Chapter 1 - Introduction
Organization of the animal cell
The organization of an animal cell is approximately analogous to that of the whole body, with specialized organelles performing the various functions required to maintain viability and perform work. The nucleus contains the set of instructions necessary to produce all of the component parts of any cell type: ribosomes, endoplasmic reticulum and golgi apparatus all participate in the synthesis and packaging of proteins produced by the cell; lysosomes and proteases degrade and recycle the component parts of the cell; mitochondria and peroxisomes use metabolic fuel that is provided via the circulatory system to continually synthesize ATP from ADP and inorganic phosphate (Pi), which in turn provides the energy to maintain all of the above processes.
All of these organelles represent discrete compartments within the cell in which specific tasks take place. They are all defined by phospholipid membranes, as the cell itself is also enclosed within a membrane.
The cell also contains a network of structural elements, called the cytoskeleton. The cytoskeleton is composed of a variety of proteins, such as actin filaments, intermediate filaments and microtubules. The figure below shows a eukaryotic cell in which actin filaments have been fluorescently labelled (green). The cytoskeleton provides rigidity and shape to the cell. For example, red blood cells maintain their biconcave shape via cytoskeletal attachments to the cell membrane.
However, the cytoskeleton is also involved in trafficking, i.e. moving materials from place to place within the cell. Intracellular vesicle and organelle transport relies on interactions between these entities and cytoskeletal elements. For example, within a neuron the transport of mitochondria from the cell body along an axon to the terminal bud relies on microtubules and a variety of molecular motors (such as dynein and kinesin) that consume ATP while driving a regulated unidirectional movement of the organelles along microtubules. The figure below (Caviston & Holzbaur) shows some of the cytoskeletal proteins involved in transport and trafficking.
Indeed, some researchers have proposed the existence of an intracellular circulation, akin to the circulatory system of the mammalian body (Hochachka). Generally, the old idea of cells relying on simple molecular diffusion to support movement, transport and exchange within the cell is gradually being replaced by an understanding of the extent to which highly organized transportation highways regulate these processes.
From Cell Biology to Cell Physiology
Cell physiology is the study of the internal workings of the cell, and how they are altered in response to changing external conditions. For example, how do cells adapt to increased work load, as in chronically exercising skeletal muscle? How do cells adapt to nutrient limitation, as during starvation or caloric restriction? How do cells adapt to temperature fluctuations? What are the relationships between organism size and cellular function? In all of the above cases, specific mechanisms to detect and respond to such changes exist and form the basis of cellular physiological adaptation. In this course we will learn to understand the basic anatomy of the animal cell as a dynamic property that is readily modified to adapt to a changing environment within the body. We will explore the mechanisms for achieving adaptation. We will identify individual organelles and proteins, study their roles in the physiology of the cell and connect their dysfunction to the pathology of disease and aging.
We have entered an age of molecular physiology, in which we can begin to appreciate the functioning of an entire animal (including a human) starting from an understanding of the functions of individual components of individual cells. We have arrived at this stage following important scientific advancements: the sequencing of the entire human genome, as well as numerous other animal genomes; the ability to delete a gene from an organism of interest and study the effect of its absence; the ability to insert a new gene into the genome of an organism and study the effect of its presence. These powerful new tools of genetic manipulation give us the ability to directly study how a single protein impacts the physiology of the host entire animal. They have become the basis of studying connections between genetic mutations and disease etiology. For example, it is now possible to remove a ‘normal’ gene and replace it with a gene that contains a single mutation, and study the effect of this mutation on the physiology of a whole animal. This basic approach, impossible a decade ago, will revolutionize our understanding of relationships between genetics and disease.
