Measuring Molecular Motor Forces In Vivo: Implications for Tug-of-War Models of Bidirectional Transport

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.     

NEX-TRAP, a Novel Method for In Vivo Analysis of Nuclear Export of Proteins

Transport of proteins between cytoplasm and nucleus is mediated by transport factors of the importin α- and β-families and occurs along a gradient of the small GTPase Ran. To date, in vivo analysis as well as prediction of protein nuclear export remain tedious and difficult. We generated a novel bipartite assay called NEX-TRAP (Nuclear EXport Trapped by RAPamycin) for in vivo analysis of protein nuclear export. The assay is based on the rapamycin-induced dimerization of the modules FRB (FK506-rapamycin (FR)-binding domain) and FKBP (FK506-binding protein-12): a potential nuclear export cargo is fused to FRB, to EYFP for direct visualization as well as to an SV40-derived nuclear localization signal (NLS) for constitutive nuclear import. An integral membrane protein that resides at the trans Golgi network (TGN) is fused to a cytoplasmically exposed FKBP and serves as reporter. EYFP-NLS-FRB fusion proteins with export activity accumulate in the nucleus at steady state but continuously shuttle between nucleus and cytoplasm. Rapamycin-induced dimerization of FRB and FKBP at the TGN traps the shuttling protein outside of the nucleus, making nuclear export permanent. Using several example cargoes, we show that the NEX-TRAP is superior to existing assays owing to its ease of use, its sensitivity and accuracy. Analysis of large numbers of export cargoes is facilitated by recombinational cloning. The NEX-TRAP holds the promise of applicability in automated fluorescence imaging for systematic analysis of nuclear export, thereby improving in silico prediction of nuclear export sequences.     

Regulation of Slow and Fast Muscle Myofibrillogenesis by Wnt/β-Catenin and Myostatin Signaling

Deviation from proper muscle development or homeostasis results in various myopathic conditions. Employing genetic as well as chemical intervention, we provide evidence that a tight regulation of Wnt/β-catenin signaling is essential for muscle fiber growth and maintenance. In zebrafish embryos, gain-of-Wnt/β-catenin function results in unscheduled muscle progenitor proliferation, leading to slow and fast muscle hypertrophy accompanied by fast muscle degeneration. The effects of Wnt/β-catenin signaling on fast muscle hypertrophy were rescued by misexpression of Myostatin or p21^CIP/WAF, establishing an in vivo regulation of myofibrillogenesis by Wnt/β-catenin signaling and Myostatin. Epistatic analyses suggest a possible genetic interaction between Wnt/β-catenin and Myostatin in regulation of slow and fast twitch muscle myofibrillogenesis.     

Prevalence of Endosymbionts in Bemisia tabaci Populations and Their In Vivo Sensitivity to Antibiotics

Bemisia tabaci can harbor both primary and secondary endosymbionts, and the specific endosymbionts can differ among different B. tabaci biotypes. This study determined (1) the prevalence of the primary endosymbiont Portiera aleyrodidarum and secondary endosymbionts Arsenophonus and Wolbachia in two invasive biotypes (B and Q) and one indigenous biotype (Cv) in China and (2) the in vivo effect of three antibiotics (tetracycline, ampicillin trihydrate, and rifampicin) against the endosymbionts; if an antibiotic substantially inhibits an endosymbiont, it could be used to determine the effect of that endosymbiont on B. tabaci. P. aleyrodidarum and Wolbachia were detected in all the three biotypes, while Arsenophonus was found only in the Q and Cv biotypes. P. aleyrodidarum was found in all tested individuals of the three biotypes. Infection rates of Wolbachia in the B, Cv, and Q biotypes were 58, 68, and 48%, respectively. The infection rate of Arsenophonus was 44% in the Q biotype but only 22% in the Cv biotype. The antibiotics failed to eliminate P. aleyrodidarum from any individual of the B, Cv, and Q biotypes but eliminated the secondary endosymbionts, Arsenophonus and Wolbachia, from 50 to 80% of the adult B. tabaci. The effect of the antibiotics depended on the species of endosymbiont, the antibiotic, the B. tabaci biotype, and various interactions between these factors. When used against Arsenophonus, the efficiency of rifampicin was better than ampicillin and tetracycline, regardless of B. tabaci biotype. When inactivating Wolbachia in Cv and Q biotypes, the efficiency tetracycline was better than ampicillin and rifampicin, and while the efficiency of tetracycline was better than rifampicin and ampicillin when they were used against Wolbachia in B biotype.     

