Tunnelling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin‐mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy, and optical tweezer‐based approaches, we demonstrate that TNTs formed through the outward extension of actin achieve distances greater than the mean length of filopodia and that branched Arp2/3‐dependent pathways attenuate the extent to which actin polymerizes in nanotubes, thus limiting their occurrence. Proteomic analysis using epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that, upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I‐BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form tunnelling nanotubes, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour tunnelling nanotube formation.

Tunneling nanotubes (TNTs) are thin membranous channels providing a direct cytoplasmic connection between remote cells. They are commonly observed in different cell cultures and increasing evidence supports their role in intercellular communication, and pathogen and amyloid protein transfer. However, the study of TNTs presents several pitfalls (e.g., difficulty in preserving such delicate structures, possible confusion with other protrusions, structural and functional heterogeneity, etc.) and therefore requires thoroughly designed approaches. The methods described in this protocol represent a guideline for the characterization of TNTs (or TNT-like structures) in cell culture. Specifically, optimized protocols to (1) identify TNTs and the cytoskeletal elements present inside them; (2) evaluate TNT frequency in cell culture; (3) unambiguously distinguish them from other cellular connections or protrusions; (4) monitor their formation in living cells; (5) characterize TNTs by a micropatterning approach; and (6) investigate TNT ultrastructure by cryo-EM are provided. Finally, this article describes how to assess TNT-mediated cell-to-cell transfer of cellular components, which is a fundamental criterion for identifying functional TNTs. (c) 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of tunneling nanotubes Alternate Protocol 1: Identifying the cytoskeletal elements present in tunneling nanotubes Alternate Protocol 2: Distinguishing tunneling nanotubes from intercellular bridges formed during cell division Basic Protocol 2: Deciphering tunneling nanotube formation and lifetime by live fluorescent microscopy Alternate Protocol 3: Deciphering tunneling nanotube formation using a live-compatible dye Basic Protocol 3: Assessing tunneling nanotubes functionality in intercellular transfer Alternate Protocol 4: Flow cytometry approach to quantify the rate of vesicle or mitochondria transfer Support Protocol: Controls to support TNT-mediated transfer Basic Protocol 4: Studies of tunneling nanotubes by cell micropatterning Basic Protocol 5: Characterization of the ultrastructure of tunneling nanotubes by cryo-EM

Filopodia are actin-rich membrane protrusions essential for cell morphogenesis, motility, and cancer invasion. How cells control filopodium initiation on the plasma membrane remains elusive. We performed experiments in cellulo, in vitro, and in silico to unravel the mechanism of filopodium initiation driven by the membrane curvature sensor IRSp53 (insulin receptor substrate protein of 53 kDa). We showed that full-length IRSp53 self-assembles into clusters on membranes depending on PIP2. Using well-controlled in vitro reconstitution systems, we demonstrated that IRSp53 clusters recruit the actin polymerase VASP (vasodilator-stimulated phosphoprotein) to assemble actin filaments locally on membranes, leading to the generation of actin-filled membrane protrusions reminiscent of filopodia. By pulling membrane nanotubes from live cells, we observed that IRSp53 can only be enriched and trigger actin assembly in nanotubes at highly dynamic membrane regions. Our work supports a regulation mechanism of IRSp53 in its attributes of curvature sensation and partner recruitment to ensure a precise spatial-temporal control of filopodium initiation.

