Transcriptional Regulation of Plant Biomass Degradation and Carbohydrate Utilization Genes in the Extreme Thermophile Caldicellulosiruptor bescii.

Extremely thermophilic bacteria from the genus Caldicellulosiruptor can degrade polysaccharide components of plant cell walls and subsequently utilize the constituting mono- and oligosaccharides. Through metabolic engineering, ethanol and other industrially important end products can be produced. Previous experimental studies identified a variety of carbohydrate-active enzymes in model species Caldicellulosiruptor saccharolyticus and Caldicellulosiruptor bescii, while prior transcriptomic experiments identified their putative carbohydrate uptake transporters. We investigated the mechanisms of transcriptional regulation of carbohydrate utilization genes using a comparative genomics approach applied to 14 Caldicellulosiruptor species. The reconstruction of carbohydrate utilization regulatory network includes the predicted binding sites for 34 mostly local regulators and point to the regulatory mechanisms controlling expression of genes involved in degradation of plant biomass. The Rex and CggR regulons control the central glycolytic and primary redox reactions. The identified transcription factor binding sites and regulons were validated with transcriptomic and transcription start site experimental data for C. bescii grown on cellulose, cellobiose, glucose, xylan, and xylose. The XylR and XynR regulons control xylan-induced transcriptional response of genes involved in degradation of xylan and xylose utilization. The reconstructed regulons informed the carbohydrate utilization reconstruction analysis and improved functional annotations of 51 transporters and 11 catabolic enzymes. Using gene deletion, we confirmed that the shared ATPase component MsmK is essential for growth on oligo- and polysaccharides but not for the utilization of monosaccharides. By elucidating the carbohydrate utilization framework in C. bescii, strategies for metabolic engineering can be pursued to optimize yields of bio-based fuels and chemicals from lignocellulose. IMPORTANCE To develop functional metabolic engineering platforms for nonmodel microorganisms, a comprehensive understanding of the physiological and metabolic characteristics is critical. Caldicellulosiruptor bescii and other species in this genus have untapped potential for conversion of unpretreated plant biomass into industrial fuels and chemicals. The highly interactive and complex machinery used by C. bescii to acquire and process complex carbohydrates contained in lignocellulose was elucidated here to complement related efforts to develop a metabolic engineering platform with this bacterium. Guided by the findings here, a clearer picture of how C. bescii natively drives carbohydrate utilization is provided and strategies to engineer this bacterium for optimal conversion of lignocellulose to commercial products emerge.

Persistent growth of microtubules at low density.

Microtubules (MTs) often form a polarized array with minus ends anchored at the centrosome and plus ends extended toward the cell margins. Plus ends display behavior known as dynamic instability-transitions between rapid shortening and slow growth. It is known that dynamic instability is regulated locally to ensure entry of MTs into nascent areas of the cytoplasm, but details of this regulation remain largely unknown. Here, we test an alternative hypothesis for the local regulation of MT behavior. We used microsurgery to isolate a portion of peripheral cytoplasm from MTs growing from the centrosome, creating cytoplasmic areas locally depleted of MTs. We found that in sparsely populated areas MT plus ends persistently grew or paused but never shortened. In contrast, plus ends that entered regions of cytoplasm densely populated with MTs frequently transitioned to shortening. Persistent growth of MTs in sparsely populated areas could not be explained by a local increase in concentration of free tubulin subunits or elevation of Rac1 activity proposed to enhance MT growth at the cell leading edge during locomotion. These observations suggest the existence of a MT density-dependent mechanism regulating MT dynamics that determines dynamic instability of MTs in densely populated areas of the cytoplasm and persistent growth in sparsely populated areas.

An Indirect Method of Micromagnetic Structure Estimation in Microwires.

