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1.
Protein Sci ; 31(6): e4334, 2022 06.
Article in English | MEDLINE | ID: mdl-35634773

ABSTRACT

Human androgen receptor contains a large N-terminal domain (AR-NTD) that is highly dynamic and this poses a major challenge for experimental and computational analysis to decipher its conformation. Misfolding of the AR-NTD is implicated in prostate cancer and Kennedy's disease, yet our knowledge of its structure is limited to primary sequence information of the chain and a few functionally important secondary structure motifs. Here, we employed an innovative combination of molecular dynamics simulations and circuit topology (CT) analysis to identify the tertiary structure of AR-NTD. We found that the AR-NTD adopts highly dynamic loopy conformations with two identifiable regions with distinct topological make-up and dynamics. This consists of a N-terminal region (NR, residues 1-224) and a C-terminal region (CR, residues 225-538), which carries a dense core. Topological mapping of the dynamics reveals a traceable time-scale dependent topological evolution. NR adopts different positioning with respect to the CR and forms a cleft that can partly enclose the hormone-bound ligand-binding domain (LBD) of the androgen receptor. Furthermore, our data suggest a model in which dynamic NR and CR compete for binding to the DNA-binding domain of the receptor, thereby regulating the accessibility of its DNA-binding site. Our approach allowed for the identification of a previously unknown regulatory binding site within the CR core, revealing the structural mechanisms of action of AR inhibitor EPI-001, and paving the way for other drug discovery applications.


Subject(s)
Prostatic Neoplasms , Receptors, Androgen , Androgen Receptor Antagonists/chemistry , Androgen Receptor Antagonists/pharmacology , DNA , Humans , Male , Prostatic Neoplasms/metabolism , Protein Domains , Receptors, Androgen/chemistry , Receptors, Androgen/genetics , Receptors, Androgen/metabolism
2.
PLoS One ; 13(8): e0200759, 2018.
Article in English | MEDLINE | ID: mdl-30110347

ABSTRACT

The use of genetically encoded 'self-labeling tags' with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved Drosophila melanogaster transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.


Subject(s)
Drosophila melanogaster/cytology , Fluorescent Dyes , Immunohistochemistry , Neurons/cytology , Staining and Labeling , Animals , Animals, Genetically Modified , Brain/cytology , Female , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Microscopy, Confocal , Molecular Structure
3.
Genetics ; 205(4): 1399-1408, 2017 04.
Article in English | MEDLINE | ID: mdl-28209589

ABSTRACT

Labeling and visualizing cells and subcellular structures within thick tissues, whole organs, and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form submicron synapses but have arbors that may span millimeters in length. Traditionally, labeling is achieved by immunofluorescence; however, diffusion of antibody molecules (>100 kDa) is slow and often results in uneven labeling with very poor penetration into the center of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week (Drosophila brain) and months (mice). Recently, we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo, and TMP for tissue labeling; this resulted in >100-fold increase in labeling speed in both mice and Drosophila, at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS- and LexA-responsive CLIPf, SNAPf, and Halo chemical labeling reagents for flies. These multimerized tags, with translational enhancers, display up to 64-fold increase in sensitivity over first-generation reagents. In addition, we developed a suite of conditional reporters (4xSNAPf tag and CLIPf-SNAPf-Halo2) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional, and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.


Subject(s)
Drosophila/cytology , Optical Imaging/methods , Staining and Labeling/methods , Alkyl and Aryl Transferases/genetics , Alkyl and Aryl Transferases/metabolism , Animals , Brain/cytology , Brain/metabolism , Drosophila/genetics , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sensitivity and Specificity
4.
Sci Rep ; 6: 38863, 2016 12 13.
Article in English | MEDLINE | ID: mdl-27958322

ABSTRACT

Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition.


