LAB 11

                                  CELL SURFACES AND SPONGE CELL REAGGREGATION

 I. Introduction

The outer surface of the cell membrane plays a major role in the assembly and maintenance of tissue integrity. The outer surfaces of developing and differentiated cells contain receptor molecules that recognize systemic signals, ligands or hormones. The binding or dissociation  of the ligands controls some of the differentiated functions of the cell, keeping it in tune with the needs of the whole system.

The outer surface is also coated with glycoproteins and proteoglycans.  These large complexes of protein and polysacchar­ides provide a tissue-specific matrix within which cells of like function can operate together as a coherent tissue.  In embryo­genesis the sorting out and tying together of cells with a common function is facilitated, and probably controlled, thorough the molecular specificities of the glycoprotein and proteoglycan surfaces of the cells.

In this experiment we will study the way in which cells interact to form functionally coherent tissues.  Sponges have been used often for this kind of work because their tissue structure is relatively simple.  There are only about five or six cell types. Sponges are sessile, aquatic animals that belong to the phylum Porifera. To make a living, they move water through pores in their body wall and phagocytose organic particles from the water.  Their body plan is relatively simple, too. 

There is a central cavity lined with "collar cells" whose beating flagella cause water to be circulated through the body wall and out of an opening in the central cavity called the osculum. The body is made up of three layers of cells which include the inner collar cells, skeleton-producing cells that form hard spicules, amoebocytes that move about aiding in nutrient distribution, archeocytes that seem to have regenerating capacities, and outer epidermal cells that cover the animal.

Interestingly, a whole animal can be converted into a suspension of individual cells, or small aggregates of cells, by simply pressing pieces of sponge through a sieve.  In such experiments, the dispersed cells still retain most of their surface coats so that when brought back together they quickly adhere into balls of cells.  If dispersed cells from different species are mixed, all cells stick together initially, but then a sorting out occurs so that only cells of one species end up within any one aggregate.  The phenomenon does not stop there.  Within the aggregates, cell movements occur.  Cells find and stick more tightly to cells of the same tissue type.  Cell sorting finally leads, in a few weeks, to whole new sponges!

Sponges can also be dispersed into individual cells by two other means.  If sponges are kept in sea water that lacks the divalent cations calcium and magnesium, they will slowly disperse into individual cells.  Moreover, under these conditions, much of the proteoglycan coat material that holds cells together goes into solution.  We learn from this that the structural integrity of the proteoglycan intercellular cements are dependent on calcium, and to a lesser extent on magnesium.

 Sponges can also be dissociated by adding proteolytic enzymes to the sea water.  These enzymes destroy the polypeptide chains within the proteoglycan complexes.  When cell dispersal is accomplished either by solubilization or destruction of proteo­glycans, reaggregation must await their resynthesis and deposi­tion onto the cell surfaces.

 

II. Procedure 

This experiment is designed to investigate the role of the cell surface in cell/cell adhesions.  We are interested in the selective adhesions of like cells that result in tissue formations in development.  We will attempt to demonstrate reaggrega­tion and show that lost surface molecules can be metabolically replaced. In the experiment today we are going to try to get quantita­tive data that will confirm or deny the proposition that sponge cells, stripped of their proteoglycan coats must resynthesize that coat in order to reaggregate.   

We will solubilize sponge cell proteoglycans by treating pieces of sponge with calcium- and magnesium-free sea water (CMF-SW).  Based on what we know, the dispersed and denuded cells must resynthesize their proteoglycan coats before they reaggregate.  If this is true, then dispersed cells incubated at low temperatures, where metabolism and macro­molecular syntheses are slow, should take longer to resynthesize and reaggregate than cells similarly treated but incubated at room temperature, where metabolic processes are generally faster. 

