Dimerization of mammalian kinesin-3 motors results in superprocessive motion

The kinesin-3 family is one of the largest among the kinesin superfamily and its members play important roles in a wide range of cellular transport activities, yet the molecular mechanisms of kinesin-3 regulation and cargo transport are largely unknown. We performed a comprehensive analysis of mammalian kinesin-3 motors from three different subfamilies (KIF1, KIF13, and KIF16). Using Forster resonance energy transfer microscopy in live cells, we show for the first time to our knowledge that KIF16B motors undergo cargo-mediated dimerization. The molecular mechanisms that regulate the monomer-to-dimer transition center around the neck coil (NC) segment and its ability to undergo intramolecular interactions in the monomer state versus intermolecular interactions in the dimer state. Regulation of NC dimerization is unique to the kinesin-3 family and in the case of KIF13A and KIF13B requires the release of a proline-induced kink between the NC and subsequent coiled-coil 1 segments. We show that dimerization of kinesin-3 motors results in superprocessive motion, with average run lengths of ∼10 μm, and that this property is intrinsic to the dimeric kinesin-3 motor domain. This finding opens up studies on the mechanistic basis of motor processivity. Such high processivity has not been observed for any other motor protein and suggests that kinesin-3 motors are evolutionarily adapted to serve as the marathon runners of the cellular world.Molecular motors of the kinesin and dynein superfamilies are responsible for a variety of microtubule-based intracellular functions such as vesicle transport, spindle assembly, and cytoskeletal organization. Several features of the kinesin and dynein mechanochemical cycles have been realized, yet much remains unknown (1, 2). For kinesin motors, much of the current knowledge is based on studies of kinesin-1, the founding member of the kinesin superfamily, where alternating catalysis by the two motor domains of the dimeric molecule results in unidirectional processive movement (the ability to take successive steps along the microtubule). Mechanochemical studies of other members of the kinesin superfamily have revealed interesting evolutionary adaptations to the kinesin motor domain that enable, for example, conversion of ATP hydrolysis into microtubule destabilizing activity (kinesin-8 and kinesin-13 families) (3, 4).The kinesin-3 family is one of the largest among the kinesin superfamily and consists of five subfamilies in mammals (KIF1, KIF13, KIF14, KIF16, and KIF28) (5). Kinesin-3 family members are characterized by high sequence conservation within their motor domains, a forkhead-associated domain, and in most cases a C-terminal lipid-binding domain such as a pleckstrin homology or Phox homology (PX) domain (5). The founding member of kinesin-3 family, CeUNC-104, was identified in Caenorhabditis elegans based on a mutation that severely affects the transport of synaptic vesicles to the axon terminal (6, 7). Since that time, mammalian kinesin-3 motors have been found to be associated with diverse cellular and physiological functions including vesicle transport (8–12), signaling (13, 14), mitosis (15–17), nuclear migration (18), viral trafficking (19, 20), and development (21). Defects in kinesin-3 transport have been implicated in a wide variety of neurodegenerative, developmental, and cancer diseases (22). However, a mechanistic understanding of this important class of cellular transporters is currently limited.To date, mechanistic studies have focused on truncated versions of mammalian KIF1A and its homolog CeUNC-104, and these studies have yielded contradictory results (23). For example, murine KIF1A has been proposed to function as a monomer that diffuses along the microtubule surface or as a processive dimer. Furthermore, dimerization has been proposed to occur before cargo binding or on the cargo surface (24–26). Thus, despite their widespread functions and clinical importance, the mechanisms of kinesin-3 motor regulation and motility remain largely enigmatic. Based on extensive characterization at the cellular and single-molecule levels of mammalian kinesin-3 motors from three subfamilies (KIF1, KIF13, and KIF16), we now provide a general model for kinesin-3 motor regulation and motility. We show that kinesin-3 motors are largely regulated by a monomer-to-dimer transition on the cargo surface that results in superprocessive motion for cargo transport.     

Loss of Miro1-directed mitochondrial movement results in a novel murine model for neuron disease

Defective mitochondrial distribution in neurons is proposed to cause ATP depletion and calcium-buffering deficiencies that compromise cell function. However, it is unclear whether aberrant mitochondrial motility and distribution alone are sufficient to cause neurological disease. Calcium-binding mitochondrial Rho (Miro) GTPases attach mitochondria to motor proteins for anterograde and retrograde transport in neurons. Using two new KO mouse models, we demonstrate that Miro1 is essential for development of cranial motor nuclei required for respiratory control and maintenance of upper motor neurons required for ambulation. Neuron-specific loss of Miro1 causes depletion of mitochondria from corticospinal tract axons and progressive neurological deficits mirroring human upper motor neuron disease. Although Miro1-deficient neurons exhibit defects in retrograde axonal mitochondrial transport, mitochondrial respiratory function continues. Moreover, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or mitochondrial calcium buffering. Our findings indicate that defects in mitochondrial motility and distribution are sufficient to cause neurological disease.Motor neuron diseases (MNDs), including ALS and spastic paraplegia (SP), are characterized by the progressive, length-dependent degeneration of motor neurons, leading to muscle atrophy, paralysis, and, in some cases, premature death. There are both inherited and sporadic forms of MNDs, which can affect upper motor neurons, lower motor neurons, or both. Although the molecular and cellular causes of most MNDs are unknown, many are associated with defects in axonal transport of cellular components required for neuron function and maintenance (1–6).A subset of MNDs is associated with impaired mitochondrial respiration and mitochondrial distribution. This observation has led to the hypothesis that neurodegeneration results from defects in mitochondrial motility and distribution, which, in turn, cause subcellular ATP depletion and interfere with mitochondrial calcium ([Ca²⁺]_m) buffering at sites of high synaptic activity (reviewed in ref. 7). It is not known, however, whether mitochondrial motility defects are a primary cause or a secondary consequence of MND progression. In addition, it has been difficult to isolate the primary effect of mitochondrial motility defects in MNDs because most mutations that impair mitochondrial motility in neurons also affect transport of other organelles and vesicles (1, 8–11).In mammals, the movement of neuronal mitochondria between the cell body and the synapse is controlled by adaptors called trafficking kinesin proteins (Trak1 and Trak2) and molecular motors (kinesin heavy chain and dynein), which transport the organelle in the anterograde or retrograde direction along axonal microtubule tracks (7, 12–24). Mitochondrial Rho (Miro) GTPase proteins are critical for transport because they are the only known surface receptors that attach mitochondria to these adaptors and motors (12–15, 18, 25, 26). Miro proteins are tail-anchored in the outer mitochondrial membrane with two GTPase domains and two predicted calcium-binding embryonic fibroblast (EF) hand motifs facing the cytoplasm (12, 13, 25, 27, 28). A recent Miro structure revealed two additional EF hands that were not predicted from the primary sequence (29). Studies in cultured cells suggest that Miro proteins also function as calcium sensors (via their EF hands) to regulate kinesin-mediated mitochondrial “stopping” in axons (15, 16, 26). Miro-mediated movement appears to be inhibited when cytoplasmic calcium is elevated in active synapses, effectively recruiting mitochondria to regions where calcium buffering and energy are needed. Despite this progress, the physiological relevance of these findings has not yet been tested in a mammalian animal model. In addition, mammals ubiquitously express two Miro orthologs, Miro1 and Miro2, which are 60% identical (12, 13). However, the individual roles of Miro1 and Miro2 in neuronal development, maintenance, and survival have no been evaluated.We describe two new mouse models that establish the importance of Miro1-mediated mitochondrial motility and distribution in mammalian neuronal function and maintenance. We show that Miro1 is essential for development/maintenance of specific cranial neurons, function of postmitotic motor neurons, and retrograde mitochondrial motility in axons. Loss of Miro1-directed retrograde mitochondrial transport is sufficient to cause MND phenotypes in mice without abrogating mitochondrial respiratory function. Furthermore, Miro1 is not essential for calcium-mediated inhibition of mitochondrial movement or [Ca²⁺]_m buffering. These findings have an impact on current models for Miro1 function and introduce a specific and rapidly progressing mouse model for MND.     

