bob mercer, [email protected] 362-6924; 991-1243 molecular cell biology 5068

Bob Mercer, [email protected]; 991-1243

Molecular Cell Biology 5068

3. Be able to critically read and evaluate the scientific literature.

1. Obtain a solid foundation of knowledge in cell biology

2. Obtain a working knowledge of available techniques.

4. Be able to define and investigate a biological problem.

Goals For MCB 5068

Visit website: www.mcb5068.wustl.eduSign up for course.

Check out Self Assessment homework

Visit Discussion Sections: Read “Official” Instructions

To Do:

Molecular Cell Biology 5068

TA’s:

Katie Campbell [email protected]

Umber Dube [email protected]

biochemistry genetics cytology

What is Cell Biology?

physiology

Molecular Cell Biology

CELL BIOLOGY/MICROSCOPE

Microscope first built in 1595 by Hans and Zacharias Jensen in Holland

Zacharias Jensen

CELL BIOLOGY/MICROSCOPERobert Hooke accomplished in physics, astronomy, chemistry, biology, geology, and architecture. Invented universal joint, iris diaphragm, anchor escapement & balance spring, devised equation describing elasticity (“Hooke’s Law”). In 1665 publishes Micrographia


Robert Hooke

. . . “I could exceedingly plainly perceive it to be all perforated and porous, much like a Honey-comb, but that the pores of it were not regular. . . . these pores, or cells, . . . were indeed the first microscopical pores I ever saw, and perhaps, that were ever seen, for I had not met with any Writer or Person, that had made any mention of them before this. . .”


Antony van Leeuwenhoek (1632-1723)


Antony van Leeuwenhoek (1632-1723)

a tradesman of Delft, Holland, in 1673, with no formal training, makes some of the most important discoveries in biology. He discovered bacteria, free-living and parasitic microscopic protists, sperm cells, blood cells and more. All of this from a very simple device that could magnify up to 300X.

Red blood cells

Spiral bacteria

THE CELL THEORYMatthias Jakob Schleiden 1804-1881Theodor Schwann 1810-1882

Schleiden Schwann

THE CELL THEORY

First coined by German physiologist, Theodore Schwann in 1839, and formed from the ideas of German botanist Matthias Schleiden, Schwann, and German Physician/Pathologist Rudolf Virchow. The theory proposes that:

1. Anything that is alive is made up of cells.

2. The chemical reactions that occur in organisms occur in cells.

3. All cells come from preexisting cells.

SPONTANEOUS GENERATION

From ancient time, through the Middle Ages, and until the late nineteenth century, it was generally accepted that some life forms arose spontaneously from non-living organic matter.

Jan Baptista van Helmont (1577-1644) Flemish physican, chemist and physiologist. Invented the word “gas”. Recipe for mice:

Place a dirty shirt or some rags in an open pot or barrel containing a few grains of wheat or some wheat bran, and in 21 days, mice will appear

SPONTANEOUS GENERATION(1668-1859)

Although the belief in the spontaneous generation of large organisms wanes after 1668, the invention of the microscope serves to enhance the belief in spontaneous generation. Microscopy revealed a whole new class of organisms (animalcules) that appeared to arise spontaneously. It was quickly learned that you needed only to place hay in water and wait a few days before examining your new creations under the microscope. This belief persisted for nearly two centuries.

SPONTANEOUS GENERATION(1668-1859)

In 1859, after years of debate The French Academy of Sciences sponsors a contest for the best experiment either proving or disproving spontaneous generation. The French chemist, Louis Pasteur (1822-1895) uses a variation of the methods of Needham and Spallanzani. He boils meat broth in a flask, heats the neck of the flask in a flame until it became pliable, and bent it into the shape of an S. Air could enter the flask, but airborne microorganisms could not - they would settle by gravity in the neck. As Pasteur had expected, no microorganisms grew. When Pasteur tilted the flask so that the broth reached the lowest point in the neck, where any airborne particles would have settled, the broth rapidly became cloudy with life. Pasteur had both refuted the theory of spontaneous generation and convincingly demonstrated that microorganisms are everywhere - even in the air.

