A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.Endocytosis mediates the internalization of cell-surface proteins and lipids in small vesicles that bud from the plasma membrane and deliver their cargo to endosomes (1). Once cargo proteins reach the endosomes, one important pathway they may follow is retrograde recycling, in which cargos are delivered from endosomes to the trans-Golgi network (TGN) (2). Many important membrane proteins, including signaling receptors and small molecular transporters, require retrograde recycling (2). Some well-studied examples include the cation-independent mannose 6-phosphate receptor, insulin-stimulated glucose transporter Glut4, and Wls/MIG-14, a protein that ferries Wnt ligands to the cell surface during their secretion (2, 3). Certain toxins and viruses co-opt such retrograde transport pathways during their toxic/infectious cycles. These include the bacterial toxins Shiga and cholera, plant exotoxins ricin and abrin, as well as adeno-associated virus type 5 (AAV5) and HIV-1 (2, 3).Relatively few studies have focused on how such recycling pathways function in polarized epithelial cells, although polarized epithelia are a very abundant and important cell type in the human body. The intestine of the nematode Caenorhabditis elegans is a powerful model system for the study of endocytic recycling in the context of polarized epithelial cells, and can be studied within their normal context of the intact living animal (4, 5). The C. elegans intestine is a simple epithelial tube composed of 20 cells arranged mostly in pairs (6). Like mammalian intestinal epithelial cells, those of the C. elegans intestine display readily apparent apicobasal polarity, with basolateral and apical domains separated by apical junctions (6). The intestinal luminal (apical) membranes display a dense microvillar brush border with an overlying glycocalyx and subapical terminal web (6). The basolateral membrane faces the body cavity, exchanging molecules between the intestine and peripheral tissues.We previously established several model transmembrane cargo markers for the analysis of basolateral endocytosis and recycling in the C. elegans intestine (5, 7–10). These include MIG-14-GFP, hTfR-GFP, and hTAC-GFP (5). MIG-14 (Wntless) and hTfR (human transferrin receptor) enter cells via clathrin-dependent endocytosis (5, 7, 11). hTAC (human IL-2 receptor α-chain) enters cells via clathrin-independent endocytosis (4, 5). hTfR and hTAC recycle to the plasma membrane via recycling endosomes, also known as the endocytic recycling compartment (4, 7). MIG-14 recycles to the TGN, but previous work had not tested whether MIG-14 transits the recycling endosome en route to the Golgi (12–14).Many studies in cultured cell lines indicate that there are multiple routes to the Golgi from endosomes, including proposed routes from the recycling endosome, in addition to the more commonly discussed early endosome and late endosome routes (15–19). For instance, CHO cell pulse–chase analysis of fusion proteins bearing the transmembrane and intracellular domains of retrograde recycling proteins, TGN38 and Furin, showed that TGN38 trafficked from the early endosome to recycling endosome to the Golgi, whereas Furin recycling involved transit from the early endosome to the late endosome to the Golgi (20, 21). TGN38 and Shiga toxin have been shown to require distinct sets of SNARE proteins to complete transport to the Golgi, also indicating that different cargos recycle to the Golgi in different types of vesicles (22). In addition, TGN38 requires recycling endosome regulator Rab11 and its effector FIP1/RCP for retrograde recycling, further indicating that the recycling endosome pathway is important in TGN38 retrieval to the Golgi (23). The recycling endosome regulator and dynamin superfamily-like ATPase EHD1/mRme-1 is also required for transport of several cargos from recycling endosomes to the Golgi (24–26). The cation-independent mannose 6-phosphate receptor has also been reported to require transport through the recycling endosome to reach the TGN (24, 25).Previous whole-genome analysis of genes required for yolk protein endocytosis in the C. elegans oocyte, a process that requires yolk receptor recycling, identified the Rho-family GTPase CDC-42 and its associated proteins PAR-3 and PAR-6 (PDZ domain proteins), as well as the C. elegans homolog of atypical protein kinase C, PKC-3 (27). Together, these proteins are often referred to as the anterior PAR complex, because they function together to establish and maintain anterior–posterior polarity in the early C. elegans embryo (28). This work showed that CDC-42 is enriched on RME-1–positive recycling endosomes in nonpolarized C. elegans coelomocytes and cultured mammalian fibroblasts (27). These and other data implicated the CDC-42/PAR complex in recycling endosome function, but further mechanistic insight was lacking (27, 29, 30). Other work showed that CDC-42–associated Bar-domain proteins TOCA-1 and TOCA-2 function redundantly in yolk endocytosis, also probably functioning at a postendocytic transport step (31).To better understand the function of the TOCA proteins, and potentially the anterior PAR complex, in membrane transport, we set out to analyze their function in the C. elegans intestine using the molecular tools that we had established in this tissue. Unlike the general recycling regulator RME-1, which affects all recycling cargo that we have tested in the C. elegans intestine, we found that toca-1; toca-2 double mutants strongly affected MIG-14 but not hTAC or hTfR. Further analysis connected TOCA-1 and TOCA-2 to the CDC-42/PAR complex in this process, and further indicated that these proteins function with WVE-1, a core subunit of the actin nucleation complex WAVE. These results indicated a requirement for TOCA/CDC-42/PAR/WAVE in retrograde recycling, in addition to the well-known retromer complex, which contains a similar complement of molecules implicated in membrane binding, membrane bending, and actin nucleation. Finally, we established the C. elegans homolog of retrograde recycling cargo TGN38 (TGN-38) as a retrograde recycling cargo in the intestine. Experiments with GFP-tagged TGN-38 allowed us to compare the cargo transport blocks imposed by depletion of a subunit of the retromer complex (SNX-3) with the block imposed by depletion of CDC-42, as a representative of the TOCA/CDC-42/PAR/WAVE group (32). These experiments indicated a specific block at the early endosome after retromer depletion, whereas CDC-42 depletion blocked TGN-38 retrograde recycling most strongly at the recycling endosome. Taken together, these results demonstrate an important role for TOCA/CDC-42/PAR/WAVE acting at the recycling endosome after retromer function at the early endosome. We also provide evidence for the physiological importance of this pathway in WNT signaling, focusing on the polarity of the ALM mechanosensory neurons.     

