Molecular motor proteins use the energy released from ATP hydrolysis to generate force and haul cargoes along cytoskeletal filaments. Thus, measuring the force motors generate amounts to directly probing their function. We report on optical trapping methodology capable of making precise in vivo stall-force measurements of individual cargoes hauled by molecular motors in their native environment. Despite routine measurement of motor forces in vitro, performing and calibrating such measurements in vivo has been challenging. We describe the methodology recently developed to overcome these difficulties, and used to measure stall forces of both kinesin-1 and cytoplasmic dynein-driven lipid droplets in Drosophila embryos. Critically, by measuring the cargo dynamics in the optical trap, we find that there is memory: it is more likely for a cargo to resume motion in the same direction-rather than reverse direction-after the motors transporting it detach from the microtubule under the force of the optical trap. This suggests that only motors of one polarity are active on the cargo at any instant in time and is not consistent with the tug-of-war models of bidirectional transport where both polarity motors can bind the microtubules at all times. We further use the optical trap to measure in vivo the detachment rates from microtubules of kinesin-1 and dynein-driven lipid droplets. Unlike what is commonly assumed, we find that dynein's but not kinesin's detachment time in vivo increases with opposing load. This suggests that dynein's interaction with microtubules behaves like a catch bond.

Summary Fracturing-fluid loss into the formation can potentially damage hydrocarbon production in shale or other tight reservoirs. Well shut-ins are commonly used in the field to dissipate the lost water into the matrix near fracture faces. Borrowing from ideas in chemical enhanced oil recovery (CEOR), surfactants have potential to reduce the effect of fracturing-fluid loss on hydrocarbon permeability in the matrix. Unconventional tight reservoirs can differ significantly from one another, which could make the use of these techniques effective in some cases but not in others. We present an experimental investigation dependent on a coreflood sequence that simulates fluid invasion, flowback, and hydrocarbon production from hydraulically fractured reservoirs. We compare the benefits of shut-ins and reduction in interfacial tension (IFT) by surfactants for hydrocarbon permeability for different initial reservoir conditions (IRCs). From this work, we identify the mechanism responsible for the permeability reduction in the matrix, and we suggest criteria that can be used to optimize fracturing-fluid additives and/or manage flowback operations to enhance hydrocarbon production from unconventional tight reservoirs.

Drosophila melanogaster is widely used as a model system for development and disease. Due to the homology between Drosophila and human genes, as well as the tractable genetics of the fly, its use as a model for neurologic disorders, in particular, has been rising. Locomotive impairment is a commonly used diagnostic for screening and characterization of these models, yet a fast, sensitive and model-free method to compare behavior is lacking. Here, we present a high throughput method to quantify the crawling behavior of larvae. We use the mean squared displacement as well as the direction autocorrelation of the crawling larvae as descriptors of their motion. By tracking larvae from wild-type strains and models of the Fragile X mental retardation as well as Alzheimer disease, we show these mutants exhibit impaired crawling. We further show that the magnitude of impairment correlates with the severity of the mutation, demonstrating the sensitivity and the dynamic range of the method. Finally, we study larvae with altered expression of the shaggy gene, a homolog of Glycogen Synthase Kinase-3 (GSK-3), which has been implicated in Alzheimer disease. Surprisingly, we find that both increased and decreased expression of dGSK-3 lead to similar larval crawling impairment. These findings have implications for the use of GSK-3 inhibitors recently proposed for Alzheimer treatment.

Summary Hydraulic fracturing is used to obtain economical rates from tight and unconventional formations by increasing the surface area of the reservoir within the flowing distance to a high-conductivity pathway. However, a significant fraction of the fracturing fluid is never recovered, and thus may reduce the hydrocarbon permeability near the fracture. Here, we experimentally mimic the water-invasion process during fracturing, and measure the effective permeability changes in a low-permeability core. Measurements of water flowback and effective permeability as a function of interfacial tension (IFT), flow rate, and shut-in time suggest that water is being held at the fracture face because of the capillary discontinuity (i.e., when the water leaves the matrix and enters a space with minimal capillary pressure). This effect arises from the capillary interaction between the matrix and the fracture, and is akin to the capillary end effect in coreflood experiments. The results show that this effect, although only a laboratory experimental artifact for conventional reservoirs, can be a significant source of effective hydrocarbon-permeability reduction by fracturing-fluid invasion into the formation in unconventional and tight reservoirs.

To complete meiosis II in animal cells, the male DNA material needs to meet the female DNA material contained in the female pronucleus at the egg center, but it is not known how the male pronucleus, deposited by the sperm at the periphery of the cell, finds the cell center in large eggs. Pronucleus centering is an active process that appears to involve microtubules and molecular motors. For small and medium-sized cells, the force required to move the centrosome can arise from either microtubule pushing on the cortex, or cortically-attached dynein pulling on microtubules. However, in large cells, such as the fertilized Xenopus laevis embryo, where microtubules are too long to support pushing forces or they do not reach all boundaries before centrosome centering begins, a different force generating mechanism must exist. Here, we present a centrosome positioning model in which the cytosolic drag experienced by cargoes hauled by cytoplasmic dynein on the sperm aster microtubules can move the centrosome towards the cell’s center. We find that small, fast cargoes (diameter ∼100 nm, cargo velocity ∼2 µm/s) are sufficient to move the centrosome in the geometry of the Xenopus laevis embryo within the experimentally observed length and time scales.

Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport.

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Significant amounts of fracturing fluid are lost during hydraulic fracturing operations and it is believed that this fluid can hinder hydrocarbon production. For this reason, higher fracturing fluid recovery (flowback) might be desirable. One way to enhance the flowback is to reduce the capillary force within the formation. While the applicability of surfactants in conventional reservoirs that operate in tertiary production phase is fairly well understood, it is unclear whether these techniques can enhance the production in unconventional reservoirs that operate in the primary phase; and the magnitude of the enhancement that can be expected by using the surfactants as fracturing fluid additives. Here, we designed a coreflood sequence that simulated fracturing fluid invasion, flowback and hydrocarbon production in order to quantify the permeability damage due to fluid invasion. Furthermore, we evaluated the enhancements in hydrocarbon permeability due to the use of IFT reducing agents. Our results revealed a mechanism of permeability damage and indicated that improvements in hydrocarbon permeability observed with surfactant-supplemented fracturing fluids were due to the reduction of water trapping through matrix-fracture interaction and not to the capillary desaturation of the rock.