Investigating mechanisms of protein transport and assembly at the node of Ranvier

E. Grunewald, E. Malavasi, A. Ghosh, M. Zagnoni, D. Sherman, P. Brophy

Research output: Contribution to journalMeeting abstract

Abstract

Action potential (AP) propagation in myelinated nerves (saltatory conduction) requires axonal clustering of voltage gated sodium and potassium channels. These sodium channels are specifically localised at the axon initial segment (AIS) and nodes of Ranvier (NoR), where the AP is generated and propagated respectively. Neuronal and glial Neurofascins are the key cell adhesion molecules required for the correct clustering of sodium channels at the node. The question is how these membrane proteins that are required for saltatory conduction are trafficked to and stabilised in CNS and PNS axonal domains. To study this, we are using transgenic mice that express the β1 subunit of the voltage-gated sodium channel (β1Nav) and neuronal Neurofascin 186 (Nfasc186) as fluorescent nodal fusion proteins. DRG neurons purified from these mice are co-cultured with myelinating glia (either Schwann cells or Oligodendrocytes) in microfluidic chambers, and the trafficking and delivery of fluorescently tagged β1Nav and Nfasc186 to the NoR and axon initial segment (AIS) is recorded by live cell imaging as myelination progresses. Moreover, we are also investigating the contribution of neuronal Nfasc186 trafficking in AIS assembly and stablization. In order to elucidate this, we employ live cell imaging in cerebellar slice culture in combination with lentiviral injection of fluorescently tagged Nfasc186. To dissect the molecular mechanisms governing their trafficking and delivery, we have purified axonal transport vesicles containing these proteins from mouse brain at various stages during myelination using biochemical methods and performed mass spectrometry. Candidates identified by this proteomics approach will be probed for their ability to modify the transport dynamics and nodal delivery of β1Nav and Nfasc186 in our co-culture system.
LanguageEnglish
Article numberT12-034C
Number of pages1
JournalGlia
Volume65
Issue numberS1
DOIs
Publication statusPublished - 12 Jun 2017

Fingerprint

Ranvier's Nodes
Protein Transport
Voltage-Gated Sodium Channels
Sodium Channels
Sodium
Proteins
Neuroglia
Action Potentials
Nodal Protein
Cluster Analysis
Voltage-Gated Potassium Channels
Imaging techniques
Transport Vesicles
Axonal Transport
Microfluidics
Diagnosis-Related Groups
Schwann Cells
Neural Conduction
Oligodendroglia
Cell Adhesion Molecules

Keywords

  • action potential
  • AP
  • sodium channels
  • cell adhesion molecules

Cite this

Grunewald, E., Malavasi, E., Ghosh, A., Zagnoni, M., Sherman, D., & Brophy, P. (2017). Investigating mechanisms of protein transport and assembly at the node of Ranvier. Glia, 65(S1), [T12-034C]. https://doi.org/10.1002/glia.23157
Grunewald, E. ; Malavasi, E. ; Ghosh, A. ; Zagnoni, M. ; Sherman, D. ; Brophy, P. / Investigating mechanisms of protein transport and assembly at the node of Ranvier. In: Glia. 2017 ; Vol. 65, No. S1.
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Grunewald, E, Malavasi, E, Ghosh, A, Zagnoni, M, Sherman, D & Brophy, P 2017, 'Investigating mechanisms of protein transport and assembly at the node of Ranvier' Glia, vol. 65, no. S1, T12-034C. https://doi.org/10.1002/glia.23157

Investigating mechanisms of protein transport and assembly at the node of Ranvier. / Grunewald, E.; Malavasi, E.; Ghosh, A.; Zagnoni, M.; Sherman, D.; Brophy, P.

In: Glia, Vol. 65, No. S1, T12-034C, 12.06.2017.

Research output: Contribution to journalMeeting abstract

TY - JOUR

T1 - Investigating mechanisms of protein transport and assembly at the node of Ranvier

AU - Grunewald, E.

AU - Malavasi, E.

AU - Ghosh, A.

AU - Zagnoni, M.

AU - Sherman, D.