A. Sample Preparation (night before lab) 

It takes at least 9 hours in CMF-SW to complete the solubi­lization of sponge proteoglycans.  Therefore, student volunteers will perform the following protocol the day before our experiment. It should be noted that it is sometimes difficult to maintain sponges in lab so sponges should only be handled with rinsed latex gloves.  

1.         Obtain about 20 grams of sponge and cut into 1-cc chunks, rinse in CMF-SW, carefully blot free of water and debris, and immerse in 1 liter of CMF-SW for each sponge at pH 7.2.  

2.         The pieces will be further cut up into 3-mm fragments during a 60-minute soak.   

3.         The CMF-SW will be decanted and 1600 ml of fresh CMF-SW added.  The mixture will be kept in the refrigerator overnight so that by the next day most of the calcium and magnesium of the pieces will be withdrawn into the CMF-SW. 

4.         Place on shaker table overnight at 15C

 

B. Generating the Cell Suspension (during scheduled lab) 

1.         Collect the pieces of sponge into a square of fine bolting cloth.  The corners are brought together in "purse string" fashion and the contained sponge pieces are placed into a beaker containing 50 ml of fresh CMF-SW depending on the condition and amount of the sponges in a given year.   

The bulge of sponge pieces is then squeezed with the fingers.  A cloud of cells will be seen coming through the bolting cloth into the CMF-SW.  When maximum pressure has squeezed out as many cells as possible, the residual sponge debris is removed from the cloth and discarded.  The cloth is washed and kept for further use. We want to generate concentrations of cell suspensions (total number of cells in the suspension) that differ for the different species we are using.  

Microciona (red sponge)                                    3.0 x 10⁸ 

Haliciona (brown sponge)                                  1.5 X 10⁸ 

Axinella (orange devils finger sponge)    2.7 x 10⁸ 

C. Counting the Cells and Clumps 

1.         Two or three students will then use hemocytometers or the Coulter Counter to determine the concentration of cells and clumps of cells.  (See the appendix at the end of this experiment for the way in which the hemocytometer is used.)  The average determination of the two or three students will be taken as the cell concentration.  Most of the surface proteoglycans should be gone from these cells and clumps.

 D. Determining the Characteristics of Cells and Clumps 

1.         Working individually, students will thoroughly stir the cells into a uniform suspension and pipette out a sufficient volume to give target cell numbers provided on the previous page.  

2.         This aliquot of suspended cells is transferred to a small beaker, and the cells and clumps are drawn in and out of a fine-tipped Pasteur pipette repeatedly to break up as many remaining clumps as possible. 

3.         The initial characteristics of this sample must now be determined.  The cells are swirled into suspension and one drop removed and placed on a hemocytometer slide. Note, you should keep the stock solution of cells cool.  A coverslip is placed over the drop for observation under a compound microscope.  A random count of objects in several fields of view is made to: 

(1) determine what percent of the objects are single cells and what percent are clumps of cells, and  

(2) measure the diameters of clumps (using an ocular micrometer) to find their average diame­ter.  In this and subsequent samples, at least 100 cells and clumps should be counted and 50 clumps measured in order to give reliable data. 

4.         Take the average diameters of the clumps at the initial and subse­quent time points of sampling may be compared directly if the measure­ments are always made with the same microscopic magnifica­tion.    

 5.         One-third of the suspension should now be pipetted into each of three centrifuge tubes and centrifuged at 2000 rpm in the clinical centrifuge for 2 minutes.  The CMF-SW supernatant is carefully removed insofar as possible from each tube with a Pasteur pipette.  

6.         The cells of one tube are resuspended in 3-10 ml of fresh CMF-SW; cells of the second and third tubes are resuspended in 3 ml each of complete artificial sea water (which contains normal calcium ion). You now have 3 ml of cell suspension at 30 x 10⁶ objects/ml (Microciona) or 15 x 10⁶ objects/ml (Haliclona) resuspended in each of three tubes. 

7.         Each suspension is swirled and poured into a 50 or 35 mm Petri dish.  The lids are put on to slow evaporation.   