In Vitro and In Vivo Neuronal Electrotaxis: A Potential Mechanism for Restoration?

Electrical brain stimulation used to treat a variety of neurological and psychiatric diseases is entering a new period. The technique is well established and the potential complications are well known and generally manageable. Recent studies demonstrated that electrical fields (EFs) can enhance neuroplasticity-related processes. EFs applied in the physiological range induce migration of different neural cell types from different species in vitro. There are some evidences that also the speed and directedness of cell migration are enhanced by EFs. However, it is still unclear how electrical signals from the extracellular space are translated into intracellular actions resulting in the so-called electrotaxis phenomenon. Here, we aim to provide a comprehensive review of the data on responses of cells to electrical stimulation and the relation to functional recovery.     

The Effect of Experimental Hyperthyroidism on Characteristics of Actin–Myosin Interaction in Fast and Slow Skeletal Muscles

The molecular mechanism of the failure of contractile function of skeletal muscles caused by oxidative damage to myosin in hyperthyroidism is not fully understood. Using an in vitro motility assay, we studied the effect of myosin damage caused by oxidative stress in experimental hyperthyroidism on the actin–myosin interaction and its regulation by calcium. We found that hyperthyroidism-induced oxidation of myosin is accompanied by a decrease in the sliding velocity of the regulated thin filaments in the in vitro motility assay, and this effect is increased with the duration of the pathological process.     

A Deconvolution-Based Method with High Sensitivity and Temporal Resolution for Detection of Spontaneous Synaptic Currents In?Vitro and?In?Vivo

Spontaneous postsynaptic currents (PSCs) provide key information about the mechanisms of synaptic transmission and the activity modes of neuronal networks. However, detecting spontaneous PSCs in vitro and in vivo has been challenging, because of the small amplitude, the variable kinetics, and the undefined time of generation of these events. Here, we describe a, to our knowledge, new method for detecting spontaneous synaptic events by deconvolution, using a template that approximates the average time course of spontaneous PSCs. A recorded PSC trace is deconvolved from the template, resulting in a series of delta-like functions. The maxima of these delta-like events are reliably detected, revealing the precise onset times of the spontaneous PSCs. Among all detection methods, the deconvolution-based method has a unique temporal resolution, allowing the detection of individual events in high-frequency bursts. Furthermore, the deconvolution-based method has a high amplitude resolution, because deconvolution can substantially increase the signal/noise ratio. When tested against previously published methods using experimental data, the deconvolution-based method was superior for spontaneous PSCs recorded in vivo. Using the high-resolution deconvolution-based detection algorithm, we show that the frequency of spontaneous excitatory postsynaptic currents in dentate gyrus granule cells is 4.5 times higher in vivo than in vitro.     

Chimeric β-Lactamases: Global Conservation of Parental Function and Fast Time-Scale Dynamics with Increased Slow Motions