The tunable magnetic properties of amorphous ferromagnetic glass-coated microwires make them suitable for a wide range of applications. Accurate knowledge of the micromagnetic structure is highly desirable since it affects almost all magnetic properties. To select an appropriate wire-sample for a specific application, a deeper understanding of the magnetization reversal process is required, because it determines the measurable response (such as induced voltage waveform and its spectrum). However, the experimental observation of micromagnetic structure of micro-scale amorphous objects has strict size limitations. In this work we proposed a novel experimental technique for evaluating the microstructural characteristics of glass-coated microwires. The cross-sectional permeability distribution in the sample was obtained from impedance measurements at different frequencies. This distribution enables estimation of the prevailing anisotropy in the local region of the wire cross-section. The results obtained were compared with the findings of magnetostatic measurements and remanent state analysis. The advantages and limitations of the methods were discussed.

From isolated diamondoids to a van-der-Waals crystal: A theoretical and experimental analysis of a trishomocubane and a diamantane dimer in the gas and solid phase.

The electronic properties of sp2/sp3 diamondoids in the crystalline state and in the gas phase are presented. Apparent differences in electronic properties experimentally observed by resonance Raman spectroscopy in the crystalline/gas phase and absorption measurements in the gas phase were investigated by density functional theory computations. Due to a reorganization of the molecular orbitals in the crystalline phase, the HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energy gaps are lowered significantly by 0.5 eV-1 eV. The π → π* transition is responsible for large absorption in both gas and crystalline phases. It further causes a large increase in the Raman intensity of the C=C stretch vibration when excited resonantly. By resonance Raman spectroscopy we were able to determine the C=C bond length of the trishomocubane dimer to exhibit 1.33 Å in the ground and 1.41 Å in the excited state.

Optogenetic activation of Plexin-B1 reveals contact repulsion between osteoclasts and osteoblasts.

During bone remodelling, osteoclasts induce chemotaxis of osteoblasts and yet maintain spatial segregation. We show that osteoclasts express the repulsive guidance factor Semaphorin 4D and induce contact inhibition of locomotion (CIL) in osteoblasts through its receptor Plexin-B1. To examine causality and elucidate how localized Plexin-B1 stimulation may spatiotemporally coordinate its downstream targets in guiding cell migration, we develop an optogenetic tool for Plexin-B1 designated optoPlexin. Precise optoPlexin activation at the leading edge of migrating osteoblasts readily induces local retraction and, unexpectedly, distal protrusions to steer cells away. These morphological changes are accompanied by reorganization of Myosin II, PIP3, adhesion and active Cdc42. We attribute the resultant repolarization to RhoA/ROCK-mediated redistribution of ß-Pix, which activates Cdc42 and promotes protrusion. Thus, our data demonstrate a causal role of Plexin-B1 for CIL in osteoblasts and reveals a previously unknown effect of Semaphorin signalling on spatial distribution of an activator of cell migration.

Stimulation of microtubule-based transport by nucleation of microtubules on pigment granules.

Microtubule (MT)-based transport can be regulated through changes in organization of MT transport tracks, but the mechanisms that regulate these changes are poorly understood. In Xenopus melanophores, aggregation of pigment granules in the cell center involves their capture by the tips of MTs growing toward the cell periphery, and granule aggregation signals facilitate capture by increasing the number of growing MT tips. This increase could be explained by stimulation of MT nucleation either on the centrosome or on the aggregate of pigment granules that gradually forms in the cell center. We blocked movement of pigment granules to the cell center and compared the MT-nucleation activity of the centrosome in the same cells in two signaling states. We found that granule aggregation signals did not stimulate MT nucleation on the centrosome but did increase MT nucleation activity of pigment granules. Elevation of MT-nucleation activity correlated with the recruitment to pigment granules of a major component of MT-nucleation templates, γ-tubulin, and was suppressed by γ-tubulin inhibitors. We conclude that generation of new MT transport tracks by concentration of the leading pigment granules provides a positive feedback loop that enhances delivery of trailing granules to the cell center.

A minimal actomyosin-based model predicts the dynamics of filopodia on neuronal dendrites.

