Cell Biology Introduction

I. Introduction
A. The Origin and Evolution of Cells
B. Cells as Experimental Models
C. Tools of Cell Biology

Cell biology (formerly cytology,
from the Greek kytos, "container") is
an academic discipline that studies
cells • their physiological properties
• their structure,
• the organelles they contain
• interactions with their
environment,
• their life cycle, division and
death.

Cell – basic unit of life structurally
and functionally.
History:
a. Robert Hooke (1665)using his microscope
discovers cells in cork
b. Schleiden ; Schwann and Virchow
Cell theory:
1. All organisms are composed of one
or more cells
2. The cell is the structural unit of life
3. Cells can arise only by division from
preexisting cells

Various cell types:
shape, size, intracellular organizations,
polarization – Functions

Dynamic Nature of Cell
-it has the capacity
 to grow
 to reproduce
 become specialized
 ability to respond to stimuli
 adapt to changes in its environment

Fundamental properties shared by all cells:
(conserved throughout evolution)
1. all cells employ DNA as their
genetic material
2. surrounded by plasma membrane
3. use the same basic mechanisms for
energy metabolism

Two Main Classes of Cells:
a.Prokaryotic cells – no nucleus
- simpler structure (bacteria)
b.Eukaryotic cells - contain nucleus
- more complex structure(protists,
fungi, plants & animals)

Fig.1.2.Average_prokaryote_cell-
_en.svg (SVG file, nominally 494 × 402
pixels, file size: 135 KB)
A bacterium

The Main Functions of the Membrane-bounded
Compartments of a Eukaryotic Cell
Compartment Main Function
Cytosol contains many metabolic pathways
protein synthesis
Nucleus contains main genome
DNA and RNA synthesis
Endoplasmic
reticulum (ER)
synthesis of most lipids
synthesis of proteins for distribution to many organelles and
plasma membrane
Golgi apparatus modification, sorting, and packaging of proteins and lipids
for either secretion or delivery to another organelle
Lysosomes intracellular degradation
Endosomes sorting of endocytosed material
Mitochondria ATP synthesis by oxidative phosphorylation
Chloroplasts (in
plant cells)
ATP synthesis and carbon fixation by photosynthesis
Peroxisomes oxidation of toxic molecules

Organisms:
1. Unicellular (eg. bacteria, amoebas &
yeasts) – capable of independent self-
replication
2. Multicellular(eg. Humans)- composed of
collection of cells w/c fxns in a
coordinated manner w/ diff cells
specialized to perform particular tasks.

All organisms: 1 or more cells
PROKARYOTESEUKARYOTES

Prokaryotes Eukaryotes
Typical organisms bacteria, archaea protists, fungi, plants, animals
Typical size ~ 1-10 µm
~ 10-100 µm (sperm cells, apart
from the tail, are smaller)
Type of nucleus nucleoid region; no real nucleus
real nucleus with double
membrane
DNA circular (usually)
linear molecules (chromosomes)
with histone proteins
RNA-/protein-synthesis coupled in cytoplasm
RNA-synthesis inside the nucleus
protein synthesis in cytoplasm
Ribosomes 50S+30S 60S+40S
Cytoplasmic structure very few structures
highly structured by
endomembranes and a
cytoskeleton
Cell movement flagella made of flagellin
flagella and cilia containing
microtubules; lamellipodia and
filopodia containing actin
Mitochondria none
one to several thousand (though
some lack mitochondria)
Chloroplasts none in algae and plants
Organization usually single cells
single cells, colonies, higher
multicellular organisms with
specialized cells
Cell division Binary fission (simple division)
Mitosis (fission or budding)
Meiosis
DNA content (base pairs) 1 × 106 to 5 × 106 1.5 × 107 to 5 × 109
Table 1: Comparison of features of prokaryotic and eukaryotic cells

The Origin and Evolution of Cells

the First Cell:
-all present day cells (both prokaryotes &
eukaryotes) descended from a single ancestor.
-the 1st cell is thought to have arisen at least
3.8 B years ago as a result of enclosure of self-
replicating RNA in a phospholipid membrane (RNA
world hypothesis)
Present-Day Prokaryotes
-divided into two groups: the archaebacteria and
the eubacteria which
diverged early in evolution
Eukaryotic Cells
-thought to have evolved from symbiotic
associations of prokaryotes (ENDOSYMBIONT
THEORY)

