Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB255

Poster Communications

Mechanism of calcium-dependent activator protein for secretion 1 and 2 differential subcellular localization in dorsal root ganglion neuron

A. Staudt1, A. Shaib1,3, O. Ratai1, A. Shaaban1, H. Bzeih1, R. Mohrmann1,2, J. Rettig1, U. Becherer1

1. CIPMM, Institute for Physiology, Saarland University, Homburg, Germany. 2. Institute of Physiology, Otto-von-Guericke University Magdeburg, Magdeburg, Germany. 3. Molecular Neurobiology, MPI for Experimental Medicine, Göttingen, Germany.

  • Figure 1: Differential CAPS2/CAPS1 localization to synapses. A: CAPS1, CAPS2 and CAPS2/CAPS1 chimera were attached to HaloTag and expressed via lentivirus. In addition, DRG neurons were infected with a lentivirus encoding for Synaptophysin-pHluorin to enable the recognition of DRG neurites. On DIV2 spinal cord neurons were added to the culture. Halo ligand chloralkane-atto590 labeling was performed at DIV6. On DIV9 cells were fixed and immuno-labeled against bassoon and synapsin. B: CAPS signal at synapses was quantified and normalized to the signal in adjacent areas of the neurites to account for variability in transfection efficiency. CAPS2 fluorescence intensity at synapses was significantly lower in comparison to CAPS1 and CAPS2-CAPS1 chimera (n = 182, 148 and 173 for CAPS1, CAPS2 and CAPS2/CAPS1 chimera, p<0.01 ANOVA on rank with Dunn's post-test, N = 3)

The calcium-dependent activator protein for secretion (CAPS) is an established priming factor for large dense core vesicles (LDCVs) and has been reported to prime synaptic vesicles (SVs) as well [1, 2]. It is a multi-domain protein consisting of two family members CAPS1 and CAPS2. Recently, we showed that in dorsal root ganglion (DRG) neurons CAPS1 exclusively mediates SV exocytosis, while CAPS2 only promote neuropeptide release from LDCVs. We further showed that their differential function is due to distinct localization. In fact, CAPS1 is highly enriched at synapses while CAPS2 has a more diffuse mainly somatic localization. To establish and maintain this differential localization, the subcellular transport mechanism of both CAPS paralogs must differ and be tightly controlled. In the peripheral nervous system, cell soma and synaptic contacts are separated by a great distance, which poses a challenge for synaptic targeting of cytoplasmic proteins. Active zone proteins overcome this problem by their association with so-called piccolo/bassoon transport vesicles. We propose that CAPS1 but not CAPS2 is able to bind to these vesicles to be directed to synapses. We investigated this question by targeted mutagenesis of CAPS2. We verified that our mutant CAPS2/CAPS1 was correctly expressed and functional. Using immunocytochemistry together with confocal microscopy we could show that the CAPS2/CAPS1 chimera protein was re-localized from the soma to synapses (Fig. 1). With this approach we identified a specific domain responsible for CAPS1 synaptic localization.

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