Dendritic filopodia are actin-filled dynamic subcellular structures that sprout on neuronal dendrites during neurogenesis. The exploratory motion of the filopodia is crucial for synaptogenesis, but the underlying mechanisms are poorly understood. To study filopodial motility, we collected and analyzed image data on filopodia in cultured rat hippocampal neurons. We hypothesized that mechanical feedback among the actin retrograde flow, myosin activity, and substrate adhesion gives rise to various filopodial behaviors. We formulated a minimal one-dimensional partial differential equation model that reproduced the range of observed motility. To validate our model, we systematically manipulated experimental correlates of parameters in the model: substrate adhesion strength, actin polymerization rate, myosin contractility, and the integrity of the putative microtubule-based barrier at the filopodium base. The model predicts the response of the system to each of these experimental perturbations, supporting the hypothesis that our actomyosin-driven mechanism controls dendritic filopodia dynamics.

Engineered Tug-of-War Between Kinesin and Dynein Controls Direction of Microtubule Based Transport In Vivo.

Bidirectional transport of membrane organelles along microtubules (MTs) is driven by plus-end directed kinesins and minus-end directed dynein bound to the same cargo. Activities of opposing MT motors produce bidirectional movement of membrane organelles and cytoplasmic particles along MT transport tracks. Directionality of MT-based transport might be controlled by a protein complex that determines which motor type is active at any given moment of time, or determined by the outcome of a tug-of-war between MT motors dragging cargo organelles in opposite directions. However, evidence in support of each mechanisms of regulation is based mostly on the results of theoretical analyses or indirect experimental data. Here, we test whether the direction of movement of membrane organelles in vivo can be controlled by the tug-of-war between opposing MT motors alone, by attaching a large number of kinesin-1 motors to organelles transported by dynein to minus-ends of MTs. We find that recruitment of kinesin significantly reduces the length and velocity of minus-end-directed dynein-dependent MT runs, leading to a reversal of the overall direction of dynein-driven organelles in vivo. Therefore, in the absence of external regulators tug-of-war between opposing MT motors alone is sufficient to determine the directionality of MT transport in vivo.

Toward an Understanding of Diamond sp(2)-Defects with Unsaturated Diamondoid Oligomer Models.

Nanometer-sized doubly bonded diamondoid dimers and trimers, which may be viewed as models of diamond with surface sp(2)-defects, were prepared from corresponding ketones via a McMurry coupling and were characterized by spectroscopic and crystallographic methods. The neutral hydrocarbons and their radical cations were studied utilizing density functional theory (DFT) and ab initio (MP2) methods, which reproduce the experimental geometries and ionization potentials well. The van der Waals complexes of the oligomers with their radical cations that are models for the self-assembly of diamondoids, form highly delocalized and symmetric electron-deficient structures. This implies a rather high degree of σ-delocalization within the hydrocarbons, not too dissimilar to delocalized π-systems. As a consequence, sp(2)-defects are thus also expected to be nonlocal, thereby leading to the observed high surface charge mobilities of diamond-like materials. In order to be able to use the diamondoid oligomers for subsequent surface attachment and modification, their C-H-bond functionalizations were studied, and these provided halogen and hydroxy derivatives with conservation of unsaturation.

Cargo transport at microtubule crossings: evidence for prolonged tug-of-war between kinesin motors.

Intracellular transport of cargos along microtubules is often complicated by the topology of the underlying filament network. The fundamental building blocks for this complex arrangement are filament intersections. The navigation of cargos across microtubule intersections remains poorly understood. Here, we demonstrate that kinesin-driven cargos are engaged in a tug-of-war at microtubule intersections. Tug-of-war events result in long pauses that can last from a few seconds to several minutes. We demonstrate that the extent of the tug-of-war and the duration of pauses change with the number of motors on the cargo and can be regulated by ionic strength. We also show that dwell times at intersections depend on the angle between crossing microtubules. Our data suggest that local microtubule geometry can regulate microtubule-based transport.

Regulation of microtubule-based transport by MAP4.

