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The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scientific network, a benchmarking initiative was designed to compare the diverse protocols established in thirteen individual laboratories. This benchmarking initiative compared the expression of four selected intracellular proteins (mouse Dicer-2, 204 kDa; human ABL1 wildtype, 126 kDa; human FMRP, 68 kDa; viral vNS1-H1, 76 kDa). Here, we present the expression and purification results on these proteins and highlight the significant differences in expression yields obtained using different commercially-packaged baculovirus vectors. The highest expression level for difficult-to-express intracellular protein candidates were observed with the EmBacY baculovirus vector system., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Proteins such as FUS phase separate to form liquid-like condensates that can harden into less dynamic structures. However, how these properties emerge from the collective interactions of many amino acids remains largely unknown. Here, we use extensive mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of proteins in the FUS family and test aspects of this grammar in cells. Phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhance the fluidity, whereas glutamine and serine residues promote hardening. We develop a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase-separation properties based on amino acid sequences., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Most vertebrate oocytes contain a Balbiani body, a large, non-membrane-bound compartment packed with RNA, mitochondria, and other organelles. Little is known about this compartment, though it specifies germline identity in many non-mammalian vertebrates. We show Xvelo, a disordered protein with an N-terminal prion-like domain, is an abundant constituent of Xenopus Balbiani bodies. Disruption of the prion-like domain of Xvelo, or substitution with a prion-like domain from an unrelated protein, interferes with its incorporation into Balbiani bodies in vivo. Recombinant Xvelo forms amyloid-like networks in vitro. Amyloid-like assemblies of Xvelo recruit both RNA and mitochondria in binding assays. We propose that Xenopus Balbiani bodies form by amyloid-like assembly of Xvelo, accompanied by co-recruitment of mitochondria and RNA. Prion-like domains are found in germ plasm organizing proteins in other species, suggesting that Balbiani body formation by amyloid-like assembly could be a conserved mechanism that helps oocytes function as long-lived germ cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)

A rapid method for temporally and spatially controlled CRISPR-mediated gene knockout in vertebrates will be an important tool to screen for genes involved in complex biological phenomena like regeneration. Here we show that in vivo injection of CAS9 protein-guide RNA (gRNA) complexes into the spinal cord lumen of the axolotl and subsequent electroporation leads to comprehensive knockout of Sox2 gene expression in SOX2 + neural stem cells with corresponding functional phenotypes from the gene knockout. This is particularly surprising considering the known prevalence of RNase activity in cerebral spinal fluid, which apparently the CAS9 protein protects against. The penetrance/efficiency of gene knockout in the protein-based system is far higher than corresponding electroporation of plasmid-based CRISPR systems. We further show that simultaneous delivery of CAS9-gRNA complexes directed against Sox2 and GFP yields efficient knockout of both genes in GFP -reporter animals. Finally, we show that this method can also be applied to other tissues such as skin and limb mesenchyme. This efficient delivery method opens up the possibility for rapid in vivo genetic screens during axolotl regeneration and can in principle be applied to other vertebrate tissue systems., Competing Interests: The authors declare no conflict of interest.

In salamanders, grafting of a left limb blastema onto a right limb stump yields regeneration of three limbs, the normal limb and two 'supernumerary' limbs. This experiment and other research have shown that the juxtaposition of anterior and posterior limb tissue plus innervation are necessary and sufficient to induce complete limb regeneration in salamanders. However, the cellular and molecular basis of the requirement for anterior-posterior tissue interactions were unknown. Here we have clarified the molecular basis of the requirement for both anterior and posterior tissue during limb regeneration and supernumerary limb formation in axolotls (Ambystoma mexicanum). We show that the two tissues provide complementary cross-inductive signals that are required for limb outgrowth. A blastema composed solely of anterior tissue normally regresses rather than forming a limb, but activation of hedgehog (HH) signalling was sufficient to drive regeneration of an anterior blastema to completion owing to its ability to maintain fibroblast growth factor (FGF) expression, the key signalling activity responsible for blastema outgrowth. In blastemas composed solely of posterior tissue, HH signalling was not sufficient to drive regeneration; however, ectopic expression of FGF8 together with endogenous HH signalling was sufficient. In axolotls, FGF8 is expressed only in the anterior mesenchyme and maintenance of its expression depends on sonic hedgehog (SHH) signalling from posterior tissue. Together, our findings identify key anteriorly and posteriorly localized signals that promote limb regeneration and show that these single factors are sufficient to drive non-regenerating blastemas to complete regeneration with full elaboration of skeletal elements.