Cell membranes are highly deformable and can be strongly curved. For instance, cells extend thin and dynamic tubular plasma membrane protrusions for sensing their surroundings and for their motion called “filopodia.” These finger-like protrusions are formed by actin parallel filaments pushing against the membrane while they polymerize. How actin is locally recruited to exert this pushing force has been debated for a long time. We have combined in cellulo and in vitro experiments with in silico multiscale computer simulations to address this issue. Our results on Rat2 cells confirm that IRSp53 clusters on the PM before filopodia growth. A subset of IRSp53 foci induce clustering of VASP to initiate filopodia growth and concomitant fascin recruitment. Our coarse-grained molecular dynamics simulations show that specific interactions between the curved I-BAR domains and PIP2 lipids induce directional membrane-mediated interactions between I-BARs, which facilitate PIP2 and I-BAR clustering, as compared to non-specific, homogeneously distributed interactions with PS lipid. In vitro reconstitution and mechanical experiments on liposomes confirm that the unique capability of IRSp53 to couple with membrane curvature induces its clustering on membranes. Moreover, we have succeeded in reconstituting filopodia formation with a minimal system: in conditions where IRSp53 clusters on GUVs but does not form tubules, the addition of low concentrations of VASP, actin and fascin induces the formation of localized tubular, actin-rich filopodia-like invaginations, However, with tube pulling experiments on Rat2 cells, we have evidenced a pivotal IRSp53 activation step is necessary in vivo to precisely regulate the time and localization of filopodia growth.

Tunneling nanotubes (TNTs) connect distant cells and mediate the transfer of cargo for intercellular communication in both normal physiological and pathological contexts. How the cell controls a common actin toolbox to generate TNTs rather than shorter filopodia that are characteristically <5 μm remains unknown. Utilizing surface micropatterning, we characterized the distances over which actin-dependent processes leads to TNT formation in a mouse neuronal cell model. Our data shows that TNT formation between micropatterned cells occurs most frequently at distances of 15 to 20 μm, while TNT occurrence becomes rarer at distances ≥30 μm.

Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions to span lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we demonstrate that TNTs forming through the outward extension of actin (not through cellular dislodgement) achieve distances greater than the mean length of filopodia, and that branched Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, limiting TNT occurrence. Proteomic analysis using Epidermal growth factor receptor kinase substrate 8 (Eps8) as a positive effector of TNTs showed that upon Arp2/3 inhibition, proteins enhancing filament turnover and depolymerization were reduced and Eps8 instead exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 that provides a direct connection with linear actin polymerases. Our data reveals how common protrusion players (Eps8 and IRSp53) form TNTs, and that when competing pathways overutilizing such proteins and monomeric actin in Arp2/3 networks are inhibited, processes promoting linear actin growth dominate to favour TNT formation. Thus, this work reinforces a general principle for actin network control for cellular protrusions where simple shifts in the balance between processes that inhibit actin growth versus those that promote growth dictate protrusion formation and the ultimate length scales protrusions achieve. ### Competing Interest Statement The authors have declared no competing interest.

Tunneling nanotubes (TNTs) connect distant cells and mediate cargo transfer for intercellular communication in physiological and pathological contexts. How cells generate these actin-mediated protrusions spanning lengths beyond those attainable by canonical filopodia remains unknown. Through a combination of micropatterning, microscopy and optical tweezer-based approaches, we found that Arp2/3-dependent pathways attenuate the extent to which actin polymerizes in nanotubes, limiting the formation and lengths of TNTs. Upon Arp2/3 inhibition, epidermal growth factor receptor kinase substrate 8 (Eps8) exhibited heightened interactions with the inverted Bin/Amphiphysin/Rvs (I-BAR) domain protein IRSp53 resulting in increased TNTs. In these conditions, Eps8 interaction with proteins enhancing filament turnover and depolymerization were reduced. Our data suggest a shift in the equilibrium (and usage of common actin proteins players) between branched and linear actin polymerization to form different cell protrusions.