Microtubule (MT)-based transport of organelles driven by the opposing MT motors kinesins and dynein is tightly regulated in cells, but the underlying molecular mechanisms remain largely unknown. Here we tested the regulation of MT transport by the ubiquitous protein MAP4 using Xenopus melanophores as an experimental system. In these cells, pigment granules (melanosomes) move along MTs to the cell center (aggregation) or to the periphery (dispersion) by means of cytoplasmic dynein and kinesin-2, respectively. We found that aggregation signals induced phosphorylation of threonine residues in the MT-binding domain of the Xenopus MAP4 (XMAP4), thus decreasing binding of this protein to MTs. Overexpression of XMAP4 inhibited pigment aggregation by shortening dynein-dependent MT runs of melanosomes, whereas removal of XMAP4 from MTs reduced the length of kinesin-2-dependent runs and suppressed pigment dispersion. We hypothesize that binding of XMAP4 to MTs negatively regulates dynein-dependent movement of melanosomes and positively regulates kinesin-2-based movement. Phosphorylation during pigment aggregation reduces binding of XMAP4 to MTs, thus increasing dynein-dependent and decreasing kinesin-2-dependent motility of melanosomes, which stimulates their accumulation in the cell center, whereas dephosphorylation of XMAP4 during dispersion has an opposite effect.

Loss of spastin function results in disease-specific axonal defects in human pluripotent stem cell-based models of hereditary spastic paraplegia.

Human neuronal models of hereditary spastic paraplegias (HSP) that recapitulate disease-specific axonal pathology hold the key to understanding why certain axons degenerate in patients and to developing therapies. SPG4, the most common form of HSP, is caused by autosomal dominant mutations in the SPAST gene, which encodes the microtubule-severing ATPase spastin. Here, we have generated a human neuronal model of SPG4 by establishing induced pluripotent stem cells (iPSCs) from an SPG4 patient and differentiating these cells into telencephalic glutamatergic neurons. The SPG4 neurons displayed a significant increase in axonal swellings, which stained strongly for mitochondria and tau, indicating the accumulation of axonal transport cargoes. In addition, mitochondrial transport was decreased in SPG4 neurons, revealing that these patient iPSC-derived neurons recapitulate disease-specific axonal phenotypes. Interestingly, spastin protein levels were significantly decreased in SPG4 neurons, supporting a haploinsufficiency mechanism. Furthermore, cortical neurons derived from spastin-knockdown human embryonic stem cells (hESCs) exhibited similar axonal swellings, confirming that the axonal defects can be caused by loss of spastin function. These spastin-knockdown hESCs serve as an additional model for studying HSP. Finally, levels of stabilized acetylated-tubulin were significantly increased in SPG4 neurons. Vinblastine, a microtubule-destabilizing drug, rescued this axonal swelling phenotype in neurons derived from both SPG4 iPSCs and spastin-knockdown hESCs. Thus, this study demonstrates the successful establishment of human pluripotent stem cell-based neuronal models of SPG4, which will be valuable for dissecting the pathogenic cellular mechanisms and screening compounds to rescue the axonal degeneration in HSP.

UV resonance Raman analysis of trishomocubane and diamondoid dimers.

We present resonance Raman measurements of crystalline trishomocubane and diamantane dimers containing a C=C double bond. Raman spectra were recorded with excitation energies between 2.33 eV and 5.42 eV. The strongest enhancement is observed for the C=C stretch vibration and a bending mode involving the two carbon atoms of the C=C bond, corresponding to the B2g wagging mode of ethylene. This is associated with the localization of the π-HOMO and LUMO and the elongation of the C=C bond length and a pyramidalization of the two sp(2)-hybridized carbon atoms at the optical excitation. The observed Raman resonance energies of the trishomocubane and diamantane dimers are significantly lower than the HOMO-LUMO gaps of the corresponding unmodified diamondoids.

Force-dependent detachment of kinesin-2 biases track switching at cytoskeletal filament intersections.

