Transcript The Cell, 5e

Overview of Cells and Cell Research
BL 424 Cell Biology
Ch 1 Overview
Student learning outcomes:
1. Describe Origin and Evolution of Cells –
prokaryote and eukaryote
2. Explain Cells as Experimental Models –
examples of model organisms
3. Describe Major Tools of Cell Biology:
microscopy, subcellular fractionation
cells and tissue culture
1. Unity and diversity of present-day cells
reflects evolution from common ancestor:
Prokaryotic cells (Bacteria and Archaea):
lack nuclear envelope
Eukaryotic cells (yeast, plants, animals):
nuclear membrane encloses genetic material
Fig 1.3 Enclosure of self-replicating RNA in a phospholipid membrane
Phospholipids:
basic
components of
present-day
biological
membranes.
RNA world theory:
Fig. 1.3
first cell arose from
self-replicating RNA
molecule in
membrane
Phospholipids are amphipathic:
Water-insoluble (hydrophobic) hydrocarbon chains
Water-soluble (hydrophilic) head groups with PO4
In water, phospholipids spontaneously aggregate into bilayers.
The Origin and Evolution of Cells
Adenosine 5′-triphosphate
(ATP) metabolic energy
3 Mechanisms of generation of ATP:
Glycolysis
Photosynthesis
Oxidative
metabolism
Fig. 1.4
The Origin and Evolution of Cells
Present-day prokaryotes:
Archaea (Archaebacteria):
many live in extreme environments
Halobacterium; Methanooccus
Bacteria (Eubacteria):
large group - many environments:
Escherichia, Bacillus
Cyanobacteria, photosynthesis evolved;
largest and most complex prokaryotes:
Anabaena
Fig. 1.5 E. coli : cell wall, plasma membrane,
circular DNA without nuclear membrane
The Origin and Evolution of Cells
Eukaryotic cells are larger, more complex:
Plasma membrane, ribosomes
Nucleus is large organelle: linear DNA molecules
Membrane-bound organelles - specialized structures
and functions
Fig. 1.6: animal cell, plant cell
Fig 1.7 Evolution of cells
Endosymbiosis: eukaryote organelles arose from
prokaryotic cells living inside ancestors of eukaryotes.
• evidence strongest for mitochondria and chloroplasts
Mosaic nature of
eukaryotic genomes:
fusion of
archaeal and
eubacterial
genomes.
Archaea more closely
related to Eukarya
Fig. 1.7
The Origin and Evolution of Cells
Unicellular eukaryotes:
Yeast is simple eukaryote
Saccharomyces cerevisiae
~ 6 µm diameter; 12x106 bp DNA
Protozoan
Plasmodium falciparium
causes malaria
Gametocyte and blood cells
The Origin and Evolution of Cells
Multicellular organisms - diversity
of cell types for specialized functions:
Animal cell tissue types:
Epithelial cells - cover surfaces, line organs.
2. Connective tissues - bone, cartilage, adipose tissue.
Loose connective tissue is formed by fibroblasts.
3. Blood cells: Red cells (erythrocytes) transport oxygen.
White blood cells inflammatory, immune response.
4. Nervous tissue – neurons, supporting cells, sensory cells.
5. Muscle cells
1.
Fig. 1.12 cells
A, epithelial
B, fibroblasts
C, blood cells
2. Cells as Experimental Models
1.2 Cells as Experimental Models:
•
Evolution conserved fundamental properties
• Basic principles from experiments on one type of cell
generally applicable to other cells
• Some cells and organisms are good experimental
models – model organisms.
Unity of molecular cell biology:
• General principles of cell structure, function from
studies of yeast apply to all eukaryotic cells.
• Understanding development of multicellular
organisms requires analysis of plants, animals
(Table 2 shows organisms vary widely in DNA content, genes)
Cells as Experimental Models
Escherichia coli – E. coli Bacteria
Basic features of DNA replication, genetic code,
gene expression, and protein synthesis
Genome only 4.6x106 bp, 4300 genes
Ease of culture in laboratory,
Elucidate biochemical pathways;
Easy to select genetic variants
Can transfer partial genome to others
Recombinant DNA, plasmids
Divides every 20 min ->
colony of 108 cells overnight
Fig. 1.13
Cells as Experimental Models
Yeast is simple eukaryote,
model for fundamental studies of eukaryotic biology.
Genome of Saccharomyces cerevisiae is 12x106 bp
of DNA, about 6000 genes.