ENDOSYMBIOSIS
A large anaerobic, heterotrophic prokaryote engulfs a
small aerobic prokaryote
The aerobic endosymbiont has evolved into a mitochondrion
A portion of the plasma membrane has invaginated
and evolved into a nuclear envelope and endoplasmic
reticulum
(primitive eukaryote)
Nonphotosynthetic protist, fungal,
animal cells
Engulfs a photosynthetic
prokaryote
Evolve into a chloroplast
Algal & plant cells

Fig.1.5. Time scale of evolution The scale indicates the approximate
times at which some of the major events in the evolution of cells are
thought to have occurred.

Figure 1.6. Generation of metabolic energy Glycolysis is the anaerobic breakdown of glucose to lactic acid.
Photosynthesis utilizes energy from sunlight to drive the synthesis of glucose from CO2 and H2O, with the release
of O2 as a by-product. The O2 released by photosynthesis is used in oxidative metabolism, in which glucose is
broken down to CO2 and H2O, releasing much more energy than is obtained from glycolysis.
The Evolution of Metabolism:

Figure 1.6. Evolution of cells Present-day cells evolved from a common prokaryotic ancestor along three
lines of descent, giving rise to archaebacteria, eubacteria, and eukaryotes. Mitochondria and chloroplasts
originated from the endosymbiotic association of aerobic bacteria and cyanobacteria, respectively, with
the ancestors of eukaryotes.

Cells as Experimental Models
E. coli S. cerevisiae Dictyostelium discoideum
Arabidopsis thaliana Caenorhabditis elegans Drosophila
melanogaster

Xenopus laevis zebrafish
House mouse

Choosing the Right Experimental Organism for
the Job

Organism Haploid DNA content (millions of base
pairs)
Bacteria
Mycoplasma 0.6
E. coli 4.6
Unicellular eukaryotes
Saccharomyces cerevisiae (yeast) 12
Dictyostelium discoideum 70
Euglena 3000
Plants
Arabidopsis thaliana 130
Zea mays (corn) 5000
Animals
Caenorhabditis elegans (nematode) 97
Drosophila melanogaster (fruit fly) 180
Chicken 1200
Zebrafish 1700
Mouse 3000
Human 3000
Table 1.2 DNA Content of Cells

Tools of Cell Biology
1. Light Microscopy
• Bright-field microscopy
• Phase-contrast microscopy
• Differential interference –contrast microscopy
• Video-enhanced differential interference-contrast microscopy
• Fluorescence microscopy
• Confocal scanning microscopy
2. Electron microscopy
• Transmission electron microscopy
• Scanning electron microscopy

- To magnify an object, it uses a system of lenses to
manipulate the path a light beam travels between the
object being studied and the eye
- Produce a maximum useful magnification of about
1000 times the original size.
- Has three lenses:
1. the condenser(focuses light on the specimen
2. the objective(s)
a. Low-power objective
b. High-power objective
c. Oil-immersion objective
3. the eyepiece (ocular)
Light Microscope
- earliest tool of cytologist
- Limit of resolution is λ/2= 0.20-0.35 um

Objective
Designation
Objective
Magnification
Eyepiece
Magnification
Total
Magnification
Low Power 10 10 100
High Power (high
dry)
40 10 400
Oil Immersion 100 10 1000
Table 2-1
Unit of
length
Meter (m) Centimeter
(cm)
Millimeter
(mm)
Micrometer
(um)
Nanometer
(nm)
Micrometer
(um)
0.000001
10-6
0.0001
10-4
0.001
10-3
1 1000
103
Nanometer
(nm)
0.000000001
10-9
0.0000001
10-7
0.000001
10-6
0.001
10-3
1
Angstrom
(Å)
0.0000000001
10-10
0.00000001
10-8
0.0000001
10-7
0.0001
10-4
0.1
10-1
Table 2-2. Metric unit equivalents in expressing cell dimensions