Structural basis of Vps33A recruitment to the human HOPS complex by Vps16

The multisubunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is required for late endosome-lysosome and autophagosome-lysosome fusion in mammals. We have determined the crystal structure of the human HOPS subunit Vps33A, confirming its identity as a Sec1/Munc18 family member. We show that HOPS subunit Vps16 recruits Vps33A to the human HOPS complex and that residues 642–736 are necessary and sufficient for this interaction, and we present the crystal structure of Vps33A in complex with Vps16(642–736). Mutations at the binding interface disrupt the Vps33A–Vps16 interaction both in vitro and in cells, preventing recruitment of Vps33A to the HOPS complex. The Vps33A–Vps16 complex provides a structural framework for studying the association between Sec1/Munc18 proteins and tethering complexes.Eukaryotic cells tightly regulate the movement of macromolecules between their membrane-bound compartments. Multiple proteins and protein complexes interact to identify vesicles or organelles destined to fuse, bring them into close proximity, and then fuse their membranes, thereby allowing their contents to mix (1). Multisubunit tethering complexes modulate key steps in these fusion events by recognizing specific Rab-family small GTPases on the membrane surfaces, physically docking the membranes and then recruiting the machinery that effects the membrane fusion (2, 3).In metazoans, the multisubunit tethering complex homologous to the yeast homotypic fusion and vacuole protein sorting (HOPS) complex (4–7) is required for the maturation of endosomes (8); the delivery of cargo to lysosomes (9) and lysosome-related organelles, such as pigment granules in Drosophila melanogaster (10); and the fusion of autophagosomes with late endosomes/lysosomes (11). The mammalian HOPS complex comprises six subunits (Vps11, Vps16, Vps18, Vps33A, Vps39, and Vps41) (4–6). Homologs of HOPS components can be identified in almost all eukaryotic genomes (12) and are thought to be essential; for example, removal of the Vps33A homolog carnation (car) in Drosophila is lethal during larval development (13).HOPS components have been identified in animal models of human disease. A missense point mutation in the murine Vps33a gene gives rise to the buff mouse phenotype, characterized by pigmentation, platelet activity, and motor deficiencies (14). This phenotype closely resembles the clinical presentation of Hermansky–Pudlak syndrome (HPS) (15), and a mutation in the human VPS33A gene has been observed in a patient with HPS who lacked mutations at other known HPS loci (14). In metazoans, there is a second homolog of yeast Vps33 called Vps33B, but disruption of the VPS33B gene in humans gives rise to a clinical phenotype distinct from HPS (16).Human Vps33A is predicted to be a member of the Sec1/Munc18 (SM) family of proteins (7, 17) that, together with SNAREs, comprise the core machinery essential for membrane fusion in eukaryotes (18). Three SNAREs with glutamine residues at the center of their SNARE domain (Q_a-, Q_b-, and Q_c-SNAREs) and one with a central arginine residue (R-SNARE) associate to form a four-helical bundle, the trans-SNARE complex. Formation of this trans-SNARE complex by SNAREs on adjacent membranes drives the fusion of these membranes (18). SM proteins are essential regulators of this process, promoting membrane fusion by correctly formed (cognate) SNARE complexes (18). Although a comprehensive understanding of how SM proteins achieve this still remains elusive, it is clear that SM proteins bind directly both to individual SNAREs and to SNARE complexes (18, 19). Most SM proteins bind strongly and specifically to an N-terminal segment of their cognate Q_a-SNARE, the N-peptide, and this interaction is thought to recruit the SM protein to the site of SNARE-mediated fusion (20, 21).When considered as a whole, the HOPS complex has the functional characteristics of an SM protein: It binds SNAREs and SNARE complexes (5, 22–24), and yeast HOPS has been shown to promote SNARE-mediated membrane fusion (25, 26). Recent biochemical analysis of Vps33, the yeast Vps33A homolog, shows it to be capable of binding isolated SNARE domains and SNARE complexes but not the N-terminal domain or full cytosolic portion of the Q_a-SNARE Vam3 (23, 24). Data from the yeast HOPS complex are consistent with a model whereby Vps33 provides the SM functionality of HOPS, accelerating SNARE-mediated fusion, whereas the rest of the HOPS complex recruits Vps33 (and thus SM function) to the site of SNARE-mediated fusion (24).Although a recent EM study has defined the overall topology of the yeast HOPS complex (27), atomic resolution insights into the assembly of the HOPS complex have thus far been unavailable. Here, we present the 2.4-Å resolution structure of human Vps33A, confirming its structural identity as an SM protein. We have mapped the HOPS epitope that binds Vps33A to a helical fragment comprising residues 642–736 of Vps16, solved the structure of this complex to 2.6-Å resolution, and identified mutations at the binding interface that disrupt the Vps33A–Vps16 complex both in vitro and in cultured cells.     