Delineating cooperative responses of processive motors in living cells

Characterizing the collective functions of cytoskeletal motors is critical to understanding mechanisms that regulate the internal organization of eukaryotic cells as well as the roles various transport defects play in human diseases. Though in vitro assays using synthetic motor complexes have generated important insights, dissecting collective motor functions within living cells still remains challenging. Here, we show that the protein heterodimerization switches FKBP-rapalog-FRB can be harnessed in engineered COS-7 cells to compare the collective responses of kinesin-1 and myosinVa motors to changes in motor number and cargo size. The dependence of cargo velocities, travel distances, and position noise on these parameters suggests that multiple myosinVa motors can cooperate more productively than collections of kinesins in COS-7 cells. In contrast to observations with kinesin-1 motors, the velocities and run lengths of peroxisomes driven by multiple myosinVa motors are found to increase with increasing motor density, but are relatively insensitive to the higher loads associated with transporting large peroxisomes in the viscoelastic environment of the COS-7 cell cytoplasm. Moreover, these distinctions appear to be derived from the different sensitivities of kinesin-1 and myosinVa velocities and detachment rates to forces at the single-motor level. The collective behaviors of certain processive motors, like myosinVa, may therefore be more readily tunable and have more substantial roles in intracellular transport regulatory mechanisms compared with those of other cytoskeletal motors.The transport of vesicles and organelles along cytoskeletal filaments by processive motor proteins is essential to physiological processes in eukaryotic cells requiring the spatial regulation of signaling complexes and other important subcellular commodities. Aberrant motor functions have also been implicated in several human diseases (1). The mechanochemical properties of motors have been studied extensively using suites of single-molecule and bulk biochemical techniques. However, many cargos are propelled in cells by systems of motors containing multiple copies of the same and even of different types of microtubule and actin-dependent motors (2). Characterizing how these motors cooperate or compete with one another is therefore critical to understanding mechanisms that regulate the internal organization of cells and how disrupted motor functions lead to diseases.Despite increased attention, current studies of collective motor behaviors are often limited by the challenges associated with analyzing or controlling the number and organization of motors on individual cargos. These issues have been addressed in part by synthetic approaches that use protein (3, 4) and DNA-based molecular scaffolds (5–8) to prepare organized multiple motor complexes of known composition. Subsequent theoretical studies of these systems have uncovered key differences in the way that classes of processive motors function in groups (9, 10). Relatively modest increases in cargo run lengths and detachment forces have been observed when multiple processive motors possessing high single-motor stalling forces, such as kinesin-1, are grouped together on a common cargo (5, 6, 8). These weak collective responses occur due to effects from motor interference as well as kinetic constraints that limit the abilities of multiple kinesin complexes to bind cytoskeletal filaments in configurations that allow motors to share applied loads equally (8, 9, 11–13). Similar studies have shown that motors whose velocities decrease more rapidly with increasing load, such as the nonprocessive, minus-end directed kinesin called NCD (kinesin-14), can cooperate much more productively via load sharing, yielding higher sensitivities of cargo transport parameters to variation in motor number (6). Along these lines, our group has demonstrated that the velocities of elastically coupled myosinVa motors are much more sensitive to changes in motor number than systems of coupled kinesins in the absence of an applied load (14). Under these conditions, coupled myosinVa motors were found to move with lower velocities than single myosinVa molecules, a response we attributed to the large step size and relatively small stalling force of myosinVa. Together, these properties lead to high sensitivity of collective motor stepping rates to strain produced when the motors step asynchronously. As with NCD, we also expect that this behavior will allow multiple myosinVa motors to cooperate more productively than kinesins in the presence of an applied load. Such distinctions are significant because they suggest cells may be able to modulate the collective functions of certain types of motors more sensitively than others, which could elevate the role of these motor systems in mechanisms that regulate cargo motion.Various signatures of the collective behaviors observed in vitro appear to be reflected in recent particle-tracking and optical-trapping analyses of cargo transport in living cells (15, 16). However, resolving the responses of cargo transport to the number and organization of motors on endogenous cargos in living cells remains challenging. It is still not clear whether the collective responses found using synthetic motor complexes, where the motors are coupled elastically, translate directly to scenarios where motors are bound to vesicular cargos that are transported along the native cytoskeleton. Resolving this issue is critical to ultimately determining how sensitively regulatory factors that affect motor functions can modulate intracellular transport processes, and how severely mutations in motor proteins can perturb a cell’s transport machinery.Here, we describe a method to configure live-cell particle (peroxisome)-tracking assays where the type of motor, motor surface density on vesicular cargos, and cargo size are controlled via the regulation of synthetic genes. Comparisons of the collective behaviors exhibited by kinesin-1 and myosinVa motors revealed several key distinctions in the way that these motors cooperate as a team. MyosinVa motors display much higher sensitivities to changes in motor density and appear to cooperate more productively when transporting vesicular cargos against cargo size-dependent viscoelastic loads imposed by the cytoplasm compared with kinesin motors. Implications of these different responses to mechanisms that regulate intracellular trafficking and transport processes are discussed.     

Switching dynamics of the bacterial flagellar motor near zero load

Switching dynamics of flagellar motors of Escherichia coli is commonly observed through markers attached to the flagellar filaments. To eliminate possible complications resulting from the conformational transitions of these filaments and to look at the output of motors more directly, we monitored motor rotation by attaching nanogold spheres to the hooks of cells lacking filaments. We observed exponentially distributed counterclockwise (CCW) and clockwise (CW) intervals and Lorentzian power spectra of the switching time series consistent with models that treat motor switching as a two-state Poisson process.Peritrichously flagellated bacteria such as Escherichia coli perform chemotaxis by a biased random walk comprising runs and tumbles, determined by the direction of rotation of the flagellar motors. When all of the motors on a cell rotate counterclockwise (CCW), the flagellar filaments form a helical bundle and the cell swims smoothly (runs); when one or more motors switch to clockwise (CW) rotation, the bundle is disrupted and the cell changes direction (tumbles) (1). The response regulator of the chemotaxis-signaling pathway, CheY-P, interacts with the motor switch protein FliM, increasing the probability that the motor spins CW (2–4). Switching dynamics of the motor has been observed by tethering single filaments to a glass slide and observing rotation of the cell body (5) or by attaching micrometer-sized beads to short (6, 7) or long (8, 9) filament stubs and watching the beads wobble. A recent model noted that conformational transitions of filaments can contribute to the observed dynamics (10). To remove this complication and observe the motor dynamics directly, we used the nanogold method developed to study motor behavior near zero load, in which 60- to 200-nm-diameter gold spheres are attached to the hooks of cells lacking flagellar filaments (11–13).     

Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8

Kinesin-8s are plus-end–directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are “ultraprocessive,” a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end–directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.Kinesins are microtubule (MT)-dependent motors that fulfill a wide range of cellular functions. By ferrying cargos through cytoplasm, transport motors help to organize the contents of a cell. MT regulatory kinesins, on the other hand, modulate the polymerization state of MT ends, thereby altering the architecture and function of MT-based structures. Kinesin-8s, motors that regulate diverse cellular processes, ranging from spindle positioning in yeast (1) to chromosome alignment (2–4) and motile cilia length in animal cells (5), exhibit features of both transport and MT-regulating kinesins (6). Yeast Kip3, the first well-studied kinesin-8, and human Kif19 are plus-end–directed motors that depolymerize MTs once they reach the plus end (5, 7, 8). Human Kif18A, also a plus-end–directed motor, does not depolymerize MTs but instead causes their plus ends to pause, a state wherein tubulin subunits neither add to nor release from the plus end (9–11). Kif18B, a third human kinesin-8, limits the length of astral MTs in the mitotic spindle (12, 13) and has been reported to do so by recruiting the MT depolymerizing kinesin-13 MCAK to MT plus ends (13). Whether Kif18B also regulates MT plus-end dynamics is unclear, but recent work showing that the motor domain of Kif18B can substitute for that of Kif18A (14) suggests that this may be the case.Among the MT-regulating kinesins, Kip3 and Kif18A are unique in that they are capable of regulating MT dynamics in a length-dependent manner. A major factor thought to underlie this property is that Kip3 and Kif18A are ultraprocessive motors (8, 10, 15), preferentially concentrating them at the ends of long MTs. Ultraprocessivity is enabled, at least in part, by MT binding of the nonmotor C-terminal tail domains of Kif18A and Kip3, an activity that tethers the motors to the MT track (10, 15, 16). In addition, Kip3 and Kif18A exhibit long dwell times at MT plus ends (8–10). Combined, these properties enrich Kip3 and Kif18A onto long, stable MTs such as those attached to kinetochores (K-MTs) during mitosis (2, 3) and are therefore well matched to their function in driving chromosome movement toward the spindle equator (2–4, 17).Whether the paradigm set by Kip3 and Kif18A will apply to all kinesin-8 motors is unclear. Kif18B localizes to astral MT plus ends, but does so in a manner that requires the plus-end tracking protein EB1 (12, 13). Heightened processivity is therefore not required to target Kif18B to MT plus ends, and it remains unclear how the motor employs plus-end–directed motility. Using a combination of high-resolution single-molecule and cellular assays, we show that Kif18B is not ultraprocessive like Kip3 and Kif18A and that it uses a hybrid form of motility involving both diffusion and plus-end–directed motility. Diffusion is promoted by a second MT-binding site located in the tail of Kif18B, a function distinct from the processivity-promoting MT-binding tail of Kif18A. We also demonstrate that Kif18B targets the extreme MT plus end, a region spatially distinct from that recognized by EB1. Kif18B thus uses a suite of unique biophysical properties to target the plus ends of astral MTs.     

The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5

Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction—including switch-1 and the neck linker—how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as “structural kinetics,” to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.There are more than 42 kinesin genes in the human genome, representing 14 distinct classes (1). All are members of the P-loop NTPase superfamily of nucleotide triphosphate hydrolases (2–4). Like other NTPases, kinesins share a conserved Walker motif nucleotide-binding fold (2, 4) that consists of a central twisted β-sheet and three nucleotide-binding loops, which are termed switch-1, switch-2, and the P-loop. Kinesins also share a common microtubule (MT) binding interface, which isomerizes between states that either bind MTs weakly or strongly, and a mechanical element, termed the neck linker (NL). The NL has been proposed to isomerize between two conformations: one that is flexible and termed undocked, and the other that is ordered and termed docked, where it interacts with a cleft in the motor domain formed by the twisted β-sheet and is oriented along the MT axis (5–7). NL isomerization (5, 8) is hypothesized to be the force-generating transition in kinesin motors (6, 7, 9–11), and its position has also been proposed to coordinate the ATPase cycles of processive kinesin dimers by regulating nucleotide binding and hydrolysis (11).Spectroscopic and structural studies have led to a model to explain how kinesins generate force (5–7, 9, 10, 12–15) (summarized in SI Appendix, Fig. S1), which proposes that the conformations of the nucleotide binding site, the MT-binding interface, and the NL are all determined by the state of the catalytic site. It predicts that when unbound to the MT, the motor contains ADP in its catalytic site and its NL is undocked. MT binding accelerates ADP dissociation, thereby allowing ATP to bind, the NL to dock, and mechanical work to be performed. ATP hydrolysis and phosphate release are then followed by dissociation from the MT to complete the cycle (5, 7–10, 14). This model also argues that: (i) NL docking of the MT-attached motor domain moves the tethered, trailing head into a forward position, where it undergoes a biased diffusional search to attach to the next MT-binding site (11, 14); (ii) switch-1, which coordinates the γ-phosphate of ATP, alternates between two conformations, referred to as “open” and “closed,” and the NL alternates between docked and undocked (5, 6, 10, 13–15); and (iii) coordination between the conformations of switch-1 and the NL regulates the timing of the ATPase cycles of the two motor domains in processive kinesin dimers (11). However, the model fails to explain several features of kinesins. For example, it predicts that ATP does not bind to kinesin when the NL is docked. This prediction is inconsistent with studies of both Eg5 and kinesin-1, which suggest ATP binds more readily when the NL is docked (11, 16, 17). The model also predicts that the NL should be docked after ATP binding. However, electron paramagnetic resonance (EPR) probes attached to the NL show a significant population of both mobile and immobile NL states in the presence of both pre- and posthydrolytic ATP analogs (5). Furthermore, the model cannot explain the load dependence of stall, detachment, and back stepping, all of which require a branched pathway (11).To resolve these uncertainties, we have measured the kinetics of the structural changes that occur in switch-1 and the NL with nucleotide binding while the motor is bound to the MT in an experimental design that we refer to as “structural kinetics.” We carried out these experiments using an novel spectroscopic approach, termed transient time-resolved fluorescence resonance energy transfer, (TR)²FRET, that allows us to monitor the kinetics and thermodynamics of both the undocked/docked transition in the NL and the open/closed transition in switch-1 that accompany the process of nucleotide binding. These experiments explain differences in the enzymologies of kinesin-1 and Eg5 and suggest an interesting role for the L5 loop in controlling the timing of conformational changes in the Eg5 switch-1 and NL.     

Quantitative analysis of vesicle recycling at the calyx of Held synapse

Vesicle recycling is pivotal for maintaining reliable synaptic signaling, but its basic properties remain poorly understood. Here, we developed an approach to quantitatively analyze the kinetics of vesicle recycling with exquisite signal and temporal resolution at the calyx of Held synapse. The combination of this electrophysiological approach with electron microscopy revealed that ∼80% of vesicles (∼270,000 out of ∼330,000) in the nerve terminal are involved in recycling. Under sustained stimulation, recycled vesicles start to be reused in tens of seconds when ∼47% of the preserved vesicles in the recycling pool (RP) are depleted. The heterogeneity of vesicle recycling as well as two kinetic components of RP depletion revealed the existence of a replenishable pool of vesicles before the priming stage and led to a realistic kinetic model that assesses the size of the subpools of the RP. Thus, our study quantified the kinetics of vesicle recycling and kinetically dissected the whole vesicle pool in the calyceal terminal into the readily releasable pool (∼0.6%), the readily priming pool (∼46%), the premature pool (∼33%), and the resting pool (∼20%).Synaptic vesicle recycling ensures synaptic transmission during sustained neuronal activity (1–3). Despite its crucial role, the cycle is poorly understood. In contrast to vesicle exocytosis and endocytosis, which can be directly assayed by presynaptic capacitance measurements and postsynaptic current recordings, vesicle recycling is usually investigated by fluorescence imaging and electron microscopy (EM) with limited signal or temporal resolution (4–7). Likely owing to technical difficulties, the basic properties of vesicle recycling, such as the size of the recycling pool (RP) (3, 6, 8–11), the kinetics of vesicle recycling (6, 8–12), and how the RP supports synaptic transmission (1, 13–15) remain to be elucidated. Classically, presynaptic vesicles can be functionally divided into three populations: the readily releasable pool (RRP), the reserve pool, and the resting pool (3, 16, 17). The RRP is defined as being composed of docked and immediately releasable vesicles (17), which are usually depleted by high-frequency stimulation, prolonged presynaptic depolarization, or the application of hypertonic solution (18–21). The reserve pool functions as a reservoir and serves to maintain vesicle refilling into the RRP (2, 3). These two pools together are commonly referred to as the RP. The resting pool serves as a depot of vesicles for backup use (16, 22). However, it has been debated for a decade whether nerve terminals use the majority (∼100%, from electrophysiology) or only a small fraction (5–40%, from fluorescence imaging and EM) of vesicles in recycling, and whether the RP size undergoes dynamic changes during varied neuronal activity (6, 7, 23–28).The use of vesicles in recycling is a critical determinant of synaptic transmission (1, 13–15). However, it has never been rigorously determined how fast recently recaptured vesicles are organized to recycle and whether vesicles in the RP are homogeneously ready for use (25). Two forms of vesicle retrieval, “kiss-and-run” and full collapse, have been reported for many years. It is still ambiguous whether the rapidly recaptured vesicles in the kiss-and-run mode can be rapidly reused (29–31).Here, we addressed the above issues by developing a new approach to quantify the basic properties of vesicle recycling with unparalleled precision. Different from previous studies in cultured cell systems, the present work combined electrophysiological measurements and EM observations at the calyx of Held synapse in acute brain slices, quantitatively analyzed synaptic vesicle recycling, and kinetically dissected the recycling vesicle pool. We propose a realistic kinetic model and provide new insights into the mechanism that ensures rate-limited but sustainable synaptic transmission.     

