BIOLOGY 468/568

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PRINCIPLES AND APPLICATIONS OF ELECTRON MICROSCOPY

STUDENT NAME:

Suzanne Delmonte

PROJECT TITLE:

Isolation of Cellular Organelles and Structures by Differential Velocity Centrifugation in Kidney tissue from Littorina littorea.

Species Identification

• Kingdom: Animalia
  
  
• Phylum: Mollusca
  
  
• Class: Gastropoda
  
  
• Order: Prosobranchia
  
  
• Family: Littorinacae
  
  
• Genus: Littorina
  
  
• Species: littorea

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Abstract

Differential velocity centrifugation is a tool used widely by biochemists and structural biologists to study cellular organelles. This technique is based on the principle that cellular components have different sedimentation values that can be fractionated out using various centrifugation speeds. The purity of such fractions may be analyzed using electron microscopy.

The purpose of this study was to use differential velocity centrifugation to isolate cellular organelles from the kidney of the marine prosobranch Littorina littorea and analyze those fractions using transmission electron microscopy. Upon such examination the 1000 g pellet was shown to be full of cellular debris along with many nuclei as expected. The 20,000 g pellet contained many vesicles and presumably nuclei and mitochondria without any clear membrane surrounding them. The 100,000 g pellet was, as expected, a very homogenous pellet consisting of microsomes and various cellular membrane remnants.

Key Words:

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INTRODUCTION

The ability to separate subcellular organelles by differential velocity centrifugation has provided scientists a valuable tool to study cell organelles in great detail. The early studies of rat livers by De Duve (1975) and others opened up the biochemical and structural analysis afforded by cell fractionation. The basis of this technique lies in the different sedimentation rates of cellular components. Organelles vary both in size and density, therefore theoretically one can pellet out specific organelles by altering centrifugation speed.

Three distinct cell fractions are identified as the nuclear, the mitochondrial, and the microsomal. The nuclear fraction is obtained at low centrifuge speeds, the mitochondrial at medium, and the microsomal at high. The initial steps for cell fractionation is homogenization of the tissue to be used. The tissue is suspended in a isotonic solution containing salt and sucrose concentrations compatible with the cells. Addition of Ca++ during homogenization has been reported to reduce nuclear breakage (Bogenhagen and Clayton 1974). Additionally, the presence of EDTA in the homogenization buffer has been reported to minimize the aggregation of mitochondria, and inhibit nuclease activity during differential centrifugation (Lansman et al., 1981). Homogenization can be by manual homogenizers (mortar and pestle) or by automated blenders. The homogenate is then subjected to various centrifugation speeds to pellet out desired fraction.

The purity of the fractionated pellet can be analyzed using marker enzymes, enzymes that are known to be concentrated in particular organelles, or by electron microscopy. Electron microscopy has revealed that the mitochondrial fraction is usually heavily contaminated with fragments of other cytoplasmic components and that the mitochondria are structurally modified when compared to those in the intact cell (Sjostrand 1967). Additionally, the microsomal fraction is often a heterogeneous fraction of cytosolic membranes including the plasma membrane.

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MATERIALS AND METHODS

Differential Centrifugation

After removal of their shells, the kidneys from 20 snails were dissected and weighed (all procedures were carried out on ice using sterile techniques). The tissue was then homogenized as described by Komm et al., (1982) using three parts homogenization buffer (0.03M Tris-HC1, 0.36 M KCI, 0.3M sucrose, 3OmM CaCl2, pH 7.6) to kidney tissue. Final homogenate was poured into appropriate tubes and centrifuged in a swinging bucket rotor at 1000 g for 10 minutes at 40 C. Supernatant was carefully decanted and EDTA was added to a final concentration of 3OmM. Resulting pellet was broken into smaller pieces and subjected to primary fixation for electron microscopy using 2% glutaraldehyde made in 0.2M sodium cacodylate, lOmM EDTA and 15 ppt NaCl, pH 7.6 . The supernatent was then centrifuged again at 1000 g for 10 minutes. After decanting supernatent, resulting pellet was placed in primary fixative as above. The supernatent was then centrifuged in Beckman SW-28 swinging bucket rotor at 20,000 for 20 minutes. Resulting pellet was carefully washed by re suspending it in original homogenization buffer to which 3OmM EDTA was added and centrifuged again at 20,000 g for 20 minutes. Resulting pellet was placed in primary fixative as above. Supernatent from first 20,000 spin was centrifuged at 100,000 g in above rotor for 90 minutes. Resulting pellet was also fixed as above.