Terms:
1. Limit of Resolution-refers to how far apart
adjacent objects must be in order to be distinguished as
separate entities. (eg. LOR of microscope is 400 nm)
2. Resolving Power-expressed in terms of λ(the
wavelength of light used to illuminate the sample)
-the smaller is the limit of resolution the
greater is the resolving power
A. Light Microscope
Resolving Power of any microscope
-a measure of its ability to discriminate between two adjacent
objects.
- is a function of the wavelength of light and the numerical
aperture of the lens system
- light microscopes (using visible light) have RP of approximately
0.25 um which means that particles of a smaller size cannot be
distinguished from one another).

Fig.1-9. Resolving Power
Of the Human Eye, the
Light microscope and the
Electron Microscope

1 um = 10-6 m (one-millionth of a meter)
1 nm = 10-9 m (one-billionth of a meter)
1000 nm = 1 um
Angstrom (Å) = 10-10 m or 0.1 nm

Type of
Microscopy
Maximum useful
magnification
Appearance of
specimen
Useful
Applications
Bright-field 1000-2000 Specimens stained
or unstained;
bacteria generally
stained and appear
color of stain
Gross
morphological
features of
bacteria, yeasts,
molds,algae and
protozoa
Dark-field 1000-2000 Generally
unstained; appears
bright or “lighted” in
an otherwise dark
field
Microorganisms
that exhibit some
characteristic
morphological
feature in the living
state and in fluid
suspension e.g.
spirochetes
Fluorescence 1000-2000 Bright and colored;
color of the
fluorescent dye
Diagnostic
techniques where
fluorescent dye
fixed to organism
reveals the
organism’s identity

a. Differential-interference-
contrast micrograph of a mitotic
yeast cell.
b. Fluorescence microscopy
c. Phase-contrast micrograph of fibroblasts
in culture.

Type of
Microscopy
Maximum useful
magnification
Appearance of
specimen
Useful
Applications
Phase-contrast 1000-2000 Varying degrees of
“darkness”
Examination of
cellular structures
in living cells of the
larger
microorganisms,
e.g yeasts, algae,
protozoa and some
bacteria
c) Dark-field photomi-
crograph of Mysis
d)Phase-contrast
micrograph of a cheek
cella) Flourescent microscope b) flourescent micrograph
of chromosomes and mitotic spindle

B. Electron Microscope
-uses a beam of electrons controlled by a system of
magnetic fields
-has high resolving power thus greater magnification
(can resolve objects separated by a distance of 0.003 um
compared to 0.25 um of light microscope).
-useful magnification is 200,000 to 400,000
-use in the examination of viruses and the ultra-
stucture of microbial cells.
- has two types:
a. Scanning electron microscopy (SEM)
-employed to study the surface structure
of a specimen(eg. Attachment of bacterial cells to
objects)
b. Transmission electron microscopy (TEM)
-used to view subcellular components
(even nucleic acid molecules)

Scanning electron
micrograph
of a flea
Transmission electron micrograph
of Bacillus anthracis

Techniques and Methods
of Studying Cells

Several different techniques exist to study
cells:
1. Cell culture
2. Cell Fractionation
3. Immunostaining
4. Computational Genomics
5. DNA MICROARRAYS
6. Gene knockdown
7. In situ hybridization
8. Polymerase Chain Reaction (PCR)

Figure 1-7. A procedure used to make a transgenic plant.

Figure 1.8. Using DNA
microarrays to monitor the
expression of thousands of
genes simultaneously.
Figure 8-63. Using cluster
analysis to identify sets of
genes that are coordinately
regulated.

Figure 8-47. Results of a BLAST search. Sequence databases can be
searched to find similar amino acid or nucleic acid sequences. Here a
search for proteins similar to the human cell-cycle regulatory protein
cdc2 (Query) locates maize cdc2 (Subject), which is 68% identical (and
82% similar) to human cdc2 in its amino acid sequence.
Basic Local Alignment Search Tool (BLAST)

-a technique that can be used to better visualize
cells and cell components under a microscope.
-by using different stains, one can preferentially
stain certain cell components, such as a nucleus or a
cell wall, or the entire cell.
- Most stains can be used on fixed, or non-
living cells, while only some can be used on living
cells; some stains can be used on either living or
non-living cells.
Cell staining

Why Stain Cells?
-most basic reason that cells are stained is
to enhance visualization of the cell or certain
cellular components under a microscope.
-Cells may also be stained to highlight
metabolic processes or to differentiate
between live and dead cells in a sample.
- to determine biomass in an environment of
interest.

