Cargo Size Research Articles

Synthetic Biology Approaches to Dissecting Multiple Motor Functions in Living Cells

Dissecting how the coordination of cytoskeletal motor proteins, (kinesin, dynein and myosin) has become increasingly important to characterizing various transport processes in eukaryotes. Most subcellular commodities (ribonucleoprotein, particles, organelles, vesicles etc.) are driven by “teams” of motor proteins that are composed of multiple copies of the same motors or even mixtures of different motors that move in opposite directions, with different velocities, or along different types of filament tracks. Consequently, numerous intracellular transport processes and regulatory mechanisms depend fundamentally on how these collections of similar and dissimilar motors either cooperate or compete with one another. Understanding these behaviors are important to elucidating various disease pathologies stemming from motor and other transport-related protein mutations since their impact on cargo transport will ultimately depend on the extent to which they perturb the composite dynamics of motor systems that also contain wild type motors. Our work has focused on developing genetically engineered cells that can be used to characterize endogenous cargo transport and trafficking response to variation in motor number, type and ratio and cargo size. While integrating multiple genetic control elements, these cells also exploit drug- and light-dependent protein dimerization switches to allow competitions between different types of wild-type and mutant motors to be adaptively regulated. We are also developing micro-fabrication techniques to pattern these cells into defined shapes as a way to control the organization of their cytoskeleton and facilitate direct comparisons of transport responses within the cell population. Given these unique handles, these cells provide a powerful platform to examine fundamental mechanisms of intracellular transport regulation and assess the impact of disease-related motor mutations.

Study of damage safety assessment for a ship carrying radioactive waste

Ship damage caused by maritime casualties leads to marine pollution and loss of life and property. To prevent serious damage from maritime casualties, several types of safety regulations are applied in ship design. Damage stability regulation is one of the most important safety issues. Designs of ships for long international voyages must comply with these regulations. Current regulations, however, do not consider the characteristics of the operating route of each ship and reflect only ship size and type of cargo. In this paper, a damage safety assessment was undertaken for a ship carrying radioactive waste in actual wave conditions. Damage cases for safety assessment were constructed on the basis of safety regulations and related research results. Hull form, internal arrangement, loading condition and damage condition were modeled for damage safety simulation. The safety simulation was performed and analyzed for 10 damage cases with various wave heights, frequency and angle of attack on an operating route. Based on evaluation results, a design alternative was generated, and it was also simulated. These results confirmed that damage safety analysis is highly important in the design stage in consideration of the operating route characteristics by simulation. Thus a ship designer can improve safety from damage in this manner.

Cargo‐Towing Fuel‐Free Magnetic Nanoswimmers for Targeted Drug Delivery

Fuel-free nanomotors are essential for future in-vivo biomedical transport and drug-delivery applications. Herein, the first example of directed delivery of drug-loaded magnetic polymeric particles using magnetically driven flexible nanoswimmers is described. It is demonstrated that flexible magnetic nickel-silver nanoswimmers (5-6 μm in length and 200 nm in diameter) are able to transport micrometer particles at high speeds of more than 10 μm s(-1) (more than 0.2 body lengths per revolution in dimensionless speed). The fundamental mechanism of the cargo-towing ability of these magnetic (fuel-free) nanowire motors is modelled, and the hydrodynamic features of these cargo-loaded motors discussed. The effect of the cargo size on swimming performance is evaluated experimentally and compared to a theoretical model, emphasizing the interplay between hydrodynamic drag forces and boundary actuation. The latter leads to an unusual increase of the propulsion speed at an intermediate particle size. Potential applications of these cargo-towing nanoswimmers are demonstrated by using the directed delivery of drug-loaded microparticles to HeLa cancer cells in biological media. Transport of the drug carriers through a microchannel from the pick-up zone to the release microwell is further illustrated. It is expected that magnetically driven nanoswimmers will provide a new approach for the rapid delivery of target-specific drug carriers to predetermined destinations.

