Announcements

• Please remove homework, exam answer sheets from your folders.– Answer key is posted outside BR 179.

• Reading for today: pp. 74-100 on cellular organization

• Reading for Friday: 154-171 on membrane lipids

Outline/Learning OutcomesI. Microscopy (cont’d)

E. Electron Microscopy1. TEM2. SEM

II. Cellular OrganizationA. “Prokaryotes” versus

EukaryotesB. Isolation and fractionation of

eukaryotic organelles and proteins

C. Eukaryotic organelles

After reading the text, attending lecture, and reviewing lecture notes, you should be able to:

• Compare and contrast scanning versus transmission EM.

• Compare and contrast “prokaryotic” and eukaryotic cells

• Explain how cells are purified and how their contents are fractionated, including techniques of differential centrifugation and column chromatography.

• State the primary function of each eukaryotic organelle.

• Apply your knowledge of these topics to cell biological problems.

Epifluorescence/Confocal light path

Conventional epifluorescence Confocal epifluorescence

Comparison of resolving power

Light microscopy Electron microscopy

r0.61NA

Why does electron microscopy have greater resolving power?

Electron Microscopy

• How do we get higher resolution than 0.2 μm?

• Wavelength () of an electron (at 1,000,000 V) = 0.001 nm (versus 450 nm for LM).

• If NA = 0.004 (versus 1.4 for LM), r = 0.61(0.001nm)/0.004 = 0.2 nm

• This is 1000-fold better than the best light microscope.

Philips CM10 TEM in CMU Biology

Microscopy Facility

Scanning EM• Primary electrons hit sample and

produce secondary electrons, which are collected and converted to photons on a video screen.

• Electron beam scans across sample to produce image.

• Large depth of field possible, good for 3-D

• Used together with freeze-fracture technique to study membranes.

JEOL JSM-840A SEM in CMUBiology Microcopy Facility

Examples of SEM

(a) Cultured human neuroblastoma, (b) pollen grain

“Prokaryotes” – a paraphyletic group (doesn’tinclude all descendants of last common ancestor)

DOMAINS

“Prokaryotic” versus Eukaryotic Cells

cytoskeleton

Limits on cell size

• How large depends on:– Sufficient surface

area/volume ratio– Rates of diffusion of

molecules– Adequate local

concentrations of substrates and enzymes

• How small depends on:– Minimal number of

substrates, enzymes– Sufficient volume to

package the DNACollisions with other moleculescause diffusion from higher tolower concentration

Typical Animal and Plant Cells

Isolation of Organelles

A. Cell purification by centrifugation, cell culture, etc.

B. Cell disruption by grinding, sonication, or osmotic lysis.

C. Cell fractionation by differential centrifugation

D. Characterization by activity of enzyme markers, EM, etc.

Fig. 12A-1

Differential Centrifugation

• Particles separate based on different rates of sedimentation due to size, shape, or density

• In velocity sedimentation: – 1000 X g, 10 min → nuclear

fraction – 20,000 X g, 20 min →

mitochondrial fraction – 80,000 X g, 1 hr → microsomal

fraction (membranes)– Remaining supernatant: cytosol

• 150,000+ X g, 3 hr → macromolecules

Fig. 12A-4

Sedimentation Coefficients

• Particles with large sedimentation coefficients (S) pellet sooner than those with smaller ones.

• Organelles, macromolecules, and viruses can be separated by their density (by density gradient centrifugation) or size:

Fig. 12A-2, 12A-35S-80S for rRNA

Enzyme Assays for Purity(Don’t memorize)

• Plasma membrane: Na/K ATPase• Nucleus: DNA• Golgi: UDP-galactosyl transferase• Mitochondria: monoamine oxidase, cytochrome oxidase,

etc.• Lysosomes: acid phosphatase• Peroxisomes: catalase• Endoplasmic reticulum (rough): glucose-6-phosphatase

Protein purification by column chromatography

Three kinds of chromatography

• Ion-exchange: separation by charge

• Gel-filtration: separation by size

• Affinity: separation by specific binding

HPLC

• Highest resolution obtained by high-performance liquid chromatography, using tiny spheres and solvent under pressure.

Learning checkThe purification of a protein usually requires multiple steps and often involves several types of chromatography. A key component of any purification is an assay for the desired protein. The assay can be a band on a gel, a structure in the electron microscope, the ability to bind to another molecule, or an enzymatic activity. The purification of an enzyme is particularly instructive because the assay allows one to quantify the extent of purification at each step. Consider the purification of the enzyme shown below. The total volume, total protein, and total enzyme activity are shown at each step. (A) For each step, calculate the specific activity. (B) Which purification step was most effective? Which step was the least effective? (C) How would S.A. change if carried through further purification steps? How would you know when the enzyme was totally pure?

Procedure Total Volume

(ml)

Total protein

(mg)

Total activity

(units *)

Specific activity

(units/mg)

1. Crude extract 2000 15,000 150,000

2. Ammonium sulfate precipitat’n

320 4000 140,000

3. Ion-exchange

Chrom.

100 550 125,000

4. Gel-filtration

Chrom.

85 120 105,000

5. Affinity chrom. 8 5 75,000

*1 unit of enzyme activity defined as amount converting 1 μmol substrate per min at 25oC.

Learning checkThe purification of a protein usually requires multiple steps and often involves several types of chromatography. A key component of any purification is an assay for the desired protein. The assay can be a band on a gel, a structure in the electron microscope, the ability to bind to another molecule, or an enzymatic activity. The purification of an enzyme is particularly instructive because the assay allows one to quantify the extent of purification at each step. Consider the purification of the enzyme shown below. The total volume, total protein, and total enzyme activity are shown at each step. (A) For each step, calculate the specific activity. (B) Which purification step was most effective? Which step was the least effective? (C) How would S.A. change if carried through further purification steps? How would you know when the enzyme was totally pure?

Procedure Total Volume

(ml)

Total protein

(mg)

Total activity

(units)

Specific activity

(units/mg)

1. Crude extract 2000 15,000 150,000 10

2. Ammonium sulfate precipitat’n

320 4000 140,000 35

3. Ion-exchange

Chrom.

100 550 125,000 227

4. Gel-filtration

Chrom.

85 120 105,000 875

5. Affinity chrom. 8 5 75,000 15,000

3.5X

6.5X

3.8X

17X

Nucleus: Cell’sInformation Center

5-10 m

Mitochondrion:Cell’s Energy Center (ATP production)

1-2 m

Chloroplast: Photosynthesis(CO2, H20 3C sugars)

5-10 m

Plant Cell Vacuole:Maintains turgor pressure, storage

The Secretory Pathway

Endoplasmic Reticulum

Transition vesicles

Golgi complex

Secretory vesicles

Plasma membrane