Enhanced receptor–clathrin interactions induced by N-glycan–mediated membrane micropatterning

Glycan–protein interactions are emerging as important modulators of membrane protein organization and dynamics, regulating multiple cellular functions. In particular, it has been postulated that glycan-mediated interactions regulate surface residence time of glycoproteins and endocytosis. How this precisely occurs is poorly understood. Here we applied single-molecule-based approaches to directly visualize the impact of glycan-based interactions on the spatiotemporal organization and interaction with clathrin of the glycosylated pathogen recognition receptor dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). We find that cell surface glycan-mediated interactions do not influence the nanoscale lateral organization of DC-SIGN but restrict the mobility of the receptor to distinct micrometer-size membrane regions. Remarkably, these regions are enriched in clathrin, thereby increasing the probability of DC-SIGN–clathrin interactions beyond random encountering. N-glycan removal or neutralization leads to larger membrane exploration and reduced interaction with clathrin, compromising clathrin-dependent internalization of virus-like particles by DC-SIGN. Therefore, our data reveal that cell surface glycan-mediated interactions add another organization layer to the cell membrane at the microscale and establish a novel mechanism of extracellular membrane organization based on the compartments of the membrane that a receptor is able to explore. Our work underscores the important and complex role of surface glycans regulating cell membrane organization and interaction with downstream partners.Glycans are fundamental cellular components ubiquitously present in the extracellular matrix and cell membrane as glycoproteins or glycolipids. Glycan-binding proteins such as galectins, siglecs, and selectins are mostly multivalent and thus thought to cross-link glycoproteins into higher-order aggregates, creating a cell surface glycan-based connectivity also called glycan lattice or network (1–3). By concentrating specific glycoproteins or glycolipids while excluding other cell surface molecules, surface glycan-based connectivity can organize the plasma membrane into specialized domains that perform unique functions (1, 3–6). Nevertheless, direct observation of glycan-mediated ligand cross-linking in living cells remains challenging (7). Notwithstanding, there is no doubt that surface glycan-based connectivity is essential in the control of multiple biological processes including immune cell activation and homeostasis, cell proliferation and differentiation, and receptor turnover and endocytosis (1, 5, 6, 8).Clathrin-mediated endocytosis (CME) constitutes the primary pathway of cargo internalization in mammalian cells regulating the surface expression of receptors (9). Formation of clathrin-coated pits (CCPs) starts by nucleation of coat assembly at distributed positions in the inner surface of the plasma membrane, where it continues to grow or dissolve rapidly unless coat stabilization occurs (10, 11). One event that clearly correlates with successful CCP stabilization is cargo loading (11). Recent studies show that cargo molecules diffuse randomly on the cell membrane until they meet growing CCPs, with the extent of cargo interactions regulating CCP maturation (12). As such, factors that affect cargo mobility within/at the cell surface will inevitably impact on CCP maturation and successful internalization. In the context of surface glycan–protein interactions, it has been shown that glycoproteins with an intact glycan-based connectivity exhibit reduced lateral mobility and this correlates with compromised endocytosis (3, 13–17). How this precisely occurs is poorly defined, although fluorescence recovery after photobleaching on the EGF receptor (EGFR) suggested that cell surface glycan-based interactions restrict EGFR dynamics and localization into membrane regions away from endocytic platforms (14, 17). Whether this is a general mechanism for glycosylated proteins or specific to EGFR is not known. Moreover, visualization of receptor interactions with the endocytic machinery under the influence of the glycan network has not yet been attained.In this work we applied superresolution nanoscopy and developed a dedicated dual-color single-molecule spatio-dynamic exploration approach to visualize the impact of glycan-based interactions on the spatiotemporal organization and clathrin interaction of a glycosylated membrane receptor involved in pathogen recognition and uptake. We focused on the transmembrane glycoprotein dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) given its importance in supporting primary immune responses such as pathogen recognition and uptake on immature dendritic cells (imDCs), signaling, and cell adhesion (6, 18–20). Moreover, DC-SIGN contains a single N-glycosylation site, organizes in nanoclusters at the cell membrane (19, 21–23), and internalizes bound antigens via CPPs for subsequent processing and presentation to T cells (20, 24–26). Our work provides insights on how surface glycan-mediated interactions tune spatiotemporal micropatterning of receptors on the cell membrane, potentially regulating interactions with the endocytic machinery and underscoring the importance and complex role of surface glycans on cell membrane organization and function.     

Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding

HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.Live-cell fluorescence microscopy of assembling HIV-1 virions has established the temporal sequence in which various viral and host molecules are recruited to the assembly site (1–6). The HIV-1 genome is recruited first to the plasma membrane by a subdetectable number of molecules of the structural protein, Gag (3), and a steady accumulation of Gag ensues for 6–10 min (1, 2, 4). After Gag recruitment is completed, members of the endosomal sorting complex required for transport III (ESCRT-III) complex and the ATPase vacuolar protein sorting-associated protein 4A (Vps4A) are recruited transiently, for just a few minutes, to the site of assembly (4, 6). The ESCRT machinery functions during membrane fission in processes such as the formation of multivesicular bodies, the terminal stages of cytokinesis (7), and the budding of enveloped viruses such as HIV-1 (8, 9). These processes all have inverted topologies compared with the topology of endocytic events at the plasma membrane. HIV-1 hijacks the ESCRT machinery by recruiting its members through specific amino acid sequences, called late domains, in the major structural protein Gag. Specifically, the PTAP motif recruits tumor susceptibility gene 101 (Tsg101) and the LXXLF motif recruits ALG-2 interacting protein X (Alix), with PTAP being the functionally more important motif (10, 11). Biochemical and genetic assays have defined specific molecular interactions between ESCRT proteins that are recruited by Gag (8, 12–14), but a fine temporal and spatial mapping of the recruitment of viral and host components relative to each other is lacking. Here, using live-cell multiple-color total internal reflection fluorescence microscopy (TIR-FM), we demonstrate that the ESCRT protein Tsg101 is corecruited with Gag and accumulates progressively, whereas charged multivesicular body protein 4b (Chmp4b) and Vps4A are recruited sequentially and transiently to Gag assembly sites. Moreover, because diffraction-limited microscopy cannot resolve spatial differences the size of an HIV-1 virion (∼100 nm) (15–17), we determined the relative spatial positions of Gag and of several members of the ESCRT machinery in nascent virions using two-color superresolution imaging.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.The cell surface mediates interactions between the cell and the outside world by serving as the site for signal transduction. It also facilitates the uptake and release of cargo and supports adhesion to substrates. These diverse roles require that the cell surface components involved in each function are spatially and temporally organized into domains spanning a few nanometers (nanoclusters) to several micrometers (microdomains). The cell surface itself may be considered as a fluid–lipid bilayer wherein proteins are embedded (1). In the living cell, this multicomponent system is supported by an actin cortex, composed of a branched network of actin and a collection of filaments (2–4).Current models of membrane organization fall into three categories: those invoking lipid–lipid and lipid–protein interactions in the plasma membrane [e.g., the fluid mosaic model (1, 5) and the lipid raft hypothesis (6)], or those that appeal to the membrane-associated actin cortex (e.g., the picket fence model) (7), or a combination of these (8, 9). Although these models based on thermodynamic equilibrium principles have successfully explained the organization and dynamics of a range of membrane components and molecules, there is a growing class of phenomena that appears inconsistent with chemical and thermal equilibrium, which might warrant a different explanation. These include aspects of the organization and dynamics of outer leaflet glycosyl-phosphatidylinositol-anchored proteins (GPI-anchored proteins) (10–13), inner leaflet Ras proteins (14), and actin-binding transmembrane proteins (13, 15, 16).Recent experimental and theoretical work has shown that these features can be explained by taking into account that many cortical and membrane proteins are driven by ATP-consuming processes that drive the system out of equilibrium (13, 15, 17). The membrane models mentioned above have by-and-large neglected this active nature of the actin cortex where actin filaments are being continuously polymerized and depolymerized (18–21), in addition to being persistently acted upon by a variety of myosin motors (22–24) that consume ATP and exert contractile stresses on cortical actin filaments, continually remodeling the architecture of the cortex (4, 21, 25). These active processes in turn can generate tangential stresses and currents on the cell surface, which could drive the dynamics and local composition of membrane components at different scales (22, 26–29).Actin polymerization is proposed to be driven at the membrane by two nucleators, the Arp2/3 complex, which creates a densely branched network, as well as formins that nucleate filaments (18, 21, 30). A number of myosin motors are also associated with the juxtamembranous actin cortex, of which nonmuscle myosin II is the major component in remodeling the cortex and creating actin flows (4, 23, 25, 26, 31, 32). Based on our observations that the clustering of cell surface components that couple directly or indirectly to cortical actin [e.g., GPI-anchored proteins, proteins of the Ezrin, Radaxin, or Moesin (ERM) family (13, 15)] depends on myosin activity, we proposed that this clustering arises from the coupling to contractile actomyosin platforms (called “actin asters”) produced at the cortex (15, 33).A coarse-grained theory describing this idea has been put forward and corroborated by the verification of its key predictions in live cells (15, 33), but a systematic identification of the underlying microscopic processes is lacking. Given the complexity of numerous processes acting at the membrane of a living cell, we use an in vitro approach to study the effect of an energy-consuming actomyosin network on the dynamics of membrane molecules that directly interact with filamentous actin.A series of in vitro studies have explored the organization of confined, dynamic filaments (both actin and microtubules) (34–39) or the role of actin architecture on membrane organization (40–46). Indeed, these studies have yielded insights into the nontrivial emergent configurations that mixtures of polar filaments and motors can adopt when fueled by ATP (34–37), in particular constitutively remodeling steady states that display characteristics of active mechanics (38, 39, 47). However, the effect of linking these mechanics to the confining lipid bilayer and its organization has not been studied.The consequences of actin polymerization on membrane organization, in particular on giant unilamellar vesicles (GUVs), have been addressed in a number of studies on the propulsion of GUVs by an actin comet tail (40, 45, 46). In those experiments, the apparent advection of membrane bound ActA or WASP toward the site of actin polymerization is mainly due to the change in binding affinity of WASP to actin through Arp2/3 (44) and the spherical geometry resulting in the drag of actin to one pole of the vesicle after symmetry break of the actin shell. That this dynamic process changes the bulk properties of the bilayer, namely the critical temperature of a phase-separating lipid bilayer, was shown by Liu and Fletcher (40) when the actin nucleator N-WASP was connected to a lipid species (PIP₂) that was capable of partitioning into one of the two phases.Besides these pioneering studies on the effects of active processes on membrane organization, little was done to directly test the effect of active lateral stresses as well as actomyosin remodeling at the membrane, particularly on the dynamics and organization of membrane-associated components.To this end, we build an active composite in vitro by stepwise addition of components: a supported lipid bilayer with an actin-binding component, actin filaments, and myosin motors. By systematically varying the concentrations of actin and myosin as well as the average actin filament length, we find distinct states of actomyosin organization at the membrane surface upon complete ATP consumption. More importantly, we find that the ATP-fueled contractile actomyosin currents induce the transient accumulation of actin-binding membrane components. As predicted, the active mechanics of actin and myosin at physiologically relevant ATP concentrations drives the system into a nonequilibrium steady state with anomalous density fluctuations and the transient clustering of actin-binding components of the lipid bilayer (15, 33). Finally, connection of this active layer of actomyosin to a phase-segregating bilayer, influences its phase behavior and coarsening dynamics.     

