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Biology 230 - Molecules and Cells

Subcellular Architecture and Experimental Approaches to Cell Biology

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Terms

  • actin filament
  • antibody
  • cell homogenate
  • cell wall
  • centrifugation
  • chloroplast
  • chromatin
  • confocal microscopy
  • cytology
  • cytoplasm
  • cytoskeleton
  • density gradient centrifugation
  • differential centrifugation
  • endomembrane system
  • endoplasmic reticulum (ER)
  • eukaryote
  • fluorescence microscopy
  • fluorescent dye
  • Golgi complex
  • homolog
  • intermediate filament
  • light microscopy
  • lysosome
  • microfilament
  • microtubule
  • mitochondrion
  • nucleolus
  • nucleus
  • pellet
  • peroxisome
  • plasma membrane
  • primary cell culture
  • prokaryote
  • ribosome
  • subcellular fractionation
  • scanning electron microscopy (SEM)
  • supernatant
  • transformed cell line
  • transmission electron microscopy (TEM)
  • vacuole

Introduction and Goals

This tutorial provides a brief description of the subcellular architectures of typical animal and plant cells. The subcellular structures and their functions will be described in greater detail in subsequent tutorials. In addition, this tutorial describes the techniques and approaches that are commonly employed in laboratories to study cellular structures and their functions.

By the end of this tutorial you should know:

  • The differences between prokaryotic and eukaryotic cells
  • The organelles of a typical animal cell and plant cell
  • The types of cell cultures used in laboratories
  • The common model systems used in the genetic analyses of cellular processes
  • Several methods of microscopy, including light, fluorescence and electron microscopy
  • The use of centrifugation to isolate organelles

Eukaryotic vs. Prokaryotic Cells

Figure 1. A typical prokaryotic cell. Most of the cell's DNA is located in the cytoplasm and compacted into a region called the nucleoid. In addition, there are freely replicating, circular, plasmid DNAs. Note that prokaryotes have cell walls.

There are two fundamentally distinct types of cells, prokaryotic and eukaryotic. Prokaryotes are generally simpler, smaller cell types than eukaryotes; however, the distinction between these two is more than just size and shape. Prokaryotic cells do not have distinct organelles and their genetic material is located in the cytoplasm (see Figure 1).

In eukaryotic cells, most of the genetic material (DNA) is partitioned into the nucleus, which is surrounded by a distinct membrane, and the DNA is tightly associated with proteins. Eukaryotes possess a variety of membrane-bound specialized organelles that are capable of distinct activities. In addition, eukaryotes possess networks of proteins, referred to collectively as the cytoskeleton, which regulate cell shape, mobility and intracellular movement. Most of the material presented in this tutorial, and in subsequent tutorials, will focus on processes and structures of eukaryotic cells. It should be noted that prokaryotes and eukaryotes share the same basic components; namely, proteins, nucleic acids, lipids and carbohydrates. They also share many of the same core metabolic pathways. Both cell types possess a plasma membrane, which acts as a barrier between the cell and its environment, and both interact with their environments, although in different ways.

A Typical Eukaryotic Cell

Figure 2. A typical animal cell.

Figure 3. A typical plant cell

There really is not a "typical" eukaryotic cell; each cell type is unique and specialized in some fashion, be it a single-cell organism (e.g. a yeast cell) or a single cell that is part of a much larger tissue or organ (e.g. a neuron in the central nervous system). Nonetheless, there are some common subcellular structures that all eukaryotic cells have, and some structures that are unique to animal or plant cells (illustrated in Figures 2 and 3, respectively).

Plant and animal cells are surrounded by a plasma membrane, which is composed of a lipid bilayer embedded with proteins. Plant cells have an additional barrier, the cell wall, which is composed primarily of cellulose. One of the largest organelles in most cells is the nucleus. The nucleus is delineated by the nuclear envelope, which is composed of two membrane layers interrupted by small openings (nuclear pores) that allow traffic in and out of the nucleus. The nucleus is where the bulk of genetic material, in the form of chromatin (a DNA protein complex), is organized into chromosomes (see Tutorial entitled DNA and Chromosomes). The nucleus is also the site of RNA synthesis. The nucleolus is a structure within the nucleus, and it is the site of ribosome assembly. Recall, ribosomesare the RNA/protein complexes needed for protein translation. They are located free in the cytoplasm or attached to the membranes.