Subdiffusion as a model of transport through the nuclear pore complex

Cargo transport through the nuclear pore complex continues to be a subject of considerable interest to experimentalists and theorists alike. Several recent studies have revealed details of the process that have still to be fully understood, among them the apparent nonlinearity between cargo size and the pore crossing time, the skewed, asymmetric nature of the distribution of such crossing times, and the non-exponentiality in the decay profile of the dynamic autocorrelation function of cargo positions. In this paper, we show that a model of pore transport based on subdiffusive particle motion is in qualitative agreement with many of these observations. The model corresponds to a process of stochastic binding and release of the particle as it moves through the channel. It suggests that the phenylalanine-glycine repeat units that form an entangled polymer mesh across the channel may be involved in translocation, since these units have the potential to intermittently bind to hydrophobic receptor sites on the transporter protein.

Biophysical Coarse-Grained Modeling Provides Insights into Transport through the Nuclear Pore Complex

The nuclear pore complex (NPC) is the gatekeeper of the nucleus, capable of actively discriminating between the active and inert cargo while accommodating a high rate of translocations. The biophysical mechanisms underlying transport, however, remain unclear due to the lack of information about biophysical factors playing role in transport. Based on published experimental data, we have established a coarse-grained model of an intact NPC structure to examine nucleocytoplasmic transport with refined spatial and temporal resolutions. Using our model, we estimate the transport time versus cargo sizes. Our findings suggest that the mean transport time of cargos smaller than 15 nm is independent of size, while beyond this size, there is a sharp increase in the mean transport time. The model confirms that kap-FG hydrophobicity is sufficient for active cargo transport. Moreover, our model predicts that during translocation, small and large cargo-complexes are hydrophobically attached to FG-repeat domains for 86 and 96% of their transport time, respectively. Inside the central channel FG-repeats form a thick layer on the wall leaving an open tube. The cargo-complex is almost always attached to this layer and diffuses back and forth, regardless of the cargo size. Finally, we propose a plausible model for transport in which the NPC can be viewed as a lubricated gate. This model incorporates basic assumptions underlying virtual-gate and reduction-of-dimensionality models with the addition of the FG-layer inside the central channel acting as a lubricant.

Theoretical Modeling of the Weaving of Clathrin into Nanoscale Baskets

Many biological systems are capable of spontaneously assembling a diverse set of molecular architectures from a single subunit, without the need to pre-pattern the assembly. Cellular uptake of external substances is accomplished by a highly adaptive endocytosis process that accommodates a wide range of cargo shapes and sizes. Clathrin-mediated endocytosis involves the formation of a pit that is surrounded by a honeycomb coating whose pinwheel-shaped subunit is a clathrin-protein complex. We develop a theoretical model for the thermodynamics and kinetics of clathrin assembly, addressing the behavior in 2 and 3 dimensions, relevant to membrane and bulk assembly, respectively. The clathrin triskelions are modeled as effective flexible pinwheels that form leg-leg associations and resist elastic deformation. Thus, the pinwheels are capable of forming a range of ring structures, including 5-, 6-, and 7-member rings that are observed experimentally. Our theoretical model employs Monte Carlo simulations to address thermodynamic behavior and Brownian dynamics simulations to track the motion of clathrin pinwheels at sufficiently long time scales to achieve complete assembly. With this theoretical model, we predict the phase diagram for clathrin assembly incorporating binding interactions, elastic deformation, and defect-pair coupling, utilizing Kosterlitz-Thouless theory of defect-induced melting in 2 dimensions. Using analytical theory and computational simulations, we explore the role of binding strength and clathrin elasticity in the ability for clathrin lattices to dynamically reorganize due to local changes in membrane elasticity and tension. We then proceed to discuss the dynamics of lattice reorganization during the process of a clathrin-coated membrane wrapping around a nanoscale cargo.

Structure of Apolipoprotein A-I N Terminus on Nascent High Density Lipoproteins

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.

Nuclear Size Is Regulated by Importin α and Ntf2 in Xenopus

The size of the nucleus varies among different cell types, species, and disease states, but mechanisms of nuclear size regulation are poorly understood. We investigated nuclear scaling in the pseudotetraploid frog Xenopus laevis and its smaller diploid relative Xenopus tropicalis, which contains smaller cells and nuclei. Nuclear scaling was recapitulated in vitro using egg extracts, demonstrating that titratable cytoplasmic factors determine nuclear size to a greater extent than DNA content. Nuclear import rates correlated with nuclear size, and varying the concentrations of two transport factors, importin α and Ntf2, was sufficient to account for nuclear scaling between the two species. Both factors modulated lamin B3 import, with importin α increasing overall import rates and Ntf2 reducing import based on cargo size. Importin α also contributes to nuclear size changes during early X. laevis development. Thus, nuclear transport mechanisms are physiological regulators of both interspecies and developmental nuclear scaling.