How Are Cells Stained and Slides Prepared?
Cell staining techniques and preparation depend on the type of stain and analysis used. One
or more of the following procedures may be required to prepare a sample:
•Permeabilization - treatment of cells, generally with a mild surfactant, which dissolves cell
membranes in order to allow larger dye molecules to enter inside the cell.
•Fixation - serves to "fix" or preserve cell or tissue morphology through the preparation
process. This process may involve several steps, but most fixation procedures involve adding a
chemical fixative that creates chemical bonds between proteins to increase their rigidity.
Common fixatives include formaldehyde, ethanol, methanol, and/or picric acid.
•Mounting - involves attaching samples to a glass microscope slide for observation and
analysis. Cells may either be grown directly to the slide or loose cells can be applied to a slide
using a sterile technique. Thin sections (slices) of material such as tissue may also be applied
to a microscope slide for observation.
•Staining - application of stain to a sample to color cells, tissues, components, or metabolic
processes. This process may involve immersing the sample (before or after fixation or
mounting) in a dye solution and then rinsing and observing the sample under a microscope.
Some dyes require the use of a mordant, which is a chemical compound that reacts with the
stain to form an insoluble, colored precipitate. The mordanted stain will remain on/in the
sample when excess dye solution is washed away.

A. Dye-cellular interactions
____________________________________
Fig. 1. Schematic representation of dye-protein interactions. At pI of protein
(center), negligible binding of charged dyes occurs. Below pI (left), protein
binds acid
(=anionic) dyes; above pI (right), protein binds basic (=cationic) dyes. At
physiologic pH, specific proteins may exhibit net (+) or net (-) charges and
are therefore characterized as acidophilic (=affinity for acid dyes) or
basophilic (=affinity for basic dyes), respectively.

Important stains and reactions
1. Cationic ("basic") Dyes: tissue components stained
with these dyes are basophilic.
a. Examples of basic dyes
i. Hematoxylins (behave as cationic dyes)
ii. Azures
iii. Methylene blue
iv. Toluidine blue
b. Examples of basophilic tissue components
i. Nuclei and nucleoli
ii. Cytoplasmic RNA (e.g . ,ergastoplasm,
Nissl)

2. Anionic ("acid") Dyes: tissue components stained
with these dyes are acidophilic.
a. Examples of acid dyes
i. Aniline blue: blue
ii. Eosin: pink-red
iii. Fast green: green
iv. Orange G: orange
v. Picric acid: yellow
b. Examples of acidophilic tissue
components
i. Most cytoplasm
ii. Hemoglobin
iii. Keratin
iv. Collagens

3. Special Stains and Reactions
a. Alcian blue (=basic dye) for polyanions
and acidic glycoproteins (alcianophilia)
b. Elastic stains (e.g., orcein, resorcin fuchsin,
Verhoeff)
c. Feulgen reaction for DNA
d. Lipid colorants/stains (e.g., Sudan black, oil
red O, osmium tetroxide)
e. Masson trichrome for differential staining of
several tissues
f. Metachromasia (e.g., toluidine blue for
polyanions)
g. PAS reaction for vic-glycols
h. Silver stains for Golgi, basement
membranes, reti- cular fibers, neurofibrils
(argyrophilia)

j. Oxidation-reduction reactions and
staining
k. Romanowsky dyes: mixtures of acid and basic dyes for
staining blood smears
i. Mechanism
• Orthochromasia -cellular components
stained either pink/red due to eosin or blue
due to basic dye component
• Polychromasia –layered staining with both
dyes
• Metachromasia -color shift of basic dye
from blue to violet-purple due to high
concentrations of polyanions
ii. Examples
• Wright stain
• Giemsa stain

Cell Biology Introduction

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