Many proteins contain disordered regions of low-sequence complexity, which cause aging-associated diseases because they are prone to aggregate. Here, we study FUS, a prion-like protein containing intrinsically disordered domains associated with the neurodegenerative disease ALS. We show that, in cells, FUS forms liquid compartments at sites of DNA damage and in the cytoplasm upon stress. We confirm this by reconstituting liquid FUS compartments in vitro. Using an in vitro "aging" experiment, we demonstrate that liquid droplets of FUS protein convert with time from a liquid to an aggregated state, and this conversion is accelerated by patient-derived mutations. We conclude that the physiological role of FUS requires forming dynamic liquid-like compartments. We propose that liquid-like compartments carry the trade-off between functionality and risk of aggregation and that aberrant phase transitions within liquid-like compartments lie at the heart of ALS and, presumably, other age-related diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Direct lineage reprogramming induces dramatic shifts in cellular identity, employing poorly understood mechanisms. Recently, we demonstrated that expression of Neurog2 or Ascl1 in postnatal mouse astrocytes generates glutamatergic or GABAergic neurons. Here, we take advantage of this model to study dynamics of neuronal cell fate acquisition at the transcriptional level. We found that Neurog2 and Ascl1 rapidly elicited distinct neurogenic programs with only a small subset of shared target genes. Within this subset, only NeuroD4 could by itself induce neuronal reprogramming in both mouse and human astrocytes, while co-expression with Insm1 was required for glutamatergic maturation. Cultured astrocytes gradually became refractory to reprogramming, in part by the repressor REST preventing Neurog2 from binding to the NeuroD4 promoter. Notably, in astrocytes refractory to Neurog2 activation, the underlying neurogenic program remained amenable to reprogramming by exogenous NeuroD4. Our findings support a model of temporal hierarchy for cell fate change during neuronal reprogramming., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Hydrogel beads as microcarriers could have many applications in biotechnology. However, bead formation by noncovalent cross-linking to achieve high cell compatibility by avoiding chemical reactions remains challenging because of rapid gelation rates and/or low stability. Here we report the preparation of homogeneous, tunable, and robust hydrogel beads from peptide-polyethylene glycol conjugates and oligosaccharides under mild, cell-compatible conditions using a noncovalent crosslinking mechanism. Large proteins can be released from beads easily. Further noncovalent modification allows for bead labeling and functionalization with various compounds. High survival rates of embedded cells were achieved under standard cell culture conditions and after freezing the beads, demonstrating its suitability for encapsulating and conserving cells. Hydrogel beads as functional system have been realized by generating protein-producing microcarriers with embedded eGFP-secreting insect cells., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The proneural transcription factor Ascl1 coordinates gene expression in both proliferating and differentiating progenitors along the neuronal lineage. Here, we used a cellular model of neurogenesis to investigate how Ascl1 interacts with the chromatin landscape to regulate gene expression when promoting neuronal differentiation. We find that Ascl1 binding occurs mostly at distal enhancers and is associated with activation of gene transcription. Surprisingly, the accessibility of Ascl1 to its binding sites in neural stem/progenitor cells remains largely unchanged throughout their differentiation, as Ascl1 targets regions of both readily accessible and closed chromatin in proliferating cells. Moreover, binding of Ascl1 often precedes an increase in chromatin accessibility and the appearance of new regions of open chromatin, associated with de novo gene expression during differentiation. Our results reveal a function of Ascl1 in promoting chromatin accessibility during neurogenesis, linking the chromatin landscape at Ascl1 target regions with the temporal progression of its transcriptional program., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The gene regulatory network (GRN) that supports neural stem cell (NS cell) self-renewal has so far been poorly characterized. Knowledge of the central transcription factors (TFs), the noncoding gene regulatory regions that they bind to, and the genes whose expression they modulate will be crucial in unlocking the full therapeutic potential of these cells. Here, we use DNase-seq in combination with analysis of histone modifications to identify multiple classes of epigenetically and functionally distinct cis-regulatory elements (CREs). Through motif analysis and ChIP-seq, we identify several of the crucial TF regulators of NS cells. At the core of the network are TFs of the basic helix-loop-helix (bHLH), nuclear factor I (NFI), SOX, and FOX families, with CREs often densely bound by several of these different TFs. We use machine learning to highlight several crucial regulatory features of the network that underpin NS cell self-renewal and multipotency. We validate our predictions by functional analysis of the bHLH TF OLIG2. This TF makes an important contribution to NS cell self-renewal by concurrently activating pro-proliferation genes and preventing the untimely activation of genes promoting neuronal differentiation and stem cell quiescence., (© 2015 Mateo et al.; Published by Cold Spring Harbor Laboratory Press.)