Easily grown as colonies from single
cell – grow as haploid or diploid
Recombinant DNA techniques
Two mating types
Has cell wall
Genetic manipulations similar to
those performed using bacteria
Fig. 1.14
Cells as Experimental Models – C. elegans
Nematode Caenorhabditis elegans is widely
used model:
Fig. 1.15
Genome ~ 19,000 genes
Adult worm only 959 somatic cells. Embryonic origin
and lineage of all cells has been traced; grows fast
Mutants with developmental abnormalities
Cells as Experimental Models – Drosophila, Arabidopsis
Developmental biology: fruitfly, mouse-ear cress
Drosophila melanogaster
Arabidopsis thaliana
Small genomes;
short reproductive cycles;
easy to find mutants
Figs. 1.16, 1.17
Cells as Experimental Models - vertebrates
Vertebrates - most complex animals, most
difficult to study from cell, molecular biology.
One approach uses cells in culture:
culture cells in chemically defined media
Highly differentiated cells are important models for
particular aspects of cell biology.
Ex. Muscle cells model for cell movement at molecular level.
Ex. Giant neurons to study ion transport, cytoskeleton function.
Cells as Experimental Models – Xenopus, Danio
Models for vertebrate development include:
Frog Xenopus laevis and Zebrafish (Danio rerio)
Frog - large eggs in large numbers
facilitates biochemical analysis
Zebrafish are small, reproduce
rapidly. Transparent embryos
develop outside of mother;
early stages of development
can be easily observed.
African
clawed frog
Figs.
1.18,
1.19
Cells as Experimental Models – Mus musculus
Mouse (Mus musculus) is mammal model.
Genetically engineer mice with specific mutant genes
to study functions of genes, development
Similarity of genomes:
Mutations in
homologous genes
result in similar
developmental defects
Ex. Piebald mutation of
gene for melanocyte
migration
Fig. 1.20
1.3 Tools of Cell Biology
1.3 Tools of Cell Biology:
Research depends on available laboratory methods
and experimental tools.
Important advances directly followed development of
new methods that opened avenues of investigation:
A. Microscopes
B. Cell fractionation
C. Cell and tissue culture
Tools of Cell Biology - microscope
Discovery of cells arose from
development of light microscope
1665 Hooke termed “cell”
after observations of cork
1670s van Leeuwenhoek
observed cells: sperm, protozoa
1838 Cell theory of Schleiden and Schwann
from studies of plant and animal cells:
Cells are not formed de novo, but arise
only from division of pre-existing cells.
Fig. 1.21
Tools of Cell Biology - microscopy
Light microscopes magnify objects
up to about 1000x:
Most cells ~1–100 µm, can be
observed by light microscopy, as
can some organelles.
Resolution: ability to distinguish
objects separated by small
distances; more important than
magnification.
Limit of resolution of light
microscope is approximately 0.2
µm. Objects separated by less
than that appear as one object.
Tools of Cell Biology
Resolution limit:
determined by wavelength of visible light (λ),
aperture (NA): light-gathering power of lens
numerical
λ is fixed at ~ 0.5 mm
NA size of cone of light that enters lens (max α is 90°, at which
sin α = 1)
η = refractive index of the medium
0.61
(1.0 for air, 1.4 oil-immersion lens)
Fig. 1.22
Resolution 
NA
NA   sin 
0.61  0.5
Re solution 
 0.22 m
1.4
Tools of Cell Biology - microscopy
Major types of light microscopy:
*Bright-field microscopy:
light passes directly through cell.
Cells often preserved with
fixatives, stained with dyes to
enhance the contrast.
Fix/stain technique can’t be
used to study living cells.
Fig. 1.23 benign kidney tumor
Tools of Cell Biology
•
•
Phase-contrast microscopy and
differential interference-contrast microscopy
convert variations in density or thickness to
differences in contrast that can be seen in final image.
Fig. 1. 24
Human cheek cells
A brightfield
B phase contrast
C differential interference
Tools of Cell Biology
** Fluorescence microscopy:
Fluorescent dye labels molecule of interest in fixed or living
cells: can have two or more labels (colors)
gene-level fusion proteins, or fluorescent antibodies
• Fluorescent dye molecules absorb light at one wavelength,
and emit light at different wavelength
• Illuminate specimen at one wavelength;
• Filters permit detect emitted wavelength
Fig. 1.26 Newt
lung:
DNA blue dye
Microtubules
green dye
Tools of Cell Biology
* Green fluorescent protein (GFP) of jellyfish
permits visualize proteins in living cells.
GFP (238-aa) fused to protein of interest using
standard recombinant DNA (gene level fusion)
Jellyfish
Aequora victoria
Fig. 1. 27 mouse neurons:
GFP- Microtubule-associated
protein; DNA stained blue
Tools of Cell Biology
** Confocal microscopy increases contrast and
detail by analyzing fluorescence from single point.
•
Emitted light passes through pin-hole aperture (confocal
aperture). Only light from plane of focus reaches detector.
