Analysis of Dysbindin-BLOC-1 Complex Mechanisms in the Neuromuscular Synapse of the Fruit Fly Drosophila melanogaster Open Access

Burch, Amelia Margaret (2015)

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The Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is an evolutionarily conserved, endosomally-localized protein complex involved in the biogenesis of lysosome-related organelles, including synaptic vesicles. Polymorphisms in the human gene DTNBP1, which encodes the BLOC-1 subunit dysbindin, confer an increased risk of schizophrenia. Moreover, defects in neurotransmission and synapse morphology have been observed in mouse and Drosophila BLOC-1 loss-of-function mutants. This suggests that molecular pathways downstream of dysbindin deficiency may intersect with schizophrenia disease mechanisms to regulate neurotransmission. Here, we studied molecular phenotypes arising from loss-of-function defects in the dysbindin-BLOC-1 schizophrenia susceptibility network. Using quantitative mass spectrometry, we measured changes in protein expression associated to dysbindin-BLOC-1 down-regulation in mammalian cells. We identified components of the actin polymerization machinery that were down-regulated in neuronal cells following reduced dysbindin-BLOC-1 content, and confirmed that these actin polymerization regulators biochemically interacted with BLOC-1. For my thesis, I tested the hypothesis that these BLOC-1 sensitive factors identified by mass spectrometry participate in the same molecular pathway to regulate synaptic morphology in the Drosophila neuromuscular junction. Here, I aimed to assess synaptic phenotypes using transheterozygotic analysis between loss-of-function alleles of the actin nucleator Arp2/3 and BLOC-1 subunits; however, I instead unintentionally tested the Arp1 null allele. Arp1 is a subunit of the dynactin complex, a protein complex critical for retrograde axonal transport. I found that Arp1 null mutants generate an abnormal synaptic morphology, which is phenocopied in BLOC-1 deficient animals. This synaptic phenotype is reproduced in animals carrying null alleles for BLOC-1 expressed in trans with Arp1 loss-of-function alleles, suggesting Arp1 acts in the same pathway as BLOC-1 to regulate synaptic architecture in Drosophila. These findings suggest an unforeseen connection between BLOC-1 complex and dynein-dynactin mechanisms.

Table of Contents

Introduction 1

Schizophrenia Susceptibility Gene 1

The BLOC-1 Complex 2

The Arp2/3 Complex 5

The function of the Arp2/3 complex at the synapse 5

Central Hypothesis 7

Predictions 7

Foundational Results 8

Methods and Results of Honor's Thesis 10

Methods 10

Drosophila Stocks 10

Immunohistochemistry and confocal microscopy 10

Quantification 11

Statistical Analysis 11

Results 12

Discussion 15

Summary 20

Tables and Figures 21

References 33

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