Direct lineage reprogramming is a promising approach for human disease modeling and regenerative medicine, with poorly understood mechanisms. Here, we reveal a hierarchical mechanism in the direct conversion of fibroblasts into induced neuronal (iN) cells mediated by the transcription factors Ascl1, Brn2, and Myt1l. Ascl1 acts as an "on-target" pioneer factor by immediately occupying most cognate genomic sites in fibroblasts. In contrast, Brn2 and Myt1l do not access fibroblast chromatin productively on their own; instead, Ascl1 recruits Brn2 to Ascl1 sites genome wide. A unique trivalent chromatin signature in the host cells predicts the permissiveness for Ascl1 pioneering activity among different cell types. Finally, we identified Zfp238 as a key Ascl1 target gene that can partially substitute for Ascl1 during iN cell reprogramming. Thus, a precise match between pioneer factors and the chromatin context at key target genes is determinative for transdifferentiation to neurons and likely other cell types., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Accurately characterizing transcription factor (TF)-DNA affinity is a central goal of regulatory genomics. Although thermodynamics provides the most natural language for describing the continuous range of TF-DNA affinity, traditional motif discovery algorithms focus instead on classification paradigms that aim to discriminate 'bound' and 'unbound' sequences. Moreover, these algorithms do not directly model the distribution of tags in ChIP-seq data. Here, we present a new algorithm named Thermodynamic Modeling of ChIP-seq (TherMos), which directly estimates a position-specific binding energy matrix (PSEM) from ChIP-seq/exo tag profiles. In cross-validation tests on seven genome-wide TF-DNA binding profiles, one of which we generated via ChIP-seq on a complex developing tissue, TherMos predicted quantitative TF-DNA binding with greater accuracy than five well-known algorithms. We experimentally validated TherMos binding energy models for Klf4 and Esrrb, using a novel protocol to measure PSEMs in vitro. Strikingly, our measurements revealed strong non-additivity at multiple positions within the two PSEMs. Among the algorithms tested, only TherMos was able to model the entire binding energy landscape of Klf4 and Esrrb. Our study reveals new insights into the energetics of TF-DNA binding in vivo and provides an accurate first-principles approach to binding energy inference from ChIP-seq and ChIP-exo data.

The mammalian nervous system is the most complex organ of any living organism. How this complexity is generated during neural development is just beginning to be elucidated. This article discusses the signaling, transcriptional, and epigenetic mechanisms that are involved in neural development. The first part focuses on molecules that control neuronal numbers through regulation of the timing of onset of neurogenesis, the timing of the neuronal-to-glial switch, and the rate of progenitor proliferation. The second part focuses on molecules that control neuronal diversity by generating spatially or temporally distinct populations of neuronal progenitors. Most of the studies discussed in this article are focused on the developing mammalian cerebral cortex, because this is one of the main model systems for neural developmental studies and many of the mechanisms identified in this tissue also operate elsewhere in the developing brain and spinal cord.

