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1

The molecular machinery that orchestrates neuromuscular junction development

100%
Proszynski T. J., Sanes J. R.
Acta Neurobiologiae Experimentalis
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2013
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tom 73
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nr Suppl.1
Neuromuscular junctions (NMJs) in mammalian skeletal muscle undergo a postnatal topological transformation from a simple oval plaque to a complex branch-shaped structure. We previously demonstrated that podosomes, actin-rich adhesive organelles, promote the remodeling process and showed a key role for one podosome component, LL5β. To better understand molecular mechanisms of postsynaptic maturation, we purified LL5β protein complex from myotubes and showed that three regulators of the actin cytoskeleton – Amotl2, Asef2 and Flii – interact with LL5β. These and other LL5β-interacting proteins are associated with conventional podosomes in macrophages and podosome-like invadopodia in fibroblasts, strengthening the close relationship between synaptic and non-synaptic podosomes. We then focused on Amotl2, showing that it is associated with synaptic podosomes in cultured myotubes and with NMJs in vivo. Depletion of Amotl2 in myotubes leads to increased size of synaptic podosomes and corresponding alterations in postsynaptic topology. Depletion of Amotl2 from fibroblasts disrupts invadopodia in these cells. These results demonstrate the role Amotl2 plays in synaptic remodeling and support the involvement of podosomes in this process.
2

The role of drebrin in the neuromuscular junction

81%
Alvarez-Suarez P., Gawor M., Proszynski T. J.
Acta Neurobiologiae Experimentalis
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2019
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tom 79
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nr Suppl.1
INTRODUCTION: Drebrin is an actin-binding protein that regulates cytoskeleton dynamics in different cell types. It plays an important role in shaping dendritic spines and facilitating the stabilization of neurotransmitter receptors at the postsynaptic membrane in the CNS. Its role at the neuromuscular junction (NMJ) in the PNS has not been investigated. AIM(S): In the present study we aimed to explore the role of Drebrin in this special type of synapse and to study its mechanism of action. METHOD(S): We used an in vitro model of C2C12‑derived myotubes in which Drebrin1 expression is silenced with siRNA or its actin-binding function is blocked by a BTP2 inhibitor. To address the role of Drebrin at the postsynaptic machinery, we used both biochemical and immunohistochemical approaches. RESULTS: We found that Drebrin colocalizes with acetylcholine receptors (AChR) at the surface of myofibers in vivo and in vitro, and its depletion causes impairments in receptor aggregation and clusters complexity, suggesting a crucial role in the regulation of these processes. We assessed whether drebrin inhibition affects the expression levels and cell surface delivery of AChRs or the microtubule organization underneath AChRs. Our experiments revealed that drebrin depletion in cultured myotubes affects the organization of cortical microtubules, which has been previously shown to be indispensable for incorporation of newly synthesized AChR into the postsynaptic specialization. CONCLUSIONS: We found that Drebrin is a component of the muscle postsynaptic machinery and it plays an important role in their organization. The mechanism through which Drebrin regulates AChR clustering appears to occur through its interaction with EB3, that leads to the recruitment of microtubules and allows the stabilization of AChRs. FINANCIAL SUPPORT: This research was supported by the National Science Centre grants: UMO‑2018/29/B/ NZ3/02675, UMO‑2016/21/D/NZ4/03069, and UMO- ‑2018/29/N/NZ3/02682.
3

Amotl1: a novel synaptic protein important for mice social behavior and brain organization

