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An Enhanced Green Fluorescence Protein-based Assay for Studying Neurite Outgrowth in Primary Neurons
Published on: October 19, 2019
Metastasis suppressor 1 regulates neurite outgrowth in primary neuron cultures
Juan Yu1, Shuyun Lin1, Mei Wang1
1The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, College of Life Sciences, Hunan Normal University, Changsha 410081, PR China.
Abstract:
Metastasis suppressor 1 (MTSS1) or missing in metastasis (MIM) is an actin- and membrane-binding protein with tumor suppressor functions. MTSS1 is important for cell morphology, motility, metastasis. The role of MTSS1 in cell morphology has been widely investigated in non-neuronal tissues; however the role of MTSS1 in neurite outgrowth remains unclear. Here we investigated the effect of MTSS1 on neurite outgrowth in primary cerebellar granule and hippocampal neurons of mouse. We found that overexpression of MTSS1 in cerebellar granule neurons significantly enhanced dendrite elaboration but inhibited axon elongation. This phenotype was significantly reduced by deletion of the Wiskott-Aldrich homology 2 (WH2) motif and point mutation in the insulin receptor substrate p53 (IRSp53) and MIM/MTSS1 homology (IMD) domain. Furthermore, inhibition of Rac1 activity or blocking of phosphatidyl inositol phosphates (PIPs) signaling decreased the effect of MTSS1 markedly. In accordance with the over-expression data, knockdown of MTSS1 in cerebellar granule neurons could increase the axon length but decrease the dendrite length and the number of dendrites. In addition, MTSS1 knock down in embryonic hippocampal neurons suppressed neurite branching and reduced dendrite length. Our findings have demonstrated that MTSS1 modulates neuronal morphology, possibly through a Rac1-PIPs signaling pathway.
Insights
Metastasis suppressor 1 (MTSS1) enhances dendrite elaboration while inhibiting axon growth in neurons. This protein modulates neuronal morphology via Rac1 and PIPs signaling pathways.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Metastasis suppressor 1 (MTSS1), also known as missing in metastasis (MIM), is an actin- and membrane-binding protein with established tumor suppressor functions.
- MTSS1 plays a crucial role in cell morphology, motility, and metastasis in various cell types.
- While MTSS1's role in non-neuronal cell morphology is well-studied, its impact on neurite outgrowth in neurons remains largely unexplored.
Purpose of the Study:
- To investigate the effect of MTSS1 on neurite outgrowth in primary mouse cerebellar granule and hippocampal neurons.
- To elucidate the molecular mechanisms underlying MTSS1's influence on neuronal morphology.
Main Methods:
- Overexpression and knockdown of MTSS1 in primary neuronal cultures.
- Analysis of neurite outgrowth, including axon elongation and dendrite elaboration.
- Site-directed mutagenesis to assess the role of specific protein domains (WH2 motif, IMD domain).
- Pharmacological inhibition of Rac1 activity and phosphatidyl inositol phosphates (PIPs) signaling.
Main Results:
- Overexpression of MTSS1 enhanced dendrite elaboration but inhibited axon elongation in cerebellar granule neurons.
- Deletion of the WH2 motif or mutation in the IMD domain significantly reduced MTSS1's effect on neuronal morphology.
- Inhibition of Rac1 or PIPs signaling markedly diminished the impact of MTSS1.
- MTSS1 knockdown led to increased axon length but decreased dendrite length and number in cerebellar granule neurons.
- MTSS1 knockdown in hippocampal neurons suppressed neurite branching and reduced dendrite length.
Conclusions:
- MTSS1 plays a significant role in modulating neuronal morphology, specifically influencing neurite outgrowth.
- The effects of MTSS1 on neuronal morphology are mediated through its WH2 motif and IMD domain.
- MTSS1 likely exerts its function via a signaling pathway involving Rac1 and PIPs.
