Adaptation to Lysozyme Does Not Occur Via Receptor-Mediated Endocytosis in Tetrahymena thermophila

Joseph M. Cantor, Stephanie R. Mace, Coriann M. Kooy, Brian D. Caldwell, and Heather G. Kuruvilla*

Cedarville College
Department of Science and Mathematics, PO Box 601, Cedarville, OH 45314
 

Correspondence should be addressed to: Heather G. Kuruvilla, Ph.D.
Email: kuruvilh@cedarville.edu

Submitted for publication: 08 July 1999


Keywords: adaptation, chemorepellent, receptor-mediated endocytosis




ABSTRACT

The free-swimming ciliate, Tetrahymena thermophila, exhibits avoidance behavior when exposed to chemorepellent compounds, such as lysozyme.  Cells fail to respond to lysozyme after prolonged exposure (10-14 minutes), a phenomenon known as adaptation.  The mechanism of chemosensory adaptation in this ciliate is unknown.  The lysozyme receptor has been affinity purified from Tetrahymena and a polyclonal antibody has been generated to the receptor.  This antibody was used to immunolocalize the receptor on the cell membrane of Tetrahymena.  Lysozyme-adapted cells showed a similar staining pattern to non-adapted cells.  These data indicate that receptor-mediated endocytosis is not the primary mechanism for lysozyme adaptation in T. thermophila.



INTRODUCTION

Signal transduction is an essential component of cellular function in prokaryotes, unicellular eukaryotes, and differentiated eukaryotic cells.  Chemoattractants allow aggregation of Dictyostelium cells to form a motile, slug-like mass [1], and help Tetrahymena find food [2]. While the effects of chemorepellents on protozoans have been studied in less detail, functions may include avoidance of predation in Paramecium [3].  Some mammalian hormones have a chemorepellent effect on certain bacteria which inhibits binding of the bacterium to the plasma membrane of the mammalian cell, thereby preventing infection [4].  Both chemorepellents and chemoattractants are involved in guidance of neuronal growth patterns during mammalian development [5] , [6].

Chemosensory adaptation has been studied in a number of systems, including the prokaryotes E. coli  [7], and Bacillus subtilus [8]Dictyostelium have a bi-phasic adaptation response to cAMP exposure.  The first phase of adaptation is defined as "a form of desensitization by which the cells lose responsiveness to trains of stimuli with equal concentrations, but remain responsive to higher cAMP stimuli"[9].  This loss of responsiveness is rapidly reversed (t1/2 of 2-4 minutes), [9].  The second phase of adaptation, which occurs via receptor phosphorylation, results in a decrease in the number of detectable binding sites in the cell, and requires a t1/2 of an hour for reversal [9].

The chemorepellent activity of lysozyme has been studied in both Paramecium [10] , [11] and Tetrahymena [12].  In both ciliates, prolonged exposure (10-14 minutes) results in a drastic decline in the number of detectable binding sites [11], [12].  This adaptation is fully reversible upon removing the ciliates from the repellent solution and placing them in buffer for about 10 minutes[11], [12].   The mechanism of this adaptation is still unknown.

Chemosensory adaptation may occur in one or more ways, including downregulation of the receptor, covalent modification of the receptor structure, or regulation of some component of the secondary messenger pathway.  In this study, we explore the possibility that adaptation to lysozyme may be occurring via receptor-mediated endocytosis.

A 42 kD lysozyme-binding protein, presumably the lysozyme receptor, has been purified from Tetrahymena by affinity chromatography and used to generate polyclonal antibodies [13].  This 5545 antibody blocks Tetrahymena avoidance to lysozyme in vivo [13].  We have used this polyclonal antibody in our current study as a means of determining whether receptor-mediated endocytosis is occurring by visualizing receptors on the surface of both lysozyme-adapted and non-adapted Tetrahymena thermophila via indirect immunofluorescence.



MATERIALS AND METHODS

Cell Cultures

Tetrahymena thermophila B,  strain SB715, a generous gift from T. M. Hennessey (SUNY at Buffalo) was used throughout the study.  Cells were grown for 72 hours in the axenic medium of Dentler (1988) without the addition of antibiotics.  Cells were incubated at 25oC during growth.

