Introduction of this Cell Biology course

1. Goals: convey the excitement and challenges of research in contemporary cell biology

A cell is the basic unit of life. Understanding how cells grow, divide and respond to environment is the major purpose of biology. The number of applications of cell biology continues to grow in medicine, agriculture, biotechnology, and biomedical engineering.

2. Organization of the textbook:

Part I: Introduction

Part II: The flow of genetic information

Part III: Cell structure and function Part IV: Cell regulation

3. Companion website: www.sinauer.com/cooper5e/

Various information (animation and video clips), Homework Have to register yourself to solve Online Quizzes (homework) Online Quizzes Create a new account bhoh@kaist.ac.kr

Introduction of this Cell Biology course

4. Lecture materials: klms.kaist.ac.kr 5. Your performance and grading Midterm exam (40%), Final exam (40%), Other activities (participation/homework/attendance: 20%) Participation: 6 Homework: 10 Attendance: 4

Chapter 1. An Overview of Cells and Cell Research

Chapter sections

The origin and evolution of cells

Cells and organisms as experimental models

Some of the properties of cells and organisms that make them valuable experimental model

Tools of cell biology

Progress in cell biology depends on the availability of experimental tools. Some of important experimental tools are discussed.

The Origin and Evolution of Cells

Two types of cells: Prokaryotic cells lack a nuclear envelope. Eukaryotic cells have a nucleus that separates genetic material from cytoplasm.

The first cell (premordal ancestor) emerged at least 3.8 billion years ago. Spontaneous synthesis of organic molecules probably provided the basic materials from which the first living cells arose (Fig. 1.1).

Macromolecules may have formed by spontaneous polymerization of monomeric building blocks under plausible prebiotic conditions. The critical characteristic of the macromolecule from which life evolved must have been the ability to replicate itself.

Nucleic acids are capable of directing self-replication (Fig.1.2). Sid Altman and Tom Cech first discovered that RNA is capable of catalyzing chemical reactions, including the polymerization of nucleotides.

RNA can serve as a template for its own replication, and it is also able to catalyze its own replication (= self-replicating RNA). Consequently, RNA is generally believed to have been the initial genetic system in evolution. This period is known as the RNA world.

The first cell probably arose by the enclosure of self-replicating RNA in a membrane composed of phospholipids. Phospholipids are the basic components of all present-day biological membranes.

Properties of phospholipids

Amphipathic When placed in water, they spontaneously aggregate into a bilayer. Forms a physical barrier against free influx of molecules from outside.

Cells needed to evolve mechanisms for generating energy and synthesizing molecules. The principal pathways of energy generation are highly conserved in present-day cells; and all cells use ATP as their source of metabolic energy.

The mechanisms of generation of ATP are thought to have evolved in three stages, corresponding to the evolution of glycolysis, photosynthesis, and oxidative metabolism (Fig. 1.4).

Glycolysis evolved when the Earths atmosphere was anaerobic.

No involvement of oxygen Breakdown of glucose to lactic acid All present-day cells carry out glycolysis.

Photosynthesis evolved more than 3 billion years ago.

It allowed some cells to harness energy from sunlight; and they no longer required preformed organic molecules. The first photosynthetic bacteria probably used H2S to convert CO2 to organic molecules: a pathway of photosynthesis still used by some bacteria. (Analysis of sedimentary rocks: completely anoxic, filamentous microbial tangles) The use of H2O evolved later (~1.5 billion years ago, as H2S diminished); it changed Earths atmosphere by making free O2 available.

O2 in the atmosphere may have allowed the evolution of oxidative metabolism.

It is much more efficient than glycolysis; the complete oxidative breakdown of glucose yields 36 to 38 ATP molecules.

Present-day prokaryotes

Archaebacteria: many live in extreme environments (e.g., hot sulfur spring). Eubacteria: a large group that live in a wide range of environments.

