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All Publications

  • The intrinsically disordered cytoplasmic tail of a dendrite branching receptor uses two distinct mechanisms to regulate the actin cytoskeleton. eLife Kramer, D. A., Narvaez-Ortiz, H. Y., Patel, U., Shi, R., Shen, K., Nolen, B. J., Roche, J., Chen, B. 2023; 12


    Dendrite morphogenesis is essential for neural circuit formation, yet the molecular mechanisms underlying complex dendrite branching remain elusive. Previous studies on the highly branched Caenorhabditis elegans PVD sensory neuron identified a membrane co-receptor complex that links extracellular signals to intracellular actin remodeling machinery, promoting high-order dendrite branching. In this complex, the claudin-like transmembrane protein HPO-30 recruits the WAVE regulatory complex (WRC) to dendrite branching sites, stimulating the Arp2/3 complex to polymerize actin. We report here our biochemical and structural analysis of this interaction, revealing that the intracellular domain (ICD) of HPO-30 is intrinsically disordered and employs two distinct mechanisms to regulate the actin cytoskeleton. First, HPO-30 ICD binding to the WRC requires dimerization and involves the entire ICD sequence, rather than a short linear peptide motif. This interaction enhances WRC activation by the GTPase Rac1. Second, HPO-30 ICD directly binds to the sides and barbed end of actin filaments. Binding to the barbed end requires ICD dimerization and inhibits both actin polymerization and depolymerization, resembling the actin capping protein CapZ. These dual functions provide an intriguing model of how membrane proteins can integrate distinct mechanisms to fine-tune local actin dynamics.

    View details for DOI 10.7554/eLife.88492

    View details for PubMedID 37555826

  • Dendrites use mechanosensitive channels to proofread ligand-mediated neurite extension during morphogenesis. Developmental cell Tao, L., Coakley, S., Shi, R., Shen, K. 2022


    Ligand-receptor interactions guide axon navigation and dendrite arborization. Mechanical forces also influence guidance choices. However, the nature of such mechanical stimulations, the mechanosensor identity, and how they interact with guidance receptors are unknown. Here, we demonstrate that mechanosensitive DEG/ENaC channels are required for dendritic arbor morphogenesis in Caenorhabditis elegans. Inhibition of DEG/ENaC channels causes reduced dendritic outgrowth and branching invivo, a phenotype that is alleviated by overexpression of the mechanosensitive channels PEZO-1/Piezo or YVC1/TrpY1. DEG/ENaCs trigger local Ca2+ transients in growing dendritic filopodia via activation of L-type voltage-gated Ca2+ channels. Anchoring of filopodia by dendrite ligand-receptor complexes is required for the mechanical activation of DEG/ENaC channels. Therefore, mechanosensitive channels serve as a checkpoint for appropriate chemoaffinity by activating Ca2+ transients required for neurite growth.

    View details for DOI 10.1016/j.devcel.2022.05.019

    View details for PubMedID 35709764

  • A two-step actin polymerization mechanism drives dendrite branching. Neural development Shi, R., Kramer, D. A., Chen, B., Shen, K. 2021; 16 (1): 3


    BACKGROUND: Dendrite morphogenesis plays an essential role in establishing the connectivity and receptive fields of neurons during the development of the nervous system. To generate the diverse morphologies of branched dendrites, neurons use external cues and cell surface receptors to coordinate intracellular cytoskeletal organization; however, the molecular mechanisms of how this signaling forms branched dendrites are not fully understood.METHODS: We performed in vivo time-lapse imaging of the PVD neuron in C. elegans in several mutants of actin regulatory proteins, such as the WAVE Regulatory Complex (WRC) and UNC-34 (homolog of Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP)). We examined the direct interaction between the WRC and UNC-34 and analyzed the localization of UNC-34 in vivo using transgenic worms expressing UNC-34 fused to GFP.RESULTS: We identify a stereotyped sequence of morphological events during dendrite outgrowth in the PVD neuron in C. elegans. Specifically, local increases in width ("swellings") give rise to filopodia to facilitate a "rapid growth and pause" mode of growth. In unc-34 mutants, filopodia fail to form but swellings are intact. In WRC mutants, dendrite growth is largely absent, resulting from a lack of both swelling and filopodia formation. We also found that UNC-34 can directly bind to the WRC. Disrupting this binding by deleting the UNC-34 EVH1 domain prevented UNC-34 from localizing to swellings and dendrite tips, resulting in a stunted dendritic arbor and reduced filopodia outgrowth.CONCLUSIONS: We propose that regulators of branched and linear F-actin cooperate to establish dendritic branches. By combining our work with existing literature, we propose that the dendrite guidance receptor DMA-1 recruits the WRC, which polymerizes branched F-actin to generate "swellings" on a mother dendrite. Then, WRC recruits the actin elongation factor UNC-34/Ena/VASP to initiate growth of a new dendritic branch from the swelling, with the help of the actin-binding protein UNC-115/abLIM. Extension of existing dendrites also proceeds via swelling formation at the dendrite tip followed by UNC-34-mediated outgrowth. Following dendrite initiation and extension, the stabilization of branches by guidance receptors further recruits WRC, resulting in an iterative process to build a complex dendritic arbor.