Immune surveillance cells such as T cells and phagocytes utilize integral plasma membrane receptors to recognize surface signatures on triggered and activated cells such as those in apoptosis. One such family of plasma membrane sensors, the transmembrane immunoglobulin and mucin domain (Tim) proteins, specifically recognize phosphatidylserine (PS) but elicit distinct immunological responses. The molecular basis for the recognition of lipid signals on target cell surfaces is not well understood. Previous results suggest that basic side chains present at the membrane interface on the Tim proteins might facilitate association with additional anionic lipids including but not necessarily limited to PS. We, therefore, performed a comparative quantitative analysis of the binding of the murine Tim1, Tim3, and Tim4, to synthetic anionic phospholipid membranes under physiologically relevant conditions. X-ray reflectivity and vesicle binding studies were used to compare the water-soluble domain of Tim3 with results previously obtained for Tim1 and Tim4. Although a calcium link was essential for all three proteins, the three homologs differed in how they balance the hydrophobic and electrostatic interactions driving membrane association. The proteins also varied in their sensing of phospholipid chain unsaturation and showed different degrees of cooperativity in their dependence on bilayer PS concentration. Surprisingly, trace amounts of anionic phosphatidic acid greatly strengthened the bilayer association of Tim3 and Tim4, but not Tim1. A novel mathematical model provided values for the binding parameters and illuminated the complex interplay among ligands. In conclusion, our results provide a quantitative description of the contrasting selectivity used by three Tim proteins in the recognition of phospholipids presented on target cell surfaces. This paradigm is generally applicable to the analysis of the binding of peripheral proteins to target membranes through the heterotropic cooperative interactions of multiple ligands.

Cytoskeletal protrusions are emerging as key elements in the development of cellular networks through which material is readily exchanged. In parallel studies, Ortin‐Martinez et al (2021) and Kalargyrou et al (2021) report for the first time a direct transfer of cytoplasmic and membrane‐bound material between photoreceptors through nanotube‐like connections, providing further evidence toward the existence of nanotube‐mediated material transfer in vivo within the central nervous system. Two new studies report for the first time a direct in vivo transfer of cytoplasmic and membrane‐bound material between photoreceptors through nanotube‐like connections

Actin remodeling is at the heart of the response of cells to external or internal stimuli, allowing a variety of membrane protrusions to form. Fifteen years ago, tunneling nanotubes (TNTs) were identified, bringing a novel addition to the family of actin-supported cellular protrusions. Their unique property as conduits for cargo transfer between distant cells emphasizes the unique nature of TNTs among other protrusions. While TNTs in different pathological and physiological scenarios have been described, the molecular basis of how TNTs form is not well understood. In this review, we discuss the role of several actin regulators in the formation of TNTs and suggest potential players based on their comparison with other actin-based protrusions. New perspectives for discovering a distinct TNT formation pathway would enable us to target them in treating the increasing number of TNT-involved pathologies.

Antimicrobial peptides (AMPs) are a promising class of innate host defense molecules for next-generation antibiotics, as they uniquely target and permeabilize membranes of pathogens. This selectivity has been explained by the electrostatic attraction between these predominantly cationic peptides and the bacterial membrane, which is heavily populated with anionic lipids. However, AMP-resistant bacteria have non-electrostatic countermeasures that modulate membrane rigidity and thickness. We explore how variations in physical properties affect the membrane affinity and disruption process of protegrin-1 (PG-1) in phosphatidylcholine (PC) membranes with altered lipid packing densities and thicknesses. From isothermal titration calorimetry and atomic force microscopy, our results showed that PG-1 could no longer insert into membranes of increasing cholesterol amounts nor into monounsaturated PC membranes of increasing thicknesses with similar fluidities. Prevention of PG-1's incorporation consequently made the membranes more resistant to peptide-induced structural transformations like pore formation. Our study provides evidence that AMP affinity and activity are strongly correlated with the fluidity and thickness of the membrane. A basic understanding of how physical mechanisms can regulate cell selectivity and resistance towards AMPs will aid in the development of new antimicrobial agents.

Some synthetic polymers can block cell death when applied following an injury that would otherwise kill the cell. This cellular rescue occurs through interactions of the polymers with cell membranes. However, general principles for designing synthetic polymers to ensure strong, but nondisruptive, cell membrane targeting are not fully elucidated. Here, we tailored biomimetic phosphorylcholine-containing block copolymers to interact with cell membranes and determined their efficacy in blocking neuronal death following oxygen-glucose deprivation. By adjusting the hydrophilicity and membrane affinity of poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC)-based triblock copolymers, the surface active regime in which the copolymers function effectively as membrane-targeting cellular rescue agents was determined. We identified nonintrusive interactions between the polymer and the cell membrane that alter the collective dynamics of the membrane by inducing rigidification without disrupting lipid packing or membrane thickness. In general, our results open new avenues for biological applications of polyMPC-based polymers and provide an approach to designing membrane-targeting agents to block cell death after injury.

