Channel width modulates the permeability of DNA origami based nuclear pore mimics.

Nucleoporins (nups) in the central channel of nuclear pore complexes (NPCs) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptors (NTRs) with bound cargos. The complex molecular interactions between nups and NTRs have been thought to underlie the gatekeeping function of the NPC. Recent studies have shown considerable variation in NPC diameter but how altering NPC diameter might impact the selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangement to mimic NPCs of different diameters. We use hepatitis B virus (HBV) capsids as a model for large-size cargos. We find that Nup62 proteins form a dynamic cross-channel meshwork impermeable to HBV capsids when grafted on the interior of 60-nm wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importin-ß1 substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through NPC mimics containing Nup62 and Nup153. Our study shows the transport channel width is critical to the permeability of nup barriers and underscores the role of NTRs in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.

Simulations suggest robust microtubule attachment of kinesin and dynein in antagonistic pairs.

Intracellular transport is propelled by kinesin and cytoplasmic dynein motors that carry membrane-bound vesicles and organelles bidirectionally along microtubule tracks. Much is known about these motors at the molecular scale, but many questions remain regarding how kinesin and dynein cooperate and compete during bidirectional cargo transport at the cellular level. The goal of the present study was to use a stochastic stepping model constructed by using published load-dependent properties of kinesin-1 and dynein-dynactin-BicD2 (DDB) to identify specific motor properties that determine the speed, directionality, and transport dynamics of a cargo carried by one kinesin and one dynein motor. Model performance was evaluated by comparing simulations to recently published experiments of kinesin-DDB pairs connected by complementary oligonucleotide linkers. Plotting the instantaneous velocity distributions from kinesin-DDB experiments revealed a single peak centered around zero velocity. In contrast, velocity distributions from simulations displayed a central peak around 100 nm/s, along with two side peaks corresponding to the unloaded kinesin and DDB velocities. We hypothesized that frequent motor detachment events and relatively slow motor reattachment rates resulted in periods in which only one motor is attached. To investigate this hypothesis, we varied specific model parameters and compared the resulting instantaneous velocity distributions, and we confirmed this systematic investigation using a machine-learning approach that minimized the residual sum of squares between the experimental and simulation velocity distributions. The experimental data were best recapitulated by a model in which the kinesin and dynein stall forces are matched, the motor detachment rates are independent of load, and the kinesin-1 reattachment rate is 50 s-1. These results provide new insights into motor dynamics during bidirectional transport and put forth hypotheses that can be tested by future experiments.

The capsid lattice engages a bipartite NUP153 motif to mediate nuclear entry of HIV-1 cores.

Increasing evidence has suggested that the HIV-1 capsid enters the nucleus in a largely assembled, intact form. However, not much is known about how the cone-shaped capsid interacts with the nucleoporins (NUPs) in the nuclear pore for crossing the nuclear pore complex. Here, we elucidate how NUP153 binds HIV-1 capsid by engaging the assembled capsid protein (CA) lattice. A bipartite motif containing both canonical and noncanonical interaction modules was identified at the C-terminal tail region of NUP153. The canonical cargo-targeting phenylalanine-glycine (FG) motif engaged the CA hexamer. By contrast, a previously unidentified triple-arginine (RRR) motif in NUP153 targeted HIV-1 capsid at the CA tri-hexamer interface in the capsid. HIV-1 infection studies indicated that both FG- and RRR-motifs were important for the nuclear import of HIV-1 cores. Moreover, the presence of NUP153 stabilized tubular CA assemblies in vitro. Our results provide molecular-level mechanistic evidence that NUP153 contributes to the entry of the intact capsid into the nucleus.

Modeling HIV-1 nuclear entry with nucleoporin-gated DNA-origami channels.

Delivering the virus genome into the host nucleus through the nuclear pore complex (NPC) is pivotal in human immunodeficiency virus 1 (HIV-1) infection. The mechanism of this process remains mysterious owing to the NPC complexity and the labyrinth of molecular interactions involved. Here we built a suite of NPC mimics-DNA-origami-corralled nucleoporins with programmable arrangements-to model HIV-1 nuclear entry. Using this system, we determined that multiple cytoplasm-facing Nup358 molecules provide avid binding for capsid docking to the NPC. The nucleoplasm-facing Nup153 preferentially attaches to high-curvature regions of the capsid, positioning it for tip-leading NPC insertion. Differential capsid binding strengths of Nup358 and Nup153 constitute an affinity gradient that drives capsid penetration. Nup62 in the NPC central channel forms a barrier that viruses must overcome during nuclear import. Our study thus provides a wealth of mechanistic insight and a transformative toolset for elucidating how viruses like HIV-1 enter the nucleus.