Enzyme engineering has been facilitated by recombination of close homologues, followed by functional screening. In one such effort, chimeras of two class-A β-lactamases – TEM-1 and PSE-4 – were created according to structure-guided protein recombination and selected for their capacity to promote bacterial proliferation in the presence of ampicillin (Voigt et al., Nat. Struct. Biol. 2002 9:553). To provide a more detailed assessment of the effects of protein recombination on the structure and function of the resulting chimeric enzymes, we characterized a series of functional TEM-1/PSE-4 chimeras possessing between 17 and 92 substitutions relative to TEM-1 β-lactamase. Circular dichroism and thermal scanning fluorimetry revealed that the chimeras were generally well folded. Despite harbouring important sequence variation relative to either of the two ‘parental’ β-lactamases, the chimeric β-lactamases displayed substrate recognition spectra and reactivity similar to their most closely-related parent. To gain further insight into the changes induced by chimerization, the chimera with 17 substitutions was investigated by NMR spin relaxation. While high order was conserved on the ps-ns timescale, a hallmark of class A β-lactamases, evidence of additional slow motions on the µs-ms timescale was extracted from model-free calculations. This is consistent with the greater number of resonances that could not be assigned in this chimera relative to the parental β-lactamases, and is consistent with this well-folded and functional chimeric β-lactamase displaying increased slow time-scale motions.     

Dimeric DOTA-α-Melanocyte-Stimulating Hormone Analogs: Synthesis and In Vivo Characteristics of Radiopeptides with High In Vitro Activity

Dimeric analogs of α-melanocyte-stimulating hormone (α-MSH) labeled with radiometals are potential candidates for diagnosis and therapy of melanoma by receptor-mediated tumor targeting. Both melanotic and amelanotic melanomas (over-)express the melanocortin-1 receptor (MC1-R), the target for α-MSH. In the past, dimerized MSH analogs have been shown to display increased receptor affinity compared to monomeric MSH, offering the possibility of improving the ratio between specific uptake of radiolabeled α-MSH by melanoma and nonspecific uptake by the kidneys. We have designed three linear dimeric analogs containing a slightly modified MSH hexapeptide core sequence (Nle-Asp-His-d-Phe-Arg-Trp) in parallel or antiparallel orientation, a short spacer, and the DOTA chelator for incorporation of the radiometal. In vitro, all three peptides were more potent ligands of the mouse B16-F1 melanoma cell melanocortin-1 receptor (MC1-R) than DOTA-NAPamide, which served as standard. The binding activity of DOTA-diHexa(NC-NC)-amide was 1.75-fold higher, that of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was 3.37-fold higher, and that of DOTA-diHexa(CN-NC)-amide was 2.34-fold higher. Using human HBL melanoma cells, the binding activity of diHexa(NC-NC)-Gly-Lys(DOTA)-amide was sixfold higher than that of DOTA-NAPamide. Uptake by cultured B16-F1 cells was rapid and almost quantitative. In vivo, however, the data were less promising: tumor-to-kidney ratios 4 hr postinjection were 0.11 for [¹¹¹In]DOTA-diHexa(NC-NC)-amide, 0.26 for diHexa(NC-NC)-Gly-Lys([¹¹¹In]DOTA)-amide, and 0.36 for [¹¹¹In]DOTA-diHexa(CN-NC)-amide, compared to 1.67 for [¹¹¹In]DOTA-NAPamide. It appears that despite the higher affinity to the MC1-R of the peptide dimers and their excellent internalization in vitro, the uptake by melanoma tumors in vivo was lower, possibly because of reduced tissue penetration. More striking, however, was the marked increase of kidney uptake of the dimers, explaining the unfavorable ratios. In conclusion, although radiolabeled α-MSH dimer peptides display excellent receptor affinity and internalization, they are no alternative to the monomeric DOTA-NAPamide for in vivo application.     

FGF21 Can Be Mimicked In Vitro and In Vivo by a Novel Anti-FGFR1c/β-Klotho Bispecific Protein

The endocrine hormone FGF21 has attracted considerable interest as a potential therapeutic for treating diabetes and obesity. As an alternative to the native cytokine, we generated bispecific Avimer polypeptides that bind with high affinity and specificity to one of the receptor and coreceptor pairs used by FGF21, FGFR1c and β-Klotho. These Avimers exhibit FGF21-like activity in in vitro assays with potency greater than FGF21. In a study conducted in obese male cynomolgus monkeys, animals treated with an FGFR1c/β-Klotho bispecific Avimer showed improved metabolic parameters and reduced body weight comparable to the effects seen with FGF21. These results not only demonstrate the essential roles of FGFR1c and β-Klotho in mediating the metabolic effects of FGF21, they also describe a first bispecific activator of this unique receptor complex and provide validation for a novel therapeutic approach to target this potentially important pathway for treating diabetes and obesity.     