Electron Microscopy of Pellets

Following primary fixation (1 hour) pelleted material underwent three 10 minute rinses with cacodylate buffer (0.2 M sodium cacodylate, lOmM EDTA, and 15 ppt NaCl pH 7.6) and then secondarily fixed in a 1:1 solution of 1% Osmium tetroxide and double strength cacodylate buffer for 1 hour. Following fixation, pellets underwent three 10 minute rinses with cacodylate buffer prior to dehydration. Dehydration consisted of a series of ethyl alcohol rinses at the following concentrations, each lasting 10 minutes : 30%, 50%, and 75%. Final dehydration was completed with four 15 minute rinses of room temperature 100% ethyl alcohol. Pellets were then rinsed in two 10 minutes washes of propylene oxide and placed in a 2:1 mixture of propylene oxide and Spurr's epoxy resin overnight. Infiltration of resin continued with an 8 hour wash of 1:2 mixture of propylene oxide and Spurr's epoxy resin and concluded with an overnight wash in 100% Spurr's epoxy resin. The pelleted material was then placed in flat embedding molds and cured overnight at 700 C.

Using a thermal advance ultramicrotome, ultrathin sections suitable for viewing were placed directly onto 200 and 300 mesh copper grids. Grids were then positively stained using the double staining technique of uranyl acetate and lead citrate developed by Reynolds (1963). Stained sections were then viewed in a Joel-1200 EXII transmission electron microscope at 80 kV.

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RESULTS

Fig. 1.



1000 g pellet. Representative micrograph of pellet. Legend: Nu=nucleus; Gly=glycogen. Scale bar = 0.5um. Magnification = 44,000.

Fig. 2.



1000 g pellet. Cluster of nuclei. Legend: Nu=nucleus; Cl=collogen fibers. Scale bar = l.0um. Magnification = 28,000.

Fig. 3.



1000 g pellet. Representative micrograph of pellet. Legend: Nu=nucleus; Gly=glycogen. Mp=mucoprotein. Scale bar = 1 um. Magnification = 21,000.

Fig. 4.



1000 g pellet. Cluster of nuclei. Legend: Nu=nucleus. Scale bar = 500nm. Magnification 32,000.

Fig. 5.



1000 g pellet. High magnification of boxed area in Fig. 4. Note absence of visible membrane around nuclei. Arrow indicates displaced membrane. Scale bar = 100nm. Magnification 144,000.

Fig. 6.



20,000 g pellet. Cluster of nuclei. Arrow indi- cates membrane. Scale bar= 500 nm. Magnification = 32,000.

Fig. 7.



20,000 g pellet. High magnification of Fig. 6. Scale bar = 100 nm. Magnification = 60,000.

Fig. 8.



20,000 g pellet. Representative micrograph of pellet. Scale bar = 500 nm. Magnification 30,000.

Fig. 9.



20,000 g pellet. Cluster of nuclei. Legend: Nu= nucleus. Scale bar = l.Oum. Magnification= 18,000.

Fig. 10.



100,000 g pellet. Representative micrograph of pellet. Scale bar = 200nm. Magnification = 90,000.

Fig. 11.



100,000 g pellet. Lower magnification of pellet. Scale bar = 1.0 um. Magnification = 25,000.

Fig. 12.



100,000 g pellet. Representative micrograph of pellet. Scale bar = 200 nm. Magnification = 90,000.