Another organelle found in animal and plant cells is the mitochondrion, which houses the enzymes and other proteins used to generate energy for the cell in the form of ATP (see Tutorial entitled Oxidative Phosphorylation). The reactions of cellular respiration and ATP synthesis are localized in the mitochondrion.

The chloroplast is an organelle unique to plant cells and other photosynthetic cells (see Tutorial entitled Photosynthesis). The proteins and other molecules required for light absorption and for the fixation of carbon from carbon dioxide into simple sugars are located in the chloroplasts.

The endomembrane system includes other prominent organelles common to animal and plant cells. The endomembrane system includes the endoplasmic reticulum (ER) and the Golgi complex, both of which are networks of specialized membranes rather than discrete organelles like the mitochondria and chloroplasts. The ER appears either smooth or rough, and this distinction reflects different structures and activities. The smooth ER is the major site of lipid synthesis and drug detoxification. The rough ER is ER membrane studded with ribosomes, and is the site of protein synthesis. Many proteins synthesized in the rough ER are transported to the Golgi complex, and will be further modified by the addition of sugars. Proteins move between the ER, the Golgi complex and many other subcellular destinations through vesicles, which are small membrane-bound organelles. Animal cells also contain additional organelles, including lysosomes, which are filled with digestive enzymes for degradation of macromolecules, and peroxisomes, which are filled with enzymes that carry out oxidation reactions (see Tutorial entitled Intracellular Compartments). Plants cells have a unique organelle termed a vacuole, which can serve as a storage compartment and as a degradation organelle; it is similar to the lysosome in an animal cell.

Figure 4. Three types of microfilaments: actin filaments, microtubules and intermediate filaments.

Finally, another common feature of animal and plant cells is the cytoskeleton, a network of microfilamentsthat runs throughout the cytoplasm (see Tutorial entitled The Cytoskeleton). There are three types of microfilaments, each composed of distinct proteins, termed microtubules, actin filaments and intermediate filaments (Figure 4.). Microtubules are found in the cytoplasm and nucleus. In the cytoplasm, they are responsible for cell motility (e.g. they drive the movement of a sperm tail) and movement of vesicles and organelles within the cell. In the nucleus, they are responsible for the separation of chromosomes during cell division (the spindle fiber is composed of microtubules). Actin filaments are used for several processes, including cell crawling and cell division. In muscle cells, the actin filaments are part of the molecular mechanism of muscle contractions. Finally, intermediate filaments are found in animal cells, where they are important for providing support and strength to the cell.

Choosing Cells To Study


An important consideration in experimental cell biology is the type of cell one chooses to investigate. _Escherichia coli (E. coli)_is a bacterial species that has been intensively studied, and much of our understanding of the mechanisms of DNA replication, RNA transcription and protein translation stems from these studies. E. coli is easy to grow, reproduces rapidly, and is generally not harmful to humans. However, if one is interested in studying organelles or processes that are unique to eukaryotes, a variety of cell types are commonly used. As stated above, there is not a typical eukaryotic cell; rather, each cell type is specialized for a distinct function and has a unique shape and composition. Therefore, the type of cell used for investigation is largely determined by the process or organelle that one wants to study. For instance, if one were interested in studying the process of cell motility, the fish keratocyte would be a common cell type to consider using - these cells can be easily isolated by plucking the scales off of the fish, which are attached to tissue that contains the keratocytes. Sometimes whole tissues can be used; however, these are often composed of numerous cells and many different cell types, which is not ideal for an investigation of the cellular and molecular mechanisms of a cell-specific process. Different types of cells can sometimes be isolated from a tissue and then cultured in an appropriate medium; this is referred to as primary cell culture. Its disadvantage is that the cells are not in their natural environment so they may not behave normally. Furthermore, these cells will not survive indefinitely and can be difficult to maintain in culture. An alternative is to use transformed cell lines, which are immortal and can grow indefinitely in culture. These cell lines are derived from tumors (or, in some cases, they arise spontaneously in a primary culture) and have the distinct property of infinite growth in culture. The first such human cell line to be established, and that is still commonly used, is the HeLA cell line, which was derived from a uterine tumor removed from a female patient. One caveat of using transformed cell lines is that they are often associated with chromosomal anomalies; however, over the years, many of these cell lines have been characterized and developed as useful tools for studying cellular processes.