Self-assembly of amphiphilic Janus dendrimers into uniform onion-like dendrimersomes with predictable size and number of bilayers

A constitutional isomeric library synthesized by a modular approach has been used to discover six amphiphilic Janus dendrimer primary structures, which self-assemble into uniform onion-like vesicles with predictable dimensions and number of internal bilayers. These vesicles, denoted onion-like dendrimersomes, are assembled by simple injection of a solution of Janus dendrimer in a water-miscible solvent into water or buffer. These dendrimersomes provide mimics of double-bilayer and multibilayer biological membranes with dimensions and number of bilayers predicted by the Janus compound concentration in water. The simple injection method of preparation is accessible without any special equipment, generating uniform vesicles, and thus provides a promising tool for fundamental studies as well as technological applications in nanomedicine and other fields.Most living organisms contain single-bilayer membranes composed of lipids, glycolipids, cholesterol, transmembrane proteins, and glycoproteins (1). Gram-negative bacteria (2, 3) and the cell nucleus (4), however, exhibit a strikingly special envelope that consists of a concentric double-bilayer membrane. More complex membranes are also encountered in cells and their various organelles, such as multivesicular structures of eukaryotic cells (5) and endosomes (6), and multibilayer structures of endoplasmic reticulum (7, 8), myelin (9, 10), and multilamellar bodies (11, 12). This diversity of biological membranes inspired corresponding biological mimics. Liposomes (Fig. 1) self-assembled from phospholipids are the first mimics of single-bilayer biological membranes (13–16), but they are polydisperse, unstable, and permeable (14). Stealth liposomes coassembled from phospholipids, cholesterol, and phospholipids conjugated with poly(ethylene glycol) exhibit improved stability, permeability, and mechanical properties (17–20). Polymersomes (21–24) assembled from amphiphilic block copolymers exhibit better mechanical properties and permeability, but are not always biocompatible and are polydisperse. Dendrimersomes (25–28) self-assembled from amphiphilic Janus dendrimers and minidendrimers (26–28) have also been elaborated to mimic single-bilayer biological membranes. Amphiphilic Janus dendrimers take advantage of multivalency both in their hydrophobic and hydrophilic parts (23, 29–32). Dendrimersomes are assembled by simple injection (33) of a solution of an amphiphilic Janus dendrimer (26) in a water-soluble solvent into water or buffer and produce uniform (34), impermeable, and stable vesicles with excellent mechanical properties. In addition, their size and properties can be predicted by their primary structure (27). Amphiphilic Janus glycodendrimers self-assemble into glycodendrimersomes that mimic the glycan ligands of biological membranes (35). They have been demonstrated to be bioactive toward biomedically relevant bacterial, plant, and human lectins, and could have numerous applications in nanomedicine (20).Open in a separate windowFig. 1.Strategies for the preparation of single-bilayer vesicles and multibilayer onion-like vesicles.More complex and functional cell mimics such as multivesicular vesicles (36, 37) and multibilayer onion-like vesicles (38–40) have also been discovered. Multivesicular vesicles compartmentalize a larger vesicle (37) whereas multibilayer onion-like vesicles consist of concentric alternating bilayers (40). Currently multibilayer vesicles are obtained by very complex and time-consuming methods that do not control their size (39) and size distribution (40) in a precise way. Here we report the discovery of “single–single” (28) amphiphilic Janus dendrimer primary structures that self-assemble into uniform multibilayer onion-like dendrimersomes (Fig. 1) with predictable size and number of bilayers by simple injection of their solution into water or buffer.     

Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle

To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the one-head–bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.Kinesin-1 is a motor protein that steps processively toward microtubule plus-ends, tracking single protofilaments and hydrolyzing one ATP molecule per step (1–6). Step sizes corresponding to the tubulin dimer spacing of 8.2 nm are observed when the molecule is labeled by its C-terminal tail (7–10) and to a two-dimer spacing of 16.4 nm when a single motor domain is labeled (4, 11, 12), consistent with the motor walking in a hand-over-hand fashion. Kinesin has served as an important model system for advancing single-molecule techniques (7–10) and is clinically relevant for its role in neurodegenerative diseases (13), making dissection of its step a popular ongoing target of study.Despite decades of work, many essential components of the mechanochemical cycle remain disputed, including (i) how much time kinesin-1 spends in a one-head–bound (1HB) state when stepping at physiological ATP concentrations, (ii) whether the motor waits for ATP in a 1HB or two-heads–bound (2HB) state, and (iii) whether ATP hydrolysis occurs before or after tethered head attachment (4, 11, 14–20). These questions are important because they are fundamental to the mechanism by which kinesins harness nucleotide-dependent structural changes to generate mechanical force in a manner optimized for their specific cellular tasks. Addressing these questions requires characterizing a transient 1HB state in the stepping cycle in which the unattached head is located between successive binding sites on the microtubule. This 1HB intermediate is associated with the force-generating powerstroke of the motor and underlies the detachment pathway that limits motor processivity. Optical trapping (7, 19, 21, 22) and single-molecule tracking studies (4, 8–11) have failed to detect this 1HB state during stepping. Single-molecule fluorescence approaches have detected a 1HB intermediate at limiting ATP concentrations (11, 12, 14, 15), but apart from one study that used autocorrelation analysis to detect a 3-ms intermediate (17), the 1HB state has been undetectable at physiological ATP concentrations.Single-molecule microscopy is a powerful tool for studying the kinetics of structural changes in macromolecules (23). Tracking steps and potential substeps for kinesin-1 at saturating ATP has until now been hampered by the high stepping rates of the motor (up to 100 s⁻¹), which necessitates high frame rates, and the small step size (8.2 nm), which necessitates high spatial precision (7). Here, we apply interferometric scattering microscopy (iSCAT), a recently established single-molecule tool with high spatiotemporal resolution (24–27) to directly visualize the structural changes underlying kinesin stepping. By labeling one motor domain in a dimeric motor, we detect a 1HB intermediate state in which the tethered head resides over the bound head for half the duration of the stepping cycle at saturating ATP. We further show that at physiological stepping rates, ATP binding is required to enter this 1HB state and that ATP hydrolysis is required to exit it. This work leads to a significant revision of the sequence and kinetics of mechanochemical transitions that make up the kinesin-1 stepping cycle and provides a framework for understanding functional diversity across the kinesin superfamily.     

