Higher-order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes

The activities of neuronal signaling receptors depend heavily on the maturation state of the endosomal compartments in which they reside. However, it remains unclear how the distribution of these compartments within the uniquely complex morphology of neurons is regulated and how this distribution itself affects signaling. Here, we identified mechanisms by which Sorting Nexin 16 (SNX16) controls neuronal endosomal maturation and distribution. We found that higher-order assembly of SNX16 via its coiled-coil (CC) domain drives membrane tubulation in vitro and endosome association in cells. In Drosophila melanogaster motor neurons, activation of Rab5 and CC-dependent self-association of SNX16 lead to its endosomal enrichment, accumulation in Rab5- and Rab7-positive tubulated compartments in the cell body, and concomitant depletion of SNX16-positive endosomes from the synapse. This results in accumulation of synaptic growth–promoting bone morphogenetic protein receptors in the cell body and correlates with increased synaptic growth. Our results indicate that Rab regulation of SNX16 assembly controls the endosomal distribution and signaling activities of receptors in neurons.

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Higher order assembly of Sorting Nexin 16 controls tubulation and distribution of neuronal endosomes

10.1101/469932 ◽

2018 ◽

Author(s):

ShiYu Wang ◽

Zechuan Zhao ◽

Avital A. Rodal

Keyword(s):

Cell Body ◽

Motor Neurons ◽

Coiled Coil ◽

Higher Order ◽

Synaptic Growth ◽

Sorting Nexin ◽

Bmp Receptors ◽

Self Association ◽

Signaling Receptors

AbstractThe activities of neuronal signaling receptors depend on the maturation state of the endosomal compartments in which they reside. However, it remains unclear how the distribution of these compartments within the uniquely complex morphology of neurons is regulated, and how this distribution itself affects signaling. Here we identified mechanisms by which Sorting Nexin 16 (SNX16) controls neuronal endosomal maturation and distribution. We found that higher-order assembly of SNX16 via its coiled-coil domain drives membrane tubulation in vitro and endosome association in cells. In Drosophila motor neurons, activation of Rab5 and coiled-coil-dependent self-association of SNX16 lead to its endosomal enrichment, concomitant with depletion of SNX16-positive endosomes from the synapse, and their accumulation as Rab5- and Rab7-positive tubulated compartments at the cell body. This leads to higher levels of synaptic growth-promoting BMP receptors at the cell body, and correlates with increased synaptic growth. Our results indicate that Rab regulation of SNX16 assembly controls the endosomal distribution and signaling activities of neuronal receptors.

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A presynaptic endosomal trafficking pathway controls synaptic growth signaling

The Journal of Cell Biology ◽

10.1083/jcb.201009052 ◽

2011 ◽

Vol 193 (1) ◽

pp. 201-217 ◽

Cited By ~ 57

Author(s):

Avital A. Rodal ◽

Aline D. Blunk ◽

Yulia Akbergenova ◽

Ramon A. Jorquera ◽

Lauren K. Buhl ◽

...

Keyword(s):

Endocytic Pathway ◽

Physical Interaction ◽

Endosomal Trafficking ◽

Synaptic Growth ◽

Recycling Endosomes ◽

Sorting Nexin ◽

Trafficking Pathway ◽

Early Endosomes ◽

Protein Receptors ◽

Escrt Complex

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila melanogaster. In this paper, we show that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)–mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments.

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The TRIM5α B-Box 2 Domain Promotes Cooperative Binding to the Retroviral Capsid by Mediating Higher-Order Self-Association

Journal of Virology ◽

10.1128/jvi.01548-08 ◽

2008 ◽

Vol 82 (23) ◽

pp. 11495-11502 ◽

Cited By ~ 84

Author(s):

Xing Li ◽

Joseph Sodroski

Keyword(s):

Virus Entry ◽

Coiled Coil ◽

Higher Order ◽

Restriction Factor ◽

Cooperative Binding ◽

Specific Function ◽

Retroviral Infection ◽

Spry Domain ◽

Dense Arrays ◽

Self Association

ABSTRACT The retroviral restriction factor, TRIM5α, blocks infection of a spectrum of retroviruses soon after virus entry into the cell. TRIM5α consists of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. The B-box 2 domain is essential for retrovirus restriction by TRIM5α, but its specific function is unknown. We show here that the B-box 2 domain mediates higher-order self-association of TRIM5αrh oligomers. This self-association increases the efficiency of TRIM5α binding to the retroviral capsid, thus potentiating restriction of retroviral infection. The contribution of the B-box 2 domain to cooperative TRIM5α association with the retroviral capsid explains the conditional nature of the restriction phenotype exhibited by some B-box 2 TRIM5α mutants; the potentiation of capsid binding that results from B-box 2-mediated self-association is essential for restriction when B30.2(SPRY) domain-mediated interactions with the retroviral capsid are weak. Thus, B-box 2-dependent higher-order self-association and B30.2(SPRY)-dependent capsid binding represent complementary mechanisms whereby sufficiently dense arrays of capsid-bound TRIM5α proteins can be achieved.

