Studying
Cell Biology |
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Heres a different kind of question! Answering it requires
you to read and understand two Abstracts of research presented at an annual
meeting of the American Society for Cell Biology. By their very
nature, such Abstracts are written in a terse, almost skeletal fashion
and their comprehension requires the reader to make use of background
information he or she has already mastered (and to read the Abstracts
carefully). Binding and Internalization of GnRH by Rat Pituitary Gonadotrophs.
T.M. Duello, T.M. Nett* and M.G. Farquahar, Section of Cell
Biology, Yale University School of Medicine, New Haven, CT and Department
of Physiology and Biophysics, Colorado State University, Fort Collins,
CO. Gonadotropin-releasing hormone (GnRH), like other peptide hormones, exerts
its initial action in regulating secretion of gonadotropins (LH and FSH)
by binding to specific plasma membrane receptors on the surface of the
pituitary cells that secrete the LH and FSH. These cells are called
gonadotrophs. Studies using fluorescent derivatives and ferritin
conjugates of GnRH analogues have demonstrated their initial, diffuse
binding to the cell surface followed by formation of aggregates which
are gradually internalized by endocytosis. In this in vivo
study we followed the fate of bound hormone by EM autoradiography using
an 125I-labeled analogue of GnRH. This analog was previously shown
(Mol. Cell. Endocrinol. 19: 601, 1980) to be taken up to a greater
extent (~ 3-fold) and retained longer (~ 4-fold) by gonadotrophs than
the native hormone. Ovariectomized rats were given intracarotid
injections of 20 ng of the labeled analogue, and the pituitaries were
fixed at 5, 15, 30, and 60 min. thereafter. A 1 min. pretreatment
with a 200-fold excess of unlabeled analogue blocked binding at the 15,
30, and 60 min. intervals as demonstrated by very low cpm/pituitary gland
and the absence of autoradiographic grains over gonadotrophs. The
major findings were as follows: 1) At early time points ~50% of
the grains were found within two half distances of the plasma membrane,
the grain density decreasing over time; 2) the labeling of lysosomes was
initially low and increased dramatically by 60 min.; 3) the grain density
over the nucleus (<0.6), Golgi elements (<1.2), and rough ER (<1.2)
remained relatively low at all time points; and 4) grain density of secretory
vesicles was high (2.2-4.8) at all time points. And -
Recently it was shown that ferritin bound electrostatically
to the plasma membrane of IgG-secreting murine myeloma cells is internalized
by endocytosis and appears in the stacked Golgi cisternae and secretory
vesicles after 60 min. (Ottosen et al, J. Exp. Med. 152:1, 1980).
In this study we covalently labeled plasma membrane components of cultured
myeloma cells using the lactoperoxidase iodination (125I) procedure
of Hubbard and Cohn and followed the subsequent fate of labeled components
by EM autoradiography. Characterization of the iodinated species
revealed that ~15% of the label was incorporated into lipids and ~85%
into proteins. Cellular incorporation of 125I was a linear
function of [125I] in the medium. In a separate experiment
it was shown that iodination did not affect significantly the rate of
release of 14C-labeled IgG (the normal secretory
product of the myeloma). In 3 experiments EM autoradiographic analysis
of cells fixed immediately after iodination at 4oC showed up
to ~80% of the total grains to be within two half distances (HD) of the
plasma membrane and only 2% over the Golgi region. After 1 hour
at 37oC, 50-60% of the label remained associated with the plasma membrane
and up to 23% was found in the Golgi region. Thus, 25-50% of the
total internalized label was found in the Golgi region. Of the latter,
the majority of the grains were found either directly over or within 2HD
of the Golgi cisternae and secretory vesicles. At no time were >
1% of the total grains found over lysosomes. (Supported by USPHS
Grant AM 17780.) 3. (8 pts) Discuss critically how both sets of results
contribute to our understanding of plasma membrane turnover and of possible
functions of the Golgi complex.
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