1. David Su)er, Koto Kikuma, Daniel Kim, Dion Dickman
Department of Biology, University of Southern California, Los Angeles, CA USA
Diverse signaling systems orchestrate the essen@al processes of synaptogenesis, yet the
homeosta@c influence in membrane trafficking events that occur in synaptogenesis
remain poorly understood. While selec@ng new genes required for synap@c architecture
and plas@city, Extended Synaptotagmins (Esyts) emerged as a gene of interest because it
shares a close structural rela@onship to Synaptotagmin, a known moderator of rapid
exocytosis at the synapse. Esyt itself is an evolu@onarily conserved endoplasmic re@culum
(ER) protein involved in tethering the ER and plasma membrane (PM). Interes@ngly,
recent studies show that Esyt dependent ER-PM tethering and lipid transfer is regulated
by cytosolic Ca2+ concentra@on. All together this suggests a possibility that Esyt
dependent lipid metabolism is regulated by neurotransmission via Ca2+ signaling, such
that Esyt may have a role in homeosta@c synap@c func@ons.
To address in vivo synap@c func@ons of Esyt, we generated Drosophila Esyt null mutants
using CRISPR/Cas-9 genome engineering technology. Here, we characterize synap@c
func@ons of Esyt mutants in vivo using Drosophila neuromuscular junc@on (NMJ) as a
model system. Surprisingly, Esyt mutants have normal basal synap@c structure and
transmission. However, we do find an increase in ac@ve zone density in the mutant and
with neuronal overexpression of Esyt. Electrophysiology results showed a corresponding
increase in the frequency of miniature excitatory junc@onal poten@als (mEJP). Using live
imaging techniques we are currently studying ER func@on and localiza@on in Esyt
mutants, and how this coordinates synap@c growth and structure. These results will help
dis@nguish Esyt’s known Ca2+-dependent regula@on of lipid exchange at synap@c
terminals from neurotransmission. This work has relevance to a variety of
neuropsychiatric diseases, such as Fragile X Syndrome, Au@sm Spectrum Disorders, and
Epilepsy, in which defects in synap@c development and func@on are fundamental to its
e@ology. Ul@mately, we aim to establish a new framework of understanding health and
disease through the lens of synap@c plas@city and its mechanisms.
[1] Synap*c Homeostasis at the Drosophila NMJ
[2] Extended Synaptotagmins Protein Domains
Introduc*on
Esyt HS C2 C2 C2 SMP
1kbp
ATG
Esyt1
Esyt2
[4] Perturba*on Expressed Presynap*cally –
Change in Ac*ve Zone Density and Bouton Count
[3] Electrophysiology Assessment of Synap*c
Transmission
[6] Perturba*on Contained Presynap*cally – GlurIII
Morphology Does Not Change
[7] Esyt Mutant Shows No Change in Homeostasis
[8] Conclusions and Future Direc*ons
[9] Acknowledgments
BRPDensity
0
1
2
* *
BRPSumIntensity
0
1500
3000
]
*
mEPSPfrequency(Hz)
0
3.0
*
2.0
mEPSPamplitude(mV)
0
1.0
*
15
30
45
0
*
quantalcontent
Wild Type
Esyt Mutant
Esyt Overexpression
Analysis of Homeosta/c Mechanisms Behind Extended
Synaptotagmin’s Involvement in Membrane Trafficking Events
0
50
100
150
Bouton#
*
wild type Esyt1/2
wild type Esyt1/2 Esyt Overexpression
Esyt Overexpression
0.0
0.5
1.0
GluRIIIdensity
0.00
0.75
1.50
GluRIIIArea
BRPOpposingGluRIII
0
350
700
0
50
100
150
200
250
GluRIIA Esyt2, GluRIIA
% baseline
0
50
100
150
200
250
% baseline
wild type + PhTx Esyt1/2 + PhTx
mEPSP Quantal Content
[10] References
1) In Esyt mutants ac@ve zone density, mEPSP frequency, and mEPSP amplitudes increase,
while quantal content decreases. This morphologic response is contained presynap@cally.
2) Despite Esyt’s role in Ca2+ dependent lipid transfer, Eyst is not essen@al for basal
synap@c func@on. The Esyt muta@on does not show any change in homeostasis.
3) Live imaging will be used to verify ER localiza@on in Esyt mutants and further explore
Ca2+ dependent regula@on of lipid exchange to help coordinate neurotransmission.
*
*
*
*
1) USC Department of Biology, USC Provost Fellowship Funding
2) Dickman Lab member who helped with @ssue dissec@ons and staining: Gandhar
Katre
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