In cells, the curved clathrin structures in vesicle budding are well characterized, while the flat ones remain poorly understood. Here, we reconstitute the flat assembly of ESCRT-0 protein HRS and clathrin onto lipid membranes in vitro. HRS forms gel-like protein condensates at micromolar concentrations in solutions. These condensates spread as a two-dimensional layer on negatively charged membranes and, together with clathrin, form multilayered coats. Importantly, the two-dimensional condensates spontaneously form only on membranes at HRS concentrations below 50 nM, its cytoplasmic concentration. Correlative cryo-electron tomography of HRS-labelled endosomes in cells reveals a multilayered structure containing a flat clathrin layer 16 nm away from the membrane, consistent with our in vitro findings. Cholesterol enhances HRS recruitment to the membrane both in cells and in supported bilayers. Furthermore, cholesterol promotes the phase separation of HRS onto membranes, which in turn concentrates cholesterol underneath. This positive feedback promotes the formation of HRS-clathrin microdomains that sorts reconstituted ubiquitinated cargoes. Altogether, our results show that the distinct architecture of ESCRT-0 is assembled by the two-dimensional phase-separation of HRS which drives the assembly of flat clathrin coats.

The emergence of cell compartmentalization depends on membrane fission to create the endomembrane compartments. In eukaryotes, membrane fission is commonly executed by ESCRT-III, a protein complex conserved in all domains of life. However, whether membrane fission was an ancestral ESCRT-III activity predating eukaryogenesis remains unknown. Here we show that ESCRT-IIIA from Asgard Heimdallarchaeota, the closest archaeal relatives of eukaryotes, performs membrane fission through an N-terminal amphipathic helix, which we term Hofund. In eukaryotes, Hofund is fragmented across ESCRT-IIIA paralogs, and disrupting these regions causes severe fission defect in yeast. Remarkably, Heimdallarchaeota Hofund restores fission when fused to defective eukaryotic paralogs. These findings suggest that ESCRT-III-mediated fission arose before eukaryogenesis and later diversified to support the regulatory complexity of eukaryotic compartmentalization. ### Competing Interest Statement The authors have declared no competing interest.

Clathrin-mediated endocytosis is a conserved eukaryotic trafficking process where an Arp2/3 complex nucleated branched actin network provides force for vesicle formation. The mechanisms that initiate endocytic actin assembly are incompletely understood. In the fission yeast, Schizosaccharomyces pombe , actin assembly is initiated by Dip1, an Arp2/3 activator. In the budding yeast, Saccharomyces cerevisiae, the initiation of actin assembly has remained a mystery. Here we show that S. cerevisiae Ldb17, the homolog of Dip1, functions as an on-switch for endocytic actin assembly. Unexpectedly, the regulation of Ldb17 is more complicated than that of constitutively active Dip1. Ldb17 is controlled by a coat protein, Sla1, via separate recruitment and activation steps. This regulation was likely lost in the S. pombe lineage and this simplification may be related to other changes in actin assembly between these species. Our findings add a key missing piece in the understanding of endocytosis in S. cerevisiae and reveal an intriguing evolutionary tinkering of the actin on-switch. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, https://ror.org/00yjd3n13, 212288

Biological membranes are partitioned into functional zones termed membrane microdomains, which contain specific lipids and proteins(1-3). The composition and organization of membrane microdomains remain controversial because few techniques are available that allow the visualization of lipids in situ without disrupting their native behaviour(3,4). The yeast eisosome, composed of the BAR-domain proteins Pil1 and Lsp1 (hereafter, Pil1/Lsp1), scaffolds a membrane compartment that senses and responds to mechanical stress by flattening and releasing sequestered factors(5-9). Here we isolated near-native eisosomes as helical tubules made up of a lattice of Pil1/Lsp1 bound to plasma membrane lipids, and solved their structures by helical reconstruction. Our structures reveal a striking organization of membrane lipids, and, using in vitro reconstitutions and molecular dynamics simulations, we confirmed the positioning of individual PI(4,5)P-2, phosphatidylserine and sterol molecules sequestered beneath the Pil1/Lsp1 coat. Three-dimensional variability analysis of the native-source eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects the sequestration of these lipids. Collectively, our results support a mechanism in which stretching of the Pil1/Lsp1 lattice liberates lipids that would otherwise be anchored by the Pil1/Lsp1 coat, and thus provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain.

