Studying Cell Biology
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D.5.b. PM Turnover II <<Previous Problem
     
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D.5.a.
D.5.b.

 

 

   Here’s a longer, less open-ended and more complex (and wordier) version of the same question (used for a take-home Final Exam!).  Work your way through the data and questions and see if you come away with a similar understanding of plasma membrane recycling.
 
(45 pts) Cells and their organelles are dynamic entities.  In particular, cellular membranes seem constantly in motion, carrying out their various functions and being newly synthesized, degraded, recycled or transformed.  The existence of these dynamic phenomena is well-document, but we do not yet completely understand how the various processes are regulated.
 
For example, under some circumstances a portion of the plasma membrane (PM) may be endocytosed and become associated with lysosomal vesicles.  In another context, labeled PM becomes associated with different, non-lysosomal organelles following endocytosis.  (See data from the previous question, reproduced in more detail in i. And ii. Below.)  Data from more recent research, carried out in a laboratory near the one that produced the data in i. And ii., are presented in iii. below.  You may assume all three data sets were obtained under similar conditions unless described differently.
 
I. Many cells that secrete hormones are themselves under hormonal control.  One such cell, the pituitary gonadotroph, secretes two polypeptide hormones (LH and FSH) when stimulated by GnRH (Gonadotropin-releasing Hormone).  To investigate the regulation of LH and FSH secretion, a biologically active, radioactively iodinated (125I) analogue of GnRH was injected into rats.  At varying periods of time post injection, pituitary glands were fixed and sectioned, and the sections were then examined by EM-autoradiography.  Photographic grains resulting from radioactive decay were counted and their location in the sections noted.  These data are presented in the table below, as the percentage of total grains per cell.

 

Time Post Injection, min
Location
 5 15 30 60
1. Plasma Membrane   60% 15% 20% 5%
2. AP' ase-positive vesicles   20 30 60 80
3. GalTran' ase-positive vesicles   2 2 2 2
4. rER   3 3 2 2
5. Nucleus   1 1 1 1
6. Secretory Vesicles   5 8 9 8
           

Note: “AP’as- positive” indicates the presence of acid phosphatase; “GalTran’ase-positive” indicates the presence of galactosyl transferase.

 

II. In another set of experiments using a culture of cancerous bone-marrow cells (myeloma), an iodination procedure was used to label plasma membranes directly, using 125I. The fate of the radioactive label was then followed with EM-autoradiography.  Chemical analysis of the iodinated species revealed 15% of the label was covalently bound to membrane lipid and 85% to IMP.  (Separate experiments confirmed the labeling process did not affect the viability of these cells or their ability to secrete IgG.)  Cells fixed 5 and 60 min after labeling showed the following distribution of grains.

 

Time Post Labeling, min
Location
 5   60  
1. Plasma Membrane   60%   5%  
2. AP' ase-positive vesicles   20   80  
3. GalTran' ase-positive vesicles   2   2  
4. rER   3   2  
5. Nucleus   1   1  
6. Secretory Vesicles   5   8  

 

III. The internalization and intracellular transport of the FC receptor (FCR was examined in cultures of mouse macrophages using 125I-labeled antibodies against the receptor.  Following a brief pulse for labeling the receptors, labeled antibodies remaining at the surface were removed with a brief wash at pH 4.0, cells were incubated for varying periods of time and the intracellular fates of the label was monitored by cell fractionation and radioisotopic assay.  The data in Table I was obtained using antibody fragments (F’ab) with only one receptor binding domain; those in Table II made use of intact IgG (with two receptor binding domains).

TABLE 1
Time Post Washing, min
Location
 5 15 30 60
1. Plasma Membrane   10% 40% 50% 60%
2.Lysosomes   8 9 7 7
3. Trans-Golgi vesicles   3 4 3 5
4. Cis-Golgi vesicles   4 5 5 4
5. rER   1 1 1 1
6. Nucleus   2 1 2 1
7. Secretory Vesicles   62 40 32 22

 

TABLE 2
Time Post Washing, min
Location
 5 15 30 60
1. Plasma Membrane   12% 8% 7% 7%
2.Lysosomes   60 60 50 50
3. Trans-Golgi vesicles   5 10 12 11
4. Cis-Golgi vesicles   4 5 5 4
5. rER   1 1 1 1
6. Nucleus   1 2 1 1
7. Secretory Vesicles   18 17 25 28

 

A. (25 pts) Discuss critically and as completely as possible what these data indicate concerning the regulation of plasma membrane flow/turnover during brief periods in the lives of cells.  Illustrate your discussion with well-labeled diagrams and attend closely to the following questions:

  • i. what components in each experiment are being labeled and traced?
  • ii. what appear to be the fate of these components?
  • iii. how do these results compare with other endocytotic experiments you have examined: e.g., LDL uptake by fibroblasts?
  • iv. are the date equivocal – that is, open to different interpretations?  How so?
  • v. what additional experiment could be performed to remove ambiguity or to test your hypothesis concerning plasma membrane turnover?

 

 

B. (10 pts) The data in all experiments represent relative amounts of label in each fraction at the indicated time points.  Considering both parts of Experiment III, would you expect the total amount of label within the cell to vary among the time periods.  Why or why not?
 
 
 
 
 
 
C. (10 pts)  Given your answer in B., hypothesize how the insertion of newly synthesized FCR into the plasma membrane might occur and how the insertion might be regulated.
 

 

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