High-Resolution Tracking of Dynein-Dynactin-BicD2 Complexes.

The adapter dynactin and the activator BicD2 associate with dynein to form the highly motile dynein-dynactin-BicD2 (DDB) complex. In single-molecule assays, DDB displays processive runs, diffusive episodes, and transient pauses. The switching rates and durations of the different phases can be determined by tracking gold nanoparticle-labeled DDB complexes with interferometric scattering (iSCAT) microscopy and using an algorithm to separate out different motility phases. Here we describe methods for purifying DDB complexes from brain lysate, labeling with gold nanoparticles, imaging by iSCAT, and analyzing the resulting trajectories.

Functionalized DNA-Origami-Protein Nanopores Generate Large Transmembrane Channels with Programmable Size-Selectivity.

The DNA-origami technique has enabled the engineering of transmembrane nanopores with programmable size and functionality, showing promise in building biosensors and synthetic cells. However, it remains challenging to build large (>10 nm), functionalizable nanopores that spontaneously perforate lipid membranes. Here, we take advantage of pneumolysin (PLY), a bacterial toxin that potently forms wide ring-like channels on cell membranes, to construct hybrid DNA-protein nanopores. This PLY-DNA-origami complex, in which a DNA-origami ring corrals up to 48 copies of PLY, targets the cholesterol-rich membranes of liposomes and red blood cells, readily forming uniformly sized pores with an average inner diameter of ∼22 nm. Such hybrid nanopores facilitate the exchange of macromolecules between perforated liposomes and their environment, with the exchange rate negatively correlating with the macromolecule size (diameters of gyration: 8-22 nm). Additionally, the DNA ring can be decorated with intrinsically disordered nucleoporins to further restrict the diffusion of traversing molecules, highlighting the programmability of the hybrid nanopores. PLY-DNA pores provide an enabling biophysical tool for studying the cross-membrane translocation of ultralarge molecules and open new opportunities for analytical chemistry, synthetic biology, and nanomedicine.

Kinesin-1, -2, and -3 motors use family-specific mechanochemical strategies to effectively compete with dynein during bidirectional transport.

Bidirectional cargo transport in neurons requires competing activity of motors from the kinesin-1, -2, and -3 superfamilies against cytoplasmic dynein-1. Previous studies demonstrated that when kinesin-1 attached to dynein-dynactin-BicD2 (DDB) complex, the tethered motors move slowly with a slight plus-end bias, suggesting kinesin-1 overpowers DDB but DDB generates a substantial hindering load. Compared to kinesin-1, motors from the kinesin-2 and -3 families display a higher sensitivity to load in single-molecule assays and are thus predicted to be overpowered by dynein complexes in cargo transport. To test this prediction, we used a DNA scaffold to pair DDB with members of the kinesin-1, -2, and -3 families to recreate bidirectional transport in vitro, and tracked the motor pairs using two-channel TIRF microscopy. Unexpectedly, we find that when both kinesin and dynein are engaged and stepping on the microtubule, kinesin-1, -2, and -3 motors are able to effectively withstand hindering loads generated by DDB. Stochastic stepping simulations reveal that kinesin-2 and -3 motors compensate for their faster detachment rates under load with faster reattachment kinetics. The similar performance between the three kinesin transport families highlights how motor kinetics play critical roles in balancing forces between kinesin and dynein, and emphasizes the importance of motor regulation by cargo adaptors, regulatory proteins, and the microtubule track for tuning the speed and directionality of cargo transport in cells.