1859: Kowalski publishes the first usable method to deduce the rotation of the Milky Way. Darwin publishes Origin of Species. Lenoir produces the first two-stroke gas engine with an electric ignition system.


Camillo Golgi (1843-1926)

In 1898, Golgi develops a staining technique (silver nitrate) that allows the identification of an "internal reticular apparatus" that now bears his name: the "Golgi complex” or the “Golgi”.


By the late 1800’s to the early 1900’s the limits to the light microscope had been reached.

Resolving ability roughly 1/2 of light used: ≈ 0.2 µm

In 1930 A.A. Lebedeff designs and builds the first interference microscope.

In 1932 Frits Zernike (1888-1966) invents the phase-contrast microscope. It is first brought to market in 1941 in Germany.

Both microscopes aid in elucidating the details in unstained living cells.


In 1932 Zernike traveling from Amsterdam, visits the Zeiss factory in Germany to present his method of phase contrast microscopy. After reviewing Zernike's method an older scientist said:

"If this really had any practical value, then we would have invented it a long time ago."

In 1953 Zernike was awarded the Nobel Prize for his phase contrast work.

Wavelength sets limitson what one can see

Light behaves as a Wave

Resolution = 0.61 x wavelength of light

NA (numerical aperture)

The effect of NAon the image ofa point.

The need forseparation to allow resolution

θθ

θ

Lower limits on spatial resolution are

defined by the Rayleigh Criterion

NA = nsinθn = refractive index of the mediumθ = semi-angle of an objective lens

Contrast in the Image is Necessary:Types of Optical Microscopy Generate Contrast in Different Ways•Bright field - a conventional light microscope

•DIC (Differential Interference Contrast - Nomarski)

•Phase contrast•Fluorescence•Polarization•Dark field

Bright-field Optics: Light Passing Straight Through the Sample•Most living cells are optically clear, so stains are essential to get bright field contrast

•Preserving cell structure during staining and subsequent observation is essential, so cells must be treated with “fixatives” that make them stable

•Fixing and staining is an art

Generating Contrast

Staining

Coefficients of absorption among different materials differ by >10,000, so contrast can be big

Without staining

Everything is brightMost biological macromolecules do not

absorb visible lightContrast depends on small differences between big numbers

Need an optical trick

Mammalian Cell: Bright-field and Phase-contrast

Optics

Principles of bright field

and phase contrast optics

Differential Interference Contrast (DIC)

• Optical trick to visualize the interference between two parts of a light beam that pass through adjacent regions of the specimen

• Small amounts of contrast can be expanded electronically

• Lots of light: Video camera with low brightness & high gain

Brightfield vs DIC

FluorescenceMicroscopy

•Absorption of high-energy (low wavelength) photon

•Loss of electronic energy (vibration)

•Emission of lower-energy (higher wavelength) photon

Design of a Fluorescence Microscope

Green Fluorescent Protein - Considerations

• Color - Not just green

• Brightness

• Size/Location 26.9 kDa

• Time for folding

• Time to bleaching

GFP-Cadherin in cultured epithelial cells

Immunofluorescence

• Primary Abs recognize the antigen (Ag)• Secondary Abs recognize the primary Ab• Secondary Abs are labeled

Immunofluorescence Example

•Ab to tubulin

•Ab to kinetochore proteins

•DNA stain (DAPI)

Biological microscopy problem: Cells are 3D objects, and pictures are 2D images.

• Single cells are thicker than the wavelength of visible light, so they must be visualized with many “optical sections”

• In an image of one section, one must remove light from other sections

• Achieving a narrow “depth-of-field”

• A “confocal light microscope”

Laser-Scanning Confocal Light

Microscopy•Laser thru pinhole

•Illuminates sample with tiny spot of light

•Scan the spot over the sample

•Pinhole in front of detector: Receive only light emitted from the spot

Light from points that are in focus versus out of focus

Spinning-disk confocal microscopy:

Higher speed and sensitivity

Example: Confocal imaging lessens

blur from out-of-focus light

Optically Sectioning a Thick Sample: Pollen Grain

Multiple optical sections assembled to form a 3D image

Fluorescence can Measure Concentration of Ca2+ Ions in Cells:

