At presynaptic nerve terminals, the precise nano-scale positioning of two protein machineries, the active zone
and the endocytic apparatus, is critical for high fidelity synaptic transmission. The active zone, which generates
release sites for synaptic vesicles, is aligned to postsynaptic receptors to ensure efficient signal transmission.
The endocytic apparatus, which restores vesicles after release, is assembled adjacent to active zones. Most
components of these machineries are associated with brain disorders, underlining their importance for brain
function. This grant generates fundamental insight into the molecular mechanisms that position the
endocytic apparatus next to the active zone at vertebrate synapses.
The presynaptic scaffold Liprin-a is an ideal candidate to spatially organize these two machineries for three
reasons. First, the broad synaptic interactome of Liprin-a and the generalized phenotypes caused by ablating
the single invertebrate gene predict that it organizes multiple presynaptic compartments. Second, my previous
work and preliminary data show that deletion of a subset of the four vertebrate Liprin-a proteins already causes
mislocalization of active zone and endocytic components, which suggests co-organizing roles for vertebrate
Liprin-a. Finally, Liprin-a undergoes phase separation, which is a potential synaptic organizing mechanism, and
my ongoing work establishes that both active zone and endocytic proteins are recruited to Liprin-a phase
condensates. Therefore, I hypothesize that Liprin-a positions the endocytic apparatus next to active zones
at vertebrate synapses.
I will use newly-generated Liprin-a quadruple mutants, in which all four Liprin-a genes can be deleted, to test
this hypothesis. Aim 1 (training) will dissect roles for Liprin-a in active zone organization by comparing the nano-
scale positioning of active zone proteins and active zone function after Liprin-a quadruple ablation in excitatory
and inhibitory synapses in cultured neurons and at the Calyx of Held. Aim 2 (training) will determine roles for
Liprin-a in organizing the endocytic apparatus by using the same approaches. Both aims will be complemented
with experiments in cell lines to assess the ability of Liprin-a to recruit endocytic and active zone proteins. I will
also do structure-function experiments to dissect the relevance of the protein sequences that mediate Liprin-a
phase separation in presynaptic assembly. These aims will establish the roles of Liprin-a in the organization of
active zones and endocytic assemblies, and will provide an essential training experience in the studies of the
endocytic machinery and in the assessment of their in vivo organization using super-resolution microscopy. Aim
3 (independent) will test the complementary model that the endocytic apparatus mediates the spatial
organization of these machineries. In particular, I will dissect structural roles for Dynamin, which is fundamental
for synaptic endocytosis, and also undergoes phase separation. I will use Dynamin triple mutants and build upon
the methodology implemented during (training) aims 1 & 2 to determine these roles.