Column generation approaches to ship scheduling with flexible cargo sizes

We present a Dantzig–Wolfe procedure for the ship scheduling problem with flexible cargo sizes. This problem is similar to the well-known pickup and delivery problem with time windows, but the cargo sizes are defined by intervals instead of by fixed values. The flexible cargo sizes have consequences for the times used in the ports because both the loading and unloading times depend on the cargo sizes. We found it computationally hard to find exact solutions to the subproblems, so our method does not guarantee to find the optimum over all solutions. To be able to say something about how good our solution is, we generate a bound on the difference between the true optimal objective and the objective in our solution. We have compared our method with an a priori column generation approach, and our computational experiments on real world cases show that our Dantzig–Wolfe approach is faster than the a priori generation of columns, and we are able to deal with larger or more loosely constrained instances. By using the techniques introduced in this paper, a more extensive set of real world cases can be solved either to optimality or within a small deviation from optimality.

More Freight, More Engines: Force Scales with Cargo Size in a Living Cell

The protein FMG-1 is transported in a saltatory manner within the flagellar membrane of Chlamydomonas by intracellular kinesin-2 and dynein-2 motors. Three models have been proposed to explain how these movements are coordinated: transport complex, biased accumulation, and molecular clutch (Laib et al., PNAS, 2009). To help discriminate between these models, we attached microspheres of varying diameter (0.5-1.4 μm) but identical surface chemistry to Chlamydomonas flagella and used a laser trap to determine whether the forces imposed on the microspheres by intracellular motors were dependent upon bead size.

Biophysical Studies of Peptides that Translocate through Cell Membranes: Induced Structures and Membrane Interactions

Biophysical techniques such as high resolution NMR, CD, linear dichroism and fluorescence spectroscopy have been used to investigate the interactions of cell penetrating peptides (CPPs) with various membrane mimetic solvent systems. A pH gradient appears to be required to drive the peptide across a unilamellar phospholipid vesicle bilayer. Membrane leakage induction by CPPs (possibly associated with transient pore formation) in unilamellar vesicles has been studied in parallel with peptide translocation. The membrane perturbation caused by the TP10 peptide depends on the type and size of cargo attached to the peptide, and the potent leakage caused by the peptide alone is lost when the peptide is attached to a large cargo. Effects of the hydrophobic negatively charged counter-ion pyrene butyrate on membrane leakage has been studied for selected CPPs and compared to its CPP-enhancing efficiency using biological assays. The different proposed mechanisms for CPP activities will be discussed based on these observations.We have also studied native peptide sequences which have CPP activities that may be related to a biological function. These are peptides derived from the N-terminal sequence of prion proteins (including the signal sequence) from mouse or cow. The prion protein derived peptides have shown an unexpected activity in counteracting scrapie infections in a neuronal cell system. We hypothesize that the CPP activity of the peptides may guide them into a specific cellular compartment where they may interfere with the prion protein aggregation and structure conversion into the scrapie form.

Probing Nuclear Localization Signal-Importin α Binding Equilibria in Living Cells

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein-import receptors. In this study, we present fluorescence-based methods to quantitatively address the physicochemical details of NLS recognition by the receptor protein importin alpha (Impalpha) in living cells. First, by combining fluorescence recovery after photobleaching measurements and protein-concentration calibration, we quantitatively define nuclear import saturability and afford an affinity value for NLS-Impalpha binding. Second, by fluorescence lifetime imaging microscopy, we directly monitor the occurrence of NLS-Impalpha interaction and measure its effective dissociation constant (K(D)) in the actual cellular environment. Our kinetic and thermodynamic analyses independently indicate that the subsaturation of Impalpha with the expressed NLS cargo regulates nuclear import rates in living cells, in contrast to what can be predicted on the basis of available in vitro data. Finally, our experiments also provide evidence for the regulation of nuclear import mediated by the intrasteric importin beta-binding domain of Impalpha and yield the first estimate of its autoinhibition energy in living cells.