Intracellular trafficking of organelles often involves cytoskeletal track switching. Organelles such as melanosomes are transported by multiple motors including kinesin-2, dynein, and myosin-V, which drive switching between microtubules and actin filaments during dispersion and aggregation. Here, we used optical trapping to determine the unitary and ensemble forces of kinesin-2, and to reconstitute cargo switching at cytoskeletal intersections in a minimal system with kinesin-2 and myosin-V motors bound to beads. Single kinesin-2 motors exerted forces up to ∼5 pN, similar to kinesin-1. However, kinesin-2 motors were more likely to detach at submaximal forces, and the duration of force maintenance was short as compared to kinesin-1. In multimotor assays, force increased with kinesin-2 density but was not affected by the presence of myosin-V. In crossed filament assays, switching frequencies of motor-bound beads were dependent on the starting track. At equal average forces, beads tended to switch from microtubules onto overlying actin filaments consistent with the relatively faster detachment of kinesin-2 at near-maximal forces. Thus, in addition to relative force, switching probability at filament intersections is determined by the dynamics of motor-filament interaction, such as the quick detachment of kinesin-2 under load. This may enable fine-tuning of filament switching in the cell.

Stimulation of the CLIP-170--dependent capture of membrane organelles by microtubules through fine tuning of microtubule assembly dynamics.

Cytoplasmic microtubules (MTs) continuously grow and shorten at their free plus ends, a behavior that allows them to capture membrane organelles destined for MT minus end-directed transport. In Xenopus melanophores, the capture of pigment granules (melanosomes) involves the +TIP CLIP-170, which is enriched at growing MT plus ends. Here we used Xenopus melanophores to test whether signals that stimulate minus end MT transport also enhance CLIP-170-dependent binding of melanosomes to MT tips. We found that these signals significantly (>twofold) increased the number of growing MT plus ends and their density at the cell periphery, thereby enhancing the likelihood of interaction with dispersed melanosomes. Computational simulations showed that local and global increases in the density of CLIP-170-decorated MT plus ends could reduce the half-time of melanosome aggregation by ~50%. We conclude that pigment granule aggregation signals in melanophores stimulate MT minus end-directed transport by the increasing number of growing MT plus ends decorated with CLIP-170 and redistributing these ends to more efficiently capture melanosomes throughout the cytoplasm.

Conformationally restricted GABA analogs: from rigid carbocycles to cage hydrocarbons.

GABA was discovered to play an important role as the major inhibitory neurotransmitter in the adult mammalian CNS 60 years ago. The conformational flexibility of GABA is important for its biological function, as it has been found to bind to different receptors with different conformations. In an effort to increase the lipophilicity and to reduce conformational flexibility of GABA itself, a polycyclic or cage hydrocarbon framework can be introduced into the 3D structure of GABA in order to better control the binding. This article explores the available synthetic methods, properties and activity of carbocyclic (cyclopropanes, cyclobutanes and cyclohexanes) and cage (adamantane and others) hydrocarbons - analogs of GABA with conformationally rigid carbon skeletons.

CK1 activates minus-end-directed transport of membrane organelles along microtubules.

Microtubule (MT)-based organelle transport is driven by MT motor proteins that move cargoes toward MT minus-ends clustered in the cell center (dyneins) or plus-ends extended to the periphery (kinesins). Cells are able to rapidly switch the direction of transport in response to external cues, but the signaling events that control switching remain poorly understood. Here, we examined the signaling mechanism responsible for the rapid activation of dynein-dependent MT minus-end-directed pigment granule movement in Xenopus melanophores (pigment aggregation). We found that, along with the previously identified protein phosphatase 2A (PP2A), pigment aggregation signaling also involved casein kinase 1ε (CK1ε), that both enzymes were bound to pigment granules, and that their activities were increased during pigment aggregation. Furthermore we found that CK1ε functioned downstream of PP2A in the pigment aggregation signaling pathway. Finally, we discovered that stimulation of pigment aggregation increased phosphorylation of dynein intermediate chain (DIC) and that this increase was partially suppressed by CK1ε inhibition. We propose that signal transduction during pigment aggregation involves successive activation of PP2A and CK1ε and CK1ε-dependent phosphorylation of DIC, which stimulates dynein motor activity and increases minus-end-directed runs of pigment granules.