Figs. 1.30,
1.31
Human cells:
Microtubules
red;
actin green
Tools of Cell Biology
Electron microscopy greater resolution (~0.2 nm)
than light microscopy - short wavelength of electrons.
*Transmission electron microscopy
Specimens fixed, stained with salts of heavy
metals: contrast by scattering electrons.
Beam of electrons passed through specimen
forms image on fluorescent screen.
Specimens stained either positive or negative
Fig. 1.33 white blood cell
Fig. 1.34 actin filaments
Tools of Cell Biology
Freeze fracturing: specimens frozen in liquid
nitrogen and fractured with knife blade; specimen is
shadowed with platinum; often splits lipid bilayer,
revealing interior faces of cell membrane.
Fig. 1.36
Tools of Cell Biology
Scanning electron microscopy
provides 3-D image of cells:
Electron beam does not
pass through specimen.
Instead, surface of cell is
coated with heavy metal,
Beam of electrons scans
across specimen ~ 10 nm
Fig. 1.37 macrophage
Figure 1.38 Subcellular fractionation (Part 1)
B. Subcellular fractionation:
Isolate organelles from cells,
then determine function
1. Differential centrifugation
separates by size and density.
Fig. 1.38
Tools of Cell Biology
2. Density-gradient centrifugation:
organelles separated by sedimentation through
gradient of dense substance, such as sucrose.
Velocity centrifugation:
Starting material is layered
on top of sucrose gradient
(e.g., 5-20%).
Particles of different sizes
sediment through gradient
at different rates.
Fig. 1.39
Tools of Cell Biology
Equilibrium centrifugation in density gradients
separates by buoyant density.
• Sample particles centrifuged until equilibrium position
at which their buoyant density is equal to surrounding
sucrose or cesium chloride solution.
Ex. Viral DNA differs host chromosomal DNA
Ex. Heavy vs light DNA in early studies
of DNA replication:
N15 vs. N14 (HH, HL, LL E. coli DNA)
Fig. 4.7
Tools of Cell Biology – cell culture
C. Cell Culture
Fig. 40
Fibroblast
In vitro culture systems of animal cells:
study cell growth, differentiation,
do genetic manipulations.
Embryos or tumors often starting material: rapidly growing
Embryo fibroblasts (connective tissue) grow particularly well
in culture; widely studied type of animal cells.
Embryonic stem cells can be established in culture; maintain
ability to differentiate into all adult cell types.
Tools of Cell Biology
Primary culture:
initial cell culture from tissue
(tumor, embryo)
Replated at lower density:
secondary cultures
Most normal cells such as
fibroblasts cannot be grown in
culture indefinitely.
Fig. 1.41
Tools of Cell Biology
Cell Lines: embryonic stem cells, tumor cells
proliferate indefinitely in culture: permanent, immortal
Permanent cell lines are very useful:
•
continuous and uniform source of cells.
Ex. HeLa cells from human cervical carcinoma
Plant cells can be grown in culture
•
Calluses can regenerate plants
Plant callus of halophyte:
Hela cells:
Ceoe.udel.edu/halophyte
Cancer_Hela_320.explorium.edu
Tools of Cell Biology - viruses
Viruses: intracellular parasites:
not replicate alone; infect host cells, take over
cellular machinery to produce virus particles.
Viruses: DNA or RNA surrounded by protein coat,
maybe envelope.
Viruses - simple systems
to study functions of cells
that they infect
Fig. 1.43 HPV
Human papillomavirus
Tools of Cell Biology
Bacterial viruses (bacteriophages) simplified
study of bacteria (bacterial genetics).
Bacteriophage T4 infects E. coli.
In bacteria on agar, T4
replication forms clear areas
of lysed cells (plaques).
Viral mutants easy to isolate.
T4 is manipulated more readily
than E. coli for molecular genetics.
Fig. 1.44 phage plaques
Tools of Cell Biology
Animal Viruses are important in studies of animal
cells: DNA viruses and RNA viruses (Table 1.3)
Retroviruses (HIV) have RNA genomes but synthesize DNA
copy of genome in infected cells; first demonstrated synthesis
of DNA from RNA templates.
Some animal viruses convert normal cells to cancer: these
viruses contribute to understanding cancer, mechanisms
control cell growth, differentiation.
.
HIV structure:
micro,.magnet.fsu.edu/cells/viruses
Review questions:
3. Discuss evidence that mitochondria and chloroplasts
originated from bacteria that were engulfed by precursors
of eukaryotic cells
6. Which model organism provides the simplest system for
studying eukaryotic DNA replication? Explain
10. What advantages does GFP have over use of fluorescentlabeled antibodies for studying location and movement of
protein in cells?
14. Why is the ability to culture ES cells important?