Genetic Analysis of Model Organisms

The brewer's yeast Saccharomyces cerevisiae is a single-cell eukaryote that has been a very important model system for studying many aspects of cell biology. It has the advantages of being a relatively simple cell type and it is easy to grow. More importantly, it is particularly well suited for genetic analyses. The strategy behind this type of analysis is to isolate a mutant that is defective in a specific cellular process, then use this mutant to tease out the components and mechanism of the normal process. It is paradoxical that cells defective in a process can be used as a tool to elucidate the workings of the normal process. For example, the process of protein secretion is a multistep pathway that requires activities in the rough ER, Golgi complex, plasma membrane and many specialized vesicles. Many mutations in yeast render them defective in protein secretion; consequently, each mutation is blocked at a different step in the pathway, resulting in an accumulation of secretory proteins in the subcellular locations before this step and an absence of secretory proteins in the subcellular locations after this step. An examination of many such yeast mutants allowed investigators to get a "snapshot" of each step in the pathway. Also, once the gene that was disrupted in a particular mutant was identified and the protein it encoded was determined, the role of that protein in protein secretion was determined. Identification of several important proteins in this pathway led to a model of the molecular mechanism of protein secretion. Furthermore, the genes identified in yeast that encode the proteins necessary for the process of protein secretion turn out to have counterparts or homologs (similar in sequence because of a common evolutionary origin) in other eukaryotic organisms, including humans.

The past few decades have revealed that the basic cellular processes, such as protein secretion, are very similar in many different types of eukaryotic cells. Increasingly, genetic and genomic approaches (analyses and comparisons of the genes of an organism) are being used in a variety of model systems to study all aspects of cell biology. Among the most commonly used multicellular model systems are the nematode worm (Caenorhabditis elegans), the fruit fly (Drosophila melanogaster), the zebrafish (Danio rerio), the mustard plant (Arabidopsis thaliana) and the mouse. For each of these species, the sequence of the genome has been determined and there are many mutants available (and even more are being isolated in research laboratories around the world) that disrupt some aspect of normal cellular function. The use of these model systems is a stepping-stone for understanding the cellular and molecular mechanisms of human cells. In fact, the completion of the human genome project revealed that of the predicted human proteins, approximately 74% have homologs in other eukaryotic organisms. Therefore, understanding the role of these proteins in the fruit fly, for instance, will advance our understanding of their role in a human cell.

Visualizing Cells and Subcellular Structures

Figure 5. Fluorescence microscopy. The cells in part of a Drosophila embryo are stained for two proteins using fluorescent dyes. Data for each protein are gathered individually as grayscale images (left and right panel) using filters to distinguish their different fluorescent colors. To compare the distributions of the two proteins the image of each individual protein is combined in a color image file using different color channels for each proteins (center panel). This is called a false color image since we can make these any color we want that will best illustrate the data- in this case red for the lefthand proteins and green for the righthand protein. An overlapping distribution gives an orange color. Provided by Dr. Graham Thomas.

Cytology, the study of cellular structures by direct visualization through microscopy, is perhaps the greatest tool that cell biologists have in their arsenal of techniques. Three types of microscopy are commonly used: light, fluorescence and electron microscopy. Light microscopy uses white light to illuminate the specimen, and can achieve magnifications of a thousand-fold. At this level of magnification, many of the larger organelles can be seen (e.g. nuclei and mitochondria). This type of illumination does not provide a great deal of contrast, however, the optics can be modified to increase the contrast. Examples are illustrated in these linked movies, where the same cheek cell is viewed via (A) brightfield microscopy, (B) phase contrast microscopy, and (C) differential interference contrast (DIC) microscopy. Often a specimen will be fixed to preserve the cell's structure, then stains may be added to enhance the contrast of structures in the cell or to detect a particular macromolecule. (For example, Feulgen stain is specific for DNA and therefore stains the nuclei.) When whole tissue is used, the sample is often sectioned to provide very thin slices of tissue that can then be fixed, stained and viewed.