Most bacterial cells are small. Cyanobacteria, the group in which photosynthesis evolved, are the largest and most complex prokaryotes (i.e., large number of genes)

Escherichia coli (E. coli) is a typical prokaryotic cell.

It has a rigid cell wall composed of polysaccharides and peptides (=peptidoglycan). Beneath the cell wall is the plasma membrane, a phospholipid bilayer with associated proteins.

Present-day eukaryotes

They are much larger and more complex, with a nucleus, other organelles, and cystoskeleton. *Organelles= subcellular organelles or compartment

The nucleus is the largest organelle; it contains the linear DNA molecules.

Mitochondria: site of oxidative metabolism. Chloroplasts: site of photosynthesis. Lysosomes and peroxisomes: specialized metabolic compartments for the digestion of macromolecules and for various oxidative reactions.

Vacuoles: in plant cells; perform a variety of functions, including digestion of macromolecules and storage of waste products and nutrients. The endoplasmic reticulum is a network of intracellular membranes, extending from the nuclear membrane throughout the cytoplasm. It functions in the processing and transport of proteins and the synthesis of lipids. In the Golgi apparatus, proteins are further processed and sorted for transport to their final destinations. It also serves as a site of lipid synthesis, and (in plant cells) the site of synthesis of some polysaccharides that compose the cell wall. The cytoskeleton is a network of protein filaments extending throughout the cytoplasm. It provides structural framework, determines cell shape and organization, and is involved in movement of whole cells, organelles, and chromosomes during cell division.

The origin of eukaryotes

Acquisition of membrane-bound subcellular organelles was a critical step. These are thought to have arisen by endosymbiosis: prokaryotic cells living inside the ancestors of eukaryotes. Evidence is especially strong for mitochondria and chloroplasts.

Mitochondria and chloroplasts are similar to bacteria in size Like bacteria, they reproduce by dividing in two. Both contain their own DNA, which encodes some of their components. The DNA is replicated when the organelle divides; the genes are transcribed within the organelle and translated on organelle ribosomes. The ribosomes and ribosomal RNAs are more closely related to those of bacteria than to those encoded by the nuclear genomes of eukaryotes.

The first eukaryote is thought to derived from the fusion of aerobic eubacterium with an archaebacterium rather than from one of the two. What is a supporting evidences?

the mosaic nature of eukaryotic genomes consisting of some genes derived from eubacteria (mostly related to metabolism, e.g., glycolysis) and others from archebacteria (mostly related to informational processes, e.g., DNA replication).

Evolution of cells
The DNA sequences of eubacteria and archaebacteria are as different as they are from those of presentday eukaryotes. Mitochondria is contained in both animal and plant cells. But, chloroplast is contained only in plant cells. Therefore, a scenario would be that an archaebacterium fused with an aerobic eubacterium first, and later the resulting cell fused with photosynthetic bacterium.

The development of multicelluar organisms

Many eukaryotes are unicellular organisms. The simplest eukaryotes are the yeasts.

Other unicellular eukaryotes are more complex. Amoeba proteus: its volume is more than 100,000 times that of E. coli, and it can exceed 1 mm in length. Amoebas use cytoplasmic extensions, called pseudopodia, to move and to engulf other organisms.

pseudopodia

Some unicellular eukaryotes form aggregates (=muticellular colonies) that may represent an evolutionary transition from single cells to multicellular organisms.

Volvox (a green algae): Thousands of cells are embedded in a gelatinous matrix. Individual cells are connected by tiny cytoplasmic bridges. Some division of labor; a small number of cells are specialized in reproduction.

Increasing cell specialization might have led to the transition from aggregates to truly multicellular organisms.

Cells of plants: Organized into 3 main systems: 1. Ground tissue (A) Parenchyma cells: site of metabolic reactions, including photosynthesis. (B) Collenchyma and sclerenchyma have thick cell walls and provide structural support. 2. Dermal tissue covers the surface of the plant (=C); forms a protective coat and allows absorption of nutrients. 3. Vascular tissue (xylem and phloem) (=D): xylem tissue- transport water mainly phloem tissue- transport sucrose mainly The both contains elongated cells.