Sea Urchin Egg Fertilization

Phase Contrast Fluorescence

Total Internal Reflection Fluorescence (TIRF) Microscopy

www.leica-microsystems.com

The penetration depth of the field typically ranges from 60 to 100 nm

Total Internal Reflection Fluorescence (TIRF) Microscopy

www.leica-microsystems.com

Summary

• Light microscopy provides sufficient resolution to observe events that occur inside cells

• Since light passes though water, it can be used to look at live as well as fixed material

• Phase contrast and DIC optics: Good contrast

• Fluorescence optics: Defined molecules can be localized within cells

• “Vital” fluorescent stains: Watch particular molecular species in live cells


In 1924 at the Faculty of Sciences at Paris University he delivers a thesis Recherches sur la Théorie des Quanta (Researches on the quantum theory), which earned him his doctorate. This thesis contained a series of important findings that he had obtained in the course of about two years. This research culminated in the de Broglie hypothesis stating that any moving particle or object had an associated wave. Therefore a moving electron has wavelike properties.

In 1929 he received the Nobel Prize for this observation.

Louis de Broglie (1892-1987)


Light Microscope TransmissionElectron

Microscope

ScanningElectron

Microscope

The Cell

1. Compartmentalized chemical reactions

2. Modify intra- and extra- cellular environment

3. Different properties and functions.

The Cell

Surface Area to Volume Ratio Limits Cell Size

In general, the surface area increases in proportion to the square of the width and volume as the cube of the width.

Xenopus oocyte

Electron micrograph of a thin section of a hormone-secreting cell from the rat pituitary, showing the subcellular features typical of many animal cells.

Membranes Define the Cell

CELL BIOLOGY/MEMBRANES

In the late 1890’s Charles Ernest Overton was working on a doctoral degree in botany at the University of Zurich. His research was related to heredity in plants and in order to complete his studies he needed to find substances that would be readily absorbed into plant cells. He found that the ability of a substance to pass through the membrane was related to its chemical nature. Nonpolar substances, would pass quickly through the membrane into the cell. This discovery was quite contrary to the prevalent view at the time that the membrane was impermeable to almost anything but water.


Based on his observations of what substances pass through the membrane, Overton proposes:

1. There are some similarities between cell membranes and lipids such as olive oil.

2. Certain molecules (i.e., lipids) pass through the membrane by "dissolving" in the lipid interior of the membrane.


Irving Langmuir (1881-1957)

Trained in physical chemistry under Nobel laureate Walther Nernst, Langmuir worked in the laboratories of General Electric doing research on molecular monolayers. His research eventually turned to lipids and the interaction of oil films with water. By improving an existing apparatus for the study of lipids (referred to today as a Langmuir trough), he was able to make careful measurements of surface areas occupied by known quantities of oil.

CELL BIOLOGY/MEMBRANESIrving Langmuir (1881-1957)

Based on his studies he proposed that the fatty acid molecules form a monolayer by orienting themselves vertically with the hydrocarbon chains away from the water and the carboxyl groups in contact with the surface of the water.

In 1932 he received the Nobel Prize for Chemistry “for his discoveries and investigations in surface chemistry.”

His improvement of vacuum techniques led to the invention of the high-vacuum tube. He and colleague Lewi Tonks discovered that the lifetime of a tungsten filament was greatly lengthened by filling the bulb with an inert gas, such as argon. He also discovered atomic hydrogen, which he put to use by inventing the atomic hydrogen welding process. During WWII Langmuir worked to develop protective smoke screens and methods for de-icing aircraft wings. This research led him to discover that the introduction of dry ice and iodide into a sufficiently moist cloud of low temperature could induce precipitation (cloud seeding).

Time MagazineAugust 28, 1950


In 1925 Evert Gorter and his research assistant, J.Grendel extracted the lipids from red blood cells with acetone and other organic solvents. Using a modified trough, similar to Langmuir, they were able to demonstrate that lipid molecules could form a double layer, or bilayer as well as a monolayer. Further, they were able to show that the surface area of the lipids extracted from the red blood cells was about twice the surface area of the cells themselves.