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The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies

eLife ◽

10.7554/elife.07236 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 30

Author(s):

Matthew A Cottee ◽

Nadine Muschalik ◽

Steven Johnson ◽

Joanna Leveson ◽

Jordan W Raff ◽

...

Keyword(s):

Point Mutations ◽

Coiled Coil ◽

Higher Order ◽

Centriole Duplication ◽

Terminal Domain ◽

Coiled Coil Domain ◽

Centriole Assembly ◽

Tetramer Structure

Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

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Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518280113 ◽

2016 ◽

Vol 113 (5) ◽

pp. 1238-1243 ◽

Cited By ~ 83

Author(s):

Sergei A. Grigoryev ◽

Gavin Bascom ◽

Jenna M. Buckwalter ◽

Michael B. Schubert ◽

Christopher L. Woodcock ◽

...

Keyword(s):

Strong Dependence ◽

Eukaryotic Cell ◽

Higher Order ◽

Linker Histone ◽

Cell Nuclei ◽

Metaphase Chromosomes ◽

Chromosome Conformation ◽

Interphase Cell ◽

Self Association

The architecture of higher-order chromatin in eukaryotic cell nuclei is largely unknown. Here, we use electron microscopy-assisted nucleosome interaction capture (EMANIC) cross-linking experiments in combination with mesoscale chromatin modeling of 96-nucleosome arrays to investigate the internal organization of condensed chromatin in interphase cell nuclei and metaphase chromosomes at nucleosomal resolution. The combined data suggest a novel hierarchical looping model for chromatin higher-order folding, similar to rope flaking used in mountain climbing and rappelling. Not only does such packing help to avoid tangling and self-crossing, it also facilitates rope unraveling. Hierarchical looping is characterized by an increased frequency of higher-order internucleosome contacts for metaphase chromosomes compared with chromatin fibers in vitro and interphase chromatin, with preservation of a dominant two-start zigzag organization associated with the 30-nm fiber. Moreover, the strong dependence of looping on linker histone concentration suggests a hierarchical self-association mechanism of relaxed nucleosome zigzag chains rather than longitudinal compaction as seen in 30-nm fibers. Specifically, concentrations lower than one linker histone per nucleosome promote self-associations and formation of these looped networks of zigzag fibers. The combined experimental and modeling evidence for condensed metaphase chromatin as hierarchical loops and bundles of relaxed zigzag nucleosomal chains rather than randomly coiled threads or straight and stiff helical fibers reconciles aspects of other models for higher-order chromatin structure; it constitutes not only an efficient storage form for the genomic material, consistent with other genome-wide chromosome conformation studies that emphasize looping, but also a convenient organization for local DNA unraveling and genome access.

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A B-Box 2 Surface Patch Important for TRIM5α Self-Association, Capsid Binding Avidity, and Retrovirus Restriction

Journal of Virology ◽

10.1128/jvi.01307-09 ◽

2009 ◽

Vol 83 (20) ◽

pp. 10737-10751 ◽

Cited By ~ 107

Author(s):

Felipe Diaz-Griffero ◽

Xu-rong Qin ◽

Fumiaki Hayashi ◽

Takanori Kigawa ◽

Andres Finzi ◽

...

Keyword(s):

Coiled Coil ◽

Higher Order ◽

Surface Patch ◽

Resonance Structure ◽

Nuclear Magnetic Resonance Structure ◽

Major Mechanism ◽

Cytoplasmic Bodies ◽

Surface Alteration ◽

Self Association ◽

Hiv 1

ABSTRACT TRIM5α is a tripartite motif (TRIM) protein that consists of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. The TRIM5αrh protein from rhesus monkeys recognizes the human immunodeficiency virus type 1 (HIV-1) capsid as it enters the host cell and blocks virus infection prior to reverse transcription. HIV-1-restricting ability can be eliminated by disruption of the B-box 2 domain. Changes in the TRIM5αrh B-box 2 domain have been associated with alterations in TRIM5αrh turnover, the formation of cytoplasmic bodies and higher-order oligomerization. We present here the nuclear magnetic resonance structure of the TRIM5 B-box 2 domain and identify an unusual hydrophobic patch (cluster 1) on the domain surface. Alteration of cluster 1 or the flanking arginine 121 resulted in various degrees of inactivation of HIV-1 restriction, in some cases depending on compensatory changes in other nearby charged residues. For this panel of TRIM5αrh B-box 2 mutants, inhibition of HIV-1 infection was strongly correlated with higher-order self-association and binding affinity for capsid complexes but not with TRIM5αrh half-life or the formation of cytoplasmic bodies. Thus, promoting cooperative TRIM5αrh interactions with the HIV-1 capsid represents a major mechanism whereby the B-box 2 domain potentiates HIV-1 restriction.

