Integrins regulate DLG/FAS2 via a CaM kinase II-dependent pathway to mediate synapse elaboration and stabilization during postembryonic development

Development ◽  
2002 ◽  
Vol 129 (14) ◽  
pp. 3381-3391 ◽  
Author(s):  
Kelly Beumer ◽  
Heinrich J. G. Matthies ◽  
Amber Bradshaw ◽  
Kendal Broadie
Keyword(s):  
Pdz Domain ◽  
Postembryonic Development ◽  
Synaptic Proteins ◽  
Scaffolding Protein ◽  
Structural Defects ◽  
Integrin Receptors ◽  
Synaptic Complex ◽  
Synaptic Localization ◽  
Calcium Calmodulin Dependent ◽  
Discs Large

Calcium/calmodulin dependent kinase II (CaMKII), PDZ-domain scaffolding protein Discs-large (DLG), immunoglobin superfamily cell adhesion molecule Fasciclin 2 (FAS2) and the position specific (PS) integrin receptors, including βPS and its alpha partners (αPS1, αPS2, αPS3/αVolado), are all known to regulate the postembryonic development of synaptic terminal arborization at the Drosophila neuromuscular junction (NMJ). Recent work has shown that DLG and FAS2 function together to modulate activity-dependent synaptic development and that this role is regulated by activation of CaMKII. We show that PS integrins function upstream of CaMKII in the development of synaptic architecture at the NMJ. βPS integrin physically associates with the synaptic complex anchored by the DLG scaffolding protein, which contains CaMKII and FAS2. We demonstrate an alteration of the FAS2 molecular cascade in integrin regulatory mutants, as a result of CaMKII/integrin interactions. Regulatory βPS integrin mutations increase the expression and synaptic localization of FAS2. Synaptic structural defects in βPS integrin mutants are rescued by transgenic overexpression of CaMKII (proximal in pathway) or genetic reduction of FAS2 (distal in pathway). These studies demonstrate that βPS integrins act through CaMKII activation to control the localization of synaptic proteins involved in the development of NMJ synaptic morphology.

scholarly journals Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Ki-Seo Yoo ◽  
Kina Lee ◽  
Jun-Young Oh ◽  
Hyoeun Lee ◽  
Hyungju Park ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Postsynaptic Density ◽  
Scaffolding Protein ◽  
Synaptic Localization ◽  
Conventional Kinesin ◽  
Dose Dependent ◽  
Postsynaptic Density Protein

AbstractPostsynaptic density protein 95 (PSD-95) is a pivotal postsynaptic scaffolding protein in excitatory neurons. Although the transport and regulation of PSD-95 in synaptic regions is well understood, dendritic transport of PSD-95 before synaptic localization still remains to be clarified. To evaluate the role of KIF5, conventional kinesin, in the dendritic transport of PSD-95 protein, we expressed a transport defective form of KIF5A (ΔMD) that does not contain the N-terminal motor domain. Expression of ΔMD significantly decreased PSD-95 level in the dendrites. Consistently, KIF5 was associated with PSD-95 in in vitro and in vivo assays. This interaction was mediated by the C-terminal tail regions of KIF5A and the third PDZ domain of PSD-95. Additionally, the ADPDZ3 (the association domain of NMDA receptor and PDZ3 domain) expression significantly reduced the levels of PSD-95, glutamate receptor 1 (GluA1) in dendrites. The association between PSD-95 and KIF5A was dose-dependent on Staufen protein, suggesting that the Staufen plays a role as a regulatory role in the association. Taken together, our data suggest a new mechanism for dendritic transport of the AMPA receptor-PSD-95.

scholarly journals Early Metazoan Origin and Multiple Losses of a Novel Clade of RIM Presynaptic Calcium Channel Scaffolding Protein Homologs

2020 ◽  
Vol 12 (8) ◽  
pp. 1217-1239 ◽  
Author(s):  
Thomas Piekut ◽  
Yuen Yan Wong ◽  
Sarah E Walker ◽  
Carolyn L Smith ◽  
Julia Gauberg ◽  
...  
Keyword(s):  
Lymnaea Stagnalis ◽  
Pdz Domain ◽  
Scaffolding Protein ◽  
Tissue Type ◽  
Ligand Specificity ◽  
Synaptic Localization ◽  
Trichoplax Adhaerens ◽  
Stem Lineage ◽  
Presynaptic Calcium

