1. The FNIII domain #1 of Robo3 and EGF domains 1-3 of NELL2 are required to form the NELL2-Robo3 receptor-ligand complex, as determined by an alkaline phosphatase binding assay. Deletion of these domains abolishes complex formation. 2. Additional EGF domains of NELL2 beyond domains 1-3 may also contribute to complex formation, as decreased signal intensity was observed upon deletion of domains beyond 1-3. 3. Future work aims to further map the binding interaction between NELL2 and Robo3 by testing additional constructs and determining the specific amino acids required for complex formation using mutational analysis and protein crystallography.

1. 1 2 3
3
2 3
1 2 3
3B. FNIII Ig-deletion (Robo3 Ig-del)
3C. FN 2 - Transmembrane
3D. FN 3 - Transmembrane
3A. Full Length Robo3
3E. Transmembrane
A wholehearted thanks to everyone in the
Jaworski Lab for their continued support and
guidance!
This project was funded by the Karen T.
Romer Undergraduate Teaching and Research
Award (UTRA).
Acknowledgments
•Many neurodevelopmental disorders,
including autism and ADHD, arise from mis-
wiring of the nervous system during
embryonic development.
•By understanding the mechanisms behind
nervous system wiring it will be possible to
elucidate the cause of mis-wiring disorders
and design treatments for these disorders.
•Exploitation of developmental wiring
pathways to promote axon growth in adults
has the potential to restore neural
connections after injury.
Motivation
The Brain, a structure of unparalleled complexity, is a vast network of interconnected neurons.
Proper development of the brain demands the formation of specific connections between
neuronal populations. To ensure proper wiring during embryonic growth, axons are guided to
their final destinations by responding to attractive and repulsive molecular cues. Neurons
detect these cues using receptors on the axon growth cone. Through integration of guidance
information, growth cones project towards their final synaptic targets. The embryonic spinal
cord is an ideal model system to study axon guidance. Spinal commissural neurons project
axons across the floor plate at the ventral midline in response to multiple cues. The growth of
these axons is partly regulated by the repulsive functions of the paracrine guidance cue, Neural
Epidermal growth factor-Like-Like 2 (NELL2). NELL2 prevents commissural axon growth into
the ventral horns through the guidance cue receptor Robo3. The Robo3 receptor is vital in
directing axons to the floor plate and is multifunctional: it directly mediates repulsion through
NELL2, indirectly potentiates attractive signaling through Netrin, and suppresses Slit repulsion.
However, it is unclear how Robo3 simultaneously mediates the response to three different cues.
To understand how Robo3 can perform these functions, the protein domains required to
mediate NELL2-facilitated repulsion must be identified. Here, we test binding between different
NELL2 and Robo3 constructs lacking specific structural motifs to determine the structures
involved in the interaction between these two proteins.
Understanding the NELL2–Robo3 Interaction in Axon Guidance
Nischal Acharya and Alexander Jaworski, PhD
Department of Neuroscience, Brown University
Introduction Future Directions
Conclusions
Robo3: Deletion of FNIII domain #1 abolishes formation of NELL2-Robo3
complex. This suggests that FNIII domain #1 mediates NELL2-Robo3 binding.
NELL2: EGF Domains 1-3 are required for the formation of the Robo3-NELL2
complex. However, decreased signal intensity suggests additional EGF regions are
implicated in the formation of the receptor-ligand complex.
FIGURE 6: Results of the Alkaline Phosphatase Binding Assay. Upon the formation of the NELL2-Robo3 complex, a purple color is
produced by an Alkaline Phosphatase-catalyzed reaction converting BCIP/NBT to an insoluble product, NBT Diformazan. Images of
NELL2 structural analysis shown at higher magnification to highlight difference in signal intensity.
Results and Discussion
FNIII Domain #1 is necessary to form NELL2-Robo3 complex
Full Length NELL2
Mock Robo3 Ig-del Robo3 Ig-del
Mock NELL2 EGF
Full Length NELL2
Robo3 Ig-del
Full Length NELL2
Robo3 2-Transmembrane
Full Length NELL2
Robo3 3-Transmembrane
Full Length NELL2
Robo3 Transmembrane
NELL2 EGF Domains 1-3 are required for NELL2-Robo3 Interaction
Full Length NELL2
Robo3 Ig-del
NELL2 EGF 1-3
Robo3 Ig-del
Mock NELL2 EGF
Robo3 Ig-del
NELL2 EGF 4-6
Robo3 Ig-del
Experimental Design
AP1 2 3
AP4 5 6
2A. Full Length NELL2
1 2 3 4 5 6
2B. NELL2 EGF Domains 1-3
2C. NELL2 EGF Domains 4-6
AP
NELL2 Constructs FIGURE 2, Left: Diagram of the NELL2
DNA constructs. Alkaline Phosphatase
is fused to both truncated and Full
length (FL) constructs, allowing for
colorimetric detection of NELL2-Robo3
interaction.
Note: Previous research determines
EGF Domains 4-6 are sufficient to
create NELL2-Robo3 complex. [4]
FIGURE 3, Right: Schematic of the
Robo3 DNA constructs.
Note: Past studies demonstrate
Fibronectin type III (FNIII) domains
are sufficient for NELL2-Robo3
binding. [4]
Robo3 Constructs
Alkaline Phosphatase Protocol
Add media
Collect
NELL2 media
Grow Cos7
cells
Transfect Hek293
Cells
Transfect Cos7
Cells
FIGURE 5: Cos7 cells expressing different
Robo3 constructs are incubated with NELL2
media. If receptor-ligand complex forms,
Alkaline Phosphatase will produce a visible
purple product.
Detect AP Activity
FIGURE 1A: Integration of attractive
and repulsive cues causing
directional axon growth. [1]
FIGURE 1B: NELL2 expression
facilitating commissural axon growth
towards ventral floor plate during
development. [2]
FIGURE 1C: Repulsive guidance cue
mediated growth cone turning. [3]
Attractive Cue
NELL2, Repulsive Cue
1A 1B
Floor plate
Commissural
interneuron
Axon
Growth Cone
Repulsive Cue
Receptor
1C
References
[1]: Image adapted from Mann F. et al. Cancer Cell, 2013
[2]: Image adapted from Jaworski et al. Science, 2015
[3]: Image adapted from Kalil K. et al. Frontiers of Neuroanatomy, 2011
[4]: Jaworski et al. Science, 2015
[5]: Image acquired from Alila Medical Images
DNA
Constructs
(Fig. 2,3)
Synthesis of
Proteins
Recombinant
DNA
DNA
Transfection
Insertion of DNA
into bacterial
plasmid
Sticky
Ends
Plasmid
Recombination
Human CellBacterial
Chromosome
Bacterial Cell
Bacterial Transformation: Creating DNA Constructs
FIGURE 4: Synthesis of expression constructs (Fig. 2,3) through
molecular cloning and introduction into mammalian cells.
[5]
• Determine if FNIII domain #1 is
sufficient to form the NELL2-Robo3
complex.
• Design different Robo3 DNA
constructs that contain the FNIII
domain #1; test binding to NELL2.
• Elucidate whether EGF Domains 4,5,6
potentiate NELL2-Robo3 binding
• Engineer NELL2 DNA constructs
that contain EGF domains 1-3 along
with combinations of EGF domains
4, 5, 6; test interaction with Robo3.
• Determine the amino acids necessary
to mediate the NELL2-Robo3
interaction through point mutations
of DNA.
• Using protein crystallography,
illuminate the three-dimensional
structure of the ligand-receptor
complex, NELL2-Robo3.