Finding the cell center by a balance of dynein and myosin pulling and microtubule pushing: a computational study.

The centrosome position in many types of interphase cells is actively maintained in the cell center. Our previous work indicated that the centrosome is kept at the center by pulling force generated by dynein and actin flow produced by myosin contraction and that an unidentified factor that depends on microtubule dynamics destabilizes position of the centrosome. Here, we use modeling to simulate the centrosome positioning based on the idea that the balance of three forces-dyneins pulling along microtubule length, myosin-powered centripetal drag, and microtubules pushing on organelles-is responsible for the centrosome displacement. By comparing numerical predictions with centrosome behavior in wild-type and perturbed interphase cells, we rule out several plausible hypotheses about the nature of the microtubule-based force. We conclude that strong dynein- and weaker myosin-generated forces pull the microtubules inward competing with microtubule plus-ends pushing the microtubule aster outward and that the balance of these forces positions the centrosome at the cell center. The model also predicts that kinesin action could be another outward-pushing force. Simulations demonstrate that the force-balance centering mechanism is robust yet versatile. We use the experimental observations to reverse engineer the characteristic forces and centrosome mobility.

Melanophores for microtubule dynamics and motility assays.

Microtubules (MTs) are cytoskeletal structures essential for cell division, locomotion, intracellular transport, and spatial organization of the cytoplasm. In most interphase cells, MTs are organized into a polarized radial array with minus-ends clustered at the centrosome and plus-ends extended to the cell periphery. This array directs transport of organelles driven by MT-based motor proteins that specifically move either to plus- or to minus-ends. Along with using MTs as tracks for cargo, motor proteins can organize MTs into a radial array in the absence of the centrosome. Transport of organelles and motor-dependent radial organization of MTs require MT dynamics, continuous addition and loss of tubulin subunits at minus- and plus-ends. A unique experimental system for studying the role of MT dynamics in these processes is the melanophore, which provides a useful tool for imaging of both dynamic MTs and moving membrane organelles. Melanophores are filled with pigment granules that are synchronously transported by motor proteins in response to hormonal stimuli. The flat shape of the cell and the radial organization of MTs facilitate imaging of dynamic MT plus-ends and monitoring of their interaction with membrane organelles. Microsurgically produced cytoplasmic fragments of melanophores are used to study the centrosome-independent rearrangement of MTs into a radial array. Here we describe the experimental approaches to study the role of MT dynamics in intracellular transport and centrosome-independent MT organization in melanophores. We focus on the preparation of cell cultures, microsurgery and microinjection, fluorescence labeling, and live imaging of MTs.

Novel isoform of the Xenopus tropicalis PKA catalytic alpha subunit: An example of alternative splicing.

The cAMP-dependent protein kinase (PKA) plays key roles in the control of various aspects of eukaryotic cellular activities by phosphorylating several proteins and is multifunctional in nature. In the case of frog, Xenopus tropicalis, a gene encoding the PKA catalytic alpha subunit has been identified which encodes a single protein. Here we report the occurrence of N-terminal alternative splicing events in X. tropicalis tadpole that, in addition to generating a myristoylatable isoforms, also generate the non-myristoylated variant of the catalytic alpha subunit as has been reported in various other organisms. In addition to the already characterized exon 1, the 5' untranslated region and first intron actually contains one more other exon, that is alternatively spliced on to exon 2 at the 5' end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with already characterized all internal exons. Thus, X. tropicalis tadpole expresses at least two different isoforms of the catalytic alpha subunit of PKA. The significance of this structural diversity in the family of PKA catalytic subunits is discussed.