Lipid binding and associating proteins are necessary components of cell signaling pathways historically overlooked for more amenably characterized protein-protein interactions. As peripheral membrane binding proteins attract more attention, reliable structural methods are needed to elucidate the protein-lipid interactions that facilitate their function. Traditional methods such as crystallography or NMR have produced structures of many peripheral membrane binding proteins in isolation, bound to a single lipid, or in a lipid cubic phase but not in complex with full lipid membranes. X-ray reflectivity provides structural characterization of lipid monolayer associated proteins assuming a known structure of the desired protein has already been obtained. Depending on the experimental conditions of the given structure, it is possible this structure is representative of the membrane associated structure. In our studies of three members of the T-cell Immunoglobulin Mucin (TIM) family of proteins, involved in the recognition of the apoptotic cellular signal phosphatidylserine (PtdSer) in lipid membranes, the crystal structure was only representative for TIM4 and not TIM1 or TIM3. TIM1 was crystallized without PtdSer in a closed conformation that cannot represent the PtdSer bound state and TIM3 has much lower affinity resulting in a weak x-ray reflectivity signal. We developed data analysis methods employing molecular dynamics to refine the structures of TIM1 and TIM3 to better represent their membrane bound conformations. The newly obtained structures provide much improved fits of the data and highlight protein-lipid interactions that can explain the differences in binding affinity between the TIM protein family members.

The dynamic nature of lipid membranes presents significant challenges with respect to understanding the molecular basis of protein/membrane interactions. Consequently, there is relatively little known about the structural mechanisms by which membrane-binding proteins might distinguish subtle variations in lipid membrane composition and/or structure. We have previously developed a multidisciplinary approach that combines molecular dynamics simulation with interfacial x-ray scattering experiments to produce an atomistic model for phosphatidylserine recognition by the immune receptor Tim4. However, this approach requires a previously determined protein crystal structure in a membrane-bound conformation. Tim1, a Tim4 homolog with distinct differences in both immunological function and sensitivity to membrane composition, was crystalized in a closed-loop conformation that is unlikely to support membrane binding. Here we have used a previously described highly mobile membrane mimetic membrane in combination with a conventional lipid bilayer model to generate a membrane-bound configuration of Tim1 in silico. This refined structure provided a significantly improved fit of experimental x-ray reflectivity data. Moreover, the coupling of the x-ray reflectivity analysis with both highly mobile membrane mimetic membranes and conventional lipid bilayer molecular dynamics simulations yielded a dynamic model of phosphatidylserine membrane recognition by Tim1 with atomic-level detail. In addition to providing, to our knowledge, new insights into the molecular mechanisms that distinguish the various Tim receptors, these results demonstrate that in silico membrane-binding simulations can remove the requirement that the existing crystal structure be in the membrane-bound conformation for effective x-ray reflectivity analysis. Consequently, this refined methodology has the potential for much broader applicability with respect to defining the atomistic details of membrane-binding proteins.