The polarity of myxobacterial gliding is regulated by direct interactions between the gliding motors and the Ras homolog MglA

Gliding motility in Myxococcus xanthus is powered by flagella stator homologs that move in helical trajectories using proton motive force. The Frz chemosensory pathway regulates the cell polarity axis through MglA, a Ras family GTPase; however, little is known about how MglA establishes the polarity of gliding, because the gliding motors move simultaneously in opposite directions. Here we examined the localization and dynamics of MglA and gliding motors in high spatial and time resolution. We determined that MglA localizes not only at the cell poles, but also along the cell bodies, forming a decreasing concentration gradient toward the lagging cell pole. MglA directly interacts with the motor protein AglR, and the spatial distribution of AglR reversals is positively correlated with the MglA gradient. Thus, the motors moving toward lagging cell poles are less likely to reverse, generating stronger forward propulsion. MglB, the GTPase-activating protein of MglA, regulates motor reversal by maintaining the MglA gradient. Our results suggest a mechanism whereby bacteria use Ras family proteins to modulate cellular polarity.Generating and maintaining polarity is fundamental to the proper functioning of cells. Eukaryotic cells generate polarity for migration and the accurate positioning of macromolecules and organelles (1, 2). For bacteria, polarity is important for motility, division, signal transduction, and pathogenesis (3, 4). The Gram-negative soil bacterium Myxococcus xanthus is a model organism for use in the study of cell polarity for its directed surface motilities.M. xanthus cells move on solid surfaces using two distinct mechanisms. The first mechanism, social motility (S-motility), is powered by the extension and retraction of type IV pili from the leading cell poles (5, 6). In contrast, the second mechanism, gliding motility (adventurous or A-motility), uses proton motive force to power the movement of motor complexes containing flagella stator homologs (7–11). Gliding M. xanthus cells on 1.5% agar plates typically reverse their polarity approximately every 8–12 min (12). The Frz chemosensory pathway regulates the reversal frequency and thus the direction of cell movements of both motility systems (12–16). MglA, a Ras family GTPase, has been identified as the central regulator of cell polarity and the principal responder to Frz pathway signaling (13–15). It has been reported that MglA is connected to the Frz pathway by the response regulator RomR (17–19). Importantly, MglA switches between an active GTP-bound form and an inactive GDP-bound form, which is regulated by MglB, the cognate GTPase-activating protein (GAP) of MglA, providing another layer of regulation (13, 14).The importance of cell polarity in S-motility is obvious, because the S-motility motors localize to cell poles in an MglA-dependent manner (5, 20). In contrast, cell polarity for gliding motility is enigmatic, because the gliding motor complexes, as represented by the MotA homolog AglR and motor-associated proteins, such as AgmU (GltD), localize in blurry patches that move simultaneously in opposite directions along a helical track (7, 8, 10, 11).The gliding complexes consist of the motor proteins AglR, AglQ, and AglS, along with numerous motor-associated proteins that localize in the cytoplasm, inner membrane, and periplasm (21). Genomic analysis has shown that the M. xanthus motor complexes, unlike the MotAB complexes of enteric bacteria, lack peptidoglycan-binding domains and thus are free to move within the membrane (7). Consistent with this idea, the motor protein AglR and the motor-associated protein AgmU (GltD) have been observed to decorate a helical macrostructure that rotates as cells move forward (7, 8). In addition, tracking the movements of single AglR molecules using single-particle photoactivatable localization microscopy (sptPALM) (22) revealed that the gliding motors containing AglR molecules move in helical trajectories. A subpopulation of motors slow down and accumulate into evenly distributed “traffic jam” clusters at the ventral sides of cells, where they contact surfaces. The traffic jam clusters appear to be stationary in relation to the substratum when cells move forward (7). These clusters were originally called “focal adhesion sites” because of some similarities with eukaryotic motility complexes (9, 23).Based on the results of our high-resolution experiments, we proposed a revised model of bacterial gliding (the helical rotor model) that envisions the distance between two adjacent traffic jam sites as corresponding to the period of the helical track (11). According to this model, motors at these sites push against the gliding surface, deform the cell membrane, and exert force against the surface slime (Fig. 1A) (7). This model explains evenly distributed traffic jam sites in gliding cells, without invoking that force is transmitted to the surface by breaching the peptidoglycan barrier (21, 23); however, how the bidirectional motion of gliding motors generates unidirectional cell movements remains unknown.Open in a separate windowFig. 1.Single molecules of AglR-pamCherry reverse their moving directions along cell bodies. (A) The helical rotor model predicts that the gliding motors move simultaneously toward opposite directions along helical tracks. (B) Kymograph of AglR-pamCherry fluorescence in a moving cell. AglR molecules move simultaneously toward the leading and lagging cell poles. Yellow arrows point to the reversal events of AglR. A significant population of AglR molecules also appear stationary (blue dots), which we attribute to the molecules that slow down at traffic jam sites owing to the resistance of the underlying gliding surfaces. The yellow lines mark the positions of the cell poles in the kymograph. An example of the cells is shown in Movie S2.In the present study, we found that MglA directly interacts with AglR. Using a combination of sptPALM and conventional fluorescence microscopy, we showed that MglA localizes not only at the cell poles, but also along the cell bodies, forming a gradient toward the lagging cell poles. We investigated the role of MglA in regulating gliding motility by tracking the movements of motors in various genetic backgrounds. We found that the probability of AglR reversal is positively correlated with the local MglA gradient. Our observations suggest that the MglA gradient dictates the polarity of gliding by triggering the reversal of gliding motors asymmetrically.     

Kinesin processivity is gated by phosphate release

Kinesin-1 is a dimeric motor protein, central to intracellular transport, that steps hand-over-hand toward the microtubule (MT) plus-end, hydrolyzing one ATP molecule per step. Its remarkable processivity is critical for ferrying cargo within the cell: over 100 successive steps are taken, on average, before dissociation from the MT. Despite considerable work, it is not understood which features coordinate, or “gate,” the mechanochemical cycles of the two motor heads. Here, we show that kinesin dissociation occurs subsequent to, or concomitant with, phosphate (P_i) release following ATP hydrolysis. In optical trapping experiments, we found that increasing the steady-state population of the posthydrolysis ADP·P_i state (by adding free P_i) nearly doubled the kinesin run length, whereas reducing either the ATP binding rate or hydrolysis rate had no effect. The data suggest that, during processive movement, tethered-head binding occurs subsequent to hydrolysis, rather than immediately after ATP binding, as commonly suggested. The structural change driving motility, thought to be neck linker docking, is therefore completed only upon hydrolysis, and not ATP binding. Our results offer additional insights into gating mechanisms and suggest revisions to prevailing models of the kinesin reaction cycle.Since its discovery nearly 30 years ago (1), kinesin-1—the founding member of the kinesin protein superfamily—has emerged as an important model system for studying biological motors (2, 3). During “hand-over-hand” stepping, kinesin dimers alternate between a two–heads-bound (2-HB) state, with both heads attached to the microtubule (MT), and a one–head-bound (1-HB) state, where a single head, termed the tethered head, remains free of the MT (4, 5). The catalytic cycles of the two heads are maintained out of phase by a series of gating mechanisms, thereby enabling the dimer to complete, on average, over 100 steps before dissociating from the MT (6–8). A key structural element for this coordination is the neck linker (NL), a ∼14-aa segment that connects each catalytic head to a common stalk (9). In the 1-HB state, nucleotide binding is thought to induce a structural reconfiguration of the NL, immobilizing it against the MT-bound catalytic domain (2, 3, 10–17). This transition, called “NL docking,” is believed to promote unidirectional motility by biasing the position of the tethered head toward the next MT binding site (2, 3, 10–17). The completion of an 8.2-nm step (18) entails the binding of this tethered head to the MT, ATP hydrolysis, and detachment of the trailing head, thereby returning the motor to the ATP-waiting state (2, 3, 10–17). Prevailing models of the kinesin mechanochemical cycle (2, 3, 10, 14, 15, 17), which invoke NL docking upon ATP binding, explain the highly directional nature of kinesin motility and offer a compelling outline of the sequence of events following ATP binding. Nevertheless, these abstractions do not speak directly to the branching transitions that determine whether kinesin dissociates from the MT (off-pathway) or continues its processive reaction cycle (on-pathway). The distance moved by an individual motor before dissociating—the run length—is limited by unbinding from the MT. The propensity for a dimer to unbind involves a competition among multiple, force-dependent transitions in the two heads, which are not readily characterized by traditional structural or bulk biochemical approaches. Here, we implemented high-resolution single-molecule optical trapping techniques to determine transitions in the kinesin cycle that govern processivity.     