CSP—A Model for In Vivo Presentation of Plasmodium berghei Sporozoite Antigens by Hepatocytes

One target of protective immunity against the Plasmodium liver stage in BALB/c mice is represented by the circumsporozoite protein (CSP), and mainly involves its recognition by IFN-γ producing specific CD8+T-cells. In a previous in vitro study we showed that primary hepatocytes from BALB/c mice process Plasmodium berghei (Pb) CSP (PbCSP) and present CSP-derived peptides to specific H-2k^d restricted CD8+T-cells with subsequent killing of the presenting cells. We now extend these observations to an in vivo infection model in which infected hepatocytes and antigen specific T-cell clones are transferred into recipient mice inducing protection from sporozoite (SPZ) challenge. In addition, using a similar protocol, we suggest the capacity of hepatocytes in priming of naïve T-cells to provide protection, as further confirmed by induction of protection after depletion of cross-presenting dendritic cells (DCs) by cytochrome c (cyt c) treatment or using traversal deficient parasites. Our results clearly show that hepatocytes present Plasmodium CSP to specific-primed CD8+T-cells, and could also prime naïve T-cells, leading to protection from infection. These results could contribute to a better understanding of liver stage immune response and design of malaria vaccines.     

Hexarelin Protects Rodent Pancreatic Β-Cells Function from Cytotoxic Effects of Streptozotocin Involving Mitochondrial Signalling Pathways In Vivo and In Vitro

Mitochondrial functions are crucial for pancreatic β-cell survival and glucose-induced insulin secretion. Hexarelin (Hex) is a synthetic small peptide ghrelin analogue, which has been shown to protect cardiomyocytes from the ischemia-reperfusion process. In this study, we used in vitro and in vivo models of streptozotocin (STZ)-induced β-cell damage to study the protective effect of Hex and the associated mechanisms. We found that STZ produced a cytotoxic effect in a dose- and time-dependent manner in MIN6 cells (a mouse β-cell line). Hex (1.0 μM) decreased the STZ-induced damage in β-cells. Rhodamine 123 assay and superoxide DHE production assay revealed that Hex ameliorated STZ-induced mitochondrial damage and excessive superoxide activity in β-cells. In addition, Hex significantly reduced STZ-induced expression of cleaved Caspases-3, Caspases-9 and the ratio of pro-apoptotic protein Bax to anti-apoptotic protein Bcl-2 in MIN6 cells. We further examined the in vivo effect of Hex in a rat model of type 1 diabetes induced by STZ injection. Hex ameliorated STZ-induced decrease in plasma insulin and protected the structure of islets from STZ-induced disruption. Hex also ameliorated STZ-induced expression of cleaved Caspase-9 and the Bax in β-cells. In conclusion, our data indicate that Hex is able to protects β-cell mass from STZ-caused cytotoxic effects involving mitochondrial pathways in vitro and in vivo. Hex may serve as a potential protective agent for the management of diabetes.     

Cleavage of Poly(A)-Binding Protein by Coxsackievirus 2A Protease In Vitro and In Vivo: Another Mechanism for Host Protein Synthesis Shutoff?

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and enterovirus groups results in the shutoff of host protein synthesis but allows viral protein synthesis to proceed. Although considerable evidence suggests that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease in the case of coxsackievirus), several experimental observations are at variance with this view. Thus, the cleavage of other cellular proteins could contribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70-kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in translation initiation. We have now found that PABP is also proteolytically cleaved during coxsackievirus infection of HeLa cells. The cleavage of PABP correlated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experiments with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate that the cleavage is catalyzed by 2A protease directly. N- and C-terminal sequencing indicates that cleavage occurs uniquely in human PABP at ⁴⁸²VANTSTQTM↓GPRPAAAAAA⁵⁰⁰, separating the four N-terminal RNA recognition motifs (80%) from the C-terminal homodimerization domain (20%). The N-terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate translation system. These results suggest that the cleavage of PABP may be another mechanism by which picornaviruses alter the rate and spectrum of protein synthesis.     

