end elongation (5), while others reported that VASP promotes the elongation of actin filament barbed ends in a processive-like manner (6,7). Several formins may also play a role in the formation and elongation of Talin head inhibits actin assembly 2 stress fibers (8). However, despite these isolated characterizations, the respective roles of the multiple ABPs and their coordination in this process are poorly understood. The actin-binding protein talin plays a major role in FAs (9) ( Figure S1). First it acts very early to activate integrins. In this process the N-terminal PTB (phosphotyrosine binding) domain, located in the head domain of talin, also known as the F3 subdomain of the ...

In plants, the topological organization of membranes has mainly been attributed to the cell wall and the cytoskeleton. Additionally, few proteins, such as plant-specific remorins have been shown to function as protein and lipid organizers. Root nodule symbiosis requires continuous membrane re-arrangements, with bacteria being finally released from infection threads into membrane-confined symbiosomes. We found that mutations in the symbiosis-specific SYMREM1 gene result in highly disorganized perimicrobial membranes. AlphaFold modelling and biochemical analyses reveal that SYMREM1 oligomerizes into antiparallel dimers and may form a higher-order membrane scaffolding structure. This was experimentally confirmed when expressing this and other remorins in wall-less protoplasts is sufficient where they significantly alter and stabilize de novo membrane topologies ranging from membrane blebs to long membrane tubes with a central actin filament. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. Taken together we describe a plant-specific mechanism that allows the stabilization of large-scale membrane conformations independent of the cell wall.

Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology. The androgen receptor (AR) binds androgens mediating gene expression for sexual, somatic, and behavioral functions, and is involved in various conditions including androgen-insensitivity-syndrome (AIS) or prostate cancer. Here we identified functional AIS-patient mutations in the formin and actin nucleator DAAM2. DAAM2 localized to the nucleus and colocalized with the AR to form actin-dependent transcriptional droplets in response to dihydrotestosterone. DAAM2-AR-droplets ranged from 0.02 to 0.06 µm3 in size and associated with active RNA polymerase II. DAAM2 polymerizes actin directly at the AR to promote droplet fusion in a highly dynamic manner and actin polymerization is required for prostate-specific antigen expression in cancer cells. Our data uncover signal-regulated nuclear actin assembly at a steroid hormone receptor necessary for transcription.

Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology.The androgen receptor (AR) binds androgens mediating gene expression for sexual, somatic, and behavioral functions, and is involved in various conditions including androgen-insensitivity-syndrome (AIS) or prostate cancer. Here we identi ed functional AIS-patient mutations in the formin and actin nucleator DAAM2. DAAM2 localized to the nucleus and colocalized with the AR to form actin-dependent transcriptional droplets in response to dihydrotestosterone. DAAM2-AR-droplets ranged from 0.02 to 0.06 µm 3 in size and associated with active RNA polymerase II. DAAM2 polymerizes actin directly at the AR to promote droplet fusion in a highly dynamic manner and actin polymerization is required for prostatespeci c antigen expression in cancer cells. Our data uncover signal-regulated nuclear actin assembly at a steroid hormone receptor necessary for transcription.