Abstract The precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3-interacting molecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channels in vitro via a PDZ domain that binds to the extreme C-termini of the channels at acidic ligand motifs of D/E-D/E/H-WC-COOH, and knockout of RIM in vertebrates and invertebrates disrupts CaV2 channel synaptic localization and synapse function. Here, we describe a previously uncharacterized clade of RIM proteins bearing domain architectures homologous to those of known RIM homologs, but with some notable differences including key amino acids associated with PDZ domain ligand specificity. This novel RIM emerged near the stem lineage of metazoans and underwent extensive losses, but is retained in select animals including the early-diverging placozoan Trichoplax adhaerens, and molluscs. RNA expression and localization studies in Trichoplax and the mollusc snail Lymnaea stagnalis indicate differential regional/tissue type expression, but overlapping expression in single isolated neurons from Lymnaea. Ctenophores, the most early-diverging animals with synapses, are unique among animals with nervous systems in that they lack the canonical RIM, bearing only the newly identified homolog. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOH like PDZ ligand motifs were present in the common ancestor of cnidarians and bilaterians, and delineate some deeply conserved C-terminal structures that distinguish CaV1 from CaV2 channels, and CaV1/CaV2 from CaV3 channels.

scholarly journals A model for regulation by SynGAP-α1 of binding of synaptic proteins to PDZ-domain 'Slots' in the postsynaptic density

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ward G Walkup ◽  
Tara L Mastro ◽  
Leslie T Schenker ◽  
Jost Vielmetter ◽  
Rebecca Hu ◽  
...  
Keyword(s):  
Pdz Domain ◽  
Synaptic Proteins ◽  
Major Constituent ◽  
Signaling Proteins ◽  
Pdz Domains ◽  
Heterozygous Deletion ◽  
Gtpase Activating Protein ◽  
Dependent Protein Kinase ◽  
Discs Large ◽  
Dependent Protein

SynGAP is a Ras/Rap GTPase-activating protein (GAP) that is a major constituent of postsynaptic densities (PSDs) from mammalian forebrain. Its α1 isoform binds to all three PDZ (PSD-95, Discs-large, ZO-1) domains of PSD-95, the principal PSD scaffold, and can occupy as many as 15% of these PDZ domains. We present evidence that synGAP-α1 regulates the composition of the PSD by restricting binding to the PDZ domains of PSD-95. We show that phosphorylation by Ca2+/calmodulin-dependent protein kinase II (CaMKII) and Polo-like kinase-2 (PLK2) decreases its affinity for the PDZ domains by several fold, which would free PDZ domains for occupancy by other proteins. Finally, we show that three critical postsynaptic signaling proteins that bind to the PDZ domains of PSD-95 are present in higher concentration in PSDs isolated from mice with a heterozygous deletion of synGAP.

scholarly journals Early metazoan origin and multiple losses of a novel clade of RIM pre-synaptic calcium channel scaffolding protein homologues

2020 ◽  
Author(s):  
Thomas Piekut ◽  
Yuen Yan Wong ◽  
Sarah E. Walker ◽  
Carolyn L. Smith ◽  
Julia Gauberg ◽  
...  
Keyword(s):  
Lymnaea Stagnalis ◽  
Pdz Domain ◽  
Scaffolding Protein ◽  
Tissue Type ◽  
Ligand Specificity ◽  
Synaptic Localization ◽  
Trichoplax Adhaerens ◽  
Stem Lineage ◽  
The Common

AbstractThe precise localization of CaV2 voltage-gated calcium channels at the synapse active zone requires various interacting proteins, of which, Rab3 interacting molecule or RIM is considered particularly important. In vertebrates, RIM interacts with CaV2 channels in vitro via a PDZ domain that binds to the extreme C-termini of the channels at acidic ligand motifs of D/E-D/E/H-WC-COOH, and knockout of RIM in vertebrates and invertebrates disrupts CaV2 channel synaptic localization and synapse function. Here, we describe a previously uncharacterized clade of RIM proteins bearing homologous domain architectures as known RIM homologues, but some notable differences including key amino acids associated with PDZ domain ligand specificity. This novel RIM emerged near the stem lineage of metazoans and underwent extensive losses, but is retained in select animals including the early-diverging placozoan Trichoplax adhaerens, and molluscs. RNA expression and localization studies in Trichoplax and the mollusc snail Lymnaea stagnalis indicate differential regional/tissue type expression, but overlapping expression in single isolated neurons from Lymnaea. Ctenophores, the most early-diverging animals with synapses, are unique among animals with nervous systems in that they lack the canonical RIM, bearing only the newly identified homologue. Through phylogenetic analysis, we find that CaV2 channel D/E-D/E/H-WC-COOH like PDZ ligand motifs were present in the common ancestor of cnidarians and bilaterians, and delineate some deeply conserved C-terminal structures that distinguish CaV1 from CaV2 channels, and CaV1/CaV2 from CaV3 channels.

scholarly journals Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis

2018 ◽  
Vol 115 (46) ◽  
pp. E10859-E10868 ◽  
Author(s):  
Yuwei Li ◽  
Jason A. Junge ◽  
Cosimo Arnesano ◽  
Garrett G. Gross ◽  
Jeffrey H. Miner ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Protein Interactions ◽  
Protein Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Explant Culture ◽  
Scaffolding Protein ◽  
Fluorescence Lifetime Imaging ◽  
Discs Large

Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.

scholarly journals Residue-Level Contact Reveals Modular Domain Interactions of PICK1 Are Driven by Both Electrostatic and Hydrophobic Forces

2021 ◽  
Vol 7 ◽  
Author(s):  
Amy O. Stevens ◽  
Yi He
Keyword(s):  
Hydrophobic Interactions ◽  
Pdz Domain ◽  
Md Simulations ◽  
Intramolecular Interactions ◽  
Coarse Grained ◽  
Scaffolding Protein ◽  
Concave Surface ◽  
Stable Complex ◽  
Bar Domain ◽  
C Terminus

PICK1 is a multi-domain scaffolding protein that is uniquely comprised of both a PDZ domain and a BAR domain. While previous experiments have shown that the PDZ domain and the linker positively regulate the BAR domain and the C-terminus negatively regulates the BAR domain, the details of internal regulation mechanisms are unknown. Molecular dynamics (MD) simulations have been proven to be a useful tool in revealing the intramolecular interactions at atomic-level resolution. PICK1 performs its biological functions in a dimeric form which is extremely computationally demanding to simulate with an all-atom force field. Here, we use coarse-grained MD simulations to expose the key residues and driving forces in the internal regulations of PICK1. While the PDZ and BAR domains do not form a stable complex, our simulations show the PDZ domain preferentially interacting with the concave surface of the BAR domain over other BAR domain regions. Furthermore, our simulations show that the short helix in the linker region can form interactions with the PDZ domain. Our results reveal that the surface of the βB-βC loop, βC strand, and αA-βD loop of the PDZ domain can form a group of hydrophobic interactions surrounding the linker helix. These interactions are driven by hydrophobic forces. In contrast, our simulations reveal a very dynamic C-terminus that most often resides on the convex surface of the BAR domain rather than the previously suspected concave surface. These interactions are driven by a combination of electrostatic and hydrophobic interactions.

Abstract P110: EBP50 Regulates Vascular Smooth Muscle Cell Growth by Skp2-Mediated Degradation of p21/cip1

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Gyun Jee Song ◽  
Stacey Barrick ◽  
Kristen L Leslie ◽  
Nathalie M Fiaschi-Taesch ◽  
Alessandro Bisello
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Pdz Domain ◽  
Scaffolding Protein ◽  
Mrna Levels ◽  
Neointima Formation ◽  
Vsmc Proliferation ◽  
Genetic Ablation ◽  
Skp2 Expression

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-binding phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout (KO) mice. The molecular mechanisms underlying EBP50 actions in VSMC are unknown. Genetic ablation of EBP50 reduced VSMC proliferation and was associated with increased (5-fold) expression of the cell cycle inhibitor p21cip1 both in vessels and in primary cells. No differences in mRNA levels of p21cip1 were observed in WT and KO cells. However, the half-life of p21cip1 in KO VSMC was significantly longer than in WT VSMC (80 min vs. 45 min) and p21cip1 levels were similar in WT and KO VSMC treated with the proteasome inhibitor MG132. These observations suggest that EBP50 regulates post-translational degradation of p21cip1. The S-phase kinase-associated protein 2 (skp2) is a component of the E3 ligase complex that degrades p21cip1. The C-terminal four amino acids of skp2 (ProSerCysLeu) are a canonical PDZ-binding sequence. Indeed, co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and skp2. Mutation of the C-terminal Leu to Ala (L424A-skp2) abrogated the interaction with EBP50. Skp2 expression was significantly lower in KO than in WT cells and inhibition of EBP50 expression by an shRNA lentivirus decreased skp2 expression in WT cells. Moreover, expression of skp2, but not of the mutant L424A-skp2, in WT cells reduced p21cip1 levels. Therefore, EBP50 regulates both expression and activity of skp2 with attendant effects on p21cip1 and VSMC proliferation. Collectively, these experiments show that EBP50, by regulating skp2 and p21cip1 expression, controls VSMC proliferation and the progression of neointima formation. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide a mechanistic basis for the remarkable effect of this scaffolding protein on vascular remodeling.

scholarly journals The CHD Protein, Kismet, is Important for the Recycling of Synaptic Vesicles during Endocytosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Nina K. Latcheva ◽  
Taylor L. Delaney ◽  
Jennifer M. Viveiros ◽  
Rachel A. Smith ◽  
Kelsey M. Bernard ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Chromatin Remodeling ◽  
Neurodevelopmental Disorders ◽  
Synaptic Vesicles ◽  
Synaptic Function ◽  
Synaptic Proteins ◽  
Critical Feature ◽  
Synaptic Localization ◽  
Transcription Start Sites ◽  
Mature Neurons

AbstractChromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. Here we show that Kismet, the Drosophila homolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound to dap160 and endophilin B transcription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition, kismet mutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored at kismet mutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.

scholarly journals Formation of a Bazooka–Stardust complex is essential for plasma membrane polarity in epithelia

2010 ◽  
Vol 190 (5) ◽  
pp. 751-760 ◽  
Author(s):  
Michael P. Krahn ◽  
Johanna Bückers ◽  
Lars Kastrup ◽  
Andreas Wodarz
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Pdz Domain ◽  
Phosphorylation Site ◽  
Direct Interaction ◽  
Epithelial Polarity ◽  
Kinase C ◽  
Discs Large ◽  
Evolutionarily Conserved ◽  
Functional Hierarchy

Apical–basal polarity in Drosophila melanogaster epithelia depends on several evolutionarily conserved proteins that have been assigned to two distinct protein complexes: the Bazooka (Baz)–PAR-6 (partitioning defective 6)–atypical protein kinase C (aPKC) complex and the Crumbs (Crb)–Stardust (Sdt) complex. These proteins operate in a functional hierarchy, in which Baz is required for the proper subcellular localization of all other proteins. We investigated how these proteins interact and how this interaction is regulated. We show that Baz recruits Sdt to the plasma membrane by direct interaction between the Postsynaptic density 95/Discs large/Zonula occludens 1 (PDZ) domain of Sdt and a region of Baz that contains a phosphorylation site for aPKC. Phosphorylation of Baz causes the dissociation of the Baz–Sdt complex. Overexpression of a nonphosphorylatable version of Baz blocks the dissociation of Sdt from Baz, causing phenotypes very similar to those of crb and sdt mutations. Our findings provide a molecular mechanism for the phosphorylation-dependent interaction between the Baz–PAR-3 and Crb complexes during the establishment of epithelial polarity.

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