61%
Krzemien J., Rojek K., Winiarski M., Boguszewski P. M., Knapska E., Proszynski T. J.
Acta Neurobiologiae Experimentalis
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2019
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tom 79
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nr Suppl.1
INTRODUCTION: The angiomotin family comprises of three scaffold proteins – Amot, Amotl1, and Amotl2 – that have been implicated in the regulation of cell polarity, migration, and proliferation. Recent in vitro studies have reported that Amot localizes to the synapses in mature neurons and regulates dendritic spine maturation. AIM(S): We have found that Amot, together with Yap1, the Hippo pathway transcription co‑activator, are critical for proper dendritic arborization and mice locomotor coordination. However, to date the function of the two other Angiomotins, Amotl1 and Amotl2, in neurons has not been investigated. METHOD(S): To study Amotl1 function in the mouse brain, we generated systemic and neuron-specific knock‑out (KO) mice. To assess general locomotion, we performed an open field test. Amotl1 KO mice sociability was evaluated with the three-chamber task, automatic Eco‑Hab approach, and nesting test. To record the animal’s anxiety response, we used the marble burying test. RESULTS: In the present study, we show that Amotl1 localizes to the synaptic compartments in neurons. Deletion of Amotl1 leads to hyperlocomotion, decreased anxiety-like behavior, and alteration in mice sociability. Amotl1 ablation causes an increase in volume of lateral ventricles in the mouse brain by 50%. These features have been previously observed in animal models of various psychiatric disorders, such as schizophrenia or autism. Interestingly, mass spectrometry analysis of neuron‑specific interactors demonstrated that Amotl1 binds to FMR1 and FXR1, mutations of which cause Fragile X syndrome. CONCLUSIONS: We identified a novel synaptic protein, Amotl1, the deletion of which causes behavioral deficits and that it could be a potential molecular target for the development of new therapeutics for neurological disorders. FINANCIAL SUPPORT: This research was supported by National Science Center (NCN) grants: UMO- ‑2018/29/B/NZ3/02675, UMO-2018/29/N/NZ3/02682.
4

Liprin-alpha-1 is a novel component of the neuromuscular junction and involved in the organization of the postysynaptic machinery

61%
Bernadzki K. M., Pezinski M., Gawor M., Mazurek P., Niewiadomski P., Redowicz J., Proszynski T. J.
Acta Neurobiologiae Experimentalis
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2017
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tom 77
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nr Suppl.1
INTRODUCTION: Neuromuscular junctions (NMJs) are specialized synapses formed between the motor neurons and skeletal muscles. The proper functioning of the NMJ is important for activities involving muscle contraction including breathing. Liprin‑α‑1 was previously identified in a biochemical screen as an interactor of α‑dystrobrevin‑1, a component of dystrophin-associated glycoprotein complex (DGC) responsible for the attachment of the postsynaptic machinery to the extracellular matrix. Liprin‑α‑1 has been shown to be an organizer of synapses in the central nervous system and our study presents an insight into the localization and function of this protein at the vertebrate NMJ. AIM(S): To unravel the localization and function of liprin‑α‑1 at the vertebrate NMJ. METHOD(S): We employed immunohistochemical, siRNA-mediated RNAi techniques and confocal microscopy techniques. RESULTS: We show that liprin‑α‑1 localizes to the postsynaptic site of NMJ in a nerve-dependent manner, and its localization is maintained throughout postnatal development. Liprin‑α‑1 plays a crucial role in the formation of the postsynaptic machinery since liprin‑α‑1 depleted myotubes failed to cluster postsynaptic acetylcholine receptors (AChRs). We provide evidence that liprin‑α‑1 plays a role in the attachment of microtubule ends to the cell cortex, where they stabilize the postsynaptic machinery and promote clustering of AChRs. Consistently with this observation abrupt disruption of microtubules with in cultured myotubes did not affected preexisting AChR clusters, but abolished formation of the new assemblies. CONCLUSIONS: Our results show that Liprin‑α‑1 is important component of the postsynaptic machinery involved in AChR clustering. FINANCIAL SUPPORT: This research was supported by the NCN grants 2012/05/E/NZ3/00487, 2015/19/N/ NZ5/02268, 2014/13/B/NZ3/00909, and 2013/09/B/ NZ3/03524.
5

Novel Alpha-Dystrobrevin interactors regulate neuromuscular junction postsynaptic machinery