Amongst the different clathrin structures in mammalian cells, bi-layered clathrin coat colocalizing with endosomal sorting complex required for transport (ESCRT)-0 remains one of the most ambiguous. Despite being observed for the first time twenty years ago, their structure and how they are assembled remains unknown. Here, we reconstituted in vitro the ESCRT-0 clathrin assembly onto various types of membranes. The ESCRT-0 protein HRS, a known clathrin adaptor on endosomes, was found to form protein condensates. These condensates spread into a thin layer on PI(3)P-rich membranes. Platinum replica electron microscopy revealed that, surprisingly, the assembly of clathrin was different depending on the HRS phase. Protein droplets recruited clathrin as a dense, curved lattice, with many cage-like structures. On two-dimensional condensates, HRS recruited clathrin as a dense flat assembly. Two-dimensional HRS-clathrin condensates promoted the clustering of cholesterol in the underlying membrane, while cholesterol enhanced PI(3)P- dependent HRS recruitment on the membrane. On free-standing membranes, two-dimensional HRS-clathrin condensates promoted membrane flattening. Overall, these results show that a two- dimensional HRS condensate creates a unique membrane structure for sorting cargo molecules, defining a new mechanism in membrane trafficking processes. ### Competing Interest Statement The authors have declared no competing interest.

The capping of barbed filament ends is a fundamental mechanism for actin regulation. Capping protein controls filament growth and actin turnover in cells by binding to the barbed ends of the filaments with high affinity and slow off-rate. The interaction between Capping protein and actin is regulated by capping protein interaction (CPI) motif proteins. We identified a novel CPI motif protein, Bsp1, which is involved in cytokinesis and endocytosis in budding yeast. We demonstrate that Bsp1 is an actin binding protein with a high affinity for capping protein via its CPI motif. In cells, Bsp1 regulates capping protein at endocytic sites and is a major recruiter of capping protein to the cytokinetic actin ring. Lastly, we define Bsp1-related proteins as a distinct fungi-specific CPI protein group. Our results suggest that Bsp1 promotes actin filament capping by the capping protein. This study establishes Bsp1 as a new capping protein regulator and promising candidate to regulate actin networks in fungi.

Biological membranes are partitioned into functional zones containing specific lipids and proteins, termed membrane microdomains. Their composition and organization remain controversial owing to a paucity of techniques that can visualize lipids in situ without disrupting their native behavior1,2. The yeast eisosome, a membrane compartment scaffolded by the BAR-domain proteins Pil1 and Lsp1, senses and responds to mechanical stress by flattening and releasing sequestered factors3–7. Here, we isolated native eisosomes as helical filaments of Pil1/Lsp1 lattice bound to plasma membrane lipids and solved their structures by helical reconstruction. We observe remarkable organization within the lipid bilayer density from which we could assign headgroups of PI(4,5)P2 and phosphatidylserine bound to Pil1/Lsp1 and a pattern of membrane voids, signatures of sterols, beneath an amphipathic helix. We verified these assignments using in vitro reconstitutions and molecular dynamics simulations. 3D variability analysis of the native eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects functionally important lipid sequestration, supporting a mechanism in which membrane stretching liberates lipids otherwise anchored by the Pil1/Lsp1 coat. Our results provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain and, more globally, resolve long-standing controversies about the architecture and nature of lipid microdomains.

Conserved protein complexes called ESCRTs (endosomal sorting complexes in retrograde transport) exert diverse membrane remodeling and repair functions in cells. Hakala and Roux discuss a novel type of ESCRT-III structure found by Stempels et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202205130) in migrating macrophages and dendritic cells, suggesting a novel, cell type-specific function for this complex.

Coordinated polymerization of actin filaments provides force for cell migration, morphogenesis and endocytosis. Capping protein (CP) is a central regulator of actin dynamics in all eukaryotes. It binds to actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. However, in cells, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism remains unclear. Here, we report that the conserved cytoskeletal regulator twinfilin is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association between CP and cellular F-actin arrays, as well as to its retrograde movement throughout leading-edge lamellipodia. These were accompanied by diminished F-actin turnover rates. In vitro single-filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while enabling subsequent filament depolymerization. These results uncover a bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells. Hakala et al. report that twinfilin dissociates capping proteins from the actin filament barbed ends to promote actin turnover at leading-edge lamellipodia.

Membrane phosphoinositides control organization and dynamics of the actin cytoskeleton by regulating the activities of several key actin-binding proteins. Twinfilin is an evolutionarily conserved protein that contributes to cytoskeletal dynamics by interacting with actin monomers, filaments, and the heterodimeric capping protein. Twinfilin also binds phosphoinositides, which inhibit its interactions with actin, but the underlying mechanism has remained unknown. Here, we show that the high-affinity binding site of twinfilin for phosphoinositides is located at the C-terminal tail region, whereas the two actin-depolymerizing factor (ADF)/cofilin-like ADF homology domains of twinfilin bind phosphoinositides only with low affinity. Mutagenesis and biochemical experiments combined with atomistic molecular dynamics simulations reveal that the C-terminal tail of twinfilin interacts with membranes through a multivalent electrostatic interaction with a preference toward phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), PI(4,5)P2, and PI(3,4,5)P3 This initial interaction places the actin-binding ADF homology domains of twinfilin in close proximity to the membrane and subsequently promotes their association with the membrane, thus leading to inhibition of the actin interactions. In support of this model, a twinfilin mutant lacking the C-terminal tail inhibits actin filament assembly in a phosphoinositide-insensitive manner. Our mutagenesis data also reveal that the phosphoinositide- and capping protein-binding sites overlap in the C-terminal tail of twinfilin, suggesting that phosphoinositide binding additionally inhibits the interactions of twinfilin with the heterodimeric capping protein. The results demonstrate that the conserved C-terminal tail of twinfilin is a multifunctional binding motif, which is crucial for interaction with the heterodimeric capping protein and for tethering twinfilin to phosphoinositide-rich membranes.

