Videos
Click on one of the small icons on
the left to see the movie with a
legend below.
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.