scholarly journals Dynein acts to cluster glutamate receptors and traffic the PIP5 kinase, Skittles, to regulate postsynaptic membrane organization at the neuromuscular junction

2021 ◽  
Author(s):  
Amanda L. Neisch ◽  
Thomas Pengo ◽  
Adam W. Avery ◽  
Min-Gang Li ◽  
Thomas S. Hays
Keyword(s):  
Neuromuscular Junctions ◽  
Cytoplasmic Dynein ◽  
Postsynaptic Membrane ◽  
Independent Function ◽  
Protein Levels ◽  
Cluster Organization ◽  
Synaptic Terminals ◽  
Cargo Transport ◽  
Spectrin Cytoskeleton ◽  
Phosphatidylinositol 4

Cytoplasmic dynein is essential in motoneurons for retrograde cargo transport that sustains neuronal connectivity. Little, however, is known about dynein's function on the postsynaptic side of the circuit. Here we report distinct postsynaptic roles for dynein at neuromuscular junctions (NMJs). Intriguingly, we show that dynein punctae accumulate postsynaptically at glutamatergic synaptic terminals. Moreover, Skittles, a phosphatidylinositol 4-phosphate 5-kinase that produces PI(4,5)P2 to organize the spectrin cytoskeleton, also localizes specifically to glutamatergic synaptic terminals. Depletion of postsynaptic dynein disrupts the accumulation of Skittles, PI(4,5)P2 phospholipid, and organization of the spectrin cytoskeleton at the postsynaptic membrane. Coincidental with dynein depletion, we observe an increase in the clusters size of ionotropic glutamate receptor (iGluR), and an increase in the amplitude and frequency of mEJPs. However, PI(4,5)P2 levels do not affect iGluR clustering and dynein does not affect the protein levels of iGluR subunits at the NMJ, suggesting a separate, transport independent function for dynein in iGluR cluster organization. As dynein punctae closely associate with iGluR clusters, we propose that dynein physically tethers iGluR clusters at the postsynaptic membrane to ensure proper synaptic transmission.

scholarly journals PBRM1 Cooperates with YTHDF2 to Control HIF-1α Protein Translation

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1425
Author(s):  
Alena Shmakova ◽  
Mark Frost ◽  
Michael Batie ◽  
Niall S. Kenneth ◽  
Sonia Rocha
Keyword(s):  
Protein Expression ◽  
Cellular Response ◽  
Transcriptional Activity ◽  
Mrna Translation ◽  
Protein Translation ◽  
Signalling Pathways ◽  
Independent Function ◽  
Protein Levels ◽  
Associated Proteins ◽  
Core Components

PBRM1, a component of the chromatin remodeller SWI/SNF, is often deleted or mutated in human cancers, most prominently in renal cancers. Core components of the SWI/SNF complex have been shown to be important for the cellular response to hypoxia. Here, we investigated how PBRM1 controls HIF-1α activity. We found that PBRM1 is required for HIF-1α transcriptional activity and protein levels. Mechanistically, PBRM1 is important for HIF-1α mRNA translation, as absence of PBRM1 results in reduced actively translating HIF-1α mRNA. Interestingly, we found that PBRM1, but not BRG1, interacts with the m6A reader protein YTHDF2. HIF-1α mRNA is m6A-modified, bound by PBRM1 and YTHDF2. PBRM1 is necessary for YTHDF2 binding to HIF-1α mRNA and reduction of YTHDF2 results in reduced HIF-1α protein expression in cells. Our results identify a SWI/SNF-independent function for PBRM1, interacting with HIF-1α mRNA and the epitranscriptome machinery. Furthermore, our results suggest that the epitranscriptome-associated proteins play a role in the control of hypoxia signalling pathways.

scholarly journals Registered report: A coding-independent function of gene and pseudogene mRNAs regulates tumour biology

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Israr Khan ◽  
John Kerwin ◽  
Kate Owen ◽  
Erin Griner ◽  
Keyword(s):  
Cell Proliferation ◽  
Tumor Cell ◽  
Cancer Biology ◽  
Open Science ◽  
Tumor Cell Proliferation ◽  
Independent Function ◽  
Pten Protein ◽  
Protein Levels ◽  
High Profile ◽  
Tumour Biology