Chemokines are small secreted proteins with important roles in immune responses. They consist of a conserved three-dimensional (3D) structure, so-called IL8-like chemokine fold, which is supported by disulfide bridges characteristic of this protein family. Sequence- and profile-based computational methods have been proficient in discovering novel chemokines by making use of their sequence-conserved cysteine patterns. However, it has been recently shown that some chemokines escaped annotation by these methods due to low sequence similarity to known chemokines and to different arrangement of cysteines in sequence and in 3D. Innovative methods overcoming the limitations of current techniques may allow the discovery of new remote homologs in the still functionally uncharacterized fraction of the human genome. We report a novel computational approach for proteome-wide identification of remote homologs of the chemokine family that uses fold recognition techniques in combination with a scaffold-based automatic mapping of disulfide bonds to define a 3D profile of the chemokine protein family. By applying our methodology to all currently uncharacterized human protein sequences, we have discovered two novel proteins that, without having significant sequence similarity to known chemokines or characteristic cysteine patterns, show strong structural resemblance to known anti-HIV chemokines. Detailed computational analysis and experimental structural investigations based on mass spectrometry and circular dichroism support our structural predictions and highlight several other chemokine-like features. The results obtained support their functional annotation as putative novel chemokines and encourage further experimental characterization. The identification of remote homologs of human chemokines may provide new insights into the molecular mechanisms causing pathologies such as cancer or AIDS, and may contribute to the development of novel treatments. Besides, the genome-wide applicability of our methodology based on 3D protein family profiles may open up new possibilities for improving and accelerating protein function annotation processes.

Purpose: To determine the influence of drug physicochemical properties on brain mitochondrial delivery of 20 drugs at physiological pH., Methods: The delivery of 8 cationic drugs (beta-blockers), 6 neutral drugs (corticosteroids), and 6 anionic drugs (non-steroidal anti-inflammatory drugs, NSAIDs) to isolated rat brain mitochondria was determined with and without membrane depolarization. Multiple linear regression was used to determine whether lipophilicity (Log D), charge, polarizability, polar surface area (PSA), and molecular weight influence mitochondrial delivery., Results: The Log D for beta-blockers, corticosteroids, and NSAIDs was in the range of -1.41 to 1.37, 0.72 to 2.97, and -0.98 to 2, respectively. The % mitochondrial uptake increased exponentially with an increase in Log D for each class of drugs, with the uptake at a given lipophilicity obeying the rank order cationic>anionic>neutral. Valinomycin reduced membrane potential and the delivery of positively charged propranolol and betaxolol. The best equation for the combined data set was Log % Uptake = 0.333 Log D + 0.157 Charge - 0.887 Log PSA + 2.032 (R(2) = 0.738)., Conclusions: Drug lipopohilicity, charge, and polar surface area and membrane potential influence mitochondrial drug delivery, with the uptake of positively charged, lipophilic molecules being the most efficient.

The human epidermal growth factor receptor (EGFR) is a key representative of tyrosine kinase receptors, ubiquitous actors in cell signaling, proliferation, differentiation, and migration. Although the receptor is well-studied, a central issue remains: How does the compositional diversity and functional diversity of the surrounding membrane modulate receptor function? Reconstituting human EGFR into proteoliposomes of well-defined and controlled lipid compositions represents a minimal synthetic approach to systematically address this question. We show that lipid composition has little effect on ligand-binding properties of the EGFR but rather exerts a profound regulatory effect on kinase domain activation. Here, the ganglioside GM3 but not other related lipids strongly inhibited the autophosphorylation of the EGFR kinase domain. This inhibitory action of GM3 was only seen in liposomes compositionally poised to phase separate into coexisting liquid domains. The inhibition by GM3 was released by either removing the neuraminic acid of the GM3 headgroup or by mutating a membrane proximal lysine of EGFR (K642G). Our results demonstrate that GM3 exhibits the potential to regulate the allosteric structural transition from inactive to a signaling EGFR dimer, by preventing the autophosphorylation of the intracellular kinase domain in response to ligand binding.

