Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals. IntroductionAcetylcholine (ACh) is a critical neurotransmitter in all animals. Among invertebrates, it is the most prevalent excitatory transmitter in the brain, sensory ganglia, and frequently the neuromuscular junction (NMJ). Among vertebrates, only a minority of neurons release ACh, but these signals play varying key roles. For instance, ACh signals at the NMJ, in the autonomic nervous system, and in subsets of the central nervous system, particularly projections arising from the brainstem and basal forebrain. Other cholinergic neuron populations in the brain include striatal interneurons, the stria vascularis-medial habenula-interpeduncular nucleus pathway, and sparse, incompletely characterized cell types such as intrinsic cholinergic interneurons in cortex 1 and hippocampus 2 . ACh helps to regulate attention 3 and wakefulness 4 , and participates in memory formation and consolidation 5 . ACh is also an important transmitter in glia, and between the nervous and immune systems 6 .Acetylcholine is synthesized pre-synaptically from choline and acetyl-CoA by choline acetyltransferase (ChAT), then packaged into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). A key, partially understood aspect of cholinergic signaling is co-release with other neurotransmitters, including GABA, ATP, and glutamate 7,8 . To understand the role of co-release, one must measure ACh release alongside emerging measurements of other neurotransmitters.Acetylcholine receptors are among the most diverse neurotransmitter receptor families. Humans possess five muscarinic G protein-coupled receptors (GPCRs) for ACh (mAChRs) with diverse expression in the brain and smooth, cardiac, and skeletal muscle. Vertebrate nicotinic ACh receptors (nAChRs) are pentameric ligand-gated cation channels. Humans have a total of 17 nAChR subunit genes, in five classes: 10 a, 4 b, and one each of g, d, and e. nAChRs occur with many subunit combinations 9 , and others may be undiscovered. Invertebrates also have AChgated chloride channels. On neurons, receptors can be localized pre-, post-, and extrasynaptically, often with different isoforms in each place 10

Anopheles mosquitoes are vectors of malaria, a potentially fatal blood disease affecting half a billion humans worldwide. These blood-feeding insects include in their antihemostatic arsenal a potent thrombin inhibitor, the flexible and cysteine-less anophelin.Here, we present a thorough structure-and-function analysis of thrombin inhibition by anophelin, including the 2.3-Å crystal structure of the human thrombin·anophelin complex. Anophelin residues 32-61 are well-defined by electron density, completely occupying the long cleft between the active site and exosite I. However, in striking contrast to substrates, the D50-R53 anophelin tetrapeptide occupies the active site cleft of the enzyme, whereas the upstream residues A35-P45 shield the regulatory exosite I, defining a unique reverse-binding mode of an inhibitor to the target proteinase. The extensive interactions established, the disruption of thrombin's active site charge-relay system, and the insertion of residue R53 into the proteinase S 1 pocket in an orientation opposed to productive substrates explain anophelin's remarkable specificity and resistance to proteolysis by thrombin. Complementary biophysical and functional characterization of point mutants and truncated versions of anophelin unambiguously establish the molecular mechanism of action of this family of serine proteinase inhibitors (I77). These findings have implications for the design of novel antithrombotics.coagulation inhibitor | macromolecular recognition | protease | X-ray structure | catalytic triad

Rab GTPases regulate discrete steps in vesicular transport pathways. Rabs require activation by specific guanine nucleotide exchange factors (GEFs) that stimulate the exchange of GDP for GTP. Rab27a controls motility and regulated exocytosis of secretory granules and related organelles. In melanocytes, Rab27a regulates peripheral transport of mature melanosomes by recruiting melanophilin and myosin Va. Here, we studied the activation of Rab27a in melanocytes. We identify Rab3GEP, previously isolated as a GEF for Rab3a, as the non-redundant Rab27a GEF. Similar to Rab27a-deficient ashen melanocytes, Rab3GEP-depleted cells show both clustering of melanosomes in the perinuclear area and loss of the Rab27a effector Mlph. Consistent with a role as an activator, levels of Rab27a-GTP are decreased in cells lacking Rab3GEP. Recombinant Rab3GEP exhibits guanine nucleotide exchange activity against Rab27a and Rab27b in vitro, in addition to its previously documented activity against Rab3. Our results indicate promiscuity in Rab GEF action and suggest that members of related but functionally distinct Rab subfamilies can be controlled by common activators.

Accurate chromosome segregation relies on microtubule end conversion, the ill-understood ability of kinetochores to transit from lateral microtubule attachment to durable association with dynamic microtubule plus-ends. The molecular requirements for this conversion and the underlying biophysical mechanisms are elusive. We reconstituted end conversion in vitro using two kinetochore components: the plus end–directed kinesin CENP-E and microtubule-binding Ndc80 complex, combined on the surface of a microbead. The primary role of CENP-E is to ensure close proximity between Ndc80 complexes and the microtubule plus-end, whereas Ndc80 complexes provide lasting microtubule association by diffusing on the microtubule wall near its tip. Together, these proteins mediate robust plus-end coupling during several rounds of microtubule dynamics, in the absence of any specialized tip-binding or regulatory proteins. Using a Brownian dynamics model, we show that end conversion is an emergent property of multimolecular ensembles of microtubule wall-binding proteins with finely tuned force-dependent motility characteristics.