Peroxisome extensions deliver the Arabidopsis SDP1 lipase to oil bodies

Lipid droplets/oil bodies (OBs) are lipid-storage organelles that play a crucial role as an energy resource in a variety of eukaryotic cells. Lipid stores are mobilized in the case of food deprivation or high energy demands—for example, during certain developmental processes in animals and plants. OB degradation is achieved by lipases that hydrolyze triacylglycerols (TAGs) into free fatty acids and glycerol. In the model plant Arabidopsis thaliana, Sugar-Dependent 1 (SDP1) was identified as the major TAG lipase involved in lipid reserve mobilization during seedling establishment. Although the enzymatic activity of SDP1 is associated with the membrane of OBs, its targeting to the OB surface remains uncharacterized. Here we demonstrate that the core retromer, a complex involved in protein trafficking, participates in OB biogenesis, lipid store degradation, and SDP1 localization to OBs. We also report an as-yet-undescribed mechanism for lipase transport in eukaryotic cells, with SDP1 being first localized to the peroxisome membrane at early stages of seedling growth and then possibly moving to the OB surface through peroxisome tubulations. Finally, we show that the timely transfer of SDP1 to the OB membrane requires a functional core retromer. In addition to revealing previously unidentified functions of the retromer complex in plant cells, our work provides unanticipated evidence for the role of peroxisome dynamics in interorganelle communication and protein transport.In seed plants, an essential function of seed reserves is to provide energy to the embryo for postgerminative growth until the seedling can perform photosynthesis. These reserves usually consist of storage proteins, carbohydrates, and lipids, with oil being the most common storage compound (1). The major storage lipids are triacylglycerols (TAGs), which are accumulated in subcellular structures called oil bodies (OBs). In Arabidopsis thaliana, OBs occupy 60% of the cell volume in the cotyledons of mature embryos and are located at the periphery of the cell (2). Upon seed germination, TAGs are hydrolyzed to release free fatty acids (FAs) and glycerol. Although the biochemical pathways that use glycerol and FAs to synthesize sugars are well documented, the initial step of oil degradation by TAG lipases has just started to be elucidated (3, 4). Although lipases have been purified from seeds of different plant species (5–7), physiological evidence for a function in TAG mobilization was clearly established only recently for the A. thaliana Sugar-Dependent 1 (SDP1) and SDP1-LIKE (SDP1L) lipases (8, 9). The sdp1 mutant was isolated from a forward genetic screen based on the observation that mutants impaired in TAG mobilization failed to develop in the absence of an exogenous carbon source (8). Analysis of sdp1 and sdp1l single and sdp1 sdp1l double mutants revealed that SDP1 activity accounts for the major degradation of TAGs after seed germination (9). SDP1 and SDP1L belong to the unorthodox class of patatin-like lipases similar to the mammalian and yeast lipases required for TAG breakdown (10). Although SDP1 was found to localize to the surface of OBs (8), it is unclear how SDP1 is targeted to OBs. Interestingly, A. thaliana mutants impaired in retromer functions exhibit severe defects in seed and plant development, including alteration in the maturation of seed storage proteins and the presence of small OBs (11, 12). The retromer is a multiprotein complex, conserved among eukaryotes, that is involved in the recycling of transmembrane receptors and retrograde transport of cargo proteins from endosomes to the trans-Golgi network (13). In mammals, the retromer consists of two distinct subcomplexes: one composed of a dimer of Sorting Nexins (SNXs) and the other, known as the core retromer, consisting of a trimer of vacuolar protein sorting (VPS) 26, VPS29, and VPS35 proteins (13, 14). The Arabidopsis genome contains genes encoding all components of the retromer complex, including three SNX genes—designated SNX1, SNX2a, and SNX2b—three genes coding for VPS35 isoforms (VPS35a, VPS35b, and VPS35c), two genes encoding VPS26 isoforms (VPS26a and VPS26b), and a single gene encoding VPS29 (12, 15–17). Here, we show that core retromer mutants display defects in OB biogenesis and storage oil breakdown during postgerminative growth in A. thaliana. We also report that the TAG lipase SDP1 initially surrounds peroxisomes and then migrates to the surface of OBs through peroxisome tubulations. In the absence of a functional core retromer complex, SDP1 migration is delayed, suggesting a function for the retromer in mediating the timely transport of SDP1—and possibly other lipases—to the OB surface, which is an essential step for seed oil mobilization. In addition, our work provides unanticipated evidence for the role of peroxisome dynamics in interorganelle communication and protein transport.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