Proneural genes such as Ascl1 are known to promote cell cycle exit and neuronal differentiation when expressed in neural progenitor cells. The mechanisms by which proneural genes activate neurogenesis--and, in particular, the genes that they regulate--however, are mostly unknown. We performed a genome-wide characterization of the transcriptional targets of Ascl1 in the embryonic brain and in neural stem cell cultures by location analysis and expression profiling of embryos overexpressing or mutant for Ascl1. The wide range of molecular and cellular functions represented among these targets suggests that Ascl1 directly controls the specification of neural progenitors as well as the later steps of neuronal differentiation and neurite outgrowth. Surprisingly, Ascl1 also regulates the expression of a large number of genes involved in cell cycle progression, including canonical cell cycle regulators and oncogenic transcription factors. Mutational analysis in the embryonic brain and manipulation of Ascl1 activity in neural stem cell cultures revealed that Ascl1 is indeed required for normal proliferation of neural progenitors. This study identified a novel and unexpected activity of the proneural gene Ascl1, and revealed a direct molecular link between the phase of expansion of neural progenitors and the subsequent phases of cell cycle exit and neuronal differentiation.

An important feature of the cerebral cortex is its layered organization, which is modulated in an area-specific manner. We found that the transcription factor AP2gamma regulates laminar fate in a region-specific manner. Deletion of AP2gamma (also known as Tcfap2c) during development resulted in a specific reduction of upper layer neurons in the occipital cortex, leading to impaired function and enhanced plasticity of the adult visual cortex. AP2gamma functions in apical progenitors, and its absence resulted in mis-specification of basal progenitors in the occipital cortex at the time at which upper layer neurons were generated. AP2gamma directly regulated the basal progenitor fate determinants Math3 (also known as Neurod4) and Tbr2, and its overexpression promoted the generation of layer II/III neurons in a time- and region-specific manner. Thus, AP2gamma acts as a regulator of basal progenitor fate, linking regional and laminar specification in the mouse developing cerebral cortex.

Rab GTPases and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are evolutionarily conserved essential components of the eukaryotic intracellular transport system. Although pairing of cognate SNAREs is sufficient to fuse membranes in vitro, a complete reconstitution of the Rab-SNARE machinery has never been achieved. Here we report the reconstitution of the early endosomal canine Rab5 GTPase, its key regulators and effectors together with SNAREs into proteoliposomes using a set of 17 recombinant human proteins. These vesicles behave like minimal 'synthetic' endosomes, fusing with purified early endosomes or with each other in vitro. Membrane fusion measured by content-mixing and morphological assays requires the cooperativity between Rab5 effectors and cognate SNAREs which, together, form a more efficient 'core machinery' than SNAREs alone. In reconstituting a fusion mechanism dependent on both a Rab GTPase and SNAREs, our work shows that the two machineries act coordinately to increase the specificity and efficiency of the membrane tethering and fusion process.

Many protein-protein interactions are mediated through independently folding modular domains. Proteome-wide efforts to model protein-protein interaction or "interactome" networks have largely ignored this modular organization of proteins. We developed an experimental strategy to efficiently identify interaction domains and generated a domain-based interactome network for proteins involved in C. elegans early-embryonic cell divisions. Minimal interacting regions were identified for over 200 proteins, providing important information on their domain organization. Furthermore, our approach increased the sensitivity of the two-hybrid system, resulting in a more complete interactome network. This interactome modeling strategy revealed insights into C. elegans centrosome function and is applicable to other biological processes in this and other organisms.

