Search

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.