Investigations into the biochemical and cellular biology of a cytoplasmic dynein mutation, abnormal rear leg (Arl)

Philpott, Amelia (2011) Investigations into the biochemical and cellular biology of a cytoplasmic dynein mutation, abnormal rear leg (Arl). Doctoral thesis (DPhil), University of Sussex.

[img]
Preview
PDF - Published Version
Download (6MB) | Preview

Abstract

The aim of this project was to investigate the effects of a novel mouse cytoplasmic dynein mutation; Abnormal rear leg (Arl). Cytoplasmic dynein is a microtubule (MT) based motor protein important for diverse cellular processes including Golgi maintenance and retrograde transport of organelles. Arl is a mouse point mutation in the heavy chain subunit of dynein (Dync1h1). Homozygous Dync1h1Arl/Arl die at embryonic day 10. Dync1h1Arl/+ heterozygotes have a normal life span, but exhibit abnormal gait and hindlimb clasping during tail suspension, typical of neuronal dysfunction. Protein purification from wildtype and heterozygous brain tissue showed increased MT binding in Dync1h1Arl/+ compared to wildtype. Delayed endosomal trafficking was observed in EGF stimulated Dync1h1Arl/+ mouse embryonic fibroblasts (MEFs) compared to wildtype, in both fixed cells and using live cell imaging. Similarly, a delay in the reassembly of the Golgi complex after disruption with a MT depolymerisation agent, nocodazole, was observed in Dync1h1Arl/+ MEFs compared to wildtype. In addition, the Golgi complex was observed as being structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons using transmission electron microscopy (TEM) compared to the wildtype. TEM also revealed that the mitochondria were structurally perturbed in Dync1h1Arl/+ lumbar spinal cord neurons compared to wildtype, and O2 consumption assays investigating their function showed the Dync1h1Arl/+ mitochondria to have increased respiration rates compared to wildtype. Thus, these data highlight the Arl mouse as an invaluable model for studying the mechanism of dynein function and the subsequent outcomes when they are compromised.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Biochemistry
Subjects: Q Science > QD Chemistry > QD0241 Organic chemistry > QD0415 Biochemistry
Q Science > QH Natural history > QH0301 Biology > QH0573 Cytology
Depositing User: Library Cataloguing
Date Deposited: 23 Jun 2011 09:09
Last Modified: 17 Aug 2015 13:18
URI: http://sro.sussex.ac.uk/id/eprint/6977

View download statistics for this item

📧 Request an update