Stomata

Cells of animals: Much more diverse than those of plants; e.g., 200 different kinds of cells in human body. Components of 5 main types of tissues: 1. Epithelial tissue forms sheets that cover the surface of the body and line internal organs. Functions: protection, nutrient absorption, secretion of molecules. 2. Connective tissue serve a connecting function- binds and support other tissues. They includes bone, cartilage, and adipose tissue. Loose connective tissue between organs and tissues is formed by fibroblasts. 3. Blood tissues contains several different types of cells: Red blood cells (erythrocytes) function in oxygen transport. White blood cells (granulocytes, monocytes, macrophages, and lymphocytes) function in inflammatory reactions and the immune response. 4. Nervous tissue is composed of supporting cells and nerve cells, or neurons, and various types of sensory cells; e.g., olfactory cells 5. Muscle tissue is responsible for the production of force and movement. *All these complex array of cells differentiate from a single fertilized egg.

Representative animal cells

Epithelial cells

Fibroblasts

Blood cells

Cells as Experimental Models

Because the fundamental properties of all cells have been conserved during evolution, the basic principles learned from experiments on one type of cell are generally applicable to other cells. Several kinds of cells and organisms are used as experimental models.

E. coli

The most thoroughly studied species of bacteria. Our understanding of DNA replication, the genetic code, gene expression, and protein synthesis derive from studies of this bacterium.

E. coli is particularly useful because of its simplicity and ease of culture in the laboratory. The genome consists of approximately 4.6 million base pairs and contains about 4300 genes. (human: 3 billion bps) The small size of the genome is an advantage in genetic analysis.

E. coli divide every 20 minutes. A clonal population can be readily isolated as a colony grown on agar medium. 8 Bacterial colonies containing as many as 10 cells can develop overnight. Selecting genetic variants of an E. coli strain is easy and rapid.
E. coli can carry out biosynthetic reactions in simple defined media; this made them extremely useful in elucidating biochemical pathways.

Yeasts

Yeasts are the simplest eukaryotes, and have been a model for fundamental studies of eukaryote biology; e.g., RNA processing, protein sorting and cell division. The genome of Saccharomyces cerevisiae consists of 12 million base pairs of DNA and contains about 6000 genes on 16 linear chromosomes. Contains a nucleus, cytoskeleton, subcellular organelles.

Yeasts can easily be grown in the laboratory as colonies from a single cell. Yeasts can be used for genetic manipulations similar to those performed using bacteria.

The unity of molecular cell biology is made clear by the fact that general principles of cell structure and function revealed by studies of yeasts apply to all eukaryotic cells.

Caenorhabditis elegans

Understanding the development of multicellular organisms requires the experimental analysis of plants and animals. The nematode C. elegans is one of the most widely used models.

The genome of C. elegans contains approximately 19,000 genes: nearly the same number of genes as in humans. C. elegans is relatively simple: adult worms consist of only 959 somatic cells. The embryonic origin and lineage of all the cells has been traced. Genetic studies have also identified many mutations responsible for developmental abnormalities. This led to isolation and characterization of genes that control development and differentiation. found that homologous genes in complex animals have similar functions

Drosophila melanogaster

The fruit fly Drosophila melanogaster has been a crucial model organism in developmental biology. Drosophila is easy to grow in the laboratory, and the short reproductive cycle (2 weeks) makes it very useful for genetic experiments. Many fundamental genetic concepts were derived from studies of Drosophila early in the 20th century.

Studies of Drosophila have led to advances in understanding the molecular mechanisms that govern animal development, particularly with respect to formation of the body plan of complex multicellular organisms. found that homologous genes and similar mechanisms exist in vertebrates

Arabidopsis thaliana

A model for plant molecular biology and development is the small mouse-ear cress, Arabidopsis thaliana. It has a small genome and is easily grown in the lab. Studies of Arabidopsis have led to the identification of genes involved in aspects of plant development, such as the development of flowers.