Characteristics of Injury and Recovery of Net NO3? Transport of Barley Seedlings from Treatments of NaCl

The nature of the injury and recovery of nitrate uptake (net uptake) from NaCl stress in young barley (Hordeum vulgare L, var CM 72) seedlings was investigated. Nitrate uptake was inhibited rapidly by NaCl, within 1 minute after exposure to 200 millimolar NaCl. The duration of exposure to saline conditions determined the time of recovery of NO₃⁻ uptake from NaCl stress. Recovery was dependent on the presence of NO₃⁻ and was inhibited by cycloheximide, 6-methylpurine, and cerulenin, respective inhibitors of protein, RNA, and sterol/fatty acid synthesis. These inhibitors also prevented the induction of the NO₃⁻ uptake system in uninduced seedlings. Uninduced seedlings exhibited endogenous NO₃⁻ transport activity that appeared to be constitutive. This constitutive activity was also inhibited by NaCl. Recovery of constitutive NO₃⁻ uptake did not require the presence of NO₃⁻.     

Fast and fuel efficient? Optimal use of wind by flying albatrosses

The influence of wind patterns on behaviour and effort of free-ranging male wandering albatrosses (Diomedea exulans) was studied with miniaturized external heart-rate recorders in conjunction with satellite transmitters and activity recorders. Heart rate was used as an instantaneous index of energy expenditure. When cruising with favourable tail or side winds, wandering albatrosses can achieve high flight speeds while expending little more energy than birds resting on land. In contrast, heart rate increases concomitantly with increasing head winds, and flight speeds decrease. Our results show that effort is greatest when albatrosses take off from or land on the water. On a larger scale, we show that in order for birds to have the highest probability of experiencing favourable winds, wandering albatrosses use predictable weather systems to engage in a stereotypical flight pattern of large looping tracks. When heading north, albatrosses fly in anticlockwise loops, and to the south, movements are in a clockwise direction. Thus, the capacity to integrate instantaneous eco-physiological measures with records of large-scale flight and wind patterns allows us to understand better the complex interplay between the evolution of morphological, physiological and behavioural adaptations of albatrosses in the windiest place on earth.     

In?Vivo Nonlinear Optical Imaging of Immune Responses: Tissue Injury and Infection

In this study, we demonstrate a noninvasive imaging approach based on multimodal nonlinear optical microscopy to in vivo image the responses of immune cells (neutrophils) to the tissue injury and bacterial infection in a zebrafish model. Specifically, the second harmonic generation from myosin thick filaments in sarcomere enabled a clear visualization of the muscle injury and infection. Two-photon excited fluorescence was used to track the behavior of the neutrophils that were transgenically labeled by red fluorescent protein. The corresponding reduced nicotinamide adenine dinucleotide (NADH) two-photon excited fluorescence images revealed a detailed morphological transformation process of individual neutrophils during muscle tissue injury and bacterial infection. The analysis of time-resolved NADH signals from the neutrophils provided important biological insights of the cellular energy metabolism during the immune responses. We found a significant increase of free/protein-bound NADH ratios in activated neutrophils in bacterial-infected tissue. In this study, we also discovered that, under 720 nm excitation, two wild-type strains (DH5α and BL21) of bacteria Escherichia coli emitted distinct endogenous fluorescence of double-peak at ∼450 and ∼520 nm, respectively. We demonstrated that the double-peak fluorescence signal could be used to differentiate the E. coli from surrounding tissues of dominant NADH signals, and to achieve label-free tracking of E. coli bacteria in vivo.