CK2 activates kinesin via induction of a conformational change

Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain. Furthermore, we show that treatment of kinesin with CK2 prevents and reverses this repositioning. Finally, we demonstrate that CK2 treatment facilitates ADP dissociation from the motor, resulting in a nucleotide-free state that promotes microtubule binding. Thus, we propose that kinesin inactivation results from neck-linker repositioning and that CK2-mediated reactivation results from CK2’s dual ability to reverse this repositioning and to promote ADP release.Intracellular microtubule-based transport is crucial for the creation and maintenance of cellular order, and altered transport is implicated in both neurodegeneration and cancer. Frequently, in vivo cargos are moved by multiple microtubule-based molecular motors (1–6), and changing the number of active motors on the cargo can change cargo force production (4) and also potentially the mean travel distance for predominantly unidirectionally moving cargos (7). However, until recently, it has been unclear how activity of cargo-bound motors might be regulated.Transport is frequently regulated by signaling cascades [see, e.g., cAMP control of pigment granule transport (8) or APP transport (9)]. Thus, multiple signaling pathways might contribute to control of transport under different conditions, and signaling altered in disease might affect transport, which could then contribute to disease progression. Nonetheless, mechanistic understanding of such effects is limited. For these reasons, we would like to understand transport roles of specific disease-relevant kinases. One such kinase is casein kinase 2 (CK2), which is involved in development (10), is up-regulated in various cancers (11), and is decreased in neurodegeneration (12). We found that, over time, kinesin loses its ability to bind microtubules (becomes “inactive”) and that this loss of activity could be reversed by CK2 (13).Mechanistically, how kinesin became inactive—and what CK2 did to reactivate it—was unknown. Here we discover that kinesin’s inactivation results from a conformational change involving repositioning of the neck linker (NL) and that reactivation reverses this conformational change. Intriguingly, the conformational change that results in reactivation causes release of ADP, converting kinesin from a weak microtubule-interacting state (K⋅ADP) to a strong one (K), so that in some ways CK2 acts like a small G-protein nucleotide-exchange factor.     

Bioactive cell-like hybrids coassembled from (glyco)dendrimersomes with bacterial membranes

A library of amphiphilic Janus dendrimers including two that are fluorescent and one glycodendrimer presenting lactose were used to construct giant dendrimersomes and glycodendrimersomes. Coassembly with the components of bacterial membrane vesicles by a dehydration–rehydration process generated giant cell-like hybrid vesicles, whereas the injection of their ethanol solution into PBS produced monodisperse nanometer size assemblies. These hybrid vesicles contain transmembrane proteins including a small membrane protein, MgrB, tagged with a red fluorescent protein, lipopolysaccharides, and glycoproteins from the bacterium Escherichia coli. Incorporation of two colored fluorescent probes in each of the components allowed fluorescence microscopy to visualize and demonstrate coassembly and the incorporation of functional membrane channels. Importantly, the hybrid vesicles bind a human galectin, consistent with the display of sugar moieties from lipopolysaccharides or possibly glycosylated membrane proteins. The present coassembly method is likely to create cell-like hybrids from any biological membrane including human cells and thus may enable practical application in nanomedicine.Naturally occurring (1), chemically modified (2, 3), and synthetic (4, 5) lipids, amphiphilic block copolymers (6, 7), polypeptides (8), Janus dendrimers (JDs) (9), and Janus glycodendrimers (JGDs) (10, 11) self-assemble into vesicles denoted as liposomes, polymersomes, dendrimersomes (DSs), and glycodendrimersomes (GDSs), respectively. These vesicles provide models for primitive (12) and contemporary (13, 14) cell membranes and drug-delivery devices (15–17). Recently, hybrid vesicles coassembled from naturally occurring phospholipids and amphiphilic block copolymers (18–20) have been described; these vesicles eliminated some of the deficiencies of liposomes, such as limited stability under oxidative conditions and general instability over time, and the deficiencies of polymersomes, which possess wide membrane thickness [8–50 nm (20)], exhibit toxicity, and can be tedious to synthesize. These hybrid vesicles combined the desirable feature of liposomes—specifically, their biologically suitable membrane thickness of 4 nm—with that of polymersomes, which are known for their stability. In addition, transmembrane proteins (21–23) could be incorporated into the phospholipid fragments of planar membranes derived from these assemblies. However, the variability in the extent of miscibility between the hydrophobic fragments of the phospholipid and the block copolymer (20) generates a complex morphology of the hybrid membrane that requires further characterization to enable practical applications both as drug-delivery devices and cell membrane models. Here, we report the coassembly of the components of DSs and GDSs with those of the bacterial membrane vesicles (BMVs) to generate functional hybrid vesicles. DSs, GDSs, and liposomes have hydrophobic fragments with similar chemical structures and similar membrane thickness (4.5–4.9 nm) (24). Therefore, the bacterial membranes with their intact native components are expected to be transferred to the hybrid vesicles, providing a new and simple method for the generation of bioactive cell-like hybrids of interest as critical nanoscale design parameters (25).     

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.     

Epileptic baboons have lower numbers of neurons in specific areas of cortex

Epilepsy is characterized by recurrent seizure activity that can induce pathological reorganization and alter normal function in neocortical networks. In the present study, we determined the numbers of cells and neurons across the complete extent of the cortex for two epileptic baboons with naturally occurring seizures and two baboons without epilepsy. Overall, the two epileptic baboons had a 37% average reduction in the number of cortical neurons compared with the two nonepileptic baboons. The loss of neurons was variable across cortical areas, with the most pronounced loss in the primary motor cortex, especially in lateral primary motor cortex, representing the hand and face. Less-pronounced reductions of neurons were found in other parts of the frontal cortex and in somatosensory cortex, but no reduction was apparent in the primary visual cortex and little in other visual areas. The results provide clear evidence that epilepsy in the baboon is associated with considerable reduction in the numbers of cortical neurons, especially in frontal areas of the cortex related to motor functions. Whether or not the reduction of neurons is a cause or an effect of seizures needs further investigation.Epilepsy is associated with structural changes in the cerebral cortex (e.g., refs. 1–6), and partial epilepsies (i.e., seizures originating from a brain region) may lead to loss of neurons (7) and altered connectivity (8). The cerebral cortex is a heterogeneous structure comprised of multiple sensory and motor information-processing systems (e.g., refs. 9 and 10) that vary according to their processing demands, connectivity (e.g., refs. 11 and 12), and intrinsic numbers of cells and neurons (13–16). Chronic seizures have been associated with progressive changes in the region of the epileptic focus and in remote but functionally connected cortical or subcortical structures (3, 17). Because areas of the cortex are functionally and structurally different, they may also differ in susceptibility to pathological changes resulting from epilepsy.The relationship between seizure activity and neuron damage can be difficult to study in humans. Seizure-induced neuronal damage can be convincingly demonstrated in animals using electrically or chemically induced status epilepticus (one continuous seizure episode longer than 5 min) to reveal morphometric (e.g., refs. 18 and 19) or histological changes (e.g., refs. 20 and 21). Subcortical brain regions are often studied for vulnerability to seizure-induced injury (21–27); however, a recent study by Karbowski et al. (28) observed reduction of neurons in cortical layers 5 and 6 in the frontal lobes of rats with seizures. Seizure-induced neuronal damage in the cortex has also been previously demonstrated in baboons with convulsive status epilepticus (29).The goal of the present study was to determine if there is a specific pattern of cell or neuron reduction across the functionally divided areas of the neocortex in baboons with epilepsy. Selected strains of baboons have been studied as a natural primate model of generalized epilepsy (30–36) that is analogous to juvenile myoclonic epilepsy in humans. The baboons demonstrate generalized myoclonic and tonic-clonic seizures, and they have generalized interictal and ictal epileptic discharges on scalp EEG. Because of their phylogenetic proximity to humans, baboons and other Old World monkeys share many cortical areas and other features of cortical organization with humans (e.g., refs. 9 and 10). Cortical cell and neuron numbers were determined using the flow fractionator method (37, 38) in epileptic baboon tissue obtained from the Texas Biomedical Research Institute, where a number of individuals develop generalized epilepsy within a pedigreed baboon colony (31–36). Our results reveal a regionally specific neuron reduction in the cortex of baboons with naturally occurring, generalized seizures.     