Vertebrates

Vertebrates are the most complex animals, and the most difficult to study due to the huge genome size and long reproductive cycle. One approach is to use isolated cells in culture. These studies have elucidated the mechanisms of DNA replication, gene expression, protein synthesis, and cell division.

The ability to culture cells in chemically defined media has allowed studies of signaling mechanisms that normally control cell growth and differentiation within the intact organism. Highly differentiated cells are important models for studying particular aspects of cell biology. e.g., Muscle cells: a model for studying cell movement at the molecular level. Giant neurons in squid: a model studying ion transport across the plasma membrane and the transport of subcellular organelles

The frog Xenopus laevis is an important model for studies of early vertebrate development. Xenopus produces large eggs in large numbers, facilitating laboratory study and biochemical analysis.

Zebrafish are small and reproduce rapidly. Embryos develop outside of the mother and are transparent; early stages of development can be easily observed. Zebrafish bridge the gap between humans and simpler invertebrate systems, like C. elegans and Drosophila.

embryo

The mouse is the most common mammal model. Many mutations affecting mouse development or behavior have been identified. Genetically engineered mice with specific mutant genes are now used to study the functions of these genes in the context of the whole animal.

The mouse and human genes are very similar with each other. Not surprising that mutations in homologous genes result in similar developmental defects in both species.

Tools of Cell Biology

Research in cell biology depends on available laboratory methods and experimental tools. Many important advances have directly followed the development of new methods that have opened novel avenues of investigation.

Light Microscopy

The discovery of cells arose from the development of the light microscope. Robert Hooke coined the term cell following his observations of a piece of cork in 1665. In the 1670s Antony van Leeuwenhoek was able to observe a variety of cells, including sperm, red blood cells, and bacteria.

The cell theory proposed by Matthias Schleiden and Theodor Schwann in 1838 resulted from their studies of plant and animal cells using microscopes. It was soon recognized that cells are not formed de novo but arise only from division of pre-existing cells.

Contemporary light microscopes can magnify objects up to about 1000x. Most cells are between 1100 m, so they can be observed by light microscopy, as can some organelles. Resolution: the ability to distinguish objects separated by small distances; is even more important than magnification.

The limit of resolution of the light microscope is approximately 0.2 m. Objects separated by less than that distance appear as one object. This limit is determined by the wavelength of visible light (), and the numerical aperture (NA): the light-gathering power of the lens.

Resolution

0.61 NA

is fixed at approximately 0.5 mm. NA can be envisioned as the size of the cone of light that enters the lens.

NA sin

= refractive index of the medium between the objective lens and specimen. = v in a vacuum v in a medium

Numerical aperture ( sin):

Larger or : Object lens is more closer to specimen

= ~1.0 for air, but can be increased to a maximum of 1.4 by using an oilimmersion lens. Maximum for is 90, at which sin = 1, so maximum possible for NA = 1.4. The theoretical limit of resolution is thus:

Resolution

0.61 0.5 0.22mm 1.4

Types of light microscopy

Bright-field microscopy:

Light passes directly through the cell. Cells are often preserved with fixatives (e.g., formaldehyde) and stained with dyes to enhance the contrast. This technique cant be used to study living cells.

(a) formaldehye-Asn adduct (b) cross-linking of Lys and Asn by formaldehyde

Fixed and stained kidney tumor

Phase-contrast microscopy and differential interference-contrast microscopy:

Both convert variations in density or thickness to differences in contrast that can be seen in the final image. (by modification of optics, e.g., different angles of incident light)

Shadow at this side

(A) Bright-field (B) Phase-contrast (C) differential interference-contrast

Video-enhanced differential interference-contrast microscopy: Modification of optics + computer-assisted image analysis and processing. It allows visualization of protein filaments with a diameter of only 0.025 m.