Anti-coincidence rejection of SiPM dark pulses for improved detection of low energy radiation

Neutron and gamma detectors of the CSIRO air cargo scanner are based on scintillators readout by photodiodes (PD). The scanner imaging resolution is limited by the scintillator size. It, in turns, is limited by the number of interactions that can be detected by the PD. Silicon photomultipliers (SiPM) could be used in place of PDs to improve the neutron and gamma detector efficiency. It would ultimately improve the scanner performance in terms of speed, cargo size and image quality. Efficient use of SiPM requires gain stabilisation and dark counts suppression. This work presents a technique to strip the dark counts and the electronic noise off the genuine source-induced spectrum. The method requires the use of a pair of SiPM coupled to the same scintillator. By rejecting the events not detected by both the SiPMs, the background counts were reduced by more than 99%.

Peroxisome Size Provides Insights into the Function of Autophagy-related Proteins

Autophagy is a major pathway of intracellular degradation mediated by formation of autophagosomes. Recently, autophagy was implicated in the degradation of intracellular bacteria, whose size often exceeds the capacity of normal autophagosomes. However, the adaptations of the autophagic machinery for sequestration of large cargos were unknown. Here we developed a yeast model system to study the effect of cargo size on the requirement of autophagy-related (Atg) proteins. We controlled the size of peroxisomes before their turnover by pexophagy, the selective autophagy of peroxisomes, and found that peroxisome size determines the requirement of Atg11 and Atg26. Small peroxisomes can be degraded without these proteins. However, Atg26 becomes essential for degradation of medium peroxisomes. Additionally, the pexophagy-specific phagophore assembly site, organized by the dual interaction of Atg30 with functionally active Atg11 and Atg17, becomes essential for degradation of large peroxisomes. In contrast, Atg28 is partially required for all autophagy-related pathways independent of cargo size, suggesting it is a component of the core autophagic machinery. As a rule, the larger the cargo, the more cargo-specific Atg proteins become essential for its sequestration.

Matrix-dependent local retention of secretory vesicle cargo in cortical neurons.

Neurons secrete many diffusible signals from synaptic and other secretory vesicles. We characterized secretion of guidance cues, neuropeptides, neurotrophins, and proteases from single secretory vesicles using pHluorin-tagged cargo in cortical neurons. Stimulation triggered transient and persistent fusion events. Transient events represented full release followed by cargo diffusion or incomplete release followed by vesicle retrieval, as previously observed in neuroendocrine cells. Unexpectedly, we also observed that certain cargo, such as Semaphorin 3A (Sema3A), was delivered at the cell surface as stable deposits. Stable deposits and transient events were observed for single cargo and both were SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) and calcium dependent. The ratio between stable and transient events did not depend on cargo size, subcellular localization (synaptic vs extrasynaptic secretion), or the presence of the extracellular matrix. Instead, the ratio is cargo specific and depends on an interaction with the vesicle matrix through a basic domain in the cargo protein. Inhibition of this interaction through deletion of the basic domain in Sema3A abolished stable deposits and rendered all events transient. Strikingly, cargo favoring transient release was stably deposited after corelease with cargo favoring stable deposit. These data argue against cargo diffusion after exocytosis as a general principle. Instead, the vesicle matrix retains secreted signals, probably for focal signaling at the cell surface.

A quantitative examination of the role of cargo-exerted forces in axonal transport

Axonal transport, via molecular motors kinesin and dynein, is a critical process in supplying the necessary constituents to maintain normal neuronal function. In this study, we predict the role of cooperativity by motors of the same polarity across the entire spectrum of physiological axonal transport. That is, we examined how the number of motors, either kinesin or dynein, working together to move a cargo, results in the experimentally determined velocity profiles seen in fast and slow anterograde and retrograde transport. We quantified the physiological forces exerted on a motor by a cargo as a function of cargo size, transport velocity, and transport type. Our results show that the force exerted by our base case neurofilament ( D NF =10 nm, L NF =1.6 μm) is ∼1.25 pN at 600 nm/s; additionally, the force exerted by our base case organelle ( D org =1 μm) at 1000 nm/s is ∼5.7 pN. Our results indicate that while a single motor can independently carry an average cargo, cooperativity is required to produce the experimental velocity profiles for fast transport. However, no cooperativity is required to produce the slow transport velocity profiles; thus, a single dynein or kinesin can carry the average neurofilament retrogradely or anterogradely, respectively. The potential role cooperativity may play in the hypothesized mechanisms of motoneuron transport diseases such as amyotrophic lateral sclerosis (ALS) is discussed.