Since the discovery of radial glia as the source of neurons, their heterogeneity in regard to neurogenesis has been described by clonal and time-lapse analysis in vitro. However, the molecular determinants specifying neurogenic radial glia differently from radial glia that mostly self-renew remain ill-defined. Here, we isolated two radial glial subsets that co-exist at mid-neurogenesis in the developing cerebral cortex and their immediate progeny. While one subset generates neurons directly, the other is largely non-neurogenic but also gives rise to Tbr2-positive basal precursors, thereby contributing indirectly to neurogenesis. Isolation of these distinct radial glia subtypes allowed determining interesting differences in their transcriptome. These transcriptomes were also strikingly different from the transcriptome of radial glia isolated at the end of neurogenesis. This analysis therefore identifies, for the first time, the lineage origin of basal progenitors and the molecular differences of this lineage in comparison to directly neurogenic and gliogenic radial glia.

Salamanders display unique regeneration abilities among adult vertebrates. An intriguing feature of salamander regeneration is the dedifferentiation of cells, such as myofibers and myotubes at the injury site, a process that involves cell cycle reentry from the differentiated state. A thrombin-activated serum factor that is distinct from conventional growth factors is known to cause S-phase reentry in salamander myotubes. While mammalian myotubes do not reenter S-phase upon serum stimulation, an upregulation of some immediate early genes such as jun and fos has been observed. Until now, it was unknown whether this transcriptional response was stimulated by conventional growth factors or by the thrombin-activated serum factor. By measuring transcriptional activity in individually purified C2C12 mouse myotubes using quantitative reverse transcription polymerase chain reactions, we show that a set of immediate early genes are activated in response to the thrombin-activated serum factor in a distinct manner from the growth factors PDGF, FGF and EGF. A partially purified fraction of the thrombin activated serum factor elicited stronger upregulation of a broader set of genes compared to individual growth factors and additionally caused downregulation of E2F6. Despite this robust transcriptional response in mammalian myotubes, we did not detect a large-scale change in histone H3K9 di-methylation or S-phase, a feature that characterizes salamander serum-stimulated myotubes. Our results indicate that mammalian myotubes have retained responsiveness to the thrombin-activated serum factor, but full reentry into S-phase is prevented by factors downstream of the immediate early genes.

The beta-secretase, BACE, is a membrane spanning aspartic protease, which cleaves the amyloid precursor protein (APP) in the first step of proteolytic processing leading to the formation of the neurotoxic beta-amyloid peptide (Abeta). Previous results have suggested that the regulation of beta-secretase and BACE access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have expressed recombinant human full-length BACE in insect cells and purified milligram amounts to homogeneity. We have studied partitioning of fluorophor-conjugated BACE between the liquid ordered and disordered phases in giant (10-150 mum) unilamellar vesicles, and found approximately 20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquid-ordered phase increased upon cross-linking of raft lipids. To examine involvement of individual lipid species in modulating BACE activity, we have reconstituted the purified BACE in large ( approximately 100 nm) unilamellar vesicles, and determined its specific activity in vesicles of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1) neutral glycosphingolipids (cerebrosides), 2) anionic glycerophospholipids, and 3) sterols (cholesterol).

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

The Dis1/XMAP215 family of microtubule-associated proteins conserved from yeast to mammals is essential for cell division. XMAP215, the Xenopus member of this family, has been shown to stabilize microtubules in vitro, but other members of this family have not been biochemically characterized. Here we investigate the properties of the Saccharomyces cerevisiae homologue Stu2p in vitro. Surprisingly, Stu2p is a microtubule destabilizer that binds preferentially to microtubule plus ends. Quantitative analysis of microtubule dynamics suggests that Stu2p induces microtubule catastrophes by sterically interfering with tubulin addition to microtubule ends. These results reveal both a new biochemical activity for a Dis1/XMAP215 family member and a novel mechanism for microtubule destabilization.