TLR-dependent phagosome tubulation in dendritic cells promotes phagosome cross-talk to optimize MHC-II antigen presentation

Dendritic cells (DCs) phagocytose large particles like bacteria at sites of infection and progressively degrade them within maturing phagosomes. Phagosomes in DCs are also signaling platforms for pattern recognition receptors, such as Toll-like receptors (TLRs), and sites for assembly of cargo-derived peptides with major histocompatibility complex class II (MHC-II) molecules. Although TLR signaling from phagosomes stimulates presentation of phagocytosed antigens, the mechanisms underlying this enhancement and the cell surface delivery of MHC-II–peptide complexes from phagosomes are not known. We show that in DCs, maturing phagosomes extend numerous long tubules several hours after phagocytosis. Tubule formation requires an intact microtubule and actin cytoskeleton and MyD88-dependent phagosomal TLR signaling, but not phagolysosome formation or extensive proteolysis. In contrast to the tubules that emerge from endolysosomes after uptake of soluble ligands and TLR stimulation, the late-onset phagosomal tubules are not essential for delivery of phagosome-derived MHC-II–peptide complexes to the plasma membrane. Rather, tubulation promotes MHC-II presentation by enabling maximal cargo transfer among phagosomes that bear a TLR signature. Our data show that phagosomal tubules in DCs are functionally distinct from those that emerge from lysosomes and are unique adaptations of the phagocytic machinery that facilitate cargo exchange and antigen presentation among TLR-signaling phagosomes.Professional phagocytes take up large particles, such as bacteria, by phagocytosis and submit them to an increasingly harsh environment during phagosome maturation (1). Phagocytes concomitantly alert the immune system that an invader is present via signaling programs initiated by pattern recognition receptors, such as Toll-like receptors (TLRs) (2). Conventional dendritic cells (DCs) also alter and optimize phagosome maturation and TLR-signaling programs to preserve bacterial antigens for loading onto MHC class I and class II (MHC-II) molecules and optimize cytokine secretion to stimulate and direct T-cell responses to the invading agent (3, 4). DC presentation of soluble antigen is facilitated by TLR-driven tubulation of lysosomes that harbor MHC-II–peptide complexes and by consequent fusion of tubulovesicular structures with the plasma membrane (5–7); however, little is known about the mechanism by which signaling pathways influence the formation or presentation of phagosome-derived MHC-II–peptide complexes, key processes in the adaptive immunity to bacterial pathogens.TLRs respond to microbial ligands at the plasma membrane and in intracellular stores (8). TLR stimulation at the plasma membrane, endosomes, or phagosomes elicits distinct signaling pathways via two sets of adaptors, TIRAP (or MAL)-MyD88 and TRAM-TRIF (8, 9), which induce proinflammatory cytokine secretion and other downstream responses. TLRs such as TLR2 and TLR4 are recruited to macrophage and DC phagosomes at least partly from an intracellular pool (10–13), and signal autonomously from phagosomes independent of plasma membrane TLRs (11, 14, 15). Autonomous phagosomal signaling from TLRs or Fcγ receptors enhances the degradation of phagocytosed proteins and assembly of MHC-II with their derived peptides (14–16). Phagosomal TLR signaling has been proposed to also promote the reorganization of phagosome-derived MHC-II-enriched compartments (MIICs) to favor the delivery of MHC-II–peptide complexes to the plasma membrane (17), analogous to TLR-stimulated formation of tubules from MIICs/lysosomes (18–20) that fuse with the plasma membrane (7) and extend toward the immunologic synapse with T cells (5). Tubules emerge from phagosomes in macrophages shortly after phagocytosis and likely function in membrane recycling during early phagosome maturation stages (21–23), but tubules at later stages that might facilitate the presentation of phagosome-derived MHC-II–peptide complexes have not been reported previously. Moreover, a role for TLR signaling in formation of phagosome-derived tubules has not been established.Herein we show that in DCs, maturing phagosomes undergo extensive tubulation up to several hours after phagocytosis, and that tubulation requires TLR and MyD88 signaling and an intact actin and microtubule cytoskeleton. Unlike lysosome tubulation, phagosome tubulation is not essential for MHC-II–peptide transport to the cell surface. Rather, it contributes to content exchange among phagosomes that carry a TLR signature, and thereby enhances presentation of phagocytosed antigens from potential pathogens.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Longitudinal spread of mechanical excitation through tectorial membrane traveling waves

