scholarly journals The C-terminal helix 9 motif in rat cannabinoid receptor type 1 regulates axonal trafficking and surface expression

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Alexandra Fletcher-Jones ◽  
Keri L Hildick ◽  
Ashley J Evans ◽  
Yasuko Nakamura ◽  
Kevin A Wilkinson ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Secretory Pathway ◽  
Cannabinoid Receptor ◽  
Surface Expression ◽  
Surface Polarity ◽  
Time Resolved ◽  
Surface Stability ◽  
C Terminus ◽  
Time Resolved Imaging ◽  
Dual Mechanism

Cannabinoid type one receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation in cultured rat hippocampal neurons. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminus and involves the Helix 9 (H9) domain. Removal of the H9 domain increases secretory pathway delivery to dendrites and decreases surface stability. Furthermore, CB1RΔH9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

scholarly journals The C-terminal Helix 9 motif regulates cannabinoid receptor type 1 trafficking and surface expression

2018 ◽  
Author(s):  
Alexandra Fletcher-Jones ◽  
Keri L Hildick ◽  
Ashley J Evans ◽  
Yasuko Nakamura ◽  
Kevin Wilkinson ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Cannabinoid Receptor ◽  
Surface Expression ◽  
Surface Polarity ◽  
Time Resolved ◽  
Surface Stability ◽  
Cannabinoid Type 1 Receptor ◽  
Time Resolved Imaging ◽  
Dual Mechanism

Cannabinoid type 1 receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminal and involves the Helix 9 (H9) domain. Removal of the H9 domain increases dendrite secretory pathway delivery and decreases in surface stability. Furthermore, CB1RΔH9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

Spastin Interacts with CRMP2 to Regulate Neurite Outgrowth by Controlling Microtubule Dynamics through Phosphorylation Modifications

2020 ◽  
Vol 19 ◽  
Author(s):  
Sumei Li ◽  
Jifeng Zhang ◽  
Jiaqi Zhang ◽  
Jiong Li ◽  
Longfei Cheng ◽  
...  
Keyword(s):  
Neurite Outgrowth ◽  
Hippocampal Neurons ◽  
Neuronal Development ◽  
Phosphorylation Site ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
C Terminus ◽  
Mediator Protein ◽  
Branch Formation

Aims: Our work aims to revealing the underlying microtubule mechanism of neurites outgrowth during neuronal development, and also proposes a feasible intervention pathway for reconstructing neural network connections after nerve injury. Background: Microtubule polymerization and severing are the basis for the neurite outgrowth and branch formation. Collapsin response mediator protein 2 (CRMP2) regulates axonal growth and branching as a binding partner of the tubulin heterodimer to promote microtubule assembly. And spastin participates in the growth and regeneration of neurites by severing microtubules into small segments. However, how CRMP2 and spastin cooperate to regulate neurite outgrowth by controlling the microtubule dynamics needs to be elucidated. Objective: To explore whether neurite outgrowth was mediated by coordination of CRMP2 and spastin. Method: Hippocampal neurons were cultured in vitro in 24-well culture plates for 4 days before being used to perform the transfection. Calcium phosphate was used to transfect the CRMP2 and spastin constructs and their control into the neurons. An interaction between CRMP2 and spastin was examined by using pull down, CoIP and immunofluorescence colocalization assays. And immunostaining was also performed to determine the morphology of neurites. Result: We first demonstrated that CRMP2 interacted with spastin to promote the neurite outgrowth and branch formation. Furthermore, our results identified that phosphorylation modification failed to alter the binding affinities of CRMP2 for spastin, but inhibited their binding to microtubules. CRMP2 interacted with the MTBD domain of spastin via its C-terminus, and blocking the binding sites of them inhibited the outgrowth and branch formation of neurites. In addition, we confirmed one phosphorylation site S210 at spastin in hippocampal neurons and phosphorylation spastin at site S210 promoted the neurite outgrowth but not branch formation by remodeling microtubules. Conclusion: Taken together, our data demonstrated that the interaction of CRMP2 and spastin is required for neurite outgrowth and branch formation and their interaction is not regulated by their phosphorylation.

scholarly journals Giant enhancement of THz-frequency optical nonlinearity by phonon polariton in ionic crystals

