Truant Lab Videos

This is a 3D isosurface reconstruction of a huntingtin 1-81 fragment with 138 glutamines expressed to form a large inclusion in the cell . Co- imaged in green is the late endosome marker, Rab5. This image was obtained by spinning disk confocal, followed by image deconvolution, then isosurfacing. We can see vesicles trapped in the aggregate.

This video is a 3D rotation of two yeast Candida Albicans species stained with Hoechst dye, which does not penetrate the capsule, and Mitotracker Green, staining the mitochondria. The image was obtained by widefield microscopy, then image deconvolution, then 3D isosurfacing. Note the fine mitochondrial structure resolution and the bud scar on the mother yeast cell.

This is a live cell movie of huntingtin Q15 1-588 fragment as a YFP fusion during 42C heat shock, in real time. Note the very fast response of huntingtin location to heat shock bodies (HSBs).

This is a 3D rotation of an image stack obtained on a spinning disk confocal in which huntingtin 1-81-mRFP was co-expressed with Rab5- eGFP. As a 3D object in space, we can observe localization from any perspective, including “fly-through”.

This movie is a 6 hour time course of huntingtin 1-81 expression in a striatal neuronal-derived cell line. This is with a 40x air objective in which we have overlayed the DIC and fluorescence channels. Note the only cell to die on this view is the cell expressing this toxic fragment of huntingtin, and the cell death is classic apoptosis, as can been seen with membrane ‘blebbing’ when the cell retracts.

This movie is a 3D rotation of an image stack obtained by immunofluorescence with antibodies against endogenous huntingtin and actin proteins, following 60 minutes of heat shock at 42C. This image was obtained by widefield microscopy and 3D image deconvolution using an iterative algorithm that determines the point spread function (how light behaves in 3D space) from the actual data set.

This movie is a 6 hour time course of huntingtin 1-171 eGFP expression in STHdH cells. We can see the onset of aggregation, the movement of aggregates towards the nucleus, and the coalescence of small aggregates into one huge aggregate. However, the point of this experiment was to compare cells that form huntingtin aggregates, with cells that do not. Cells that form aggregates in fact live much longer than cells that do not (upper left hand corner). This is among the first evidence that the toxic species of huntingtin is the soluble polyglutamine expanded protein, and not the aggregated protein.

This movie is a 4 hour time course of huntingtin 1-81 mRFP expressed in a STHdh cell. Typically, this fragment forms one large inclusion that increases with size over time. The important aspect of this video is that in the last few minutes, all of the soluble mutant huntingtin fragment is concentrated into the central mass. Despite the huge size of this 5um ball of protein, this cell does not die. This suggests that a the mechanism of aggregation of huntingtin to protect cells is to concentrate all mutant protein into an innocuous inclusion. This hypothesis is contrary to the amyloid hypothesis of Huntington’s disease.

This is a 3D isosurfacing and reconstruction after deconvolution of a single fibrillar type huntingtin fragment inclusion. This is thought to be a less toxic conformer of huntingtin.

This is a 3D isosurfacing and reconstruction after deconvolution of a single globular type huntingtin fragment inclusion. This is thought to be a more toxic conformer of huntingtin.

This is a two-color FRAP experiment with GFP-NLS and hnRNPA1-RFP expressed in a bi-karyon (merge red-green is yellow). All the area except the donor nucleus is bleached, then allowed to recover. We can see in 30 second increments that only the red is recovered, and not the green. This is the only assay to establish that a protein does not shuttle, as well as measure the dynamics of shuttling. This was developed in 2002 by Jenny Howell.

This is a FRAP experiment with bikaryons (two nuclei per cell) in which we have expressed GCN5-eYFP. the entire area of the cell except the bottom nucleus is photobleached. This movie shows recovery of fluorescence after photobleaching to the cytoplasm and the other nucleus, demonstrating that GCN5 is a nuclear shuttle protein.