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DISCUSSION

Figure 1 represents the general appearance of the sections obtained from the initial 1000 g pellet. Because of the nature of homogenization and centrifugation, all structures are artificially construed together. This makes analysis of micrograph somewhat difficult. Various vesicles are present along with glycogen rosettes. It is noteworthy that the nucleus maintains a nuclear membrane at this state. In most cases the nuclei appeared as aggregate as shown in Figure 2. This was typical of all sections from the 1000g pellet. In most cases, the nuclei were in close proximity to each other. Surrounding the nuclei is an artificial membrane which has wrapped itself around the nuclei. Also visible in this pellet were collagen fibers.

Vast amounts of what appears to be mucoprotein was also typical of the 1000 g pellet as is shown in Figure 3. This would be consistent with the organism used for this study. Being an intertidal marine gastropod, Littorina heavily produces mucus to prevent itself from desiccation.

Also in the 1000 g pellet were nuclei that had lost their nuclear membranes as is shown in Figure 4. These nuclei were identified based on their large size (approximately 2.5 um). Higher magnification on the electron microscope of the area boxed in Figure 4 did not reveal membranes immediately surrounding nuclei (Figure 5). Misleading however, is a membrane in close proximity.

Analysis of the 20,000 g pellet showed a decrease in fibrous material and mucoprotein among other things. Prominent in the sections from this pellet are structures resembling nuclei and mitochondria without clear membranes as shown in Figure 6. Again, although there is a membrane nearby (arrow) its proximity to the organelle is probably artificial. These structures are probably nuclei, but because of the lack of a clear membrane (Figure 7) it cannot be certain. Their size does not exclude the possibility that they are mitochondria.

Figures 8 and 9 are representative of the sections from the 20,000 g pellet. Scattered throughout various vesicles are nuclei and what may perhaps be mitochondria. Also present in Figure 9 what appears to be cilia (arrow).

The 100,000 g pellet was very homogeneous (Figures 10,11,and 12). What is left in the cytosol is a conglomeration of microsomal bodies and various cytoplasmic membranes. In none of the sections obtained were there any large organelles.

These results agree with what is normally expected by differential velocity centrifugation. The absence of intact organelles, primarily mitochondria, in the 20,000 g pellet is not however consistent with current research. Although care was taken during the homogenization process and with respect to osmolarity, it appears that all of the mitochondria have been disrupted.

The nature of studying the purity of cell fractions with an electron is perhaps the reason for this 'negative' result. One important aspect regarding the sectioning of the pellets was overlooked in the present study and that is: where in the pellet the sections actually came from. As noted by Sjostrand , in his book entitled Electron Microscopy of Cells and tissues, "The electron micrographs are mostly of sections through a regular pellet that have been fixed and embedded like a piece of tissues. The composition Of such a pellet varies with the distance from the bottom Surface where heavier components are more numerous than in the top layers".

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FUTURE WORK

Differential velocity centrifugation is a widely successful means of studying cell organelles. The ability to isolate particular organelles and study their biochemical and structural nature has led to many important advances in science. By combining this technique with density-gradient centrifugation, cell fractions may be further purified. Additionally, properly sectioning the pellet so as to ensure representative micrographs is necessary.

It has also been suggested (Douglass, personal communication) that the mucosal nature of Littorina littorea may be interfering with the natural sedimentation of cell organelles. If one could eliminate the mucus from the tissue before homogenization, perhaps more accurate results would be obtained.

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CITATIONS

Bogenhagen D.,Clayton DA (1974) J. Biol, Chem. 249:7991- 7995.

De Duve, C. (1975) Lysosomes 189: 186-194.

Komm et al., (1982) Comp, Biochem, Physiol, vol 73B, no. 4, 923-929.

Lansman et al., (1981) J. Mol, Evol, 17: 214-226.

Reynolds, E. S. (1963) J. Cell Biol, 17: 208-212.

Sjostrand, F.S.1(967) Electron Microscopy of Cells and Tissues 436-447 Academic Press Inc, New York, New York.