Visualisation of dynein complexes in vitro and inside cells
type: CreativeWork
description: Proper neuronal function relies on efficient transport within axons and dendrites, the long processes which extend from neuronal cell bodies. Within neuronal processes, the microtubule cytoskeleton provides tracks for motors to carry cargo over long distances. In axons, cytoplasmic dynein-1 (dynein) is the microtubule motor which carries cargo back towards the cell body. Dynein cargo include endolysosomal vesicles, autophagosomes and mitochondria. Dynein is activated by binding to a co-factor called dynactin. How dynactin binding activates dynein is unknown. How multiple dynein/dynactin complexes are arranged on cargo for long range transport in cells is also not clear. To understand this, I used cryo-electron microscopy (EM) combined with biochemical analysis and fluorescence microscopy to study dynein in vitro and in cells. In Chapter 2, I present the cryo-EM structure of purified full-length human cytoplasmic-1 in an inhibited state. By making structure-guided point mutations, I show that dynactin binding is the key step to promote long-range movement of dynein on microtubules and propose a model for how dynactin binding activates dynein. I then focus on visualising dynein/dynactin complexes in neurons using cryo-electron tomography. My initial aim was to perform cryo-correlative light and electron microscopy (cryo-CLEM) of dynein cargo in axons to determine if it is possible to visualise dynein/dynactin in cells and show their distribution. In Chapter 3, I show different strategies for labelling dynein cargo in primary neurons grown on EM grids for cryo-fluorescence microscopy. I describe the challenge to obtain samples with both sufficient fluorescent signal and suitable thickness for cryo-CLEM imaging. This work led me to alter my strategy and image the thin parts of mouse dorsal root ganglion (DRG) axons without guidance from cryo-FM signal. In Chapter 4, I present a survey of the intracellular architecture of thin parts of DRG axons using cryo-electron tomog…
abstract: Proper neuronal function relies on efficient transport within axons and dendrites, the long processes which extend from neuronal cell bodies. Within neuronal processes, the microtubule cytoskeleton provides tracks for motors to carry cargo over long distances. In axons, cytoplasmic dynein-1 (dynein) is the microtubule motor which carries cargo back towards the cell body. Dynein cargo include endolysosomal vesicles, autophagosomes and mitochondria. Dynein is activated by binding to a co-factor called dynactin. How dynactin binding activates dynein is unknown. How multiple dynein/dynactin complexes are arranged on cargo for long range transport in cells is also not clear. To understand this, I used cryo-electron microscopy (EM) combined with biochemical analysis and fluorescence microscopy to study dynein in vitro and in cells. In Chapter 2, I present the cryo-EM structure of purified full-length human cytoplasmic-1 in an inhibited state. By making structure-guided point mutations, I show that dynactin binding is the key step to promote long-range movement of dynein on microtubules and propose a model for how dynactin binding activates dynein. I then focus on visualising dynein/dynactin complexes in neurons using cryo-electron tomography. My initial aim was to perform cryo-correlative light and electron microscopy (cryo-CLEM) of dynein cargo in axons to determine if it is possible to visualise dynein/dynactin in cells and show their distribution. In Chapter 3, I show different strategies for labelling dynein cargo in primary neurons grown on EM grids for cryo-fluorescence microscopy. I describe the challenge to obtain samples with both sufficient fluorescent signal and suitable thickness for cryo-CLEM imaging. This work led me to alter my strategy and image the thin parts of mouse dorsal root ganglion (DRG) axons without guidance from cryo-FM signal. In Chapter 4, I present a survey of the intracellular architecture of thin parts of DRG axons using cryo-electron tomog…
author: Foster, Helen
language: eng
keywords: Structural biology, Biochemistry, Cryo-electron microscopy
license: https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/36132c69-05e3-4b33-896b-5d7602802560/download
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2020-05-01
date modified: 2026-07-22T22:24:18Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.65348