Single microtuble can be observed

Fluorescence microscopy:

Used for molecular analysis (e.g., location of a protein). A fluorescent dye is used to label the molecule of interest (called staining) in fixed or living cells. The fluorescent dye molecules absorb light at one wavelength and emit light at a different wavelength.

This fluorescence is detected by illuminating the specimen with a wavelength of light that excites the fluorescent dye, then using filters to detect the specific wavelength of light that the dye emits.

The green fluorescent protein (GFP) of jellyfish: widely used to visualize proteins in living cells, by fusing it to a protein of interest. (no need for staining)

Methods using GFP:

Fluorescence recovery after photobleaching (FRAP): This is used to study rate of protein movement in living cells. Fluorescence resonance energy transfer (FRET): This is used to study interactions between proteins in a cell. The light emitted by one GFP variant excites the second.

High-intensity light destroying the chromophore

Confocal microscopy:

Increases contrast and detail by analyzing fluorescence from a single point. A small point of light from a laser is focused on the specimen at a particular depth. The emitted fluorescent light is collected by detector such as a video camera.

The emitted light must pass through a pinhole aperture (confocal aperture). Thus only light emitted from the plane of focus is able to reach the detector. Scanning across the specimen generates a two-dimensional image of the plane of focus. A series of images can be used to reconstruct a three-dimensional image. *In fluorescence microscopy, out-of-focus emitted lights give a blurred image.

Multi-photon excitation microscopy:

Excitation of a fluorescent dye is achieved by 2 or more photons. Therefore, excitation occurs only at the point in the specimen where the laser beams are focused. No need for passing the emitted light through a pinhole aperture. The localization of excitation minimizes damage to the specimen, allowing three-dimensional imaging of living cells.

Fluorophore can absorb multiple lowenergy photons simultaneously and be excited. The total energy equals its onephoton excitation energy.

Electron microscopy:

Electron microscopy can achieve much greater resolution (0.2 nm) than light microscopy because of the short wavelength of electrons (0.004 nm; 5 10 times shorter than the visible light). o The aperture angle of the electromagnetic lens is ~0.5 . The max. resolution is about 0.2 nm (~0.61x0.004/1xsin0.5). Resolution for biological samples is about 1 to 2 nm because of their inherent lack of contrast. But, it is still 100x better resolution than light microscopy.

Transmission electron microscopy

Specimens are fixed and stained with salts of heavy metals, which provide contrast by scattering electrons. A beam of electrons is passed through the specimen and forms an image on a fluorescent screen. Electron beams deflected by heavy metal ions do not contribute to the final image, so that the stained area appears dark. Specimens can be prepared by either positive or negative staining.

Negative staining of actin filament

Electron tomography generates three-dimensional images by computer analysis of multiple two-dimensional images obtained over a range of viewing directions.

Metal shadowing is another technique used to visualize the surface of subcellular structures or macromolecules. The specimen is sprayed with evaporated metal, such as platinum. Surfaces facing the evaporated metal are coated more heavily than other surfaces, which results in a shadowing effect.

Actin/myosine filaments

Freeze fracturing: specimens are frozen in liquid nitrogen and then fractured with a knife blade.

This often splits the lipid bilayer, revealing the interior faces of a cell membrane. The specimen is then shadowed with platinum.

Membranes of two adjacent cells; membrane proteins are observed as particles

Scanning electron microscopy provides a three-dimensional image of cells.

The surface of the cell is coated with a heavy metal, and a beam of electrons is used to scan across the specimen.

The electron beam does not pass through the specimen. The electrons that are scattered from the sample surface are collected to generate a 3D image. The resolution of scanning is ~10 nm, it is restricted to studying whole cell rather than subcellular organelles or macromolecules.