Based on these two observations (i.e., that lipid molecules can form bilayers, and that the surface area of the monolayer extracted from the cells is approximately equal to twice the surface area of the cells) and repeated studies with red blood cells from several animals (human, rabbit, dog, guinea pig, sheep, and goat) Gorter and Grendel concluded that "chromocytes [red blood cells] are covered by a layer of fatty substances that is “two molecules thick”


Lipid Monolayer

Lipid Bilayer

CELL BIOLOGY/MEMBRANE MODELS

In 1935 James F. Danielli and Hugh Davson propose the first widely accepted membrane model. The model proposed by Danielli and Davson was basically a "sandwich" of lipids (arranged in a bilayer) covered on both sides with proteins. Later versions of the model included "active patches" and protein lined pores.


In 1957 J.D. Robertson proposed a modified version of the membrane model, based primarily on EM studies, which he called the "unit membrane".

Under the high magnification of the TEM, membranes have a characteristic "trilaminar" appearance consisting of two darker outer lines and a lighter inner region. According to the unit membrane model, the two outer, darker lines are the protein layers and the inner region the lipid bilayer.

CELL BIOLOGY/MEMBRANE MODELSIn the early 1970’s the unit membrane model was replaced by the fluid mosaic model. This model was first proposed by biochemists S.J. Singer and Garth L. Nicolson. The model retains the basic lipid bilayer structure, however, proteins are thought to be globular and to float within the lipid bilayer.

As in the other models, the hydrophobic tails of the phospholipids face inward, away from the water. The hydrophilic heads of the phospholipids are on the outside where they interact with water molecules in the fluid environment of the cell. Floating within this bilayer are the proteins, some of which span the entire bilayer and may contain channels, or pores, to allow passage of molecules through the membrane. The entire membrane is fluid—the lipid molecules move within the layers of the bilayer while the "floating" proteins also freely move within the bilayer.


The fluid-mosaic model of membrane structure as initially proposed by Singer and Nicolson in 1972.


Left. Image of the upper surface of a lipid bilayer containing phosphatidylcholine (black background), and sphingomyelin molecules, which organize themselves spontaneously into the orange-colored rafts. The yellow peaks represent a GPI-anchored protein, which is almost exclusively raft-associated. This image is provided by an atomic force microscope,which measures the height of various parts of the specimen at the molecular level. Right. Schematic model of a lipid raft within a cell. The outer leaflet of the raft consists primarily of cholesterol and sphingolipids (red head groups). Phosphatidylcholine molecules (blue head groups) with long saturated fatty acids also tend to concentrate in this region. A GPI-anchored protein is localized in the raft. The lipids in the outer leaflet of the raft have an organizing effect on the lipids of the inner leaflet. As a result, the inner leaflet raft lipids consist primarily of cholesterol and glycerophospholipids with saturated fatty acyl tails. The inner leaflet tends to concentrate lipid-anchored proteins, such as src kinase, that are involved in cell signaling.

FUNCTIONS OF MEMBRANES

1.Compartmentalization

2.Permeability barrier - regulate what

gets through

3.Selective pumps & gates - regulate & accelerate molecular passage

4.Generate signals for cell

communication

5.Flow of information between cells & between environment & cells

6. Surfaces for ordered array of

reactions

The properties of membranes derive from both lipids and proteins

Fluidity of membranes is determined by both temperature and composition.

Temperature:

Left. Above the transition temperature, the lipid molecules and their hydrophobic tails, although ordered are free to move in certain directions. Right. Below the transition temperature, the movement of the lipid molecules is greatly restricted and the bilayer takes on properties of a crystalline gel.

Fluidity of membranes is determined by both temperature and composition.

Unsaturated vs. saturated fatty acids.Concentration of cholesterol.

The cholesterol molecules (green) in the lipid bilayer, interfere with the tight packing of the phospholipids, making the bilayer more fluid.

Crooked, unsaturated fatty acids interfere with tight packing of the phospholipids, making the bilayer more fluid.