Hybrid-fuel bacterial flagellar motors in Escherichia coli

The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H⁺ or Na⁺ ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H⁺- and Na⁺-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na⁺ concentration ([Na⁺]), using speed changes of single motors driving 1-μm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na⁺ and H⁺ stator components expressed at high and medium levels, respectively, Na⁺ stator units dominate at high [Na⁺] and are replaced by H⁺ units when Na⁺ is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H⁺ and Na⁺ stator components sharing a common rotor.Molecular motors are tiny machines that perform a wide range of functions in living cells. Typically each motor generates mechanical work using a specific chemical or electrochemical energy source. Linear motors such as kinesin on microtubules or myosin on actin filaments and rotary motors such as F₁-ATPase, the soluble part of ATP-synthase, run on ATP, whereas the rotary bacterial flagellar motor embedded in the bacterial cell envelope is driven by the flux of ions across the cytoplasmic membrane (1–4). Coupling ions are known to be either protons (H⁺) or sodium ions (Na⁺) (5, 6).The bacterial flagellar motor consists of a rotor ∼50 nm in diameter surrounded by multiple stator units (7–10). Each unit contains two types of membrane proteins forming ion channels: MotA and MotB in H⁺ motors in neutrophiles (e.g., Escherichia coli and Salmonella) and PomA and PomB in Na⁺ motors in alkalophiles and Vibrio species (e.g., Vibrio alginolyticus) (1, 11). Multiple units interact with the rotor to generate torque independently in a working motor (9, 10, 12, 13). The structure and function of H⁺ and Na⁺ motors are very similar, to the extent that several functional chimeric motors have been made containing different mixtures of H⁺- and Na⁺-motor components (11). One such motor that runs on Na⁺ in E. coli combines the rotor of the H⁺-driven E. coli motor with the chimeric stator unit PomA/PotB, containing PomA from V. alginolyticus and a fusion protein between MotB from E. coli and PomB from V. alginolyticus (14).In most flagellated bacteria, motors are driven by ion-specific rotor–stator combinations. However, some species (e.g., Bacillus subtilis and Shewanella oneidensis) combine a single set of rotor genes with multiple sets of stator genes encoding both H⁺ and Na⁺ stator proteins, and it has been speculated that these stator components may interact with the rotor simultaneously, allowing a single motor to use both H⁺ and Na⁺. An appealing hypothesis that the mixture of stator components is controlled dynamically depending on the environment has arisen from the observation that the localization of both stator components depends upon Na⁺ (15). However, despite some experimental effort there is as yet no direct evidence of both H⁺ and Na⁺ stator units interacting with the same rotor (16).The rotation of single flagellar motors can be monitored in real time by light microscopy of polystyrene beads (diameter ∼1 μm) attached to truncated flagellar filaments (17). Under these conditions, the E. coli motor torque and speed are proportional to the number of stator units in both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB (17–19) motors. The maximum number of units that can work simultaneously in a single motor has been shown to be at least 11 by “resurrection” experiments, in which newly produced functional units lead to restoration of motor rotation in discrete speed increments in an E. coli strain lacking functional stator proteins (19). Stator units are not fixed permanently in a motor: Each dissociates from the motor with a typical rate of ∼2 min⁻¹, exchanging between the motor and a pool of diffusing units in the cytoplasmic membrane (20). Removal of the relevant ion gradient inactivates both H⁺ and Na⁺ stator units, most likely leading to dissociation from the motor into the membrane pool (2, 21, 22).Here we demonstrate a hybrid-fuel motor containing both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB stator components, sharing a common rotor in E. coli. We control the expression level of each stator type by induced expression from plasmids, and the affinity of Na⁺-driven stator units for the motor by external [Na⁺]. Units of each type compete for spaces around the rotor, and the motor torque is simply the sum of the independent contributions, with no evidence of direct interaction between units. Thus, we demonstrate the possibility of modularity in the E. coli flagellar motor, with ion selectivity determined by the choice of stator modules interacting with a common rotor. Our artificial hybrid motor demonstrates that species with multiple types of stator gene and a single set of rotor genes could contain natural hybrid motors that work on a similar principle (15, 16, 23).     

From the Cover: PNAS Plus: Calcium can mobilize and activate myosin-VI

The ability to coordinate the timing of motor protein activation lies at the center of a wide range of cellular motile processes including endocytosis, cell division, and cancer cell migration. We show that calcium dramatically alters the conformation and activity of the myosin-VI motor implicated in pivotal steps of these processes. We resolved the change in motor conformation and in structural flexibility using single particle analysis of electron microscopic data and identified interacting domains using fluorescence spectroscopy. We discovered that calcium binding to calmodulin increases the binding affinity by a factor of 2,500 for a bipartite binding site on myosin-VI. The ability of calcium-calmodulin to seek out and bridge between binding site components directs a major rearrangement of the motor from a compact dormant state into a cargo binding primed state that is nonmotile. The lack of motility at high calcium is due to calmodulin switching to a higher affinity binding site, which leaves the original IQ-motif exposed, thereby destabilizing the lever arm. The return to low calcium can either restabilize the lever arm, required for translocating the cargo-bound motors toward the center of the cell, or refold the cargo-free motors into an inactive state ready for the next cellular calcium flux.In human cells, cytoskeletal motor proteins move along microtubules and actin filaments to generate complex cellular functions that require a precise timing of motor activation and inactivation. Myosin-VI is thought to have unique properties because it is the only myosin in the human genome shown to move toward the minus end of actin filaments (1). Apart from its roles in the formation of stereocilia in cells of the auditory system (2, 3), membrane internalization (4–6), and delivery of membrane to the leading edge in migratory cells (7), myosin-VI is an early marker of cancer development, aggressiveness, and cancer–cell invasion because of its dramatically up-regulated expression in breast, lung, prostate, ovary, and gastresophagus carcinoma cells (7–11). How this motor might promote cancer–cell migration, proliferation, and survival is unknown.In migrating cells, localized calcium transients (∼50 nM to ∼10 μM) (12, 13) have been reported to play a multifunctional role in steering directional movement (14), cytoskeleton redistribution, and relocation of focal adhesions (15). The effect of calcium transients on the mobilization and cargo binding of myosin-VI and on its mechanical activation, however, are not understood. In the current model, the catalytic head domain hydrolyzes ATP, whereas the tail domain anchors the motor to specific compartments. In vitro studies have shown that calcium affects myosin-VI binding to phospholipids (6), as well as the kinetics and motility rate of the motor (16, 17). The underlying molecular mechanisms, however, are unknown. It has also been discussed that myosin-VI might be able to adopt an inactive folded state (18, 19), perhaps similar to nonmuscle myosin II and myosin-V (20–22), with folding and unfolding regulated by some unknown mechanism. When activated, the myosin-VI head domain binds to actin, generating conformational changes that are transduced by the converter to the lever arm or neck domain and amplified to nanometer displacements. The neck consists of an extended α-helix stabilized by the binding of calmodulin (23), which pointed to the intriguing possibility that the calcium sensor calmodulin bound to the myosin-VI neck domain might constitute a molecular mechanism to control both the cellular mobilization and activation of myosin-VI in migrating cells. We therefore set out to investigate the effect of calcium on the structural conformation, mechanical properties, and activation of single myosin-VI motor molecules using electron microscopy (EM), spectroscopic, and mechanical experiments.     