Subject(s)
Drosophila/genetics , Imaging, Three-Dimensional/methods , Microscopy, Electron/methods , Neurons/ultrastructure , Animals , Animals, Genetically Modified , Axons/ultrastructure , Dendrites/ultrastructure , Drosophila/ultrastructure , Image Enhancement , X-Rays
5.
Proc Natl Acad Sci U S A ; 111(36): E3805-14, 2014 Sep 09.
Article in English | MEDLINE | ID: mdl-25157152

ABSTRACT

Genetically encoded fluorescent proteins and immunostaining are widely used to detect cellular and subcellular structures in fixed biological samples. However, for thick or whole-mount tissue, each approach suffers from limitations, including limited spectral flexibility and lower signal or slow speed, poor penetration, and high background labeling, respectively. We have overcome these limitations by using transgenically expressed chemical tags for rapid, even, high-signal and low-background labeling of thick biological tissues. We first construct a platform of widely applicable transgenic Drosophila reporter lines, demonstrating that chemical labeling can accelerate staining of whole-mount fly brains by a factor of 100. Using viral vectors to deliver chemical tags into the mouse brain, we then demonstrate that this labeling strategy works well in mice. Thus this tag-based approach drastically improves the speed and specificity of labeling genetically marked cells in intact and/or thick biological samples.


Subject(s)
Brain/metabolism , Fluorescent Dyes/metabolism , Staining and Labeling/methods , Animals , Drosophila , Mice, Inbred C57BL , Neurons/cytology , Neurons/metabolism
6.
J Cell Sci ; 126(Pt 3): 838-49, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-23264732

ABSTRACT

Axon degeneration is observed at the early stages of many neurodegenerative conditions and this often leads to subsequent neuronal loss. We previously showed that inactivating the c-Jun N-terminal kinase (JNK) pathway leads to axon degeneration in Drosophila mushroom body (MB) neurons. To understand this process, we screened candidate suppressor genes and found that the Wallerian degeneration slow (Wld(S)) protein blocked JNK axonal degeneration. Although the nicotinamide mononucleotide adenylyltransferase (Nmnat1) portion of Wld(S) is required, we found that its nicotinamide adenine dinucleotide (NAD(+)) enzyme activity and the Wld(S) N-terminus (N70) are dispensable, unlike axotomy models of neurodegeneration. We suggest that Wld(S)-Nmnat protects against axonal degeneration through chaperone activity. Furthermore, ectopically expressed heat shock proteins (Hsp26 and Hsp70) also protected against JNK and Nmnat degeneration phenotypes. These results suggest that molecular chaperones are key in JNK- and Nmnat-regulated axonal protective functions.


Subject(s)
Axons/metabolism , Drosophila melanogaster/metabolism , Molecular Chaperones/metabolism , Nerve Tissue Proteins/metabolism , Wallerian Degeneration/metabolism , ADP Ribose Transferases/metabolism , Animals , Axons/pathology , Drosophila Proteins/metabolism , HSP72 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , MAP Kinase Kinase 4/metabolism , Mushroom Bodies/pathology , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Signal Transduction , Wallerian Degeneration/pathology
7.
Development ; 139(1): 165-77, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22147954

ABSTRACT

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, I analyzed the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially. I found that components of the Wnt/Planar Cell Polarity (PCP) pathway control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. I found that Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus.


Subject(s)
Axons/physiology , Drosophila melanogaster/embryology , Mushroom Bodies/cytology , Neurogenesis/physiology , Wnt Signaling Pathway/physiology , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cell Polarity/physiology , DNA-Binding Proteins/metabolism , Dishevelled Proteins , Drosophila Proteins/metabolism , Frizzled Receptors/metabolism , Immunohistochemistry , LIM Domain Proteins/metabolism , Membrane Proteins/metabolism , Mushroom Bodies/embryology , Phosphoproteins/metabolism
8.
Dev Biol ; 339(1): 65-77, 2010 Mar 01.
Article in English | MEDLINE | ID: mdl-20035736

ABSTRACT

Signaling proteins often control multiple aspects of cell morphogenesis. Yet the mechanisms that govern their pleiotropic behavior are often unclear. Here we show activity levels and timing mechanisms determine distinct aspects of Jun N-terminal kinase (JNK) pathway dependent axonal morphogenesis in Drosophila mushroom body (MB) neurons. In the complete absence of Drosophila JNK (Basket), MB axons fail to stabilize, leading to their subsequent degeneration. However, with a partial loss of Basket (Bsk), or of one of the upstream JNK kinases, Hemipterous or Mkk4, these axons overextend. This suggests that Bsk activity prevents axons from destabilizing, resulting in degeneration and overextension beyond their terminal targets. These distinct phenotypes require different threshold activities involving the convergent action of two distinct JNK kinases. We show that sustained Bsk signals are essential throughout development and act additively but are dispensable at adulthood. We also suggest that graded Bsk inputs are translated into AP-1 transcriptional outputs consisting of Fos and Jun proteins.