AU - Brophy, P.

N1 - Volume 65, Issue S1, GLIA Edinburgh 2017: Abstracts Oral Presentations, Posters, Indexes

PY - 2017/6/12

Y1 - 2017/6/12

N2 - Action potential (AP) propagation in myelinated nerves (saltatory conduction) requires axonal clustering of voltage gated sodium and potassium channels. These sodium channels are specifically localised at the axon initial segment (AIS) and nodes of Ranvier (NoR), where the AP is generated and propagated respectively. Neuronal and glial Neurofascins are the key cell adhesion molecules required for the correct clustering of sodium channels at the node. The question is how these membrane proteins that are required for saltatory conduction are trafficked to and stabilised in CNS and PNS axonal domains. To study this, we are using transgenic mice that express the β1 subunit of the voltage-gated sodium channel (β1Nav) and neuronal Neurofascin 186 (Nfasc186) as fluorescent nodal fusion proteins. DRG neurons purified from these mice are co-cultured with myelinating glia (either Schwann cells or Oligodendrocytes) in microfluidic chambers, and the trafficking and delivery of fluorescently tagged β1Nav and Nfasc186 to the NoR and axon initial segment (AIS) is recorded by live cell imaging as myelination progresses. Moreover, we are also investigating the contribution of neuronal Nfasc186 trafficking in AIS assembly and stablization. In order to elucidate this, we employ live cell imaging in cerebellar slice culture in combination with lentiviral injection of fluorescently tagged Nfasc186. To dissect the molecular mechanisms governing their trafficking and delivery, we have purified axonal transport vesicles containing these proteins from mouse brain at various stages during myelination using biochemical methods and performed mass spectrometry. Candidates identified by this proteomics approach will be probed for their ability to modify the transport dynamics and nodal delivery of β1Nav and Nfasc186 in our co-culture system.

AB - Action potential (AP) propagation in myelinated nerves (saltatory conduction) requires axonal clustering of voltage gated sodium and potassium channels. These sodium channels are specifically localised at the axon initial segment (AIS) and nodes of Ranvier (NoR), where the AP is generated and propagated respectively. Neuronal and glial Neurofascins are the key cell adhesion molecules required for the correct clustering of sodium channels at the node. The question is how these membrane proteins that are required for saltatory conduction are trafficked to and stabilised in CNS and PNS axonal domains. To study this, we are using transgenic mice that express the β1 subunit of the voltage-gated sodium channel (β1Nav) and neuronal Neurofascin 186 (Nfasc186) as fluorescent nodal fusion proteins. DRG neurons purified from these mice are co-cultured with myelinating glia (either Schwann cells or Oligodendrocytes) in microfluidic chambers, and the trafficking and delivery of fluorescently tagged β1Nav and Nfasc186 to the NoR and axon initial segment (AIS) is recorded by live cell imaging as myelination progresses. Moreover, we are also investigating the contribution of neuronal Nfasc186 trafficking in AIS assembly and stablization. In order to elucidate this, we employ live cell imaging in cerebellar slice culture in combination with lentiviral injection of fluorescently tagged Nfasc186. To dissect the molecular mechanisms governing their trafficking and delivery, we have purified axonal transport vesicles containing these proteins from mouse brain at various stages during myelination using biochemical methods and performed mass spectrometry. Candidates identified by this proteomics approach will be probed for their ability to modify the transport dynamics and nodal delivery of β1Nav and Nfasc186 in our co-culture system.

KW - action potential

KW - AP

KW - sodium channels

KW - cell adhesion molecules

U2 - 10.1002/glia.23157

DO - 10.1002/glia.23157

M3 - Meeting abstract

VL - 65

JO - Glia

T2 - Glia

JF - Glia

SN - 0894-1491

IS - S1

M1 - T12-034C

ER -

Grunewald E, Malavasi E, Ghosh A, Zagnoni M, Sherman D, Brophy P. Investigating mechanisms of protein transport and assembly at the node of Ranvier. Glia. 2017 Jun 12;65(S1). T12-034C. https://doi.org/10.1002/glia.23157