    View details for DOI 10.1186/s13064-021-00154-0

    View details for PubMedID 34281597

  • The Golgi Outpost Protein TPPP Nucleates Microtubules and Is Critical for Myelination. Cell Fu, M. M., McAlear, T. S., Nguyen, H. n., Oses-Prieto, J. A., Valenzuela, A. n., Shi, R. D., Perrino, J. J., Huang, T. T., Burlingame, A. L., Bechstedt, S. n., Barres, B. A. 2019


    Oligodendrocytes extend elaborate microtubule arbors that contact up to 50 axon segments per cell, then spiral around myelin sheaths, penetrating from outer to inner layers. However, how they establish this complex cytoarchitecture is unclear. Here, we show that oligodendrocytes contain Golgi outposts, an organelle that can function as an acentrosomal microtubule-organizing center (MTOC). We identify a specific marker for Golgi outposts-TPPP (tubulin polymerization promoting protein)-that we use to purify this organelle and characterize its proteome. In in vitro cell-free assays, recombinant TPPP nucleates microtubules. Primary oligodendrocytes from Tppp knockout (KO) mice have aberrant microtubule branching, mixed microtubule polarity, and shorter myelin sheaths when cultured on 3-dimensional (3D) microfibers. Tppp KO mice exhibit hypomyelination with shorter, thinner myelin sheaths and motor coordination deficits. Together, our data demonstrate that microtubule nucleation outside the cell body at Golgi outposts by TPPP is critical for elongation of the myelin sheath.

    View details for DOI 10.1016/j.cell.2019.08.025

    View details for PubMedID 31522887

  • SAP102 regulates synaptic AMPAR function through a CNIH-2-dependent mechanism JOURNAL OF NEUROPHYSIOLOGY Liu, M., Shi, R., Hwang, H., Han, K., Wong, M., Ren, X., Lewis, L. D., Brown, E. N., Xu, W. 2018; 120 (4): 1578–86


    The postsynaptic density (PSD)-95-like, disk-large (DLG) membrane-associated guanylate kinase (PSD/DLG-MAGUK) family of proteins scaffold α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) complexes to the postsynaptic compartment and are postulated to orchestrate activity-dependent modulation of synaptic AMPAR functions. SAP102 is a key member of this family, present from early development, before PSD-95 and PSD-93, and throughout life. Here we investigate the role of SAP102 in synaptic transmission using a cell-restricted molecular replacement strategy, where SAP102 is expressed against the background of acute knockdown of endogenous PSD-95. We show that SAP102 rescues the decrease of AMPAR-mediated evoked excitatory postsynaptic currents (AMPAR eEPSCs) and AMPAR miniature EPSC (AMPAR mEPSC) frequency caused by acute knockdown of PSD-95. Further analysis of the mini events revealed that PSD-95-to-SAP102 replacement but not direct manipulation of PSD-95 increases the AMPAR mEPSC decay time. SAP102-mediated rescue of AMPAR eEPSCs requires AMPAR auxiliary subunit cornichon-2, whereas cornichon-2 knockdown did not affect PSD-95-mediated regulation of AMPAR eEPSC. Combining these observations, our data elucidate that PSD-95 and SAP102 differentially influence basic synaptic properties and synaptic current kinetics potentially via different AMPAR auxiliary subunits. NEW & NOTEWORTHY Synaptic scaffold proteins postsynaptic density (PSD)-95-like, disk-large (DLG) membrane-associated guanylate kinase (PSD-MAGUKs) regulate synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function. However, the functional diversity among different PSD-MAGUKs remains to be categorized. We show that distinct from PSD-95, SAP102 increase the AMPAR synaptic current decay time, and the effect of SAP102 on synaptic AMPAR function requires the AMPAR auxiliary subunit cornichon-2. Our data suggest that PSD-MAGUKs target and modulate different AMPAR complexes to exert specific experience-dependent modification of the excitatory circuit.

    View details for DOI 10.1152/jn.00731.2017

    View details for Web of Science ID 000451350100012

    View details for PubMedID 30067114

    View details for PubMedCentralID PMC6230800

  • A Dendritic Guidance Receptor Complex Brings Together Distinct Actin Regulators to Drive Efficient F-Actin Assembly and Branching DEVELOPMENTAL CELL Zou, W., Dong, X., Broederdorf, T. R., Shen, A., Kramer, D. A., Shi, R., Liang, X., Miller, D. M., Xiang, Y. K., Yasuda, R., Chen, B., Shen, K. 2018; 45 (3): 362-+


    Proper morphogenesis of dendrites plays a fundamental role in the establishment of neural circuits. The molecular mechanism by which dendrites grow highly complex branches is not well understood. Here, using the Caenorhabditis elegans PVD neuron, we demonstrate that high-order dendritic branching requires actin polymerization driven by coordinated interactions between two membrane proteins, DMA-1 and HPO-30, with their cytoplasmic interactors, the RacGEF TIAM-1 and the actin nucleation promotion factor WAVE regulatory complex (WRC). The dendrite branching receptor DMA-1 directly binds to the PDZ domain of TIAM-1, while the claudin-like protein HPO-30 directly interacts with the WRC. On dendrites, DMA-1 and HPO-30 form a receptor-associated signaling complex to bring TIAM-1 and the WRC to close proximity, leading to elevated assembly of F-actin needed to drive high-order dendrite branching. The synergistic activation of F-actin assembly by scaffolding distinct actin regulators might represent a general mechanism in promoting complex dendrite arborization.

    View details for PubMedID 29738713

  • Shank Proteins Differentially Regulate Synaptic Transmission (vol 4, e0163-15.2017, 2017) ENEURO Shi, R., Redman, P., Ghose, D., Hwang, H., Liu, Y., Ren, X., Ding, L. J., Liu, M., Jones, K. J., Xu, W. 2018; 5 (1)
  • Dissecting the Role of P/Q-Type Calcium Channels in Corticothalamic Circuit Dysfunction and Absence Epilepsy. The Journal of neuroscience : the official journal of the Society for Neuroscience Shi, R. n., Schroeder, G. M., Nimarko, A. F. 2016; 36 (21): 5677–79

    View details for PubMedID 27225758

  • Differential requirement for NMDAR activity in SAP97 beta-mediated regulation of the number and strength of glutamatergic AMPAR-containing synapses JOURNAL OF NEUROPHYSIOLOGY Liu, M., Lewis, L. D., Shi, R., Brown, E. N., Xu, W. 2014; 111 (3): 648-658


    PSD-95-like, disc-large (DLG) family membrane-associated guanylate kinase proteins (PSD/DLG-MAGUKs) are essential for regulating synaptic AMPA receptor (AMPAR) function and activity-dependent trafficking of AMPARs. Using a molecular replacement strategy to replace endogenous PSD-95 with SAP97β, we show that the prototypic β-isoform of the PSD-MAGUKs, SAP97β, has distinct NMDA receptor (NMDAR)-dependent roles in regulating basic properties of AMPAR-containing synapses. SAP97β enhances the number of AMPAR-containing synapses in an NMDAR-dependent manner, whereas its effect on the size of unitary synaptic response is not fully dependent on NMDAR activity. These effects contrast with those of PSD-95α, which increases both the number of AMPAR-containing synapses and the size of unitary synaptic responses, with or without NMDAR activity. Our results suggest that SAP97β regulates synaptic AMPAR content by increasing surface expression of GluA1-containing AMPARs, whereas PSD-95α enhances synaptic AMPAR content presumably by increasing the synaptic scaffold capacity for synaptic AMPARs. Our approach delineates discrete effects of different PSD-MAGUKs on principal properties of glutamatergic synaptic transmission. Our results suggest that the molecular diversity of PSD-MAGUKs can provide rich molecular substrates for differential regulation of glutamatergic synapses in the brain.

    View details for DOI 10.1152/jn.00262.2013

    View details for Web of Science ID 000331215500019

    View details for PubMedID 24225540

    View details for PubMedCentralID PMC3921414