Subject(s)
Axons , MAP Kinase Kinase 4/metabolism , Animals , Drosophila , Morphogenesis , Phosphorylation
9.
Development ; 135(24): 4025-35, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19004854

ABSTRACT

Proper nerve connections form when growing axons terminate at the correct postsynaptic target. Here I show that Transforming growth factor beta (TGFbeta) signals regulate axon growth. In most contexts, TGFbeta signals are tightly linked to Smad transcriptional activity. Although known to exist, how Smad-independent pathways mediate TGFbeta responses in vivo is unclear. In Drosophila mushroom body (MB) neurons, loss of the TGFbeta receptor Baboon (Babo) results in axon overextension. Conversely, misexpression of constitutively active Babo results in premature axon termination. Smad activity is not required for these phenotypes. This study shows that Babo signals require the Rho GTPases Rho1 and Rac, and LIM kinase1 (LIMK1), which regulate the actin cytoskeleton. Contrary to the well-established receptor activation model, in which type 1 receptors act downstream of type 2 receptors, this study shows that the type 2 receptors Wishful thinking (Wit) and Punt act downstream of the Babo type 1 receptor. Wit and Punt regulate axon growth independently, and interchangeably, through LIMK1-dependent and -independent mechanisms. Thus, novel TGFbeta receptor interactions control non-Smad signals and regulate multiple aspects of axonal development in vivo.


Subject(s)
Axons/ultrastructure , Drosophila Proteins/physiology , Drosophila/growth & development , Drosophila/physiology , Neurogenesis/physiology , Smad Proteins/physiology , Transforming Growth Factor beta/physiology , Actin Depolymerizing Factors/genetics , Actin Depolymerizing Factors/physiology , Activin Receptors/genetics , Activin Receptors/physiology , Animals , Animals, Genetically Modified , Axons/physiology , Drosophila/genetics , Drosophila Proteins/genetics , Genes, Insect , Lim Kinases/genetics , Lim Kinases/physiology , Models, Neurological , Mushroom Bodies/growth & development , Mushroom Bodies/physiology , Mushroom Bodies/ultrastructure , Mutation , Neurogenesis/genetics , Receptors, Cell Surface/genetics , Receptors, Cell Surface/physiology , Signal Transduction , Smad Proteins/genetics , Transforming Growth Factor beta/genetics , rac GTP-Binding Proteins/genetics , rac GTP-Binding Proteins/physiology , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/physiology
10.
Neuron ; 44(5): 779-93, 2004 Dec 02.
Article in English | MEDLINE | ID: mdl-15572110

ABSTRACT

Rho GTPases are essential regulators of cytoskeletal reorganization, but how they do so during neuronal morphogenesis in vivo is poorly understood. Here we show that the actin depolymerization factor cofilin is essential for axon growth in Drosophila neurons. Cofilin function in axon growth is inhibited by LIM kinase and activated by Slingshot phosphatase. Dephosphorylating cofilin appears to be the major function of Slingshot in regulating axon growth in vivo. Genetic data provide evidence that Rho or Rac/Cdc42, via effector kinases Rok or Pak, respectively, activate LIM kinase to inhibit axon growth. Importantly, Rac also activates a Pak-independent pathway that promotes axon growth, and different RacGEFs regulate these distinct pathways. These genetic analyses reveal convergent and divergent pathways from Rho GTPases to the cytoskeleton during axon growth in vivo and suggest that different developmental outcomes could be achieved by biases in pathway selection.