Rab geranylgeranyl transferase (RGGT) catalyzes the posttranslational geranylgeranyl (GG) modification of (usually) two C-terminal cysteines in Rab GTPases. Here we studied the mechanism of the Rab geranylgeranylation reaction by bisphosphonate analogs in which one phosphonate group is replaced by a carboxylate (phosphonocarboxylate, PC). The phosphonocarboxylates used were 3-PEHPC, which was previously reported, and 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid ((؉)-3-IPEHPC), a >25-fold more potent related compound as measured by both IC 50 and K i . (؉)-3-IPEHPC behaves as a mixed-type inhibitor with respect to GG pyrophosphate (GGPP) and an uncompetitive inhibitor with respect to Rab substrates. We propose that phosphonocarboxylates prevent only the second GG transfer onto Rabs based on the following evidence. First, geranylgeranylation of Rab proteins ending with a single cysteine motif such as CAAX, is not affected by the inhibitors, either in vitro or in vivo. Second, the addition of an -AAX sequence onto Rab-CC proteins protects the substrate from inhibition by the inhibitors. Third, we demonstrate directly that in the presence of (؉)-3-IPEHPC, Rab-CC and Rab-CXC proteins are modified by only a single GG addition. The presence of (؉)-3-IPEHPC resulted in a preference for the Rab N-terminal cysteine to be modified first, suggesting an order of cysteine geranylgeranylation in RGGT catalysis. Our results further suggest that the inhibitor binds to a site distinct from the GGPP-binding site on RGGT. We suggest that phosphonocarboxylate inhibitors bind to a GG-cysteine binding site adjacent to the active site, which is necessary to align the mono-GG-Rab for the second GG addition. These inhibitors may represent a novel therapeutic approach in Rab-mediated diseases.Most proteins of the Ras-like GTPase superfamily need to be post-translationally modified by prenyl groups to associate with cellular membranes and to activate downstream effectors (1). Protein prenylation involves the formation of a thioether link between conserved C-terminal cysteines in protein substrates and farnesyl pyrophosphate (FPP) 7 or geranylgeranyl pyrophosphate (GGPP) (2, 3). These prenyl pyrophosphates, which originate from the mevalonate pathway are utilized by three different prenyltransferase enzymes (2, 3). Farnesyl transferase (FT) and geranylgeranyl transferase type I (GGT-I) transfer FPP or GGPP, respectively, to a cysteine residue in the context of a C-terminal CAAX motif, where C is a cysteine, A is an aliphatic residue, and X is any amino acid. X contributes significantly to substrate specificity in FT and GGT-I (2). Rab geranylgeranyl transferase (RGGT) is distinct from FT and GGT-I in that it specifically recognizes Rab proteins, which vary in their C terminus containing CCXX, XCXC, XXCC, CCXXX, CAAX, and other motifs (3). RGGT exhibits exquisite specificity for Rabs due to the strict requirement of Rab escort protein (REP), which is a general Rab-GDP binding protein (4). RGGT is a heterodimeric enzyme ...

The coordination between late mitotic events such as poleward chromosome motion, spindle elongation, DNA decondensation, and nuclear envelope reformation (NER) is crucial for the completion of chromosome segregation at the anaphase-telophase transition. Mitotic exit is driven by a decrease of Cdk1 kinase activity and an increase of PP1/PP2A phosphatase activities. More recently, Aurora kinases have also emerged as master regulators of late mitotic events and cytokinesis. Aurora A is mainly associated with spindle poles throughout mitosis and midbody during telophase, whereas Aurora B re-localizes from centromeres in early mitosis to the spindle midzone and midbody as cells progress from anaphase to the completion of cytokinesis. Functional studies, together with the identification of a phosphorylation gradient during anaphase, established Aurora B as a major player in the organization of the spindle midzone and in the spatiotemporal coordination between chromosome segregation and NER. Aurora A has been less explored, but a cooperative role in spindle midzone stability has also been proposed, implying that both Aurora A and B contribute to accurate chromosome segregation during mitotic exit. Here, we review the roles of the Aurora kinases in the regulation of late mitotic events and discuss how they work together with other mitotic players to ensure an error-free mitosis.

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Rab GTPases are important determinants of organelle identity and regulators of vesicular transport pathways. Consequently, each Rab occupies a highly specific subcellular localization. However, the precise mechanisms governing Rab targeting remain unclear. Guanine nucleotide exchange factors (GEFs), putative membrane-resident targeting factors and effector binding have all been implicated as critical regulators of Rab targeting. Here, we address these issues using Rab27a targeting to melanosomes as a model system. Rab27a regulates motility of lysosome-related organelles and secretory granules. Its effectors have been characterized extensively, and we have identified Rab3GEP as the non-redundant Rab27a GEF in melanocytes (Figueiredo AC et al. Rab3GEP is the non-redundant guanine nucleotide exchange factor for Rab27a in melanocytes. J Biol Chem 2008;283:23209–23216). Using Rab27a mutants that show impaired binding to representatives of all four Rab27a effector subgroups, we present evidence that effector binding is not essential for targeting of Rab27a to melanosomes. In contrast, we observed that knockdown of Rab3GEP resulted in mis-targeting of Rab27a, suggesting that Rab3GEP activity is required for correct targeting of Rab27a. However, the identification of Rab27a mutants that undergo efficient GDP/GTP exchange in the presence of Rab3GEP in vitro but are mis-targeted in a cellular context indicates that nucleotide loading is not the sole determinant of subcellular targeting of Rab27a. Our data support a model in which exchange activity, but not effector binding, represents one essential factor that contributes to membrane targeting of Rab proteins.