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Biology 230 - Molecules and Cells

Subcellular Architecture and Experimental Approaches to Cell Biology

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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.

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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.

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Figure 2. A typical animal cell.

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Figure 3. A typical plant cell

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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.

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

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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.

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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.

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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.

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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.

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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.

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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.

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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).

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