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Structure-function analysis of endogenous lectin mind-the-gap in synaptogenesis.

Emma Rushton¹, Jeffrey Rohrbough, Kalie Deutsch

¹Department of Biological Sciences, Kennedy Center for Research on Human Development, Vanderbilt University, Nashville, TN 37232, USA.

Developmental Neurobiology

|January 12, 2012

Summary

Mind-the-Gap protein is secreted to the synapse and organizes the extracellular matrix. Its carbohydrate-binding domain restricts diffusion and is vital for viability, revealing a complex role in regulating glutamate receptor localization at the Drosophila neuromuscular junction.

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Area of Science:

Neuroscience
Developmental Biology
Molecular Biology

Background:

Mind-the-Gap (MTG) is crucial for organizing postsynaptic domains at the Drosophila neuromuscular junction (NMJ), including glutamate receptor (GluR) localization.
A prior hypothesis suggested MTG is secreted presynaptically to bind glycans in the synaptic cleft, organizing the extracellular synaptomatrix for transsynaptic signaling.

Purpose of the Study:

To investigate the structure-function of MTG's predicted signal peptide (SP) and carbohydrate-binding domain (CBD).
To test the hypothesis that MTG's SP and CBD are essential for its secretion, localization, and function in NMJ development.

Main Methods:

Structure-function analysis of MTG using deletion and point-mutant transgenic constructs in mtg null mutants.
In vivo assessment of MTG secretion, synaptic localization, and effects on GluR localization during synaptogenesis.
Biochemical assays to determine MTG's glycan-binding properties, including calcium dependence and specific glycan interactions.

Main Results:

The SP is essential for MTG secretion and synaptic localization.
The CBD is required for restricting MTG diffusion, ensuring postembryonic viability, and surprisingly, its mutation elevated GluR localization.
MTG binds N-acetylglucosamine (GlcNAc) and HRP-epitope glycans in a Ca(2+)-dependent manner, though not requiring the CBD for these interactions.

Conclusions:

MTG's SP mediates its secretion and synaptic targeting.
MTG's CBD plays a critical role in regulating its diffusion and is essential for viability, with its absence leading to altered GluR localization.
MTG exhibits complex glycan-binding interactions that both facilitate and limit GluR localization during NMJ synaptogenesis, challenging initial hypotheses.