Dataset Information

Proteomic dissection of substrate nanotopography-sensitive mechanotransductive signalling hubs that foster neuronal differentiation.

ABSTRACT: Neuronal cells are competent in precisely sensing nanotopographical features of their microenvironment and the intrinsic information impacts on neuronal functioning and differentiation “interpreted” by mechanotransductive processes. Attempts to influence and direct neuronal differentiation behaviour by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the difficulty since many details of mechanosensing/-transduction are still elusive. Introducing omics methods into these biomaterial approaches has the potential to provide a deeper insight into the molecular processes and signalling cascades underlying mechanosensing/-transduction but their exigence in cellular material is often opposed by technical limitations of the biomaterial fabrication methods. Supersonic cluster beam deposition (SCBD) of zirconia nanoparticles allows instead the realisation of large macroscopic areas with biocompatible nanostructured films equipped with controllable and reproducible ECM-like nanoroughness that have been recently shown to foster neuron differentiation and maturation. Exploiting this capacity of SCBD, we challenged mechanosensing/-transduction and differentiative behaviour of neuron-like PC12 cells interacting with diverse nanotopographies and/or by changing their biomechanical status, to analyse their phosphoproteomic profiles in these settings. Versatile proteins were found to be affected that can be associated to significant processes along the mechanotransductive signal sequence, i.e. cell/cell interaction, glycocalyx and ECM, membrane/f-actin linkage and integrin activation, cell/substrate interaction, IAC, actomyosin organisation/cellular mechanics, nuclear organisation and transcription. The phosphoproteomic data suggested furthermore an involvement of ILK, mTor, Wnt and calcium signalling in these nanotopography- and/or cell mechanics-related processes. Altogether, potential nanotopography-sensitive, mechanotransductive signalling hubs participating to neuronal differentiation were dissected.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cell Culture

SUBMITTER: gabriella tedeschi

LAB HEAD: Gabriella Tedeschi

PROVIDER: PXD007644 | Pride | 2018-01-24

REPOSITORIES: Pride

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Publications

Methionine supplementation stimulates mitochondrial respiration.

Tripodi Farida F Castoldi Andrea A Nicastro Raffaele R Reghellin Veronica V Lombardi Linda L Airoldi Cristina C Falletta Ermelinda E Maffioli Elisa E Scarcia Pasquale P Palmieri Luigi L Alberghina Lilia L Agrimi Gennaro G Tedeschi Gabriella G Coccetti Paola P

Biochimica et biophysica acta. Molecular cell research 20181002 12

Mitochondria play essential metabolic functions in eukaryotes. Although their major role is the generation of energy in the form of ATP, they are also involved in maintenance of cellular redox state, conversion and biosynthesis of metabolites and signal transduction. Most mitochondrial functions are conserved in eukaryotic systems and mitochondrial dysfunctions trigger several human diseases. By using multi-omics approach, we investigate the effect of methionine supplementation on yeast cellular ...[more]

PMID: 30290237

Publication: 1/2

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Project description:The subcellular localization of specific mRNAs is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. In neurons, mRNA trafficking and local protein translation in dendrites provides an important mechanism that mediates synaptic development and plasticity. The significance of mRNA targeting and protein synthesis in axons, however, is still unclear. Only a small number of transcripts have been identified in axons to date, and their contribution to axon growth and neuronal survival remains largely unknown. Here, we report the results of a novel screen that allowed the separate identification of mRNAs localized in cell bodies and in axons of developing neurons. Using compartmentalized cultures of sympathetic neurons and Sequential Analysis of Gene Expression (SAGE), the screen identified more than 200 axonal mRNAs, including ones that encode cytoskeletal proteins and proteins that function in neural development and signal transduction. Importantly, several classes of transcripts were selectively enriched in axons, indicating that an active process drives the targeting of specific mRNAs from the cell bodies to the axons. This study is the first comprehensive and unbiased analysis of mRNA localization in subcellular domains of any neuronal cell type. We used compartmentalized chambers to culture neonatal rat sympathetic neurons (Campenot, 1977). In these cultures, the cell bodies are separated from the distal axons by a 1 mm wide Teflon divider, which maintains the cell bodies and axon terminals in separate fluid compartments. Primary rat sympathetic neurons are especially suitable for compartmentalized culture because they can be grown as a highly homogeneous population without glial cells. Neurons were seeded in the central compartment with nerve growth factor (NGF), and after a few days, the NGF was lowered in this compartment and supplied only to the peripheral compartment to stimulate axon growth. The anti-mitotic agent cytosine arabinoside C (Ara-C) was added to both compartments to remove non-neuronal cells. mRNA was then isolated after 12 days in culture (DIV) from cell body or axon compartments. As the initial mRNA content in axons was not sufficient to perform the SAGE analysis, both axon and cell body mRNAs were subjected to two rounds of linear amplification to obtain antisense RNA (aRNA). The amplified aRNA was then reverse transcribed and second strand synthesis was performed to proceed with the SAGE assay using the LongSAGE kit (Invitrogen) according to the manufacturer’s protocol.

Project description:Nerve growth factor (NGF) pays a role in neuronal differentiation and may also play a role in hematopoietic differentiation as shown by synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with M-CSF or SCF. However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells. To study the signal transduction pathways induced by NGF treatment in hematopoietic cells, we utilized the mastocytoma cell line HMC-1 (V560G c-Kit) which expresses the NGF receptor, TrkA, as well as the constitutively activated SCF receptor, V560G c-Kit, which can be inhibited by treatment with tyrosine kinase inhibitor imatinib mesylate. NGF rescues HMC-1 (V560G c-Kit) from imatinib induced cell death and promotes proliferation. To examine the NGF mediated proliferation and survival in these cells, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit activity for 4 h) by transcriptome analysis. The following conclusions can be drawn from the microarray data: Firstly, gene expression profiling reaveals 50% overlap of genes induced by NGF-TrkA with genes expressed downstream of V560G c-Kit. Secondly, NGF treatment does not enhance expression of genes involved in immune related functions that were down modulated by imatinib. Thirdly, more than 55% of common upregulated genes are involved in cell proliferation and survival. Forthly, we found KLF2 and SMAD7 as the NGF mediated novel down stream genes in hematopoietic cells that may play a role in NGF mediated survival signal in HMC-1 (V560G c-Kit) cells. Keywords: Transcriptome analysis - comparison of TrkA and V560G c-Kit downstream genes in HMC-1 (V560G c-Kit) cells In total, 4 samples were analyzed, one of which served as a common reference (HMC1 17h SerumStarv 4h Imatinib). This sample was labeled with Cy3. All additional samples were labeled with Cy5 and were cohybridized against the common reference. The first hybridization (M2370) represents a self-self-hybridization using the common reference.

Dataset Information

Proteomic dissection of substrate nanotopography-sensitive mechanotransductive signalling hubs that foster neuronal differentiation.

Publications

Methionine supplementation stimulates mitochondrial respiration.

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