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (<xref ref-type="bibr" rid="bib9">Errington et al., 2014</xref>). This Registered report describes the proposed replication plan of key experiments from ‘A coding-independent function of gene and pseudogene mRNAs regulates tumour biology’ by <xref ref-type="bibr" rid="bib26">Poliseno et al. (2010)</xref>, published in Nature in 2010. The key experiments to be replicated are reported in Figures 1D, 2F-H, and 4A. In these experiments, Poliseno and colleagues report microRNAs miR-19b and miR-20a transcriptionally suppress both PTEN and PTENP1 in prostate cancer cells (Figure 1D; <xref ref-type="bibr" rid="bib26">Poliseno et al., 2010</xref>). Decreased expression of PTEN and/or PTENP1 resulted in downregulated PTEN protein levels (Figure 2H), downregulation of both mRNAs (Figure 2G), and increased tumor cell proliferation (Figure 2F; <xref ref-type="bibr" rid="bib26">Poliseno et al., 2010</xref>). Furthermore, overexpression of the PTEN 3′ UTR enhanced PTENP1 mRNA abundance limiting tumor cell proliferation, providing additional evidence for the co-regulation of PTEN and PTENP1 (Figure 4A; <xref ref-type="bibr" rid="bib26">Poliseno et al., 2010</xref>). The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published in eLife.

scholarly journals Emerging mechanisms of dynein transport in the cytoplasm versus the cilium

2018 ◽  
Vol 46 (4) ◽  
pp. 967-982 ◽  
Author(s):  
Anthony J. Roberts
Keyword(s):  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Mechanisms Of Action ◽  
Cell Interior ◽  
Long Distance ◽  
Cargo Transport ◽  
Recent Advances ◽  
Human Disorders ◽  
Cilia And Flagella

Two classes of dynein power long-distance cargo transport in different cellular contexts. Cytoplasmic dynein-1 is responsible for the majority of transport toward microtubule minus ends in the cell interior. Dynein-2, also known as intraflagellar transport dynein, moves cargoes along the axoneme of eukaryotic cilia and flagella. Both dyneins operate as large ATP-driven motor complexes, whose dysfunction is associated with a group of human disorders. But how similar are their mechanisms of action and regulation? To examine this question, this review focuses on recent advances in dynein-1 and -2 research, and probes to what extent the emerging principles of dynein-1 transport could apply to or differ from those of the less well-understood dynein-2 mechanoenzyme.

Membrane potential fluctuations produced by glutamate in nerve cells of the squid

1975 ◽  
Vol 191 (1105) ◽  
pp. 561-565 ◽  
Keyword(s):  
Membrane Potential ◽  
Neuromuscular Junctions ◽  
Noise Analysis ◽  
Postsynaptic Membrane ◽  
Nerve Cells ◽  
Voltage Fluctuations ◽  
Potential Fluctuations ◽  
Synaptic Inputs ◽  
Small Voltage ◽  
Membrane Potential Fluctuations

Glutamate-induced potential changes have been recorded with intracellular electrodes in nerve cells of the squid. The responses are accompanied by small voltage fluctuations which resemble postsynaptic ‘membrane noise’ observed at neuromuscular junctions. Certain limitations are discussed in extending the noise analysis to neurons with multiple synaptic inputs.

scholarly journals Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes

2001 ◽  
Vol 154 (4) ◽  
pp. 707-718 ◽  
Author(s):  
Leana M. Topper ◽  
Holger Bastians ◽  
Joan V. Ruderman ◽  
Gary J. Gorbsky
Keyword(s):  
Mammalian Cells ◽  
Proteasome Inhibitors ◽  
Metaphase Plate ◽  
Cytoplasmic Dynein ◽  
Mitogen Activated Protein Kinase ◽  
Protein Levels ◽  
Mitogen Activated Protein ◽  
Associated Proteins ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E–associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.

scholarly journals Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions.

1981 ◽  
Vol 88 (2) ◽  
pp. 453-458 ◽  
Author(s):  
Y Nakajima ◽  
P C Bridgman
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Freeze Fracture ◽  
Cholesterol Content ◽  
Postsynaptic Membrane ◽  
Large Particles ◽  
Glutaraldehyde Solution ◽  
Active Zones ◽  
Low Cholesterol

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze-fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.

scholarly journals Dynein-Mediated Cargo Transport in Vivo

2000 ◽  
Vol 148 (5) ◽  
pp. 945-956 ◽  
Author(s):  
Steven P. Gross ◽  
Michael A. Welte ◽  
Steven M. Block ◽  
Eric F. Wieschaus
Keyword(s):  
Lipid Droplets ◽  
Developmental Stages ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Unit Distance ◽  
Cargo Transport ◽  
Single Droplets ◽  
Drosophila Embryos