The Cell

1. Compartmentalized chemical reactions

2. Modify intra- and extra- cellular environment

3. Different properties and functions.

Individual cells will direct the function of tissues and organs

Approaches to Cell Biology Research

Genetics

•Screen for mutants with a phenotype.

•Crosses to define complementation groups.

•Details of the phenotypes. Divide into classes.

•Order the classes by epistasis.

•Clone the genes.


Anatomy

•Structure of cells and tissues.

•Ultrastructure (EM), to detect fine structures, such as filaments or membranes.

•Correlate structures with function.

•Identify molecules if possible.


Biochemistry

•Purify molecules, such as metabolites, proteins, or even membranes.

•Study their chemical properties in vitro.

•Attempt to re-create in vitro a phenomenon observed in vivo.

•Reconstitution as an ultimate test for “sufficiency.”


Physiology

•Observe the phenomena exhibited by living cells or organisms, such as movement.

•Quantify parameters such as rate of movement and ask how they correlate with each other factors.

•Decrease or increase the activity of a component.


Pharmacology

•Find drugs (chemicals) that inhibit or enhance a phenomenon (growth, movement, etc.)

•Identify their molecular targets, such as proteins.

•Use in physiology studies to inhibit a process acutely.

Hypothesis-Driven Experiments

•State the hypothesisNot a “straw man” or trivial

•State the experiment

•Possible outcomes

•Interpretation of each outcome

•Controls - positive, negative

•Limitations and Alternative Interpretations

“Proof” of a Hypothesis or Model

Observed Results as Predicted

What Alternatives are Excluded?

•Karl Popper, The Logic of Scientific Discovery, 1934

•How strong is the evidence against the alternatives?

•Obligation to raise and test credible alternatives

•Or the ones that others find compelling

Revolutions and Paradigms

•Thomas Kuhn, The Structure of Scientific Revolutions, 1962

•Evidence against the current paradigm is the most interesting and important

Kinetic analysis• How cells change over long time periods (development, long term adaptive changes; hours - years)

• Movement of proteins and membranes within cells - dynamics of cellular events (sec - hrs)– Pulse chase analyses– Real time imaging: GFP and other fluorophores allow measurement of trafficking, diffusion, etc. (time-lapse, fluorescence recovery after photobleaching (FRAP), etc.)

• Kinetics of molecular interactions, enzyme reactions (msec - min)

Catalyst (enzyme): increases rate of a reactionSubstrate: molecule on which enzyme acts to form product

S ------> P enzyme Free energy of reaction not changed by enzyme. For a favored reaction (ΔG negative), enzymeaccelerates reaction.

Graph:ΔG* = activation energyΔG negative overall for forward reaction

Enzymes are catalysts for chemical reactions in cells

Active Site: Region of the enzyme that does the work. Amino acid residues in this site assume certain 3D conformation, which promotes the desired reaction.

What does the Enzyme do to cause catalysis?

• High affinity for substrate in its transition state, facilitating transition to product• Increased probability of proper orientation of substrates• Increased local concentration of substrates• Has atoms in places that push the reaction forward• Change hydration sphere of substrates

Enzymes as Catalysts

Phases of Enzyme Reactions• Transient phase

– Accelerating Velocity– Short (<1s)– Formation Enzyme-Substrate

Intermediates• Steady-state phase

– May Not Occur– Constant Velocity– Duration up to Several Minutes– Little Change Levels of Enzyme– Small Fraction Substrate

Consumed– Small Levels Product Formed

• Exhaustion phase– Decreasing Velocity– Depletion of Substrate– Accumulation of Product– Inactivation of Unstable Enzyme

Need an assay that measures the product of the chemical reaction. For example...

Enzyme β-galactosidase catalyzes this reaction: lactose --------------------> glucose + galactose

Measure the amount of glucose or galactose over time.

Trick - use a substrate that produces a reaction product that absorbs light (creates color). Measure absorbance.