Total reflection x-ray fluorescence (TXRF) is a widely applicable experimental technique for studying chemical element distributions across finely layered structures at extremely high sensitivity. To promote and facilitate scientific discovery using TXRF, we developed a MATLAB-based software package with a graphical user interface, named XeRay, for quick, accurate, and intuitive data analysis. XeRay lets the user model any layered system, each layer with its independent chemical composition and thickness, and enables fine-tuned data fitting. The accuracy of XeRay has been tested in the analysis of TXRF data from both air/liquid interface and liquid/liquid interfacial studies and has been compared to literature results. In an air/liquid interface study, Ca2+ sequestration was measured at a Langmuir monolayer of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (SOPA) on a buffer solution of 1 mM CaCl2 at pH 7.5. Data analysis with XeRay reveals that each 1 nm2 of interfacial area contains 2.38 ± 0.06 Ca2+ ions, which corresponds to a 1:1 ratio between SOPA headgroups and Ca2+ ions, consistent with several earlier reports. For the liquid/liquid interface study of Sr2+ enrichment at the dodecane/surfactant/water interface, analysis using XeRay gives a surface enrichment of Sr2+ at 68-5+6 Å2 per ion, consistent with the result published for the same dataset.

Biological membranes have a central role in mediating the organization of membrane-curving proteins, a dynamic process that has proven to be challenging to probe experimentally. Using atomic force microscopy, we capture the hierarchically organized assemblies of Bin/amphiphysin/Rvs (BAR) proteins on supported lipid membranes. Their structure reveals distinct long linear aggregates of proteins, regularly spaced by up to 300 nm. Employing accurate freeenergy calculations from large-scale coarse-grained computer simulations, we found that the membrane mediates the interaction among protein filaments as a combination of short-and long-ranged interactions. The long-ranged component acts at strikingly long distances, giving rise to a variety of micron-sized ordered patterns. This mechanism may contribute to the long-ranged spatiotemporal control of membrane remodeling by proteins in the cell.

Total reflection x-ray fluorescence (TXRF) at the air/liquid interface is a widely applicable experimental technique for studying chemical elements accumulated at the air/liquid interface, with extremely high sensitivity at brilliant synchrotron x-ray sources. To promote and facilitate scientific discovery using this sensitive technique, we constructed an experimental setup for TXRF atop an existing liquid surface x-ray scattering spectrometer. We also developed a MATLAB-based software package with a graphical user interface (GUI), named XeRay, for quick, accurate, and intuitive data analysis. The experimental setup and software package have been tested in the study of Ca2+ accumulation at a Langmuir monolayer of an anionic lipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphate (SOPA), on a buffer solution of 1mM CaCl2 at pH 7.5. Analysis with XeRay has shown that each 1 nm2 of interfacial area contains 1.23 Ca2+ ions, which corresponds to a 2:1 ratio between SOPA headgroups and Ca2+ ions, consistent with our intuition of a divalent ion bridging two singly charged lipids.

While molecular structures are available for many proteins, rarely is a structure determined in a protein's functional context. For proteins that bind with lipid membranes, traditional structural methods cannot probe the protein-membrane complex. X-ray reflectivity allows us to characterize the dynamic structure of these systems in physiologically relevant conditions. Given a structure of the protein, derived from techniques such as crystallography or nuclear magnetic resonance (NMR), it is possible to determine the structure of the protein-membrane complex using X-ray reflectivity. T-cell Immunoglobulin Mucin (TIM) family of proteins are involved in the recognition of phosphatidylserine (PtdSer), a cellular signal for apoptosis, in lipid membranes. The family has a conserved binding pocket that selectively recognizes PtdSer in the presence of calcium. While each member has varying binding sensitivity to the density of PtdSer in membranes, TIMs’ structural variations, as solved by crystallography and NMR, do not explain the variance in binding sensitivity. We have previously studied TIM4 and showed that peripheral interactions of particular residues on TIM4 with the membrane can explain TIM4's higher affinity for membranes with high densities of PtdSer. We have now extended the work to TIM1 and TIM3 in a similar effort to elucidate the structural basis of their binding sensitivities. Unlike TIM4, the crystal and NMR structures of TIM1 and TIM3, respectively, do not fit the X-ray reflectivity data well. We employ molecular dynamics (MD) to find the most likely conformations of TIM1 and TIM3 in complex with a lipid membrane and use the MD solved structures to fit X-ray reflectivity data. Using this combination of computational, experimental and statistical techniques, we are able to determine the most likely conformations of TIM1 and TIM3 in complex with a lipid film.