Overview of Cells and Cell Research BL 424 Cell Biology Ch 1 Overview Student learning outcomes: 1....

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: first cell arose from self-replicating RNA molecule in membranePhospholipids are amphipathic:

Water-insoluble (hydrophobic) hydrocarbon chainsWater-soluble (hydrophilic) head groups with PO4

In water, phospholipids spontaneously aggregate into bilayers.

Fig. 1.3

The Origin and Evolution of Cells

Adenosine 5′-triphosphate (ATP) metabolic energy3 Mechanisms of generation of ATP:

Glycolysis

Photo-synthesis Oxidative metabolism

Fig. 1.4


Present-day prokaryotes:Archaea (Archaebacteria): many live in extreme environmentsHalobacterium; 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


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 ofarchaeal andeubacterial genomes.

Archaea more closely related to Eukarya

Fig. 1.7


Unicellular eukaryotes:

Yeast is simple eukaryote

Saccharomyces cerevisiae

~ 6 µm diameter; 12x106 bp DNA

Protozoan Plasmodium falciparium

causes malariaGametocyte and blood cells


Multicellular organisms - diversity of cell types for specialized functions:

Animal cell tissue types:1.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

Fig. 1.12 cellsA, epithelialB, fibroblastsC, 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 diploidRecombinant DNA techniquesTwo mating typesHas cell wallGenetic manipulations similar to those performed using bacteria

Fig. 1.14

Cells as Experimental Models – C. elegans

Nematode Caenorhabditis elegans is widely used model:

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

Fig. 1.15

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)

African clawed frog

Figs. 1.18, 1.19

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.

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

λ is fixed at ~ 0.5 mmNA size of cone of light that enters lens (max α is 90°, at which sin α = 1)η = refractive index of the medium (1.0 for air, 1.4 oil-immersion lens)

NA

0.61Resolution

sin NA

Resolution limit:determined by wavelength of visible light (λ), numerical aperture (NA): light-gathering power of lens

Fig. 1.22

m22.04.1

5.061.0Re

solution

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


• 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. 24Human cheek cellsA brightfieldB phase contrastC differential interference


Fig. 1.26 Newt lung:DNA blue dyeMicrotubules green dye

** 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


* 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


** 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


*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

Electron microscopy greater resolution (~0.2 nm) than light microscopy - short wavelength of electrons.


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


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 function1. Differential centrifugation separates by size and density.

Fig. 1.38


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


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 cultureFig. 40

FibroblastC. Cell Culture

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.


Fig. 1.41

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.


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

Hela cells:Cancer_Hela_320.explorium.edu

Plant callus of halophyte:Ceoe.udel.edu/halophyte

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 systemsto study functions of cells that they infect

Fig. 1.43 HPVHuman papillomavirus


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


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 fluorescent-labeled antibodies for studying location and movement of protein in cells?

14. Why is the ability to culture ES cells important?

Overview of Cells and Cell Research BL 424 Cell Biology Ch 1 Overview Student learning outcomes: 1....

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Transcript of Overview of Cells and Cell Research BL 424 Cell Biology Ch 1 Overview Student learning outcomes: 1....