FLIM FRET Visualization of Cdc42 Activation by Netrin-1 in Embryonic Spinal Commissural Neuron Growth Cones

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Abstract

Netrin-1 is an essential extracellular chemoattractant that signals through its receptor DCC to guide commissural axon extension in the embryonic spinal cord. DCC directs the organization of F-actin in growth cones by activating an intracellular protein complex that includes the Rho GTPase Cdc42, a critical regulator of cell polarity and directional migration. To address the spatial distribution of signaling events downstream of netrin-1, we expressed the FRET biosensor Raichu-Cdc42 in cultured embryonic rat spinal commissural neurons. Using FLIM-FRET imaging we detected rapid activation of Cdc42 in neuronal growth cones following application of netrin-1. Investigating the signaling mechanisms that control Cdc42 activation by netrin-1, we demonstrate that netrin-1 rapidly enriches DCC at the leading edge of commissural neuron growth cones and that netrin-1 induced activation of Cdc42 in the growth cone is blocked by inhibiting src family kinase signaling. These findings reveal the activation of Cdc42 in embryonic spinal commissural axon growth cones and support the conclusion that src family kinase activation downstream of DCC is required for Cdc42 activation by netrin-1.

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Most cited references 73

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All cell types polarize, at least transiently, during division or to generate specialized shapes and functions. This capacity extends from yeast to mammals, and it is now clear that many features of the molecular mechanisms controlling polarization are conserved in all eukaryotic cells. At the centre of the action is Cdc42, a small GTPase of the Rho family. Its activity is precisely controlled both temporally and spatially, and this can be achieved by a wide variety of extracellular cues in multicellular organisms. Moreover, although the functional characteristics of cell polarity are extremely variable (depending on the cell type and the biological context), Cdc42 has an amazing capacity to co-ordinate the control of multiple signal transduction pathways.

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Fluorescence lifetime imaging--techniques and applications.

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Fluorescence lifetime imaging (FLIM) uses the fact that the fluorescence lifetime of a fluorophore depends on its molecular environment but not on its concentration. Molecular effects in a sample can therefore be investigated independently of the variable, and usually unknown concentration of the fluorophore. There is a variety of technical solutions of lifetime imaging in microscopy. The technical part of this paper focuses on time-domain FLIM by multidimensional time-correlated single photon counting, time-domain FLIM by gated image intensifiers, frequency-domain FLIM by gain-modulated image intensifiers, and frequency-domain FLIM by gain-modulated photomultipliers. The application part describes the most frequent FLIM applications: Measurement of molecular environment parameters, protein-interaction measurements by Förster resonance energy transfer (FRET), and measurements of the metabolic state of cells and tissue via their autofluorescence. Measurements of local environment parameters are based on lifetime changes induced by fluorescence quenching or conformation changes of the fluorophores. The advantage over intensity-based measurements is that no special ratiometric fluorophores are needed. Therefore, a much wider selection of fluorescence markers can be used, and a wider range of cell parameters is accessible. FLIM-FRET measures the change in the decay function of the FRET donor on interaction with an acceptor. FLIM-based FRET measurement does not have to cope with problems like donor bleedthrough or directly excited acceptor fluorescence. This relaxes the requirements to the absorption and emission spectra of the donors and acceptors used. Moreover, FLIM-FRET measurements are able to distinguish interacting and noninteracting fractions of the donor, and thus obtain independent information about distances and interacting and noninteracting protein fractions. This is information not accessible by steady-state FRET techniques. Autofluorescence FLIM exploits changes in the decay parameters of endogenous fluorophores with the metabolic state of the cells or the tissue. By resolving changes in the binding, conformation, and composition of biologically relevant compounds FLIM delivers information not accessible by steady-state fluorescence techniques. © 2012 The Author Journal of Microscopy © 2012 Royal Microscopical Society.

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Here, we have studied the activity of a novel protein-tyrosine kinase inhibitor that is selective for the Src family of tyrosine kinases. We have focused our study on the effects of this compound on T cell receptor-induced T cell activation, a process dependent on the activity of the Src kinases Lck and FynT. This compound is a nanomolar inhibitor of Lck and FynT, inhibits anti-CD3-induced protein-tyrosine kinase activity in T cells, demonstrates selectivity for Lck and FynT over ZAP-70, and preferentially inhibits T cell receptor-dependent anti-CD3-induced T cell proliferation over non-T cell receptor-dependent phorbol 12-myristate 13-acetate/interleukin-2 (IL-2)-induced T cell proliferation. Interestingly, this compound selectively inhibits the induction of the IL-2 gene, but not the granulocyte-macrophage colony-stimulating factor or IL-2 receptor genes. This compound offers a useful new tool for examining the role of the Lck and FynT tyrosine kinases versus ZAP-70 in T cell activation as well as the role of other Src family kinases in receptor function.

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Author and article information

Contributors

Brian Key: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 2 August 2016

Publication date Collection: 2016

Volume: 11

Issue: 8

Electronic Location Identifier: e0159405

Affiliations

[1 ]Program in NeuroEngineering, McGill University, Montreal, QC, H3A 2B4, Canada

[2 ]Department of Neurology and Neurosurgery, Montréal Neurological Institute, McGill University, Montreal, QC, H3A 2B4, Canada

[3 ]Department of Physics, McGill University, Montreal, QC, H3A 2T8, Canada

[4 ]Department of Chemistry, McGill University, Montreal, QC, H3A 0B8, Canada

School of Biomedical Sciences, The University of Queensland, AUSTRALIA

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: BR KLWS JPC PWW TEK.
Performed the experiments: BR KLWS JPC.
Analyzed the data: BR JPC KLWS PWW TEK.
Contributed reagents/materials/analysis tools: PWW TEK.
Wrote the paper: BR KLWS PWW TEK.

* E-mail: timothy.kennedy@ 123456mcgill.ca

Article

Publisher ID: PONE-D-15-54402

DOI: 10.1371/journal.pone.0159405

PMC ID: 4970703

PubMed ID: 27482713

SO-VID: ba37feba-fff3-4365-9dcc-9e2cf5820a85

License:

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

History

Date received : 16 December 2015

Date accepted : 2 July 2016

Page count

Figures: 3, Tables: 0, Pages: 13

Funding

Funded by: funder-id http://dx.doi.org/10.13039/501100000038, Natural Sciences and Engineering Research Council of Canada;

Award ID: 250013-12

Award Recipient : Paul W Wiseman

Funded by: funder-id http://dx.doi.org/10.13039/501100000026, Institute of Aging;

Award ID: 126109