Figure 6. Transmission electron microscopy (TEM). This micrograph illustrates the fine detail that can be seen by TEM. What we can see is part of a spiral membrane structure in the same Drosophila cells seen in Figure 5. In Figure 5 these are seen merely as red 'blobs' at cell membrane - here we can see individual membrane bilayers (red arrow). Electron microscopy can achieve this because the wavelength of the electrons is much much shorter than that of light. However, samples must be extremely thin for electrons to pass through them to the detector. Provided by Dr. Graham Thomas.


Fluorescence microscopy is similar to light microscopy, however, light of a defined wavelength is passed through the stained specimen. In this case the cells or tissue are stained with a fluorescent dye that absorbs light at a particular wavelength and then emits light at another, longer wavelength. Only the emitted light can pass through the eyepiece of the microscope, therefore, only the stained portion of the cell is visible (see Figure 5). There are fluorescent stains specific for some macromolecules (e.g. phalloidin is a fluorescent dye specific for actin filaments). Greater specificity and diversity can be achieved when using antibody molecules that are covalently linked to fluorescent dyes. Antibody molecules are produced by the immune system of all vertebrates, including humans. They are part of the body's immune system for protection against infection. Antibodies are protein complexes circulating in the blood, and they normally recognize and bind to specific macromolecules that are recognized as being foreign. Within every individual, there is a huge diversity of antibody molecules that each bind to and recognize a different macromolecule. Cell biologists have generated a battery of antibodies (derived from animals such as rabbits, rats and mice) directed against a variety of cellular proteins. These can be linked to fluorescent dyes and used to localize a particular protein within the cell. For example, an antibody specific for a protein that normally resides in the Golgi complex could be used as a specific marker for the Golgi complex, thereby allowing the detection of this organelle. A further technical advance is the use of confocal microscopy, a type of fluorescence microscopy that uses a powerful laser to illuminate a sample and take optical sections through the specimen. This is achieved by focusing on a spot within the specimen at a specific depth and allowing only the light emitted from that spot to be included in the image (excluding fluorescence from above or below the spot that is in focus). A series of images from a region at different depths can be generated and used to create a three-dimensional view of the sample.

Figure 7. Scanning electron microscopy (SEM). This micrograph illustrates the surface of an adult Drosophila's eye. As with TEM (Figure 6) very fine detail is visible (although the magnification here is much lower). In SEM electrons do not pass through the specimen from, but bounce off the surface to the detector so only surface details are seen. To get the electrons to bounce the specimen must be dried and coated in a thin layer of metal atoms all over its surface. Provided by Dr. Graham Thomas.

Light microscopy and fluorescence microscopy are limited by the resolution of light (~200 nm). Electron microscopy is an alternative type of microscopy that provides greater resolution and magnification. In electron microscopy a beam of electrons is used, instead of light, to illuminate the sample. Transmission electron microscopy (TEM) captures the electrons that have been transmitted through a very thin section of the sample, which has usually been stained with an electron-dense dye to enhance contrast. TEM has a magnification of approximately a million-fold and provides a view of the interior of the cell, including structures such as the ER, the Golgi complex, ribosomes and microfilaments (see Figure 6). Scanning electron microscopy (SEM) is a procedure used to visualize the three-dimensional structure of a sample. The sample is coated with a heavy metal, then a beam of electrons is focused on the sample. The pattern of electron scattering provides a view of the topology of the sample (see Figure 7).