8.         The control cells in CMF-SW and the cells in one dish of normal sea water are put onto a rotating shaker at room temperature (about 25^oC).  Cells in the other dish of normal sea water are put onto a rotating shaker at 15^oC.  All three dishes are appropriately labeled. 

 

III. Data Analysis 

1.         The suspensions are incubated on rotators to facilitate getting the cells and clumps into physical contact with each other, a condition necessary for adhesions to take place. You must now monitor what happens through time in all three dishes.  Monitoring at 12- or 24-hour intervals of time will be sufficient. You should continue to take samples until you are certain of the changes taking place in the three samples. 

             2.         To monitor each dish, one drop of swirled suspension is placed under a coverslip on a  icroscope slide and observed under a compound microscope.  The percentage of single cells and clumps, the mean clump size, and variance are calculated.

  

The mean normally gives us the peak                                         x = ∑ x

value in the distribution                                                                       N

 

 

Variance is a measure of the spread of                                      s² = ∑ (x - x)²

the distribution, it measures the extent to                                               N - 1

which samples are clustered around

the mean

 

 

Standard deviation puts values in the                                         S  =   

same units of dimension as the mean 

 

 

You can also enter you data in SigmaPlot program obtain means and basic descriptive statistics on your samples.

Additional Notes:  

It is best to order sponges to be delivered a day or two before they are to be used.  They often come with much adherent and debris that must be removed before they can be cut up. Incubation can be done on rotary shakers that move just fast enough to cause cells and clumps to come into contact.   

In these long-term experiments students should mark the sea water level in their culture dishes with a marking pencil so that distilled water can be added periodically to prevent the cultures from evaporating due to dryness or excessive salt concentration.    

Use the usual report format for your lab write-up.  Your data should include a table giving the percentage of individual cells and clumps of cells in each sample at each time point--as well as the M and SEM of clump size in each sample at each time point.  Sum up the data of the table in a graph(s). Explain the purpose of the control sample in CMF-SW. The hypothesis tested and your conclu­sions should be clearly stated.

V. References 

1.         Merriam R.W., 1988. Experiments in Animal Development, Sinauer Pub. Sunderland, Mass. Most of today's lab is derived from experiment number 10 in this lab manual. 

2.         Graham, C. F., and P. F. Wareing, 1984, Developmental Control in Animals and Plants, 2nd ed., Blackwell, Oxford, pp. 101-116. 

3.         Hopper, A. F., and N. H. Hart, 1985, Foundations of Animal Development, 2nd ed., Oxford University Press, New York, pp. 310-315, 333-335. 

4.         Monroy, S., and A. A. Moscona, 1979, Introductory Concepts in Developmental Biology, University of Chicago Press, pp. 155-205. 

5.         Henkart, P., S. Humphreys, and T. Humphreys, 1973, Characteris­tics of a sponge aggregation factor: a unique proteoglycan complex.  Biochem. 12: 3045-3050. 

6.         Humphreys, T., 1963, Chemical dissolution and in vitro reconsti­tution of sponge cell adhesions.  Isolation and functional demonstration of the components involved.  Devel. Biol. 8: 27-47. 

7.         Townes, P. L., and J. Holtfreter, 1955, Directed movements and selective adhesion of embryonic amphibian cells.  J. Exptl. Zool. 128: 53-120. 

 

Directions for using the Coulter Counter Particle Counter

 

1.         Turn switch to “I” position to turn instrument on 

2.         Lower specimen tray and place sample on tray 

3.         Carefully raise specimen tray till aperture is in sample  

4.         Press the “Setup” button – the “S1” screen should come up 

5.         Press the “Setup” button again to bring up the “S2” screen 

6.         Press “Start” to read your sample 

7.         Record your count and be sure to take into account the dilution   factor, instrument is set up to sample 0.5 ml and typically there is a total volume of 10 mls of sample.