Allosteric activation of M4 muscarinic receptors improve behavioral and physiological alterations in early symptomatic YAC128 mice

Mutations that lead to Huntington’s disease (HD) result in increased transmission at glutamatergic corticostriatal synapses at early presymptomatic stages that have been postulated to set the stage for pathological changes and symptoms that are observed at later ages. Based on this, pharmacological interventions that reverse excessive corticostriatal transmission may provide a novel approach for reducing early physiological changes and motor symptoms observed in HD. We report that activation of the M₄ subtype of muscarinic acetylcholine receptor reduces transmission at corticostriatal synapses and that this effect is dramatically enhanced in presymptomatic YAC128 HD and BACHD relative to wild-type mice. Furthermore, chronic administration of a novel highly selective M₄ positive allosteric modulator (PAM) beginning at presymptomatic ages improves motor and synaptic deficits in 5-mo-old YAC128 mice. These data raise the exciting possibility that selective M₄ PAMs could provide a therapeutic strategy for the treatment of HD.Huntington’s disease (HD) is a rare and fatal neurodegenerative disease caused by an expansion of a CAG triplet repeat in Htt, the gene that encodes for the protein huntingtin (1, 2). HD is characterized by a prediagnostic phase that includes subtle changes in personality, cognition, and motor function, followed by a more severe symptomatic stage initially characterized by hyperkinesia (chorea), motor incoordination, deterioration of cognitive abilities, and psychiatric symptoms. At later stages of disease progression, patients experience dystonia, rigidity, and bradykinesia, and ultimately death (3–7). The cortex and striatum are the most severely affected brain regions in HD and, interestingly, an increasing number of reports suggest that alterations in cortical and striatal physiology are present in prediagnostic individuals and in young HD mice (6–16).Striatal spiny projection neurons (SPNs) receive large glutamatergic inputs from the cortex and thalamus, as well as dopaminergic innervation from the substantia nigra. In the healthy striatum, the interplay of these neurotransmitters coordinates the activity of SPNs and striatal interneurons, regulating motor planning and execution as well as cognition and motivation (17, 18). Htt mutations lead to an early increase in striatal glutamatergic transmission, which begins during the asymptomatic phase of HD (12–14) and could contribute to synaptic changes observed in later stages of HD (19, 20). Based on this, pharmacological agents that reduce excitatory transmission in the striatum could reduce or prevent the progression of alterations in striatal synaptic function and behavior observed in symptomatic stages of HD.Muscarinic acetylcholine receptors (mAChRs), particularly M₄, can inhibit transmission at corticostriatal synapses (21–25). Therefore, it is possible that selective activation of specific mAChR subtypes could normalize excessive corticostriatal transmission in HD. Interestingly, previous studies also suggest that HD is associated with alterations of striatal cholinergic markers, including mAChRs (26–29). We now provide exciting new evidence that M₄-mediated control of corticostriatal transmission is increased in young asymptomatic HD mice and that M₄ positive allosteric modulators (PAMs) may represent a new treatment strategy for normalizing early changes in corticostriatal transmission and reducing the progression of HD.     

Nonequilibrium dynamics and ultraslow relaxation of confined DNA during viral packaging

Many viruses use molecular motors that generate large forces to package DNA to near-crystalline densities inside preformed viral proheads. Besides being a key step in viral assembly, this process is of interest as a model for understanding the physics of charged polymers under tight 3D confinement. A large number of theoretical studies have modeled DNA packaging, and the nature of the molecular dynamics and the forces resisting the tight confinement is a subject of wide debate. Here, we directly measure the packaging of single DNA molecules in bacteriophage phi29 with optical tweezers. Using a new technique in which we stall the motor and restart it after increasing waiting periods, we show that the DNA undergoes nonequilibrium conformational dynamics during packaging. We show that the relaxation time of the confined DNA is >10 min, which is longer than the time to package the viral genome and 60,000 times longer than that of the unconfined DNA in solution. Thus, the confined DNA molecule becomes kinetically constrained on the timescale of packaging, exhibiting glassy dynamics, which slows the motor, causes significant heterogeneity in packaging rates of individual viruses, and explains the frequent pausing observed in DNA translocation. These results support several recent hypotheses proposed based on polymer dynamics simulations and show that packaging cannot be fully understood by quasistatic thermodynamic models.DNA packaging is both a critical step in viral assembly and a unique model for understanding the physics of polymers under strong confinement. Before packaging, the DNA (∼6–60 µm long) forms a loose random coil of diameter ∼1–3 µm. After translocation into the viral prohead (∼50–100 nm in diameter), a ∼10,000-fold volume compaction is achieved. Packaging is driven by a powerful molecular motor that must work against the large forces resisting confinement arising from DNA bending, repulsion between DNA segments, and entropy loss (1–8).DNA packaging in bacteriophages phi29, lambda, and T4 has been directly measured via single-molecule manipulation with optical tweezers and the packaging motors have been shown to generate forces of >60 pN, among the highest known for biomotors, while translocating DNA at rates ranging from ∼100 bp (for phage phi29, which packages a 19.3-kbp genome into a 42 × 54-nm prohead shell) up to as high as ∼2,000 bp/s (for phage T4, which packages a 171-kbp genome into a 120 × 86-nm prohead) (9–15). The force resisting packaging rises steeply with prohead filling and has been proposed to play an important role in driving viral DNA ejection (16).Recently, a variety of theoretical models for viral DNA packaging have been proposed (3–5, 17–21). The simplest treat DNA as an elastic rod with repulsive self-interactions and assume that packaging is a quasistatic thermodynamic process, i.e., that the DNA is able to continuously relax to a free-energy minimum state (3–5, 19–21). The DNA arrangement is generally assumed to be an inverse spool with local hexagonal close packing between DNA segments, as suggested by electron microscopy and X-ray scattering studies (22, 23). Such models yield exact analytical predictions that reproduce many of the experimental trends, including the sharp rise in resistance during the latter stages of packaging (3–5, 20).Dynamic simulations, however, predict differing results. Depending on model and simulation protocol, some predict rapid equilibration into ordered spool or folded toroid conformations, whereas others predict nonequilibrium dynamics and disordered conformations (3, 6, 24–31). The packaged DNA conformation also depends on ionic conditions, capsid size and shape, and shape of the internal core structure found in some phages (6, 30). Notably, some electron microscopy studies have also been interpreted as suggesting ordered spooled conformations (22), whereas others have been interpreted as suggesting partly disordered conformations (29). Although some simulations predict nonequilibrium dynamics, several potential caveats are that (i) the DNA has been represented by coarse-grained polymer models with various approximations for physical interactions (6), (ii) the packaging rate used in the simulations is >10⁵ times higher than the measured packaging rate due to computational constraints (3, 26–28), and (iii) it has been pointed out by some authors that simulation timescale cannot be directly related to experimental timescale because of the use of coarse-grained models for DNA (25, 28). As noted in early modeling studies, the calculations based on quasistatic models may represent a lower bound on the required packaging forces due to dissipative dynamic losses (4). Whether nonequilibrium dynamics play a significant role in real systems has thus remained an important open question.