Chemicals and Solutions

Cells were harvested into a buffer containing 1 mM CaCl2, 1 mM MOPS, pH 7.2 (Tris-base) and washed three times in the same buffer.  Cells were then fixed in a solution of 3.7% formaldehyde in this buffer.  Blocking, washing, and incubation with antibodies was done a solution of 3% BSA (bovine serum albumin) in this buffer.

All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO), unless otherwise noted.

Indirect Immunofluorescence

In the case of non-adapted cells, 72-hour cultures of SB715 were washed 3 times in buffer, and the cells were fixed for 30 minutes in the solution described above.  Cells were blocked overnight in 3% BSA at 4oC.  Cells were then washed twice in BSA and incubated with a 1:100 dilution of the 5545 polyclonal anti-lysozyme receptor antibody (courtesy of T. M. Hennessey) for 60 minutes at 25oC.  After incubation, cells were washed 3 times in BSA, then incubated for 30 minutes with a 1:100 dilution of FITC-conjugated goat anti-rabbit IgG (Sigma).  Labelled cells were then washed once in BSA and viewed under a Nikon Alphaphot-2 YS2 microscope.  Fluorescence images were acquired through a Javelin SmartCam digital camera.

In the case of preimmune controls, the same steps were followed as with the non-adapted cells, except that cells were incubated with 5545 preimmune serum instead of the anti-lysozyme receptor antibody.

In the case of lysozyme adapted cells, 72-hour cultures of SB715 were washed 3 times in buffer, then the cell pellet was exposed to 100 mM lysozyme in buffer for 10 minutes.  Cells were then washed in buffer, fixed, and treated as indicated for the non-adapted cells.
 



RESULTS

Immunofluorescence labeling with a 1:100 dilution of the 5545 anti-lysozyme antibody showed similar staining patterns in both non-adapted (Figure 1 B) and lysozyme-adapted cells (Figure 1 C).   Both experimental groups showed punctate staining of the body membrane. Cells labelled with the preimmune serum (Figure 1 A) did not show this punctate staining pattern.  Staining is primarily localized to the body membrane of the cells, with occasional clusters of staining near the basal bodies of cilia or on the cilia themselves.  Internal, organellar staining was not observed.

Staining intensity is also similar in both non-adapted (Figure 1 B) and lysozyme-adapted cells (Figure 1 C).  This intensity was not observed with preimmune serum (Figure 1 A).


DISCUSSION

Receptor-mediated endocytosis is an intricate process, involving ligand binding to the extracellular portion of a receptor, binding of clathrin adaptor proteins to the cytosolic face of the receptor, formation of a clathrin-coated pit, uncoating of the vesicle, fusion with an endosome, degradation of the ligand, and degradation or recycling of the receptor.  This process may result in a rapid loss of receptors from the plasma membrane, and a reduction of the total cellular receptor pool by as much as 70-90%, as seen in epidermal growth factor (EGF) receptors [14] [15] [16].  The biological function of this process is a down-regulation, or adaptation to the ligand.

Tetrahymena adapt to lysozyme over a time course of 10-14 minutes[12].  This adaptation is accompanied by a drastic decrease in Bmax, or the total number of available binding sites [12].  The same phenomenon has been observed in Paramecium [11].  If this adaptation was occurring via receptor-mediated endocytosis, one would expect to see a marked difference in the number of binding sites visualized by indirect immunofluorescence.  However, we observed neither a significant decrease in fluorescence intensity nor a change in the punctate staining pattern between non-adapted and lysozyme-adapted cells, as shown in Figure 1-B and 1-C.  This cannot be attributed to mere non-specific binding of the antibody, since control experiments with preimmune sera show no detectable fluorescence (Figure 1 A).  The data indicate that receptor-mediated endocytosis is not the primary mechanism for lysozyme adaptation in this organism.  This is an important finding, because it narrows the field of possible mechanisms of adaptation.

Possible mechanisms for down-regulation or adaptation include covalent modification, such as phosphorylation, of the receptor or the second messenger pathway.  Preliminary studies indicate that this does not occur via tyrosine phosphorylation (Kuruvilla, unpublished data).  Other mechanisms of down-regulation are currently being explored in our laboratory.



