Chapter 1 - An Overview of Cells and Cell Research

Prokaryotic vs Eukaryotic: Nucleus

P: Absent

E: Present

Prokaryotic vs Eukaryotic: Diameter of a typical cell

P: about 1 um

E: 10-100 um

Prokaryotic vs Eukaryotic: Cytoplasmic organelles

P: Absent

E: Present

Prokaryotic vs Eukaryotic: DNA content (base paris)

P: 1×10⁶ × 5×10⁶

E: 1.5×10⁷ × 5×10⁹

Prokaryotic vs Eukaryotic: Chromosomes

P: Singular circular DNA molecule

E: Multiple linear DNA molecules

What is in all present day cells?

DNA

Mechanisms for replication and gene expression

What is a gene?

Segments of DNA that encode proteins or RNA

• Functional units of inheritance

Transcription

nucleotide gene sequence is copied into RNA

Translation

nucleotide sequence of RNA is used to specify the order of amino acids in a protein

All present-day biological membranes are composed of _____

phospholipids

Amphipathic

Water-insoluble (hydrophobic) hydrocarbon chains are joined to a water-soluble (hydrophilic) head groups

True or False: Cells evolved mechanisms for generating energy and synthesizing molecules

True

True or False: Energy metabolism is highly conserved

True

What is the main source of metabolic energy by glycolysis

ATP (adenosine 5’- triphosphate)

Photosynthesis

harness energy from sunligh

• no longer need organix molecules

Oxidative metabolism

• much more efficient than glycolysis

• 36 to 38 ATP molecules

What are the two present day prokaryotes?

• Archaebacteria

  • many live in extreme environments

• Bacteria

  • wide range of environments

Prokaryotic Cell Characteristics

• Small (1-10 um in diameter)

• DNA ranges from 0.6 mill to 5 mill base pairs

Cyanobacteria

• photosynthesis evolved

• largest and most complex prokaryotes

Cell genomes

Escherichia coli (E.coli)

• typical prokaryotic cell

• rigid cell wall

• plasma membrane

• circular DNA

• cytoplasm contains ribosomes

Eukaryotic Cell Characteristics

much larger and more complex

Nucleus (Eukaryotic Cells)

• largest organelle

• linear DNA molecules

  • site of DNA replication and RNA synthesis

Compartments for metabolic activities in Plants

• Chloroplasts (Photosynthesis)

• Vacuoles (digestion of macromolecules and storage)

List the structures of a Plant Cell

• Cell Wall

• Vacuole

• Peroxisome

• Plasma membrane

• Chloroplast

• Cytoskeleton

• Plasmodesmata

• Smooth ER

• Rough ER

• Mitochondrion

• Golgi apparatus

• Ribosome

• Nucleolus

• Nucleus

Compartments for metabolic activities in Animals

Mitochondria = oxidative metabolism

Lysosomes and Peroxisomes = digestion of macromolecules

Endoplasmic reticulum function

processing and transport of proteins and lipid synthesis

Golgi apparatus function

proteins are further processed and sorted

lipid synthesis

Cytoskeleton function

structural framework

determines cell shape and organization

List the structures in an Animal Cell

• Cytoskeleton

• Nucleolus

• Nucleus

• Rough ER

• Smooth ER

• Lysosome

• Plasma membrane

• Golgi apparatus

• Centrioles

• Peroxisome

• Mitochondrion

• Ribosomes

True or False: Many eukaryotes are not unicellular organisms

False, many ARE unicellular

What is the simplest eukaryote?

Yeasts (Saccharomyces cervisiae)

Paramecium

• Ciliated protozoan

• Specialized for movement and for feeding on bacteria and years

Chlamydomonas

• Green algae

• Can carry out photosynthesis

When did multicellular organisms evolve, and can they transition from single cel to multicellular? Name an example.

• Evolved 1-2 billion years ago

• Some multicellular algae have evolutionarily transition from single cells to multicellular organisms

  • Volvox

Five main tissue types and function

1. Epithelial cells

   1. Cover the surface of the body and line internal organs

2. Connective tissues

   1. bone, cartilage, adipose tissue

   2. loose CT formed by fibroblasts

3. Blood cell types

   1. RBC (erythrocytes) = O2 transport

   2. WBC (granulocytes, monocytes, macrophages, lymphocytes) = inflammatory reactions and the immune response

4. Nervous tissue

   1. supporting cells, neurons, and sensory cells

5. Muscle cells

   1. production of force and movement

Experimental models of cells

• Fundamental properties of all cells have been conserved during evolution

• Experimental models

  • E. coli

  • Yeasts

  • C. elegans

  • Drosophila melanogaster

  • Arabidopsis thaliana

  • Vertebrates

Experimental Models: E. Coli

• most throughly studied species

• simple and easy to culture

• small genome size

• selection of genetic variants of a strain are east and rapid

• divide every 20 minutes

  • facilitates fundamental experiments

Experimental Models: Yeasts

• Simplest eukaryotes and are a model for eukaryote biology

• easily grown and genetic manipulations are feasible

Experimental Models: Caenorhabditis elegans

• Nematode

• one of the most widely used models

Experimental Models: Drosphila melanogaster

• fruit fly

• model organism in developmental biology

• short reproductive cycle

• useful for genetic experiments

Experimental Models: Arabidopsis thaliana

• model for plant molecular biology and development

• small genome and is easily grown

Experimental Models: Vertebrates

Most complex animals

• Xenopus laevis

• Zebrafish

One approach for study - use isolated cells in culture

Highly differentiated cells important

Experimental Models: Xenopus laevis

• frog

• studies of early vertebrate development

• produces large numbers of large eggs

Experimental Models: Zebrafish

• small and reproduce rapidly

• early stages of development easily observed

Experimental Models: Mouse

• most common mammal model

• genetically engineered mice used to study the functions of genes

• high similarity of mouse and human genomes

• similar developmental defects

What are the tools used for cell biology?