Subcellular fractionation

In order to determine the function of organelles, they were isolated from the cell. Differential centrifugation was developed in the 1940s and 1950s to separate cell components on the basis of size and density.

Larger and more dense ones at lower speed

Breaks the plasma membranes and ER into small fragments without breaking up other cell compartments

ER at 200,000g ribosomes at > 200,000g

Greater purification can be achieved by density-gradient, in which organelles are separated by sedimentation through a gradient of a dense substance, such as sucrose.

In velocity centrifugation in density-gradient, the starting material is layered on top of the sucrose gradient. Particles of different sizes sediment through the gradient at different rates.

Sample at the top Sedimentation velocity

Equilibrium centrifugation in density gradient is used to separate subcellular components on the basis of their buoyant density.

Sample is mixed together with sucrose or cesium chloride in a centrifuge tube; centrifugation forms a concentration gradient of the solutes.

Centrifugal force

The sample particles are centrifuged until they reach an equilibrium position at which their buoyant density is equal to that of the surrounding sucrose or cesium chloride solution. 14 15 Example: separation of N or N labeled DNA molecules

Growth of animal cells in culture

In vitro culture systems of plant and animal cells enable scientists to study cell growth and differentiation, and perform genetic manipulations. Most animal cell types attach and grow on the plastic surface of dishes used for cell culture.

The culture media for animal cells are complex and must include salts and glucose, and various amino acids and vitamins that cells cant make for themselves. Serum provides polypeptide growth factors. The identification of individual growth factors makes it possible to culture cells in serum-free media.

Harry Eagle was the first researcher to describe a defined medium for animal cells, in 1955. This has enabled scientists to grow a wide variety of cells under defined experimental conditions, which is critical to studies of animal cell growth and differentiation.

An initial cell culture from tissue is a primary culture. They can be replated at a lower density to form secondary cultures many times. Most normal cells such as fibroblasts cannot be replated and grown indefinitely. They stop growing and die.

Embryonic stem cells and tumor cells can proliferate indefinitely in culture and are referred to as permanent or immortal cell lines. Permanent cell lines have been particularly useful for many types of experiments because they provide a continuous and uniform source of cells. *Doubling time of most actively growing animal cells is ~20 hrs. That of E. coli is 20 min.

Plant cells: Plant cells can also be cultured. In contrast to polypeptide growth factors of animal cells, the growth factors of plant cells are small molecules. Given appropriate growth factors, plant cells produce a mass of undifferentiated cells called a callus. Many plant cells are capable of differentiation into many different cell types. (Pluripotency)

Sometimes an entire plant can be propagated from a single cell. This makes it easy to introduce genetic alterations, opening possibilities for agricultural genetic engineering.

Viruses:

Viruses reproduce by infecting host cells and usurping the cellular machinery to produce more virus particles. Viruses consist of DNA or RNA surrounded by a protein coat.

Viruses provide simple systems that can be used to investigate the functions of cells. Bacterial viruses (bacteriophages) have simplified the study of bacterial genetics.

Bacteriophage T4 infects E. coli. In a culture of bacteria on agar, the replication of T4 leads to the formation of clear areas of lysed cells (plaques). Viral mutants (e.g. that will grow in one strain of E. coli but not another) are easy to isolate. Thus, T4 is manipulated even more readily than E. coli for studies of molecular genetics.

The genome of T4 is 23 times smaller than that of E. coli, further facilitating genetic analysis. Bacterial viruses have provided extremely useful experimental systems for molecular genetics and have led to understanding many fundamental principles.

Viruses are also important in studies of animal cells. There are many diverse animal viruses. The retroviruses have RNA genomes but synthesize a DNA copy of their genome in infected cells. These viruses first demonstrated the synthesis of DNA from RNA templates.

Some animal viruses convert normal cells to cancer cells. This was first described by Peyton Rous in 1911. Studies of these viruses have contributed to our current understanding of cancer, and many of the molecular mechanisms that control animal cell growth and differentiation.