Subject(s)
Axons/physiology , Signal Transduction/physiology , rho GTP-Binding Proteins/physiology , Actin Depolymerizing Factors , Actins/metabolism , Animals , Cell Division/physiology , DNA-Binding Proteins/metabolism , Drosophila , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Intracellular Signaling Peptides and Proteins , Lim Kinases , Microfilament Proteins/metabolism , Microfilament Proteins/physiology , Mushroom Bodies/innervation , Neurons/cytology , Phenotype , Phosphoprotein Phosphatases , Phosphoric Monoester Hydrolases/physiology , Phosphorylation , Polymers/metabolism , Protein Kinases/physiology , Protein Serine-Threonine Kinases/metabolism , cdc42 GTP-Binding Protein/metabolism , p21-Activated Kinases , rac GTP-Binding Proteins/metabolism , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases
11.
Development ; 130(7): 1419-28, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12588856

ABSTRACT

Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. We used the tracheal system of Drosophila to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. We found that a reduced Rac activity led to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibited dynamic epithelial cell rearrangement. Hyperactivation of Rac, on the other hand, inhibited assembly of newly synthesized E-Cadherin into cell junctions and caused loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules.


Subject(s)
Cell Movement/physiology , Drosophila/embryology , Epithelium/embryology , rac GTP-Binding Proteins/metabolism , Animals , Cadherins/metabolism , Cell Adhesion/physiology , Embryonic Induction/genetics , Embryonic Induction/physiology , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Up-Regulation/physiology , rac GTP-Binding Proteins/genetics
12.
Nature ; 416(6879): 438-42, 2002 Mar 28.
Article in English | MEDLINE | ID: mdl-11919634

ABSTRACT

Rac GTPases regulate the actin cytoskeleton to control changes in cell shape. To date, the analysis of Rac function during development has relied heavily on the use of dominant mutant isoforms. Here, we use loss-of-function mutations to show that the three Drosophila Rac genes, Rac1, Rac2 and Mtl, have overlapping functions in the control of epithelial morphogenesis, myoblast fusion, and axon growth and guidance. They are not required for the establishment of planar cell polarity, as had been suggested on the basis of studies using dominant mutant isoforms. The guanine nucleotide exchange factor, Trio, is essential for Rac function in axon growth and guidance, but not for epithelial morphogenesis or myoblast fusion. Different Rac activators thus act in different developmental processes. The specific cellular response to Rac activation may be determined more by the upstream activator than the specific Rac protein involved.


Subject(s)
Drosophila Proteins/physiology , Drosophila/physiology , rac GTP-Binding Proteins/physiology , rac1 GTP-Binding Protein/physiology , Animals , Axons/physiology , Cell Movement , Cell Polarity , Drosophila/embryology , Drosophila/genetics , Drosophila Proteins/genetics , Female , Genes, Insect , Male , Mutation , Nervous System/embryology , rac GTP-Binding Proteins/genetics , rac1 GTP-Binding Protein/genetics , RAC2 GTP-Binding Protein
13.
Nature ; 416(6879): 442-7, 2002 Mar 28.
Article in English | MEDLINE | ID: mdl-11919635

ABSTRACT

Growth, guidance and branching of axons are all essential processes for the precise wiring of the nervous system. Rho family GTPases transduce extracellular signals to regulate the actin cytoskeleton. In particular, Rac has been implicated in axon growth and guidance. Here we analyse the loss-of-function phenotypes of three Rac GTPases in Drosophila mushroom body neurons. We show that progressive loss of combined Rac1, Rac2 and Mtl activity leads first to defects in axon branching, then guidance, and finally growth. Expression of a Rac1 effector domain mutant that does not bind Pak rescues growth, partially rescues guidance, but does not rescue branching defects of Rac mutant neurons. Mosaic analysis reveals both cell autonomous and non-autonomous functions for Rac GTPases, the latter manifesting itself as a strong community effect in axon guidance and branching. These results demonstrate the central role of Rac GTPases in multiple aspects of axon development in vivo, and suggest that axon growth, guidance and branching could be controlled by differential activation of Rac signalling pathways.


Subject(s)
Axons/physiology , Drosophila Proteins/physiology , rac GTP-Binding Proteins/physiology , rac1 GTP-Binding Protein/physiology , Amino Acid Sequence , Animals , Axons/enzymology , Cell Division , Cell Movement , Drosophila , Drosophila Proteins/genetics , Genes, Insect , Molecular Sequence Data , Mutation , rac GTP-Binding Proteins/genetics , rac1 GTP-Binding Protein/genetics , RAC2 GTP-Binding Protein
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