• microscopy

• subcellular fractionation

• cell culture

• types of microorganisms used for studies

Light Microscopy

• led to discovery of cells

• Robert Hooke coined the term “cell”

• 1670s, Antony van Leeuwenhoek observed a variety of cells

• cell theory

• cells arise from preexisting cells

Modern light microscopes

• Can magnify objects up to 1000x

Resolution

ability to distinguish objects separated by small distances

• more important than magnification

What determines the limit of resolution?

• Wavelength of visible light

  • lambda is fixed at approximately 0.5 um

• Numerical aperture (NA), the light-gathering power of the lens

  • size of the cone of light that enters the lens

What does each variable stand for?

η = refractive index of the medium

η - 0.1 for air

Maximum for α is 90 degrees,

at which sinα = 1

Theoretical limit of resolution is….

Bright-field microscopy

• light passes directly though cell

  • preserved with fixatives and stained with dyes

  • techniques can’t be used to study living cells

Phase-contrast microscopy and differential interference-contrast microscopy

• convert variations in density or thickness to differences in contrast

• these can be used for living cells

Video cameras and computers

• image analysis and processing

• enhance the contrast of images

Video-enhanced differential interference — contrast microscopy

visualization of the movement of organelles

Fluorescent microscopy

• molecular analysis

• fluorescent dye is attached to a molecule of interest

• the fluorescent dye molecules absorb light at one wavelength and emit light at a different wavelength

Green fluorescent protein (GFP)

• fused to any protein of interest using recombinant DNA

• tagged protein is expressed in cells and detected by fluorescence microscopy

• no need to stain or fix the cells

Fluorescence resonance energy transfer (FRET)

• two proteins coupled to different fluorescent dyes

• light emitted by one GFP variant excited the second

• used to study interactions between proteins in a cell

Confocal microscopy

• increases contrast and detail by analyzing fluorescence from a single point

Multi-photon excitation microscopy

• excitation of a fluorescent dye at the point upon which the laser beam is focused

• localization of excitation minimizes damage to the specimen

Electron microscopy

much greater resolution than light microscopy because of the short wavelength of electrons

Transmission electron microscopy

• specimens fixed and stained with salts of heavy metals provide contrast

• beam of electrons is passed through the specimen

Electron tomography

3D images by computer analysis of multiple 2D images obtained

Metal shadowing

visualize the surface fo subcellular structures or macromolecules

Freeze fracturing

• specimens frozen in liquid nitrogen and then fractured with a knife blade

• often splits the lipid bilayer

• reveals the interior faces of a cell membrane

Scanning electron microscopy

• 3D image of cells

• electron beam does not pass through specimen

• surface of the cell coated with a heavy metal

• beam of electrons is used to scan across the specimen

Super-resolution light microscopy

increases resolution of fluorescence microscopy to 10-100 nm

STORM (stochastic optical reconstruction microscopy)

Other types of microscopy

Atomic Force Microscopy

X-ray microscopy

Cryo-electron microscopy (cryo-EM)

Subcellular fractionation

organelles must be isolated from cell to determine the function

Differential centrifugation

• separate cell components by size and density

• ultracentrifuge spins at very high speeds

Density-gradient centrifugation

organelles are separated by sedimentation through a gradient of a dense substance

Velocity centrifugation

particles of different sizes sediment through the gradient at different rates

Equilibrium centrifugation

separates subcellular components on basis of buoyant density

Viruses

• intracellular parasites

• cannot replicate on their own

• very small

• need a host to survive

• consist of DNA or RNA surrounded by a protein coat

Bacteriophages

• bacterial viruses

• simplified the study of bacterial genetics

  • T4 infects E/coli

Retroviruses

have RNA genome but synthesize a DNA copy of their genome

Cell culture

• in vitro culture systems of cells enable cell growth and differentiation studies, and perform genetic manipulations

• embryo fibroblasts grow particularly well in culture and a widely studied type of animal cell

HeLa immortal cell line

Henrietta Lacks

• durable, prolific and do not die

Embryonic stem cells

• differentiate into all of the cell types of adult organisms

• gene function in mouse development and can possibly treat diseases by transplantation therapies

Serum-free media

makes culturing cells possible by identifying individual growth factors

Primary culture

• initial cell culture from tissues

• most normal cells cannot be grown in culture indefinitely

• cells can be replated to form secondary cultures

Plant cell cultures

• with appropriate growth factors, these produce a mass of undifferentiated cells called a callus

• many plant cells capable of differentiation into many different cell types