The Use of Centrifugation to Isolate Organelles

In addition to the genetic and cytological approaches used to study the cellular processes described above, sometimes cell biologists employ biochemical techniques to study the chemistry of reactions or processes. These techniques require the isolation and purification of a single organelle or subcellular structure to investigate a specific biological activity. This can be achieved by subcellular fractionation, whereby cells are disrupted and subjected to centrifugation to separate different components of the cell. Centrifugation is a procedure in which the disrupted and homogenized cells (cell homogenate) are subjected to centrifugal force and organelles are separated based on their size and density. The cell homogenate is placed in a tube that is spun around rapidly in a centrifuge (a motorized rotor within a refrigerated chamber). As the contents of the cells are subjected to the centrifugal force, the larger and heavier organelles (e.g. nuclei) will begin to travel down to the bottom of the tube and sediment, generating a pellet. The higher the speed of centrifugation, the smaller the particle that can be pelleted. The supernatant(the remaining solution) contains the organelles and subcellular components that did not sediment. Components of the cell can be separated based on different rates of sedimentation - larger and denser organelles sediment faster than smaller organelles. In differential centrifugation, the cell homogenate is subjected to a series of sequential centrifugations at ever-increasing speeds. The first centrifugation is at a low speed for a short period of time, bringing down large organelles such as nuclei. The supernatant is then transferred to a fresh tube and centrifuged again at a higher speed for a longer period of time, this time bringing down smaller organelles such as mitochondria. The supernatant is again transferred and subjected to further centrifugation at an even higher speed, resulting in the sedimentation of subcellular structures such as the ER membrane. With each successive centrifugation, different components of the cell sediment, thereby allowing for the separation and isolation of distinct organelles. An alternative method is density gradient centrifugation. Here, the cell homogenate is layered over a solution of differing densities of some solute (usually sucrose) - the bottom of the tube has a higher density of sucrose than the top of the tube. When the cell homogenate is applied to the gradient and centrifuged at a constant speed for a fixed period of time, the subcellular components move through the sucrose densities at different rates; organelles with greater densities travel further down the tube, whereas particles with lower densities do not travel as far. Organelles stop traveling through the sucrose gradient when they encounter a solution of greater density. This results in a discrete band of organelles of the same sedimentation rate. Each band in the gradient is enriched for a particular organelle and can be collected as a purified fraction enriched for that particular organelle. In addition, various biochemical analyses can be performed, such as the detection of a particular enzymatic activity, to determine the composition and properties of each fraction.

Summary

Eukaryotic cells are distinguished from prokaryotic cells by the presence of membrane-bound organelles, including the nuclei, mitochondria and the endomembrane system (which is composed of endoplasmic reticulum), the Golgi complex and lysosomes (animal cells only). Plant cells have several unique organelles, including chloroplasts and vacuoles. In addition, eukaryotic cells have microfilaments that are important for motility of cells, movement within cells, and the general strength and structure of cells. The microfilaments are composed of either microtubules, actin filaments or intermediate filaments (animal cells only). _Escherichia coli (E. coli)_is a common bacterial cell species used in laboratory studies. There are a variety of eukaryotic cells commonly used for cell biological studies, including the microorganism yeast, primary cells derived from tissues, and transformed cell lines. Increasingly, cell biologists are using genetic analyses of model organisms (e.g. the fruit fly, nematode worm and mouse) to investigate the molecular mechanisms of normal cellular functions. These types of studies have revealed that many of the mechanisms of basic cellular function, and the proteins that carry them out, are conserved between eukaryotic species.

Microscopy is a very powerful tool, used to study cell structure and function. Cell biologists employ a variety of different types of microscopy (including light microscopy, fluorescence microscopy, transmission electron microscopy and scanning electron microscopy). Cells or tissues are often fixed, sectioned and stained in preparation for use in microscopy. Light microscopy produces a thousand-fold magnification and allows the visualization of larger organelles. Contrast within the cell, or between organelles, can be increased through the use of dyes that are specific for a particular macromolecule. In addition, there are several optical modifications that increase contrast. Fluorescence microscopy can be used with specific fluorescent dyes of fluorescent antibodies to highlight particular components of the cell. Confocal microscopy allows for much sharper images, in part, due to the ability to perform optical sectioning. For greater magnifications, transmission electron microscopy is used, allowing resolution of smaller organelles such as the Golgi complex and small cytoplasmic components such as microfilaments. Scanning electron microscopy provides a 3-D image of the cell (usually the cell's surface). Cells can be fractionated, then organelles can be isolated based on their densities through the use of centrifugation. Two commonly used methods of centrifugation are differential centrifugation and density gradient centrifugation.