How to Measure Enzyme Activity at Steady State

ONPG = ONP-galactose (ONP = o-nitro-phenol) ONPG --------------> galactose + ONP

(colorless) (colorless) (yellow)

X-gal = X-galactose (X = 4-chloro-3-bromo indole) X-gal ---------------> galactose + 4-Cl-3-Br-indigo (colorless) (colorless) (deep blue)

Measure absorbance with a spectrophotometer•Beer’s law - concentration proportional to absorbance•96-well format instruments

Color-Producing Substrates for β-galactosidase

Optimizing assay

• No Enzyme -> No Product• Optimize pH, salt, other buffer conditions

• Optimize temperature• Choose set of conditions to be kept constant

• Amount of enzyme – Linear range of assay– More is better

Run the Assay at Different Substrate Concentrations

Plot initial rate (v0)

vsConcentration of Substrate

[S]

Michaelis-Menten Plot

• What’s interesting or useful about this plot?

• Can we use this plot to compare results for different enzymes or conditions?

• Can we derive an equation for the curve?

How Km values affect metabolism

• Glucose + ATP --> glucose-6-P + ADP + H+

• Typical cell [glucose] = 5 mM• Two enzymes catalyze above reaction

– Hexokinase• Km (glucose) = 0.1 mM• Km << [S], so velocity independent of [glucose]• Reaction is inhibited by product--regulated by product utilization

– Glucokinase• Km (glucose) = 10 mM• Km > [S], promotes glucose utilization only when [glucose] is high

• Reaction not inhibited by product--regulated by substrate availability

Determining Km and Vmax

• Estimate Vmax from asymptote, Km from conc. at Vmax/2

• Curve fitting w/ computer programs, inc Excel• Visual inspection (Graph paper)• Lineweaver-Burke plot and others

Michaelis-Menten equation can be rearranged into “Lineweaver-Burke” equation

From this graph, visually estimate Km and Vmax.

Regulating enzyme activity

• Allosteric regulation• Reversible covalent modifications• Enzyme availability (synthesis, degradation, localization)

• Substrate availability (synthesis, degradation, localization)

• Inhibition– By specific metabolites within the cell– By drugs, toxins, etc.– By specific analogues in study of reaction mechanism

Competitive inhibitor:

• binds to free enzyme

• prevents simultaneous binding of substrate

-i.e. competes with substrate

• Apparent Km of the substrate is therefore increased

• High substrate concentration:- substrate overcomes inhibition by mass action- v0 approaches Vmax (which does not change)

Competitive Inhibition

Example of Competitive Inhibition

• EtOH Rx for MeOH poisoning

• Methanol (ingested from solid alcohol, paint strippers, windshield washer fluid, etc.) is metabolized by alcohol dehydrogenase to formaldehyde and formic acid. Leads to metabolic acidosis and optic neuritis (from formate) that can cause blindness.

• Treatment: Infuse EtOH to keep blood concentration at 100-200 mg/dL (legally intoxicated) for long enough to excrete the MeOH.

• EtOH serves as a competitive inhibitor. Ethylene glycol poisoning is treated in the same way.

Noncompetitive Inhibition

Noncompetitive inhibitor :

• Binds to a site on the enzyme (E or ES) that inactivates the enzyme

• Decreases total amount of enzyme available for catalysis, decreasing Vmax

• Remaining active enzyme molecules are unaffected, so Km is unchanged

Uncompetitive Inhibition

Uncompetitive inhibitor:

• Binds specifically to the [ES] complex (and inactivates it

• Fraction of enzyme inhibited increases as [S] increases

• So both Km and Vmax are affected

Summary: Types of Inhibitors

• Competitive– Binds Free Enzyme Only– Km Increased

• Noncompetitive– Binds E and ES– Vmax Decreased

• Uncompetitive– Binds ES only– Vmax Decreased– Km Decreased

Plots to Distinguish Types of Inhibitors

• Competitive

• Uncompetitive

• Noncompetitive

No inhibitor

No inhibitor

No inhibitor

Lineweaver-Burke Plots show curves with no inhibitor vs. presence of two different concentrations of inhibitor

Reading and Homework for Kinetics

• Alberts (5th edition) pp. 159-166• Lodish (6th edition) pp. 79-85• See website for self-assessment assignments

bob mercer, [email protected] 362-6924; 991-1243 molecular cell biology 5068

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