Nerve cells in the human body can reach up to one meter in length. Different regions of a nerve cell require different materials to perform their roles. The motor proteins kinesins and dynein help to transport the required 'cargo', by moving in opposite directions along tracks called microtubules. However, many cargos have both motors attached, resulting in a tug-of-war to determine which direction and how fast the cargo will travel. In many neurodegenerative diseases, including Alzheimer's, this cargo transport goes awry, so a better understanding of exactly how this process works may help to develop new therapies. There are three families of kinesin motors, for a total of about a dozen different kinesins that engage in this process. Motors in each of the three families have different mechanical properties. Specific cargos also tend to have specific kinesins attached to them. Here Gicking et al. hypothesized that when pulling against dynein in a tug-of-war, kinesins from the three families would behave differently. To test this hypothesis, Gicking et al. linked one kinesin to one dynein motor, one at a time in a test tube, and then observed how these two-motor complexes moved using fluorescence microscopy techniques. Unexpectedly, kinesins from the three different families competed similarly against dynein: there were no clear winners and losers. By incorporating previously published data describing the different motor behaviors, Gicking et al. developed a computational model that provided deeper insight into how this mechanical tug-of-war works. The modeling indicated that kinesins from the three families use different approaches for competing against dynein. Kinesin-1 motors tended to pull steadily against dynein, only detaching relatively rarely, but then take some time to attach back to the microtubule track. In contrast, kinesin-3 motors detached easily when they pull against dynein, but they attach back to the microtubule track quickly, taking only about a millisecond to start moving again. Kinesin-2 motors exhibited an intermediate behavior. Overall, these experiments suggest that the mechanical properties of the motor proteins are not the main factors determining the direction and speed of the cargo. In other words, the outcome of this molecular tug-of-war does not necessarily depend on which motor is stronger or faster. Rather, further mechanisms, including regulation of the adapter molecules that connect the motors to their cargo, may help to regulate which cargo go where in branched nerve cells. A better knowledge of how all these different factors work together will be important for understanding how cargo transport in nerve cells is disrupted in neurodegenerative diseases.

Copper-catalyzed asymmetric 1,6-conjugate addition of in situ generated para-quinone methides with ß-ketoesters.

A Cu-catalyzed asymmetric 1,6-conjugate addition of in situ generated para-quinone methides (p-QMs) with ß-ketoester has been developed to construct a ketoester skeleton bearing an adjacent tertiary-quaternary carbon stereocenter in good yields and high enantioselectivities. This is the first example of metal-catalyzed asymmetric transformations of the in situ generated p-QMs, avoiding using pre-synthesized p-QMs requiring bulky 2,6-substitutions and highlighting a new dual catalytic activation with the chiral bis(oxazoline)-metal complex acting as a normal Lewis acid to activate the ß-ketoesters and a source of Brønsted acid responsible for generating the p-QMs in situ.

A change point analysis protocol for comparing intracellular transport by different molecular motor combinations.

Intracellular transport by microtubule-based molecular motors is marked by qualitatively different behaviors. It is a long-standing and still-open challenge to accurately quantify the various individual-cargo behaviors and how they are affected by the presence or absence of particular motor families. In this work we introduce a protocol for analyzing change points in cargo trajectories that can be faithfully projected along the length of a (mostly) straight microtubule. Our protocol consists of automated identification of velocity change points, estimation of velocities during the behavior segments, and extrapolation to motor-specific velocity distributions. Using simulated data we show that our method compares favorably with existing methods. We then apply the technique to data sets in which quantum dots are transported by Kinesin-1, by Dynein-Dynactin-BicD2 (DDB), and by Kinesin-1/DDB pairs. In the end, we identify pausing behavior that is consistent with some tug-of-war model predictions, but also demonstrate that the simultaneous presence of antagonistic motors can lead to long processive runs that could contribute favorably to population-wide transport.

The Tail of Kinesin-14a in Giardia Is a Dual Regulator of Motility.

Kinesin-14s are microtubule-based motor proteins that play important roles in mitotic spindle assembly [1]. Ncd-type kinesin-14s are a subset of kinesin-14 motors that exist as homodimers with an N-terminal microtubule-binding tail, a coiled-coil central stalk (central stalk), a neck, and two identical C-terminal motor domains. To date, no Ncd-type kinesin-14 has been found to naturally exhibit long-distance minus-end-directed processive motility on single microtubules as individual homodimers. Here, we show that GiKIN14a from Giardia intestinalis [2] is an unconventional Ncd-type kinesin-14 that uses its N-terminal microtubule-binding tail to achieve minus-end-directed processivity on single microtubules over micrometer distances as a homodimer. We further find that although truncation of the N-terminal tail greatly reduces GiKIN14a processivity, the resulting tailless construct GiKIN14a-Δtail is still a minimally processive motor and moves its center of mass via discrete 8-nm steps on the microtubule. In addition, full-length GiKIN14a has significantly higher stepping and ATP hydrolysis rates than does GiKIN14a-Δtail. Inserting a flexible polypeptide linker into the central stalk of full-length GiKIN14a nearly reduces its ATP hydrolysis rate to that of GiKIN14a-Δtail. Collectively, our results reveal that the N-terminal tail of GiKIN14a is a de facto dual regulator of motility and reinforce the notion of the central stalk as a key mechanical determinant of kinesin-14 motility [3].