The helicity dependence of the gamma-->p-->-->ppi(0) reaction has been measured for the first time in the photon-energy range from 550 to 790 MeV. The experiment, performed at the Mainz microtron MAMI, used a 4pi-detector system, a circularly polarized, tagged photon beam, and a longitudinally polarized frozen-spin target. These data are predominantly sensitive to the D13(1520) resonance and are used to determine its helicity amplitudes.

In a p((-->)e,e'p)pi(0) out-of-plane coincidence experiment at the three-spectrometer setup of the Mainz Microtron MAMI, the beam-helicity asymmetry has been precisely measured around the energy of the Delta(1232) resonance and Q(2) = 0.2(GeV/c)(2). The results are in disagreement with three up-to-date model calculations. This is interpreted as a lack of understanding of the nonresonant background, which in dynamical models is related to the pion cloud.

The recoil proton polarization has been measured in the p(e-->,e'p-->)pi(0) reaction in parallel kinematics around W = 1232 MeV, Q2 = 0.121 (GeV/c)2, and epsilon = 0.718 using the polarized cw electron beam of the Mainz Microtron. All three proton polarization components, Px/P(e) = (-11.4+/-1.3+/-1.4)%, P(y) = (-43.1+/-1.3+/-2.2)%, and P(z)/P(e) = (56.2+/-1.5+/-2.6)%, could be measured simultaneously. The Coulomb quadrupole to magnetic dipole ratio, CMR = (-6.4+/-0.7(stat)+/-0.8(syst))%, was determined from Px in the framework of the Mainz Unitary Isobar Model. The consistency among the reduced polarizations and the extraction of the ratio of longitudinal-to-transverse response is discussed.

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein-coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Galpha12 and Galpha13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity.

Rho, Rac, and Cdc42 are small GTPases that regulate the formation of a variety of actin structures and the assembly of associated integrin complexes, but little is known about the target proteins that mediate their effects. Here we have used a motif-based search method to identify putative effector proteins for Rac and Cdc42. A search of the GenBankTM data base for similarity with the minimum Cdc42/Rac interactive binding (CRIB) region of a potential effector protein p65PAK has identified over 25 proteins containing a similar motif from a range of different species. These candidate Cdc42/Rac-binding proteins include family members of the mixed lineage kinases (MLK), a novel tyrosine kinase from Drosophila melanogaster (DPR2), a human protein MSE55, and several novel yeast and Caenorhabditis elegans proteins. Two murine p65PAK isoforms and a candidate protein from C. elegans, F09F7.5, interact strongly with the GTP form of both Cdc42 and Rac, but not Rho in a filter binding assay. Three additional candidate proteins, DPR2, MSE55, and MLK3 showed binding to the GTP form of Cdc42 and weaker binding with Rac, and again no interaction with Rho. These results indicate that proteins containing the CRIB motif bind to Cdc42 and/or Rac in a GTP-dependent manner, and they may, therefore, participate in downstream signaling.

The microtubule array in neuronal cells undergoes extensive growth, dynamics and rearrangements during neurite outgrowth. While little is known about how these changes are regulated, microtubule-associated proteins (MAPs) including tau protein are likely to perform an important role. Tau is one of the MAPs in mammalian brain. When isolated it is usually a mixture of several isoforms containing between 341 and 441 residues that arise from alternative splicing. Tau can be phosphorylated by several protein kinases. Phosphorylation at certain sites results in major structural and functional changes, as seen by changes in electrophoretic mobility, interaction with microtubules, molecular length and elasticity. Here we show that the sites of phosphorylation by four kinases (PKA, PKC, CK and CaMK) all lie in the C-terminal microtubule-binding half of tau, but only the phosphorylation by CaM kinase shows the pronounced shift in electrophoretic mobility characteristic for tau from Alzheimer neurofibrillary tangles. By using a combination of limited proteolysis, protein sequencing and protein engineering we show that a single phosphorylation site is responsible for this shift, located at Ser 405 in the C-terminal tail of the protein outside the region of internal repeats. Phosphorylation at this site not only reduces the electrophoretic mobility of tau, it also makes the protein long and stiff, as shown earlier. The site is likely to be phosphorylated in tau from Alzheimer neurofibrillary tangles.