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Phospholamban and sarcolipin share similar transmembrane zipper motifs that control self-association affinity and oligomer stoichiometry

10.1101/701797 ◽

2019 ◽

Cited By ~ 1

Author(s):

Nicholas R. DesLauriers ◽

Bengt Svensson ◽

David D. Thomas ◽

Joseph M. Autry

Keyword(s):

Self Assembly ◽

Resonance Energy ◽

Higher Order ◽

Site Directed Mutagenesis ◽

Heptad Repeat ◽

Steric Interaction ◽

Structural Determinants ◽

Sds Page ◽

Self Association

AbstractWe have characterized the structural determinants of phospholamban (PLB) and sarcolipin (SLN) self-association using site-directed mutagenesis, SDS-PAGE, and fluorescence resonance energy transfer (FRET) microscopy. PLB and SLN are single-pass transmembrane (TM) peptides that are critically involved in regulation of contractility in cardiac and skeletal muscle via reversible inhibition of calcium (Ca) transport by SERCA. PLB and SLN also exhibit ion channel activity in vitro, yet the physiological significance of these functions is unknown. Here we have determined that structural insights offered by the tetrameric PLB Cys41 to Leu (C41L) mutation, a mutant with four possible leucine/isoleucine zipper interactions for stabilizing PLB tetramers. Using scanning alanine mutagenesis and SDS-PAGE, we have determined the C41L-PLB tetramer is destabilized by mutation of Leu37 to Ala (L37A) or Ile40 to Ala (I40A), which are the same a- and d-arm residues stabilizing the PLB pentamer via leucine/isoleucine zippers, highlighting the importance of these two zippers in PLB higher-order oligomerization. The new possible zipper arm in C41L-PLB (N34, C41L, I48) did not contribute to tetramerization. On the other hand, we determined that tetramer conversion back to pentamer was induced by alanine mutation of Ile48, a residue located on the e-arm below C41L, implicating steric interaction and restriction are the stabilizing and destabilizing forces that control the distribution between pentamer and tetramer populations. We propose that the e-arm and hydrophobic residues in the adjacent b-arm act as secondary structural motifs that help control the stoichiometry of PLB oligomerization. FRET microscopy and alanine mutagenesis of SLN residues Val14 (V14A) or Leu21 (L21A) decreased the binding affinity of the SLN‒SLN complex, demonstrating the importance of each residue in mediating self-association. Helical wheel analysis supports a heptad-repeat TM zipper mechanism of SLN oligomerization, similar to the 3.5 residue/turn Leu and Ile zippers found in PLB pentamers. Collectively, our studies add new insights on the conservation of homologous hydrophobic 3-4 pattern of residues in zipper motifs that mediate PLB and SLN self-assembly. We propose that the importance of these apolar, steric interactions in the TM domain are widespread in stabilizing higher-order oligomerization of membrane proteins.

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Structure of the tripartite motif of KAP1/TRIM28 identifies molecular interfaces required for transcriptional silencing of retrotransposons

10.1101/505677 ◽

2018 ◽

Author(s):

Guido A. Stoll ◽

Shun-ichiro Oda ◽

Zheng-Shan Chong ◽

Minmin Yu ◽

Stephen H. McLaughlin ◽

...

Keyword(s):

Self Assembly ◽

Coiled Coil ◽

Transcriptional Silencing ◽

Higher Order ◽

Significant Loss ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Krab Domain ◽

Tripartite Motif ◽

Self Association

AbstractTranscription of transposable elements is tightly regulated to prevent damage to the genome. The family of KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, which controls the assembly of an epigenetic silencing complex including histone H3K9 methyltransferase SETDB1. The chromatin remodeling activities of this complex repress transcription of the targeted transposable element and any adjacent genes. Here, we use biophysical and structural approaches to show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevented higher-order oligomerization without a significant loss of retrotransposon silencing activity in a cell-based assay, indicating that, in contrast to other TRIM family members, self-assembly is not essential for the function of KAP1. The crystal structure of the KAP1 RBCC dimer identifies the KRAB domain binding site, in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 RBCC motif responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.