Aircraft cargo compartment fire detection and smoke transport modeling

The US Federal Aviation Administration, along with other regulatory agencies, requires that cargo compartments on passenger carrying aircraft be equipped with fire detection and suppression systems. Current regulations require that the detection system alarms within 1 min of the start of a fire and flight tests are required to demonstrate compliance with these regulations. Due to the high costs of flight tests, extensive ground certification tests are typically conducted to ensure that the detection system will meet the time to alarm requirements during the flight tests. For the purpose of improving the detection system design and certification process, a transient computational fluid dynamics computer code for the prediction of smoke, heat, and gas species transport in cargo compartments has been developed. This simulation tool couples heat, mass, and momentum transfer in a body-fitted coordinate system in order to handle a variety of cargo bay shapes and sizes. Ideally, such a physics-based simulation tool can be used during the certification process to identify worst case locations for fires, optimum placement of detector sensors within the cargo compartment, and sensor alarm levels and algorithms needed to achieve detection within the required time. Validation of the model was completed, and comparison of the predicted results with the results obtained from full-scale fire tests in a variety of actual aircraft cargo compartments provides insight into the model capabilities.

CHEMICAL ENGINEERING Cooper Union wins Chem-E-Car competition

Baking soda and vinegar helped a team of chemical engineering students from Cooper Union for the Advancement of Science & Art, in New York City, win the 2007 Chem-E-Car competition on Nov. 4. The national finals for the ninth annual alternative- fuel-vehicle competition were held in Salt Lake City at the meeting of the American Institute of Chemical Engineers (AIChE). At this year’s event, the shoebox-sized vehicles had to transport 350 mL of water exactly 67 feet. But the teams weren’t told the cargo size or the distance until shortly before the start. The challenge was to calculate how much fuel would be needed to power the cars so that the chemical reaction would end and the vehicles would stop on the finish line. Remote controls or other devices were not permitted. With 28 schools competing, Cooper Union won the $2,000 top prize. Team captain Mukarram Razvi says the five-person team had worked on their ...

Ship routing and scheduling with flexible cargo sizes

Here, we describe a real planning problem in the tramp shipping industry. A tramp shipping company may have a certain amount of contract cargoes that it is committed to carry, and tries to maximize the profit from optional cargoes. For real long-term contracts, the sizes of the cargoes are flexible. However, in previous research within tramp ship routing, the cargo quantities are regarded as fixed. We present an MP-model of the problem and a set partitioning approach to solve the multi-ship pickup and delivery problem with time windows and flexible cargo sizes. The columns are generated a priori and the most profitable ship schedule for each cargo set–ship combination is included in the set partitioning problem. We have tested the method on several real-life cases, and the results show the potential economical effects for the tramp shipping companies by utilizing flexible cargo sizes when generating the schedules.

In Vivo Study of HIV-1 Tat Arginine-rich Motif Unveils Its Transport Properties

Tat-derived peptides have attracted much interest as molecular carriers for intracellular delivery as they incorporate specific attributes required for efficient cargo delivery to sub-cellular domains. Little is known, however, about intracellular trafficking and interactions of Tat peptide-tagged cargoes, although some in vitro studies have suggested the relevance of active processes in Tat peptide-driven nuclear translocation. These issues are addressed by comparing Tat peptide-induced transport properties with well-established passive diffusion and active import benchmarks in living cells. Specifically, we examine several constructs of increasing molecular weight (MW) both below and above the threshold for passive diffusion through the nuclear pore. The resulting sub-cellular localization is analyzed by confocal imaging, and construct intracellular dynamics is investigated by fluorescence recovery after photobleaching (FRAP) real-time imaging. Our experiments yield the characteristic transport parameters of Tat peptide intra-cytoplasm dynamics and nucleus/cytoplasm shuttling. These results allow us to elucidate the mechanism of Tat peptide-driven nuclear permeation, demonstrating that it crosses the nuclear envelope (NE) by passive diffusion. Finally, we discuss the limitations of this route in terms of acceptable cargo size.