Fluorescence polarization has been used to study the interaction of human alpha1-protease inhibitor (alpha1 PI; also called alpha1-antitrypsin) with two active site modified chymotrypsins (CT), dehydroalaninyl-195-alpha-CT (AnhCT) and N-methylhistidinyl-57-alpha-CT (MeCT). For the reaction of the fluorescein-labeled AnhCT (FAnhCT) with alpha 1 PI (Pi type MM, the predominant allelic form), a Kassoc of 1.8 x 10(7) M-1 was obtained by Scatchard analysis, which also indicated 1.3 binding sites. An alternate analysis using a direct dissociation plot, which assumes 1:1 binding, gave a Kassoc of 2.2 x 10(7) M-1. Fluorescein-labeled MeCT (FMeCT) binds somewhat more weakly to alpha PT (Kassoc = 1.2 x 10(6) M-1; 0.87 binding site). Similar results were obtained by using the proflavin displacement method to determine the binding constant for MeCT with alpha 1 PI (Kassoc = 1.0 x 10(6) M-1). With alpha 1 PI (ZZ type) in which the serum level is reduced and there is a strong tendency to develop chronic obstructive pulmonary disease, the Kassoc found by the fluorescence polarization method was similar to that for alpha 1 PI (MM type) for both CT derivatives. Alpha 1 PI (MM type), modified by oxidation with N-chlorosuccinimide, shows a reduced binding affinity for FAnhCT (Kassoc = 6.5 x 10(5) M-1) and no measurable binding with FMeCT (Kassoc less than 1 x 10(4) M-1). Previous studies have demonstrated that bovine CT forms very stable complexes with alpha 1 PI. In contrast, complexes formed with both active site modified CT derivatives undergo rapid dissociation as shown by the drop in the polarization value on dilution or on the addition of excess unlabeled chymotrypsin derivative. This weakened association suggests that, for reaction with alpha 1 PI, the enzyme active site serine is important in stabilizing the enzyme-inhibitor complex.

Tau proteins consist of a family of proteins, heterogeneous in size, which associate with microtubules in vivo and are induced during neurite outgrowth. In humans, tau is one of the major components of the pathognomonic neurofibrillary tangles in Alzheimer's disease brain. Screening of a cDNA library prepared from bovine brain led to the isolation of several cDNA clones encoding tau proteins with different N termini and differing by insertions or deletions, suggesting differential splicing of the tau transcripts. One of the N-terminal domains and the repeated C-terminal domain of the encoded tau proteins are recognized by polyclonal antibodies to bovine tau. The bovine tau proteins are highly homologous to murine and human tau, especially within the repeated C-terminal domain. Compared with murine and human tau, bovine tau contains the insertion of three longer segments, one of which is an additional characteristic repeat. Portions of tau proteins generated by in vitro translation were used to show that these repeats represent tubulin-binding domains, two of which are sufficient to bind to microtubules assembled from purified tubulin in the presence of taxol.

Sepharose 4B-bound bovine anhydrochymotrypsin (AnhCT), a catalytically inactive form of chymotrypsin, was shown to be effective for retaining active alpha-1-protease inhibitor (alpha-1-PI, also alpha-1-antitrypsin) from human plasma, while showing no measurable affinity for oxidized or protease complexed alpha-1-PI, or for most other plasma proteins. alpha-1-PI eluted from this resin with 0.1 M chymostatin retained full activity against trypsin, chymotrypsin, and elastase. In addition to alpha-1-PI, AnhCT-Sepharose binds a limited number of other plasma proteins. Using monospecific antisera to plasma protease inhibitors, one of these proteins was identified as inter-alpha-trypsin inhibitor, and it was recoverable in active form. Therefore, an AnhCT-Sepharose 4B resin has been demonstrated to be of value for isolating active forms of alpha-1-PI from solutions, and may also be useful for the isolation of inter-alpha-trypsin inhibitor.