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yao Lu ◽  
Qi Zhang ◽  
Qiang Wu ◽  
Zhigang Chen ◽  
Xueming Liu ◽  
...  
Keyword(s):  
Near Infrared ◽  
Optical Nonlinearity ◽  
Ionic Crystals ◽  
Nonlinear Optical Susceptibility ◽  
Time Resolved ◽  
Light Coupling ◽  
Tremendous Progress ◽  
Fundamental Research ◽  
Ionic Contribution ◽  
Time Resolved Imaging

AbstractThe field of nonlinear optics has grown substantially in past decades, leading to tremendous progress in fundamental research and revolutionized applications. Traditionally, the optical nonlinearity for a light wave at frequencies beyond near-infrared is observed with very high peak intensity, as in most materials only the electronic nonlinearity dominates while ionic contribution is negligible. However, it was shown that the ionic contribution to nonlinearity can be much larger than the electronic one in microwave experiments. In the terahertz (THz) regime, phonon polariton may assist to substantially trigger the ionic nonlinearity of the crystals, so as to enhance even more the nonlinear optical susceptibility. Here, we experimentally demonstrate a giant second-order optical nonlinearity at THz frequency, orders of magnitude higher than that in the visible and microwave regimes. Different from previous work, the phonon-light coupling is achieved under a phase-matching setting, and the dynamic process of nonlinear THz generation is directly observed in a thin-film waveguide using a time-resolved imaging technique. Furthermore, a nonlinear modification to the Huang equations is proposed to explain the observed nonlinearity enhancement. This work brings about an effective approach to achieve high nonlinearity in ionic crystals, promising for applications in THz nonlinear technologies.

scholarly journals Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 715
Author(s):  
Tamara Tomanić ◽  
Claire Martin ◽  
Holly Stefen ◽  
Esmeralda Parić ◽  
Peter Gunning ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hippocampal Neurons ◽  
Molecular Mechanisms ◽  
Growth Cones ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
C Terminus ◽  
Primary Mouse ◽  
Terminal Amino

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.

scholarly journals Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy

2011 ◽  
Vol 16 (12) ◽  
pp. 120510 ◽  
Author(s):  
Matthew T. Rinehart ◽  
Tyler K. Drake ◽  
Francisco E. Robles ◽  
Lisa C. Rohan ◽  
David Katz ◽  
...  
Keyword(s):  
Time Resolved ◽  
Quantitative Phase ◽  
Phase Microscopy ◽  
Quantitative Phase Microscopy ◽  
Time Resolved Imaging

scholarly journals An interdomain bridge influences RNA binding of the human La protein

2018 ◽  
Vol 294 (5) ◽  
pp. 1529-1540 ◽  
Author(s):  
Stefano A. Marrella ◽  
Kerene A. Brown ◽  
Farnaz Mansouri-Noori ◽  
Jennifer Porat ◽  
Derek J. Wilson ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Polymerase Iii ◽  
Conformational Dynamics ◽  
Direct Interaction ◽  
Rna Recognition Motif ◽  
Binding Modes ◽  
Nuclear Retention ◽  
Time Resolved ◽  
La Protein ◽  
C Terminus

La proteins are RNA chaperones that perform various functions depending on distinct RNA-binding modes and their subcellular localization. In the nucleus, they help process UUU-3′OH–tailed nascent RNA polymerase III transcripts, such as pre-tRNAs, whereas in the cytoplasm they contribute to translation of poly(A)-tailed mRNAs. La accumulation in the nucleus and cytoplasm is controlled by several trafficking elements, including a canonical nuclear localization signal in the extreme C terminus and a nuclear retention element (NRE) in the RNA recognition motif 2 (RRM2) domain. Previous findings indicate that cytoplasmic export of La due to mutation of the NRE can be suppressed by mutations in RRM1, but the mechanism by which the RRM1 and RRM2 domains functionally cooperate is poorly understood. In this work, we use electromobility shift assays (EMSA) to show that mutations in the NRE and RRM1 affect binding of human La to pre-tRNAs but not UUU-3′OH or poly(A) sequences, and we present compensatory mutagenesis data supporting a direct interaction between the RRM1 and RRM2 domains. Moreover, we use collision-induced unfolding and time-resolved hydrogen–deuterium exchange MS analyses to study the conformational dynamics that occur when this interaction is intact or disrupted. Our results suggest that the intracellular distribution of La may be linked to its RNA-binding modes and provide the first evidence for a direct protein–protein interdomain interaction in La proteins.

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