61%
Gawor M., Gingras J., Bernadzki K., Niewiadomski P., Sanes R., Proszynski T. J.
Acta Neurobiologiae Experimentalis
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2015
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tom 75
|
nr Supl.
BACKGROUND AND AIMS: Alpha-Dystrobrevin-1 (aDB1) is a component of the dystrophin-associated glycoprotein complex (DGC), which stabilizes the neuromuscular junction (NMJ) postsynaptic machinery. The goal of our research is to gain insight into the mechanism of aDB-1 function at the NMJ, with special focus on the role of aDB1 phosphorylation, which was previously shown to be critical for proper synaptic organization. METHODS: To determine the importance of each tyrosine phosphorylation site, we expressed mutant proteins lacking individual sites in myotubes. Next, we developed phospho-specific antibodies and used them to analyze the level of aDB1 phosphorylation during NMJ remodeling. We also performed a biochemical screen to identify general and phospho-specific aDB1-binding proteins. Finally, we performed RNAi experiments on cultured myotubes to demonstrate the importance of these proteins in the organization of AChR clusters. RESULTS: Our experiments identified Liprin-α1, α-Catulin and Usp9x as novel aDB1-interacting proteins. We have demonstrated that aDB1 phosphorylation is dynamically regulated during NMJ remodeling in development and upon denervation and that the phosphorylation at the most critical tyrosine Y713 triggers recruitment of aDB1 phospho-specific interacting proteins, including Grb2, SH3BP2, Arhgef5 and PI3K. Subsequently, we demonstrated that Liprin-α1, α-Catulin and Grb2 are associated with the postsynaptic NMJ machinery and are indispensable for proper AChR organization. CONCLUSIONS: Our research highlights the importance of aDB1 phosphorylation in the remodeling of neuromuscular synapses. We identify several novel aDB1-interacting components of the postsynaptic machinery, which play important roles in its organization. This research was supported by the grant 2013/09/B/NZ3/03524 from the National Science Centre (NCN).
6

The role of Amot and Yap1 in the regulation of dendritic tree morphogenesis and cerebellar functions

61%
Rojek K., Krzemien J., Dolezyczek H., Rylski M., Kaczmarek L., Jaworski J., Proszynski T. J.
Acta Neurobiologiae Experimentalis
|
2019
|
tom 79
|
nr Suppl.1
INTRODUCTION: The Amot‑Yap1 complex plays a major role in the regulation of cell contact inhibition, cellular polarity and growth in many cell types. However, the function of the Amot and Hippo pathway transcription co‑activator Yap1 in the CNS remains unclear. Recent studies have demonstrated that, in mature hippocampal neurons, Amot localizes to dendritic spines where it associates with synaptic protein and regulates actin cytoskeleton. However, its function during neuronal development has not been studied. METHOD(S): Cultured primary neurons were used for RNAi experiments. For in vivo functional analysis, we used Amot and Yap1 conditional KO mice. For deletion in single sparse neurons, mice were injected with low doses of AAV‑CRE. For behavioral analysis, we used rotarod, catwalk, and foot fault tests. RESULTS: We demonstrate that Amot is a critical mediator of dendritogenesis in cultured hippocampal cells and Purkinje cells in the brain. Amot function in developing neurons depends on interactions with Yap1, which is also indispensable for dendrite growth and arborization in vitro. Conditional deletion of Amot or Yap1 in neurons leads to decreased Purkinje cell dendritic tree complexity, abnormal cerebellar morphology, and impaired motor coordination. The ability of Amot and Yap1 to regulate dendritic growth depends on regulation of S6 kinase activity and phosphorylation of S6 ribosomal protein. Hence, we suggest that Amot and Yap1 control dendritic tree morphogenesis through a cross‑talk with the PI3K/mTOR pathway, a known regulator of dendritogenesis. CONCLUSIONS: We identify a novel role for the scaffold protein Amot and the Hippo pathway transcription co‑activator Yap1 in dendritic morphogenesis. FINANCIAL SUPPORT: This research was supported by National Science Center grants 2012/05/E/ NZ3/00487 and 2015/19/N/NZ3/02346.
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