CONCLUSIONS

Our data showsno significant change in immunofluorescence staining patterns between cells which have been adapted to lysozyme, and cells which have not been adapted.  Since in vivo behavioral adaptation and lysozyme binding studies show a decrease in the number of functional receptors after 10 minutes of adaptation to lysozyme [12], but receptor staining patterns appear similar before and after lysozyme exposure, adaptation must be occurring by a mechanism other than receptor-mediated endocytosis.  Our laboratory is currently investigating other possible mechanisms for adaptation to lysozyme in this organism.



ACKNOWLEDGEMENTS

We would like to thank T.M. Hennessey for his donation of cells and antibodies.  I would also like to thank my Signal Transduction class for their participation in this class project.



REFERENCES

1. Devreotes, P. and S. Zigmond.  Chemotaxis in eucaryotic cells: a focus on leukocytes and Dictyostelium. (1988) Ann. Rev. Cell Biol. 4. 649-686. MEDLINE

2. Leick, V. Chemotactic protperties, cellular binding, and uptake of peptides and peptide derivatives: studies with Tetrahymena thermophila. (1992) J. Cell. Sci. 103(2). 565-570. MEDLINE

3. Harumoto, T. The role of trichocyst discharge and backward swimming in escaping behavior of Paramecium from Dileptus margaritifer.  (1994) J. Euk. Microbiol. 41(6). 560-564.

4. Sugarman, B, and N. Mummaw.  The effect of hormones on Trichomonas vaginalis.  (1998) J. Gen. Microbiol. 134(6). 1623-1628.  MEDLINE

5. Colamarino, S. and M. Tessier-Lavigne.  The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. (1995) Cell 81. 621-629. MEDLINE

6. Messersmith, E., Leonardo, E., Shatz, C., Tessier-Lavigne, M., Goodman, C. and A. Kolodkin. Semaphorin III can function as a selective chemorepellent to pattern sensory projections in the spinal cord. (1995) Neuron 14. 949-959. MEDLINE

7. Asakura, S. and H. Honda, Two-state model for bacterial chemoreceptor proteins: The role of multiple methylation. (1984) J. Mol. Biol.  176(3). 349-367.  MEDLINE

8. Kirsch, M., Zuberi, A.,  Henner, D., Peters, P., Yadzi, M., amd G. Ordal.  Chemotactic methyltransferase promotes adaptation to repellents in Bacillus subtilis.  (1993) J. Biol. Chem. 268(34). 25350-25356.  MEDLINE

9. Van Haastert,P.  Intracellular adenosine 3'-5'-phosphate formation is essential for down-regulation of surface adenosine 3'-5'-phosphate receptors in Dictyostelium.  (1994) Biochem. J. 303(2).539-545.  MEDLINE

10. Hennessey, T., Kim, M. and B. Satir.  Lysozyme acts as a chemorepellent and secretagogue in Paramecium by activating a novel receptor-operated Ca++ conductance.  (1995) J. Memb. Biol.,  98. 145-155.  MEDLINE

11. Kim, M., Kuruvilla, H, and T. Hennessey. Chemosensory adaptation in Paramecium involves changes in both repellent binding and the consequent receptor potentials. (1997) Comp. Biochem. Physiol, 118A (3). 589-597.

12. Kuruvilla, H., Kim, M., and T. Hennessey.  Chemosensory adaptation to lysozyme and GTP involves independently regulated receptors in Tetrahymena thermophila. (1997) J. Euk. Microbiol. 44(3). 263-268.

13. Kuruvilla, H. and T. Hennessey. Purification and characterization of a novel chemorepellent receptor from Tetrahymena thermophila. (1998) J. Membr. Biol. 162. 51-57. MEDLINE

14. Carpenter, G. and S. Cohen.  125I-labeled human epidermal growth factor: binding, internalization, and degradation in human fibroblasts.  (1976) J. Cell Biol. 71(1). 159-171. MEDLINE

15. Beguinot, L., Liall, R., Willingham, M. and I. Pastan.  Down-regulation of the epidermal growth factor in KB cells is due to receptor internalization and subsequent degradation to lysosomes.  (1984) Proc. Natl. Acad. Sci. USA 81. 2384-2388.  MEDLINE

16. Stoscheck, C. and G. Carpenter.  'Down-regulation' of EGF receptors: direct demonstration of receptor degradation in human fibroblasts.  (1984)  J. Cell Biol. 98(3).1048-1053. MEDLINE



 
 


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