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Global transcriptome profile of the developmental principles of in vitro iPSC-to-motor neuron differentiation

BMC Molecular and Cell Biology ◽

10.1186/s12860-021-00343-z ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Emilia Solomon ◽

Katie Davis-Anderson ◽

Blake Hovde ◽

Sofiya Micheva-Viteva ◽

Jennifer Foster Harris ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Motor Neuron ◽

Motor Neurons ◽

Acetylcholine Receptors ◽

Gene Networks ◽

Spinal Cord Injuries ◽

Regulatory Gene ◽

Transcriptome Profile

Abstract Background Human induced pluripotent stem cells (iPSC) have opened new avenues for regenerative medicine. Consequently, iPSC-derived motor neurons have emerged as potentially viable therapies for spinal cord injuries and neurodegenerative disorders including Amyotrophic Lateral Sclerosis. However, direct clinical application of iPSC bears in itself the risk of tumorigenesis and other unforeseeable genetic or epigenetic abnormalities. Results Employing RNA-seq technology, we identified and characterized gene regulatory networks triggered by in vitro chemical reprogramming of iPSC into cells with the molecular features of motor neurons (MNs) whose function in vivo is to innervate effector organs. We present meta-transcriptome signatures of 5 cell types: iPSCs, neural stem cells, motor neuron progenitors, early motor neurons, and mature motor neurons. In strict response to the chemical stimuli, along the MN differentiation axis we observed temporal downregulation of tumor growth factor-β signaling pathway and consistent activation of sonic hedgehog, Wnt/β-catenin, and Notch signaling. Together with gene networks defining neuronal differentiation (neurogenin 2, microtubule-associated protein 2, Pax6, and neuropilin-1), we observed steady accumulation of motor neuron-specific regulatory genes, including Islet-1 and homeobox protein HB9. Interestingly, transcriptome profiling of the differentiation process showed that Ca2+ signaling through cAMP and LPC was downregulated during the conversion of the iPSC to neural stem cells and key regulatory gene activity of the pathway remained inhibited until later stages of motor neuron formation. Pathways shaping the neuronal development and function were well-represented in the early motor neuron cells including, neuroactive ligand-receptor interactions, axon guidance, and the cholinergic synapse formation. A notable hallmark of our in vitro motor neuron maturation in monoculture was the activation of genes encoding G-coupled muscarinic acetylcholine receptors and downregulation of the ionotropic nicotinic acetylcholine receptors expression. We observed the formation of functional neuronal networks as spontaneous oscillations in the extracellular action potentials recorded on multi-electrode array chip after 20 days of differentiation. Conclusions Detailed transcriptome profile of each developmental step from iPSC to motor neuron driven by chemical induction provides the guidelines to novel therapeutic approaches in the re-construction efforts of muscle innervation.

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Structures of mouse and human GITR–GITRL complexes reveal unique TNF superfamily interactions

Nature Communications ◽

10.1038/s41467-021-21563-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Feng Wang ◽

Bryant Chau ◽

Sean M. West ◽

Christopher R. Kimberlin ◽

Fei Cao ◽

...

Keyword(s):

T Cells ◽

Cell Signaling ◽

Tumor Necrosis ◽

Immune Cell ◽

Linear Chain ◽

Tumor Necrosis Factor Receptor ◽

Higher Order ◽

Membrane Structures ◽

Receptor Interface

AbstractGlucocorticoid-induced tumor necrosis factor receptor-related protein (GITR) and GITR ligand (GITRL) are members of the tumor necrosis superfamily that play a role in immune cell signaling, activation, and survival. GITR is a therapeutic target for directly activating effector CD4 and CD8 T cells, or depleting GITR-expressing regulatory T cells (Tregs), thereby promoting anti-tumor immune responses. GITR activation through its native ligand is important for understanding immune signaling, but GITR structure has not been reported. Here we present structures of human and mouse GITR receptors bound to their cognate ligands. Both species share a receptor–ligand interface and receptor–receptor interface; the unique C-terminal receptor–receptor enables higher order structures on the membrane. Human GITR–GITRL has potential to form a hexameric network of membrane complexes, while murine GITR–GITRL complex forms a linear chain due to dimeric interactions. Mutations at the receptor–receptor interface in human GITR reduce cell signaling with in vitro ligand binding assays and minimize higher order membrane structures when bound by fluorescently labeled ligand in cell imaging experiments.

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