This
figure provides an overview of our project of the 3-D structure of the Golgi
complex in the beta cell of the pancreas. The pancreas of the Visible Man is
highlighted in green (middle left). The beta cells within the isolated Islets
of Langerhans are labeled in green (upper right). The Golgi ribbon within the
beta cells has seven cisternae (cis-most is light blue and trans-most is red)(lower
right). The transport vesicles (white) and dense core granules (blue) are shown
in the context of the surrounding microtubules (green). Finally, the crystal
structure of the insulin hexamer, the major component of the dense core granules,
is shown with the coordinating zinc molecule (lower left).
This
image is from the same data set as the Golgi ribbon in the above panel however
all the tomographic data has been modeled and is displayed. In addition to the
Golgi ribbon and associated vesicles and granules, the ER (yellow), post-Golgi
compartments (purple and red), mitochondria (green), and the ribosomes are (yellow
dots) are displayed. (From Marsh et al., 2001a). This view gives one a good
impression of the complexity of the cytoplasm. It is from information obtained
from dissection of images such as this that we have formulated out hypotheses
of the mechanisms molecules are sorted and move out of the Golgi.
Model
of Golgi ribbon in HIT-cells (pancreatic beta cell line). This model clearly
shows that the penultimate(bronze) and trans-most (red) cisternae have fragmented.
All vesicles and tubules forming from the trans most cisternae are clathrin-coated
and those from the penultimate-trans cisternae are non-clathrin coated. These
features are observed in a number of different cell types. From this and other
data the hypothesis we have formulated is that molecules leave the Golgi from
multiple trans-cisternae and that only molecules destined to the endocytic-lysosomal
pathway leave the Golgi from the trans-most cisternae.
Model of the trans-most cisternae (red) and the penultimate trans-cisternae
(bronze) from HIT-cells. This view displays the tubular-reticular structure
of the penultimate trans-cisternae and clearly shows the tubules extending from
it. It is these cisternae we immunoisolate for proteomic analysis.
Digital
deconvolved image of the Golgi ribbon in NRK cells labeled with antibodies against
TGN38 (green) and the cationic independent mannose-6-phosphate receptor (red).
TGN38 although predominately localized to the Golgi moves to the plasma membrane
and back. CI-MPR is an abundant transmembrane protein that exits the Golgi in
clathrin-coated vesicles. Antibodies against the cytoplasmic domain of each
of these transmembrane proteins are used to immunoisolate the compartments in
which these proteins traffic for proteomic analysis.
Filipin
is a polyene antibiotic that binds to cholesterol and results in this characteristic
"hole-like" pattern. This micrograph demonstrates the cholesterol
rich domains within Golgi membranes. An isolated stacked Golgi fraction from
rat liver has been filipin treated and visualized with the electron microscope
after negative staining.
Model of Golgi ribbon in HIT-cells (pancreatic beta cell line). This model clearly
shows that the penultimate(bronze) and trans-most (red) cisternae have fragmented.
All vesicles and tubules forming from the trans most cisternae are clathrin-coated
and those from the penultimate-trans cisternae are non-clathrin coated. These
features are observed in a number of different cell types. From this and other
data the hypothesis we have formulated is that molecules leave the Golgi from
multiple trans-cisternae and that only molecules destined to the endocytic-lysosomal
pathway leave the Golgi from the trans-most cisternae.