Dynactin p150 promotes processive motility of DDB complexes by minimizing diffusional behavior of dynein.

Cytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used interferometric scattering (iSCAT) microscopy to track dynein-dynactin-BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive, and stuck motility states. We find that DDB spends 65% of its time undergoing processive stepping, 4% undergoing 1D diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB-kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity of the complexes, in quantitative agreement with the shift of isolated DDB into the diffusive state. The data suggest a DDB activation model in which dynactin p150 enhances dynein processivity not solely by acting as a diffusive tether that maintains microtubule association, but rather by acting as an allosteric activator that promotes a conformation of dynein optimal for processive stepping. In bidirectional cargo transport driven by the opposing activities of kinesin and dynein-dynactin-BicD2, the dynactin p150 subunit promotes retrograde transport and could serve as a target for regulators of transport.

Motor Reattachment Kinetics Play a Dominant Role in Multimotor-Driven Cargo Transport.

Kinesin-based cargo transport in cells frequently involves the coordinated activity of multiple motors, including kinesins from different families that move at different speeds. However, compared to the progress at the single-molecule level, mechanisms by which multiple kinesins coordinate their activity during cargo transport are poorly understood. To understand these multimotor coordination mechanisms, defined pairs of kinesin-1 and kinesin-2 motors were assembled on DNA scaffolds and their motility examined in vitro. Although less processive than kinesin-1 at the single-molecule level, addition of kinesin-2 motors more effectively amplified cargo run lengths. By applying the law of total expectation to cargo binding durations in ADP, the kinesin-2 microtubule reattachment rate was shown to be fourfold faster than that of kinesin-1. This difference in microtubule binding rates was also observed in solution by stopped-flow. High-resolution tracking of a gold-nanoparticle-labeled motor with 1 ms and 2 nm precision revealed that kinesin-2 motors detach and rebind to the microtubule much more frequently than does kinesin-1. Finally, compared to cargo transported by two kinesin-1, cargo transported by two kinesin-2 motors more effectively navigated roadblocks on the microtubule track. These results highlight the importance of motor reattachment kinetics during multimotor transport and suggest a coordinated transport model in which kinesin-1 motors step effectively against loads whereas kinesin-2 motors rapidly unbind and rebind to the microtubule. This dynamic tethering by kinesin-2 maintains the cargo near the microtubule and enables effective navigation along crowded microtubules.

Desiccation and cold storage of Galleria mellonella cadavers and effects on in vivo production of Steinernema carpocapsae.

BACKGROUND: Direct application of insect cadavers infected with entomopathogenic nematodes (EPN) can successfully control target pest insects. Little is known about the effects of environmental factors (desiccation and temperature) on the production process for infective juveniles (IJ) in insects. RESULTS: We examined the effects of desiccation time and cold storage (6.7 °C) on IJ production of the nematode Steinernema carpocapsae in Galleria mellonella cadavers at 30.8 and 57% humidity. Under desiccation, the IJ yield in cadavers increased gradually and reached a maximum on day 5. IJ yield gradually declined from day 6 onwards and was almost zero by day 15. In general, cold storage at 6.7 °C caused negative effects on IJ production in desiccated cadavers. Approximately 56 h post infection was the time at which nematodes were most sensitive to low temperatures during development in cadavers. Five-day desiccated cadavers generated higher mortality and more rapid death of Galleria mellonella larvae than using newly (day 0) desiccated cadavers. CONCLUSION: This study describe methods of optimizing rearing techniques such as desiccation and cold storage to promote the mass production and application of EPN- infected host cadavers for the field control of insect pests.