Clokey and Urven Lysosome Lab

Endocytosis and Cellular Digestion

Reading pp. 323-341, 773 of Karp¹.

Rationale: Biological imaging has undergone a technological revolution in the last twenty years, leading to countless discoveries that were previously impossible. The long list of novel imaging methods extend from gross anatomical levels using nuclear magnetic resonance imaging and positron emission tomography, to cellular levels with confocal and Nomarski interference microscopy, and to molecular levels with atomic force microscopy and scanning tunneling microscopy. In this laboratory exercise, we will use fluorescence microscopy, one of the earlier innovations in light microscopy, to study lysosomal and endosomal distribution and function in the cells of the digestive system of the nematode Caenorhabditis elegans. For this laboratory exercise, we will attempt to replicate studies by Dr. Clokey2 that provided evidence that the intestinal cells of C. elegans are pinocytic but not phagocytic.

Protocol Background:

Control C.elegans were grown on a lawn of Escherichia coli. Experimental animals were fluorescently labeled in one of three ways.

They may have been pulsed by feeding them rhodamine isothiocyanate (RITC)-conjugated protein or RITC-conjugated dextran (a large carbohydrate) for four hours, then washed in phosphate buffered saline. Proteins and dextran are water soluble, and can enter the cell only by pinocytosis or receptor-mediated endocytosis. The RITC label remains intact and functional even within the cytoplasm. Rhodamine fluoresces at red wavelengths.
They may have been fed fluorescein isothiocyanate (FITC)-conjugated microspheres, which are roughly the size of bacteria (0.5 µm). This size particle can only enter cells by phagocytosis.
They may been incubated in a 0.05 mg/ml of acridine orange solution. Acridine orange is hydrophobic in neutral pH, but is protonated in acidic conditions. Protonation makes it cationic, which prevents it from passing through the hydrophobic layer of membranes. As a consequence, it enters membranous organelles easily, but if the contents of an organelle is acidic, it will be trapped within the organelle. Since lysosomes are acidic, membrane-bound organelles, acridine orange serves as a fluorescent label specific for lysosomes.

Procedure: Make a wet mount of a few control group nematodes in phosphate -buffered saline (PBS) with an 18 mm coverslip. Working under a dissecting microscope, lower the coverslip slightly by touching a lab tissue to the edge of the coverslip, until the worms just stop moving. Careful! Take too much saline and the worms may be damaged! Check them under your phase contrast microscope to make sure the coverslip has not killed the worms. If your wet mount is satisfactory, seal the coverslip by placing a bead of immersion oil around its edges. This will prevent excessive evaporation. See the instructor for assistance in observing the nematodes under ultraviolet epi-illumination.

On our model fluorescence microscope, the "G" filter blocks green light and shorter wavelengths, to allow observations of the red fluorescence of RITC-conjugated protein or acridine orange. The "B" filter on the fluorescence microscope blocks blue light and shorter wavelengths, allowing green fluorescence observations of FITC-conjugated microspheres. Examine the worms under each filter, then return to room 342 to record your observations in writing and in the form of sketches. Make additional wet mounts and fluorescence observations for each experimental group of nematodes, and record them as above. Are the intestinal epithelial cells phagocytic? Are they pinocytotic? Write a formal laboratory report including introduction, methods, results, discussion, and literature cited sections to describe your observations and conclusions.

¹Karp, G. 1999. Cell and Molecular Biology Concepts and Experiments, 2^nd edition. New York: John Wiley and Sons, Inc.

²Clokey, G.V. and Jacobson, L.A. 1986. The autofluorescent "lipofuscin granules" in the intestinal cells of Caenorhabditis elegans are secondary lysosomes. Mech. Ageing Dev. 35: 79-94.

Lab Preparation

Nutrient agar plates
E. coli cultured in lawns
C. elegans
Phosphate Buffered Saline (PBS)
RITC dextran, RITC IgG, or RITC-BSA at 10 mg/ml in BU buffer or just dissolve one crystal in 50 µl PBS until it takes on a pinkish tinge
Fluoresbrite Carboxylate Microspheres (2.5% Solids Latex), Polysciences #15700, 0.50 µm YG, 10 ml, $82.70: Make one ml of 1:10 to 1:20 dilution in PBS
5 ml Acridine orange: 0.05 mg/ ml in PBS buffer
Fluorescence microscope with fluorescein and rhodamine filters
To label worms:
1. 50 µl drop of # RITC protein, microspheres, or acridine
2. orange in 35 mm Petri dish
3. 25-50 adult worms in drop
4. cover and incubate four hours or o/n at 15oC in a humidified 50 mm dish
5. wash in one ml PBS buffer
6. centrifuge 500-1000 rpm, 1 minute
7. transfer to clean PBS in 35 mm dishes, one dish/group

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