Fourth International Symposium on Neuroacanthocytosis

Abstracts from the Fourth International Symposium on Neuroacanthocytosis

July 1-2, 2008
London and Oxford

Chairs: Prof. Kailash Bhatia, MD, FRCP, Institute of Neurology, University College London; Prof. Anthony P. Monaco, MD, PhD, Wellcome Trust Centre for Human Genetics, University of Oxford

Organizers: Antonio Velayos-Baeza, PhD; Susanne Schneider, MD; Glenn Irvine

4-2 The Tetrahymena thermophila Vacuolar Protein Sorting 13A protein (VPS13A) localizes to the membrane of phagosomes
H. Samaranayake, A. E. Cowan, and L. A. Klobutcher
Dept. of Molecular, Microbial, and Structural Biology, University of Connecticut Health Center, Farmington, CT, 06032, USA

The ciliated protozoan Tetrahymena thermophila utilizes phagocytosis as a means of ingesting other microorganisms that serve as its food source. To learn more about the molecular mechanisms of phagocytosis, we recently carried out a mass spectrometry-based analysis of the Tetrahymena phagosome proteome, identifying 73 putative protein components (Jacobs et al., 2006, Euk. Cell, 5:1990). One of the identified proteins was VPS13A. Mutations in VPS13 gene homologues have been implicated in the human genetic disorders Chorea-acanthocytosis and Cohen syndrome, but the VPS13 protein has not been previously linked to phagocytosis. As a first step to study the function of this gene and document its role in phagocytosis, we characterized the VPS13A gene structure. RLM-RACE (RNA ligase mediated Rapid Amplification of cDNA Ends) and RT-PCR were performed to confirm/revise the Tetrahymena Genome Database VPS13A gene prediction. The results of these analyses eliminate 12 predicted exons and alter 3 predicted exon/intron boundaries, so that the revised gene structure consists of 17 exons that encode a protein of 3475 amino acids. In addition, there was no evidence of major mRNA splice variants, which has been observed in other systems. To localize the VPS13A protein, we constructed a strain of Tetrahymena in which the endogenous VPS13A genes have been replaced with copies bearing a green fluorescent protein fusion at their carboxy termini (VPS13A-GFP). Live-cell confocal microscopy indicates that the VPS13A-GFP protein localizes to the membrane of phagosomes within the cell. In time course analyses, VPS13A-GFP is present on the phagosome membrane at the earliest time points that we are able to analyze (1 min.), and remains associated with phagosomes throughout their passage through the cell. Phagosome association of VPS13A-GFP occurs well before acidification of the phagosome, which begins at ~20 min. These cytological analyses provide confirmation of the phagosome proteome analysis, and further implicate VPS13A in a phagocytosis related process. We are now pursuing genetic analyses to further study the role of VPS13A in phagocytosis. A VPS13A gene construct with a selectable drug resistance gene interrupting its coding region is being constructed and will be used to generate a Tetrahymena VPS13A knock-out strain to assess its function in phagocytosis. In addition, we are preparing a construct that will allow the inducible over-expression of the well-conserved amino terminal domain of VPS13A as a means of generating a potential dominant negative mutant. These, and other future studies in Tetrahymena, should provide insights into the function of the VPS13A protein, and may provide new insights into how mutations in similar genes result in human genetic diseases.