The mammalian inner ear separates sounds by their frequency content, and this separation underlies important properties of human hearing, including our ability to understand speech in noisy environments. Studies of genetic disorders of hearing have demonstrated a link between frequency selectivity and wave properties of the tectorial membrane (TM). To understand these wave properties better, we developed chemical manipulations that systematically and reversibly alter TM stiffness and viscosity. Using microfabricated shear probes, we show that (i) reducing pH reduces TM stiffness with little change in TM viscosity and (ii) adding PEG increases TM viscosity with little change in TM stiffness. By applying these manipulations in measurements of TM waves, we show that TM wave speed is determined primarily by stiffness at low frequencies and by viscosity at high frequencies. Both TM viscosity and stiffness affect the longitudinal spread of mechanical excitation through the TM over a broad range of frequencies. Increasing TM viscosity or decreasing stiffness reduces longitudinal spread of mechanical excitation, thereby coupling a smaller range of best frequencies and sharpening tuning. In contrast, increasing viscous loss or decreasing stiffness would tend to broaden tuning in resonance-based TM models. Thus, TM wave and resonance mechanisms are fundamentally different in the way they control frequency selectivity.The sharp frequency selectivity of auditory nerve fiber responses to sound is a hallmark of mammalian cochlear function. This remarkable signal processing originates in the mechanical stage of the cochlear signal processing chain (1–7), as evidenced by measured motions and mechanical properties of the basilar membrane (BM) (2–9) and tectorial membrane (TM) (10–24). Although the hydromechanical mechanisms underlying BM motions have been characterized based on experimental and theoretical studies, the mechanisms underlying TM motions remain unclear.The TM is an acellular matrix that overlies the hair bundles of sensory receptor cells. Based on its strategic position above the organ of Corti, conventional cochlear models (25–29) have implicated local mechanical properties (i.e., mass, stiffness) of the TM in stimulating the sensory hair bundles of hair cells and in cochlear tuning. Recent dynamic measurements of the TM, in vitro (17, 30–33) and in vivo (34), suggest that the TM supports longitudinal coupling, with large spatial extents across a broad range of frequencies. This longitudinal coupling manifests in the form of propagating traveling waves that are thought to contribute to hearing mechanisms (17, 21, 30, 35–40). Genetic modification studies provide further support that the spatial extent of TM waves may play a significant role in cochlear tuning (30, 32). Although these measurements, models, and genetic modification studies have confirmed the importance of TM mechanical properties in hearing, they have not isolated the distinct roles of TM stiffness and viscosity in generating longitudinally propagating traveling waves of the TM.To understand the contributions of TM material properties on traveling waves better, we developed chemical manipulations to alter the stiffness and viscosity of the TM selectively and reversibly. Because the TM is poroelastic (32, 41), we expect that changes in bath composition can have a direct effect on the mechanical properties of the TM mechanical matrix and its interstitial fluid, which makes up 97% of TM wet weight (42). The addition of PEG has previously been shown to generate an osmotic response that could be accounted for by the permeability of these molecules through the matrix rather than by direct changes to the matrix itself (41). In contrast, changing bath pH has little effect on the osmotic pressure or viscosity of the bath but has been shown to have a direct effect on the macromolecular matrix (43). In this paper, we apply these physicochemical manipulations to alter TM material properties reversibly, and thereby probe their role in controlling longitudinal spread of excitation through the TM.     

Nanomechanical mechanism for lipid bilayer damage induced by carbon nanotubes confined in intracellular vesicles

Understanding the behavior of low-dimensional nanomaterials confined in intracellular vesicles has been limited by the resolution of bioimaging techniques and the complex nature of the problem. Recent studies report that long, stiff carbon nanotubes are more cytotoxic than flexible varieties, but the mechanistic link between stiffness and cytotoxicity is not understood. Here we combine analytical modeling, molecular dynamics simulations, and in vitro intracellular imaging methods to reveal 1D carbon nanotube behavior within intracellular vesicles. We show that stiff nanotubes beyond a critical length are compressed by lysosomal membranes causing persistent tip contact with the inner membrane leaflet, leading to lipid extraction, lysosomal permeabilization, release of cathepsin B (a lysosomal protease) into the cytoplasm, and cell death. The precise material parameters needed to activate this unique mechanical pathway of nanomaterials interaction with intracellular vesicles were identified through coupled modeling, simulation, and experimental studies on carbon nanomaterials with wide variation in size, shape, and stiffness, leading to a generalized classification diagram for 1D nanocarbons that distinguishes pathogenic from biocompatible varieties based on a nanomechanical buckling criterion. For a wide variety of other 1D material classes (metal, oxide, polymer), this generalized classification diagram shows a critical threshold in length/width space that represents a transition from biologically soft to stiff, and thus identifies the important subset of all 1D materials with the potential to induce lysosomal permeability by the nanomechanical mechanism under investigation.The interactions of low-dimensional materials with the external or plasma membrane of living cells have been the subject of prior studies due to their importance in uptake and delivery, antibacterial action, and nanomaterial safety (1–6). Following uptake, nanomaterials may also interact with internal membranes while under confinement in intracellular vesicles (7–10), but the biophysics of these geometrically constrained systems is poorly understood. Low-dimensional materials interact with biological systems in complex ways dictated by their 1D nanofibrous or 2D nanosheet geometries (7, 11–20). These interactions typically begin when materials encounter the plasma membrane and initiate phenomena that can include adhesion, membrane deformation, penetration, lipid extraction, entry, frustrated uptake, or cytotoxicity (4, 11–14, 19–21). Recent experimental data suggest that the cellular response to some 1D materials is governed by their interaction with the internal lipid-bilayer membranes of endosomes and lysosomes following nanomaterial uptake (7–10). The resulting geometry is fundamentally different in that the fibrous materials are confined within a vesicle, imposing geometric constraints and introducing mechanical forces that act bidirectionally––i.e., on both the thin fibrous structure and the inner leaflet of the soft membrane. The fundamental biophysics of this tube-in-vesicle system is virtually unexplored, yet may be critical for understanding the cellular response to nanotubes/fibers, where shape and stiffness are among the known determinants of toxicity (13, 21). The technique of coarse-grained molecular dynamics (MD), demonstrated to be effective in the study of complex biomolecular systems (22, 23), has been applied to whole lipid-bilayer patches to reveal a biophysical mechanism for carbon nanotube interaction with the plasma membranes leading to tip entry and uptake (4, 19, 20). The same technique may also provide insight relevant to internal membrane interactions, although whole vesicle MD is a significant challenge. Here we use a complement of techniques including coarse-grained MD, all-atom MD, in vitro bioimaging, and carbon nanotube length modification to reveal the behavior of vesicle-encapsulated carbon nanotubes and identify the conditions and carbon nanotube (CNT) types that lead to mechanical stress and membrane damage following cellular uptake and packaging in lysosomes (8).     