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice (Twf2a-/-) display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. Twf2a-/- platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of Twf2a-/- platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein, which inhibits actin filament assembly by sequestering actin monomers and by capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here we report that constitutive Twf2a-deficient mice (Twf2a) display a mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. Twf2a platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem)ITAM and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyper-reactivity of Twf2a platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilinand profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice. For personal use only. on July 26, 2017. by guest www.bloodjournal.org From

Cell migration is necessary for several developmental processes in multicellular organisms. Furthermore, many physiological processes such as wound healing and immunological events in adult animals are dependent on cell migration. Consequently, defects in cell migration are linked to various diseases including immunological disorders as well as cancer progression and metastasis formation. Cell migration is driven by specific protrusive and contractile actin filament structures, but the types and relative contributions of these actin filament arrays vary depending on the cell type and the environment of the cell. In this chapter, we introduce the most important actin filament structures that contribute to mesenchymal and amoeboid cell migration modes and discuss the mechanisms by which the assembly and turnover of these structures are controlled by various actin-binding proteins.

Lamellipodia are dynamic actin-rich cellular extensions that drive advancement of the leading edge during cell migration. Lamellipodia undergo periodic extension and retraction cycles, but the molecular mechanisms underlying these dynamics and their role in cell migration have remained obscure. We show that glia-maturation factor (GMF), which is an Arp2/3 complex inhibitor and actin filament debranching factor, regulates lamellipodial protrusion dynamics in living cells. In cultured S2R(+) cells, GMF silencing resulted in an increase in the width of lamellipodial actin filament arrays. Importantly, live-cell imaging of mutant Drosophila egg chambers revealed that the dynamics of actin-rich protrusions in migrating border cells is diminished in the absence of GMF. Consequently, velocity of border cell clusters undergoing guided migration was reduced in GMF mutant flies. Furthermore, genetic studies demonstrated that GMF cooperates with the Drosophila homolog of Aip1 (flare) in promoting disassembly of Arp2/3-nucleated actin filament networks and driving border cell migration. These data suggest that GMF functions in vivo to promote the disassembly of Arp2/3-nucleated actin filament arrays, making an important contribution to cell migration within a 3D tissue environment.

Actin-depolymerizing-factor (ADF)/cofilins have emerged as key regulators of cytoskeletal dynamics in cell motility, morphogenesis, endocytosis, and cytokinesis. The activities of ADF/cofilins are regulated by membrane phospholipid PI(4,5)P2 in vitro and in cells, but the mechanism of the ADF/cofilin-PI(4,5)P2 interaction has remained controversial. Recent studies suggested that ADF/cofilins interact with PI(4,5)P2 through a specific binding pocket, and that this interaction is dependent on pH. Here, we combined systematic mutagenesis with biochemical and spectroscopic methods to elucidate the phosphoinositide-binding mechanism of ADF/cofilins. Our analysis revealed that cofilin does not harbor a specific PI(4,5)P2-binding pocket, but instead interacts with PI(4,5)P2 through a large, positively charged surface of the molecule. Cofilin interacts simultaneously with multiple PI(4,5)P2 headgroups in a cooperative manner. Consequently, interactions of cofilin with membranes and actin exhibit sharp sensitivity to PI(4,5)P2 density. Finally, we show that cofilin binding to PI(4,5)P2 is not sensitive to changes in the pH at physiological salt concentration, although the PI(4,5)P2-clustering activity of cofilin is moderately inhibited at elevated pH. Collectively, our data demonstrate that ADF/cofilins bind PI(4,5)P2 headgroups through a multivalent, cooperative mechanism, and suggest that the actin filament disassembly activity of ADF/cofilin can be accurately regulated by small changes in the PI(4,5)P2 density at cellular membranes.

Minna Poukkula,1 Markku Hakala,1 Nalle Pentinmikko,1 Meredith O. Sweeney,2 Silvia Jansen,2 Jaakko Mattila,1,3 Ville Hietakangas,1,3 Bruce L. Goode,2 and Pekka Lappalainen1,* 1Institute of Biotechnology, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland 2Rosenstiel Center for Basic Biomedical Research, Brandeis University, Waltham, MA 02453, USA 3Department of Biosciences, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland