scholarly journals Calsyntenin-1 Docks Vesicular Cargo to Kinesin-1

2006 ◽  
Vol 17 (8) ◽  
pp. 3651-3663 ◽  
Author(s):  
Anetta Konecna ◽  
Renato Frischknecht ◽  
Jochen Kinter ◽  
Alexander Ludwig ◽  
Martin Steuble ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Transmembrane Protein ◽  
Point Mutations ◽  
Direct Interaction ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Tetratricopeptide Repeats ◽  
Fiber Tracts ◽  
Anterograde Axonal Transport

We identified a direct interaction between the neuronal transmembrane protein calsyntenin-1 and the light chain of Kinesin-1 (KLC1). GST pulldowns demonstrated that two highly conserved segments in the cytoplasmic domain of calsyntenin-1 mediate binding to the tetratricopeptide repeats of KLC1. A complex containing calsyntenin-1 and the Kinesin-1 motor was isolated from developing mouse brain and immunoelectron microscopy located calsyntenin-1 in association with tubulovesicular organelles in axonal fiber tracts. In primary neuronal cultures, calsyntenin-1–containing organelles were aligned along microtubules and partially colocalized with Kinesin-1. Using live imaging, we showed that these organelles are transported along axons with a velocity and processivity typical for fast axonal transport. Point mutations in the two kinesin-binding segments of calsyntenin-1 significantly reduced binding to KLC1 in vitro, and vesicles bearing mutated calsyntenin-1 exhibited a markedly altered anterograde axonal transport. In summary, our results indicate that calsyntenin-1 links a certain type of vesicular and tubulovesicular organelles to the Kinesin-1 motor.

scholarly journals Disease-associated mutations hyperactivate KIF1A motility and anterograde axonal transport of synaptic vesicle precursors

2019 ◽  
Vol 116 (37) ◽  
pp. 18429-18434 ◽  
Author(s):  
Kyoko Chiba ◽  
Hironori Takahashi ◽  
Min Chen ◽  
Hiroyuki Obinata ◽  
Shogo Arai ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neuron Disease ◽  
Synaptic Vesicle ◽  
Axonal Transport ◽  
Point Mutations ◽  
Loss Of Function ◽  
In Vitro Motility ◽  
Anterograde Axonal Transport ◽  
Abnormal Accumulation

KIF1A is a kinesin family motor involved in the axonal transport of synaptic vesicle precursors (SVPs) along microtubules (MTs). In humans, more than 10 point mutations inKIF1Aare associated with the motor neuron disease hereditary spastic paraplegia (SPG). However, not all of these mutations appear to inhibit the motility of the KIF1A motor, and thus a cogent molecular explanation for howKIF1Amutations lead to neuropathy is not available. In this study, we established in vitro motility assays with purified full-length human KIF1A and found thatKIF1Amutations associated with the hereditary SPG lead to hyperactivation of KIF1A motility. Introduction of the corresponding mutations into theCaenorhabditis elegans KIF1Ahomologunc-104revealed abnormal accumulation of SVPs at the tips of axons and increased anterograde axonal transport of SVPs. Our data reveal that hyperactivation of kinesin motor activity, rather than its loss of function, is a cause of motor neuron disease in humans.

scholarly journals Novel Membrane Protein shrew-1 Targets to Cadherin-Mediated Junctions in Polarized Epithelial Cells

2004 ◽  
Vol 15 (1) ◽  
pp. 397-406 ◽  
Author(s):  
Sanita Bharti ◽  
Heike Handrow-Metzmacher ◽  
Silvia Zickenheiner ◽  
Andreas Zeitvogel ◽  
Rudolf Baumann ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Protein ◽  
Transmembrane Protein ◽  
Adherens Junctions ◽  
Direct Interaction ◽  
Potential Candidate ◽  
Putative Open Reading Frame ◽  
Polarized Epithelial Cells ◽  
E Cadherin

While searching for potential candidate molecules relevant for the pathogenesis of endometriosis, we discovered a 2910-base pair cDNA encoding a novel putative 411-amino acid integral membrane protein that we called shrew-1. The putative open-reading frame was confirmed with antibodies against shrew-1 peptides that labeled a protein of ∼48 kDa in extracts of shrew-1 mRNA-positive tissue and also detected ectopically expressed shrew-1. Expression of epitope-tagged shrew-1 in epithelial cells and analysis by surface biotinylation and immunoblots demonstrated that shrew-1 is indeed a transmembrane protein. Shrew-1 is able to target to E-cadherin-mediated adherens junctions and interact with the E-cadherin–catenin complex in polarized MCF7 and Madin-Darby canine kidney cells, but not with the N-cadherin–catenin complex in nonpolarized epithelial cells. Direct interaction of shrew-1 with β-catenin in in vitro pull-down assay suggests that β-catenin might be one of the proteins that targets and/or retains shrew-1 in the adherens junctions. Interestingly, shrew-1 was partially translocated in response to scatter factor (ligand of receptor tyrosine kinase c-met) from the plasma membrane to the cytoplasm where it still colocalized with endogenous E-cadherin. In summary, we introduce shrew-1 as a novel component of adherens junctions, interacting with E-cadherin–β-catenin complexes in polarized epithelial cells.

scholarly journals Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

2012 ◽  
Vol 196 (4) ◽  
pp. 451-467 ◽  
Author(s):  
Veronica Krenn ◽  
Annemarie Wehenkel ◽  
Xiaozheng Li ◽  
Stefano Santaguida ◽  
Andrea Musacchio
Keyword(s):  
Crystal Structure ◽  
Structural Analysis ◽  
Convex Surface ◽  
Point Mutations ◽  
Tetratricopeptide Repeats ◽  
Checkpoint Protein ◽  
Checkpoint Kinases ◽  
Shed Light

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.

scholarly journals A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

1997 ◽  
Vol 17 (11) ◽  
pp. 6683-6692 ◽  
Author(s):  
A J Peterson ◽  
M Kyba ◽  
D Bornemann ◽  
K Morgan ◽  
H W Brock ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Point Mutations ◽  
Direct Interaction ◽  
Molecular Complexes ◽  
Polycomb Group ◽  
Ph Domain ◽  
Binding Studies

The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

scholarly journals Interaction and Subcellular Association of PRRT1/SynDIG4 With AMPA Receptors

2021 ◽  
Vol 13 ◽  
Author(s):  
Emily Eischen Martin ◽  
Erica Wleklinski ◽  
Hanh T. M. Hoang ◽  
Mohiuddin Ahmad
Keyword(s):  
Synaptic Plasticity ◽  
Ampa Receptors ◽  
Transmembrane Protein ◽  
Brain Regions ◽  
Membrane Topology ◽  
Neuronal Cultures ◽  
Physical Interaction ◽  
Primary Neuronal Cultures ◽  
Recycling Endosomes ◽  
Localization Analysis

AMPA receptors (AMPAR) are organized into supramolecular complexes in association with other membrane proteins that provide exquisite regulation of their biophysical properties and subcellular trafficking. Proline-rich transmembrane protein 1 (PRRT1), also named as SynDIG4, is a component of native AMPAR complexes in multiple brain regions. Deletion of PRRT1 leads to altered surface levels and phosphorylation status of AMPARs, as well as impaired forms of synaptic plasticity. Here, we have investigated the mechanisms underlying the observed regulation of AMPARs by investigating the interaction properties and subcellular localization of PRRT1. Our results show that PRRT1 can interact physically with all AMPAR subunits GluA1-GluA4. We decipher the membrane topology of PRRT1 to find that contrary to the predicted dual membrane pass, only the second hydrophobic segment spans the membrane completely, and is involved in mediating the interaction with AMPARs. We also report a physical interaction of PRRT1 with phosphatase PP2B that dephosphorylates AMPARs during synaptic plasticity. Our co-localization analysis in primary neuronal cultures identifies that PRRT1 associates with AMPARs extrasynaptically where it localizes to early and recycling endosomes as well as to the plasma membrane. These findings advance the understanding of the mechanisms by which PRRT1 regulates AMPARs under basal conditions and during synaptic plasticity.

scholarly journals α-Synuclein oligomers induce early axonal dysfunction in human iPSC-based models of synucleinopathies

2018 ◽  
Vol 115 (30) ◽  
pp. 7813-7818 ◽  
Author(s):  
Iryna Prots ◽  
Janina Grosch ◽  
Razvan-Marius Brazdis ◽  
Katrin Simmnacher ◽  
Vanesa Veber ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Disease Patient ◽  
In Vivo Models ◽  
Human Neurons ◽  
Anterograde Axonal Transport ◽  
Axonal Dysfunction ◽  
Underlying Mechanisms ◽  
Human Ipsc

α-Synuclein (α-Syn) aggregation, proceeding from oligomers to fibrils, is one central hallmark of neurodegeneration in synucleinopathies. α-Syn oligomers are toxic by triggering neurodegenerative processes in in vitro and in vivo models. However, the precise contribution of α-Syn oligomers to neurite pathology in human neurons and the underlying mechanisms remain unclear. Here, we demonstrate the formation of oligomeric α-Syn intermediates and reduced axonal mitochondrial transport in human neurons derived from induced pluripotent stem cells (iPSC) from a Parkinson’s disease patient carrying an α-Syn gene duplication. We further show that increased levels of α-Syn oligomers disrupt axonal integrity in human neurons. We apply an α-Syn oligomerization model by expressing α-Syn oligomer-forming mutants (E46K and E57K) and wild-type α-Syn in human iPSC-derived neurons. Pronounced α-Syn oligomerization led to impaired anterograde axonal transport of mitochondria, which can be restored by the inhibition of α-Syn oligomer formation. Furthermore, α-Syn oligomers were associated with a subcellular relocation of transport-regulating proteins Miro1, KLC1, and Tau as well as reduced ATP levels, underlying axonal transport deficits. Consequently, reduced axonal density and structural synaptic degeneration were observed in human neurons in the presence of high levels of α-Syn oligomers. Together, increased dosage of α-Syn resulting in α-Syn oligomerization causes axonal transport disruption and energy deficits, leading to synapse loss in human neurons. This study identifies α-Syn oligomers as the critical species triggering early axonal dysfunction in synucleinopathies.

Differential addressing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons

1999 ◽  
Vol 112 (6) ◽  
pp. 967-976
Author(s):  
A. Ghavami ◽  
K.L. Stark ◽  
M. Jareb ◽  
S. Ramboz ◽  
L. Segu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neuronal Cultures ◽  
Striatal Neurons ◽  
Primary Neuronal Cultures ◽  
Axon Terminals ◽  
In Vivo Experiments ◽  
In Vitro Systems ◽  
The Central Nervous System

The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors.

scholarly journals Quantifying Amyloid Beta (Aβ)–Mediated Changes in Neuronal Morphology in Primary Cultures

2012 ◽  
Vol 17 (6) ◽  
pp. 835-842 ◽  
Author(s):  
Lan Nguyen ◽  
Sarah Wright ◽  
Mike Lee ◽  
Zhao Ren ◽  
John-Michael Sauer ◽  
...  
Keyword(s):  
Amyloid Beta ◽  
Cell Injury ◽  
Primary Cultures ◽  
Neuronal Morphology ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Blocking Antibody ◽  
Cortical Cultures ◽  
Hippocampal Cultures

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aβ) in the brain. Herein, the authors quantify Aβ-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aβ caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aβ-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aβ-mediated cytotoxicity. Finally, we showed that Aβ challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aβ-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.

scholarly journals Non-proliferative neurogenesis in human periodontal ligament stem cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Carlos Bueno ◽  
Marta Martínez-Morga ◽  
Salvador Martínez
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Periodontal Ligament ◽  
Cell Biology ◽  
Basic Knowledge ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Periodontal Ligament Stem Cells ◽  
Sequence Of Events

AbstractUnderstanding the sequence of events from undifferentiated stem cells to neuron is not only important for the basic knowledge of stem cell biology, but also for therapeutic applications. In this study we examined the sequence of biological events during neural differentiation of human periodontal ligament stem cells (hPDLSCs). Here, we show that hPDLSCs-derived neural-like cells display a sequence of morphologic development highly similar to those reported before in primary neuronal cultures derived from rodent brains. We observed that cell proliferation is not present through neurogenesis from hPDLSCs. Futhermore, we may have discovered micronuclei movement and transient cell nuclei lobulation coincident to in vitro neurogenesis. Morphological analysis also reveals that neurogenic niches in the adult mouse brain contain cells with nuclear shapes highly similar to those observed during in vitro neurogenesis from hPDLSCs. Our results provide additional evidence that it is possible to differentiate hPDLSCs to neuron-like cells and suggest the possibility that the sequence of events from stem cell to neuron does not necessarily requires cell division from stem cell.

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