Cell receptors.

Chitin Microparticles (CMPs, 1-10um), a special form of the ubiquitous and nontoxic
polysaccharide Chitin (GlcNAc), is capable of inducing a switch in macrophages
from the wound-healing M2 phenotype to the classically activated pro-inflammatory M1
phenotype; which has therapeutic implications in allergy and cancer. We hypothesized
that TLR2 forms a complex with CMPs and Chitin-Binding Proteins (CBPs) at the
surface of peritoneal macrophages and remains with that complex after internalization to
initiate downstream signaling events, leading to the production of the M1 cytokine, TNFalpha.
Our results from experiments performed in RAW 264.7 cells show that TLR2 and
TLR1, but not TLR6, are associated with the CMP binding fraction, and that both TLR1
and TLR2 might be important for M1 activation as a result of CMP phagocytosis. This
project sheds light on CMP as a potential therapeutic agent and provides more evidence
for a phagocytosis-dependent TLR2 signaling pathway.

In the developing CNS, presynaptic neurons often have exuberant overgrowth and
form excess (and overlapping) postsynaptic connections. Importantly, these excess
connections are refined during circuit maturation so that only the appropriate connections
remain. This synaptic rearrangement phenomenon has been studied extensively in
vertebrates but many of those models involve complex neuronal circuits with multiple
presynaptic inputs and postsynaptic outputs. Using a simple escape circuit in Drosophila
melanogaster (the giant fiber circuit), we developed tools that enabled us to study the
molecular development of this circuit; which consists of a bilaterally symmetrical pair of
presynaptic interneurons and postsynaptic motorneurons. In the adult circuit, each
presynaptic interneuron (giant fiber) forms a single connection with the ipsilateral,
postsynaptic motorneuron (TTMn). Using new tools that we developed we labeled both
giant fibers throughout their development and saw that these neurons overgrew their targets and formed overlapping connections. As the circuit matured, giant fibers pruned
their terminals and refined their connectivity such that only a single postsynaptic
connection remained with the ipsilateral target. Furthermore, if we ablated one of the two
giant fibers during development in wildtype animals, the remaining giant fiber often
retained excess connections with the contralateral target that persisted into adulthood.
After demonstrating that the giant fiber circuit was suitable to study synaptic
rearrangement, we investigated two proteins that might mediate this process. First, we
were able to prevent giant fibers from refining their connectivity by knocking out
highwire, a ubiquitin ligase that prevented pruning. Second, we investigated whether
overexpressing Netrin (or Frazzled), part of a canonical axon guidance system, would
affect the refinement of giant fiber connectivity. We found that overexpressing Netrin (or
Frazzled) pre- & postsynaptically resulted in some giant fibers forming or retaining
excess connections, while exclusively presynaptic (or postsynaptic) expression of either
protein had no effect. We further showed that by simultaneously reducing (Slit-Robo)
midline repulsion and elevating Netrin (or Frazzled) pre- & postsynaptically, we
significantly enhanced the proportion of giant fibers that formed excess connections. Our
findings suggest that Netrin-Frazzled and Slit-Robo signaling play a significant role in
refining synaptic circuits and shaping giant fiber circuit connectivity.

PTP69D is a receptor protein tyrosine phosphatase (RPTP) with two intracellular catalytic domains (Cat1 and Cat2), which has been shown to play a role in axon
outgrowth and guidance of embryonic motorneurons, as well as targeting of photoreceptor neurons in the visual system of Drosophila melanogaster. Here, we
characterized the developmental role of PTP69D in the giant fiber (GF) neurons; two
interneurons in the central nervous system (CNS) that control the escape response of the fly. In addition to guidance and targeting functions, our studies reveal an additional role for PTP69D in synaptic terminal growth in the CNS. We found that inhibition of
phosphatase activity in catalytic domain (Cat1) proximal to the transmembrane domain
did not affect axon guidance or targeting but resulted in stunted terminal growth of the
GFs. Cell autonomous rescue and knockdown experiments demonstrated a function for
PTP69D in the GFs, but not its postsynaptic target neurons. In addition,complementation studies and structure-function analyses revealed that for GF terminal growth, Cat1 function of PTP69D requires the immunoglobulin and the Cat2 domain but not the fibronectin type III repeats nor the membrane proximal region. In contrast, the fibronectin type III repeats, but not the immunoglobulin domains, were previously shown to be essential for axon targeting of photoreceptor neurons. Thus, our studies uncover a novel role for PTP69D in synaptic terminal growth in the CNS that is mechanistically distinct from its function during earlier developmental processes.