Motor coupling through lipid membranes enhances transport velocities for ensembles of myosin Va

Myosin Va is an actin-based molecular motor responsible for transport and positioning of a wide array of intracellular cargoes. Although myosin Va motors have been well characterized at the single-molecule level, physiological transport is carried out by ensembles of motors. Studies that explore the behavior of ensembles of molecular motors have used nonphysiological cargoes such as DNA linkers or glass beads, which do not reproduce one key aspect of vesicular systems—the fluid intermotor coupling of biological lipid membranes. Using a system of defined synthetic lipid vesicles (100- to 650-nm diameter) composed of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (fluid at room temperature) or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) (gel at room temperature) with a range of surface densities of myosin Va motors (32–125 motors per μm²), we demonstrate that the velocity of vesicle transport by ensembles of myosin Va is sensitive to properties of the cargo. Gel-state DPPC vesicles bound with multiple motors travel at velocities equal to or less than vesicles with a single myosin Va (∼450 nm/s), whereas surprisingly, ensembles of myosin Va are able to transport fluid-state DOPC vesicles at velocities significantly faster (>700 nm/s) than a single motor. To explain these data, we developed a Monte Carlo simulation that suggests that these reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas faster transport velocities are consistent with a model wherein the normal stepping behavior of the myosin is supplemented by the preferential detachment of the trailing motor from the actin track.Myosin Va is a processive, actin-based molecular motor critical for transport, morphology, and positioning of a wide variety of intracellular cargoes and organelles (1, 2). Although a single myosin Va can transport cargo in vitro, intracellular membrane-bound vesicular cargoes have a high surface density of motor proteins (3–5). Ensembles of motors that transport cargo in vitro have been shown to demonstrate enhanced run lengths (6–9) and slower movement, relative to the behavior of a single motor (6, 8–13). Slower velocities are generally attributed to negative interference between motors that arise when rigidly coupled, high duty ratio motors step asynchronously (12). The stepping kinetics of myosin Va are load sensitive, whereby resistive loads slow the stepping rate, whereas assistive loads produce only a modest acceleration (14–16). This asymmetric load dependence causes an overall slowing for a motor ensemble. Indicative of the complexity of the intracellular environment, Efremov et al. (17) have shown that physiological vesicle transport (whether microtubule- or actin-based) does not directly mirror the behavior of the responsible molecular motor but is sensitive to aspects of the cargo itself. In some microtubule-based transport systems, velocities faster than the capacity of the transporting motor have been observed in cells (18, 19). However, motion of the cytoskeletal track may contribute to this enhanced motion (20).Most studies of in vitro cargo transport have a motor protein ensemble coupled to rigid cargoes such as quantum dots (Qdots) (8, 21), silica beads (12), DNA scaffolds (9, 22, 23), or directly to glass substrates (24). However, intracellular vesicles have fluid membranes that would be expected to allow the vesicle-attached motors (or coupled groups of motors) to diffuse within the membrane, so that motor ensemble transport of lipid-bound vesicles may be distinct from that observed with rigid cargo. Thus, more physiologically relevant studies have used liposomes (25) or isolated vesicles (26) with their in vivo complement of motors (26–28), or even intracellular organelles artificially coupled to a known motor type (17). Here, we attached recombinant myosin Va heavy meromyosin (myoVa) molecules at varying motor density to 100- to 650-nm lipid vesicles with membrane characteristics that were either fluid- or gel-like. When vesicles composed of fluid membranes are transported by an ensemble of myoVa molecules, they demonstrate velocities exceeding both those of identical vesicles transported by a single motor and those of individual, unloaded motors. Gel-like vesicles demonstrate velocities equal to or slower than a single motor. We developed a simulation of vesicular transport that supports at least one simple model wherein reductions in velocity can be attributed to two distinct mechanisms of intermotor interference (i.e., load-dependent modulation of stepping kinetics and binding-site exclusion), whereas enhanced transport velocities result from the concerted effects of myoVa’s stepping and the preferential detachment of the trailing motor in the ensemble from the actin track. This proposed bias in motor detachment and subsequent recentering of the vesicle above the remaining motors would generate an additional forward-directed displacement of the vesicle, providing the additional vesicular velocity. Therefore, we propose that the physical properties of the cargo itself contribute to the emergent transport behavior.