Cytoplasmic dynein is a microtubule-based motor with diverse cellular roles. Here, we use mutations in the dynein heavy chain gene to impair the motor's function, and employ biophysical measurements to demonstrate that cytoplasmic dynein is responsible for the minus end motion of bidirectionally moving lipid droplets in early Drosophila embryos. This analysis yields an estimate for the force that a single cytoplasmic dynein exerts in vivo (1.1 pN). It also allows us to quantitate dynein-mediated cargo motion in vivo, providing a framework for investigating how dynein's activity is controlled. We identify three distinct travel states whose general features also characterize plus end motion. These states are preserved in different developmental stages. We had previously provided evidence that for each travel direction, single droplets are moved by multiple motors of the same type (Welte et al. 1998). Droplet travel distances (runs) are much shorter than expected for multiple motors based on in vitro estimates of cytoplasmic dynein processivity. Therefore, we propose the existence of a process that ends runs before the motors fall off the microtubules. We find that this process acts with a constant probability per unit distance, and is typically coupled to a switch in travel direction. A process with similar properties governs plus end motion, and its regulation controls the net direction of transport.

scholarly journals Fission yeast myosin I facilitates PI(4,5)P2-mediated anchoring of cytoplasmic dynein to the cortex

2017 ◽  
Vol 114 (13) ◽  
pp. E2672-E2681 ◽  
Author(s):  
Jerrin Mathew Thankachan ◽  
Stephen Sukumar Nuthalapati ◽  
Nireekshit Addanki Tirumala ◽  
Vaishnavi Ananthanarayanan
Keyword(s):  
Fission Yeast ◽  
Total Internal Reflection ◽  
Nuclear Material ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Binding Partner ◽  
Myosin I ◽  
Phosphatidylinositol 4

Several key processes in the cell, such as vesicle transport and spindle positioning, are mediated by the motor protein cytoplasmic dynein, which produces force on the microtubule. For the functions that require movement of the centrosome and the associated nuclear material, dynein needs to have a stable attachment at the cell cortex. In fission yeast, Mcp5 is the anchor protein of dynein and is required for the oscillations of the horsetail nucleus during meiotic prophase. Although the role of Mcp5 in anchoring dynein to the cortex has been identified, it is unknown how Mcp5 associates with the membrane as well as the importance of the underlying attachment to the nuclear oscillations. Here, we set out to quantify Mcp5 organization and identify the binding partner of Mcp5 at the membrane. We used confocal and total internal reflection fluorescence microscopy to count the number of Mcp5 foci and the number of Mcp5 molecules in an individual focus. Further, we quantified the localization pattern of Mcp5 in fission yeast zygotes and show by perturbation of phosphatidylinositol 4-phosphate 5-kinase that Mcp5 binds to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Remarkably, we discovered that the myosin I protein in fission yeast, Myo1, which is required for organization of sterol-rich domains in the cell membrane, facilitates the localization of Mcp5 and that of cytoplasmic dynein on the membrane. Finally, we demonstrate that Myo1-facilitated association of Mcp5 and dynein to the membrane determines the dynamics of nuclear oscillations and, in essence, dynein activity.

scholarly journals Salbutamol modifies the neuromuscular junction in a mouse model of ColQ myasthenic syndrome

2019 ◽  
Vol 28 (14) ◽  
pp. 2339-2351 ◽  
Author(s):  
Grace M McMacken ◽  
Sally Spendiff ◽  
Roger G Whittaker ◽  
Emily O’Connor ◽  
Rachel M Howarth ◽  
...  
Keyword(s):  
Muscle Strength ◽  
Neuromuscular Junction ◽  
Structural Changes ◽  
Neuromuscular Junctions ◽  
Skeletal Muscle Fibre ◽  
Postsynaptic Membrane ◽  
Adrenergic Agonists ◽  
Muscle Fibre Size ◽  
Gradual Improvement ◽  
Functional Benefit

Abstract The β-adrenergic agonists salbutamol and ephedrine have proven to be effective as therapies for human disorders of the neuromuscular junction, in particular many subsets of congenital myasthenic syndromes. However, the mechanisms underlying this clinical benefit are unknown and improved understanding of the effect of adrenergic signalling on the neuromuscular junction is essential to facilitate the development of more targeted therapies. Here, we investigated the effect of salbutamol treatment on the neuromuscular junction in the ColQ deficient mouse, a model of end-plate acetylcholinesterase deficiency. ColQ−/− mice received 7 weeks of daily salbutamol injection, and the effect on muscle strength and neuromuscular junction morphology was analysed. We show that salbutamol leads to a gradual improvement in muscle strength in ColQ−/− mice. In addition, the neuromuscular junctions of salbutamol treated mice showed significant improvements in several postsynaptic morphological defects, including increased synaptic area, acetylcholine receptor area and density, and extent of postjunctional folds. These changes occurred without alterations in skeletal muscle fibre size or type. These findings suggest that β-adrenergic agonists lead to functional benefit in the ColQ−/− mouse and to long-term structural changes at the neuromuscular junction. These effects are primarily at the postsynaptic membrane and may lead to enhanced neuromuscular transmission.

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