Experimental Study of Molecular Cell Physiology
Schematic representations of two of the more common genetic manipulations employed are shown below. Engineering of a gene knockout mouse begins with deletion of the gene of interest in an embryonic stem cell, followed by injection of these genetically modified cells into a developing blastocyst and selected breeding combined with genotyping to identify offspring carrying two deleted genes (i.e. homozygous for the deletion). Occasionally, deletion of a gene is lethal to a mouse, though this is a relatively rare event. If this occurs, it is still possible to study the heterozygotes, i.e. those mice containing one copy of the gene of interest. Depending upon the kinetics of gene expression, these mice may have reduced levels of the encoded protein, and the effect of this reduced function can be studied.
An alternative approach to deleting or reducing the amount of a particular protein from an animal is the use of RNAi. This strategy involves allowing the gene to be transcribed to mRNA and then interfering with protein synthesis from the mRNA, either by causing it to be ‘diced’ or to not progress through the ribosome. In either case, it is possible to fully or partially deplete the cell of the protein encoded by the targeted gene and study the resultant function.
Transgenic technology can be considered a complementary approach, and its application is methodologically quite similar. In this case, a new gene is being inserted into the mouse genome, so that the function of the encoded protein can be studied. This is commonly a human gene, and the function of the human protein in mouse cells is studied. As with gene deletion, it involves an initial manipulation of the genetic material of mouse embryonic stem (ES) cells, and then transfer of these cells to a developing blastocyst, followed by a program of selective breeding and genotyping to identify individuals containing the new gene. These individuals are then studied.
This synthesis of genetic (often referred to as ‘molecular biology’) with physiological approaches relies heavily on tools for gene sequencing and comparison. The appearance of whole animal genome sequences on public databases has made this possible. The creation of algorithms for statistical comparisons of genes between species (such as BLAST) has made finding the mouse version of a particular human gene a trivial task. Programs such as BLAST are freely available on the World Wide Web, so that any researcher can use them. For example, a typical experiment may follow this pattern: a human patient is diagnosed with a heritable disease and his DNA collected for genotyping; the sequencing reveals the presence of a single substitution mutation within gene A; a similarity search (eg. BLAST) identifies the mouse version of gene A in a database of the mouse genome; gene A may then be knocked out in the mouse to reveal the function of that protein; alternatively, the mutated copy of the human gene may be introduced into the mouse genome and the resultant physiology of the mouse closely studied. This entire sequence of events might require one or two years to complete, which is quite different from the decades of inferential research that is often required without this ability to isolate individual genes and their proteins. It is important to note, however, that simply knowing the sequencing of genes and proteins is usually insufficient to instruct us about the impact on physiology. Ultimately, the physiological measurements must be made directly. Of course, mice and humans are also different, so that results always need to be interpreted with this in mind.
Measuring Cellular Activities
Cellular responses to experimental manipulations can be measured in an infinite variety of ways. However, there are some particularly common approaches. One robust approach that is taken with isolated live cells is the use of fluorescent microscopy, particularly confocal microscopy, to directly visualize specific parts of a cell. This strategy makes use of fluorescent molecules that interact with specific proteins or membranes within the cell. For example, a fluorescent probe called MitoTrackerTM localizes to mitochondrial membranes, thus showing the outline of mitochondria in live cells. Green fluorescent protein, produced by some species of jellyfish, is often used by fusing it to other cellular proteins, rendering them visible by fluorescent microscopy. The intracellular localization of the protein of interest can then be followed. The image below shows two cells that have been labelled with MitoTracker and other fluorescent probes, including Hoescht, which stains nuclear DNA blue. In the foreground, mitochondria are labelled green and have a reticular organization.
Specific fluorescent probes can be purchased from commercial suppliers to detect membrane potentials, cytosolic calcium concentrations, free radicals and many other parameters.
Indirect measurement of the amount of a specific protein is commonly done by Western blot, an approach in which cellular proteins are separated electrophoretically and then quantified using an antibody that is specific for that protein. The antibody is conjugated to a reporter molecule, which can be fluorescent. This enables both detection on the basis of size and recognition by the antibody, and quantification. mRNA levels are similarly investigated by electrophoretically separating cellular mRNA on the basis of size and then using specific oligonucleotide probes conjugated to either fluorescent or radioactive molecules.