cell structure exam 1

Robert Hooke

developed compound microscope, famous for microscopical slices of cork, coined the term “cell”

Leeuwenhoek “simple” microscope

could magnify 275x and discovered bacteria, protists, blood cells, sperm, nematodes

Zeiss oil immersion

oil matched refractive index of glass, made NA 1.4 and had resolving distance of 0.2 microns

Nomarski differential interference contrast (DIC) microscopy

studies live specimens and unstained tissues, gives 3D structure

Cell Theory

all living organisms consist of cells
cells arise by division from other cells

Coagulating fixatives (chemical)

ethanol, methanol, acetone

Pros:

• fixes specimens rapidly by changing hydration state

• proteins coagulated/extracted

• antigen recognition preserved

Cons:

• shrinkage of specimens

• inaccurate 3D imaging

Cross-linking fixatives (chemical)

glutaraldehyde, formaldehyde (preferred), ethelene glycol-bis-succinimidyl succinate (EGS)

forms covalent cross-links that are determined by active groups of compound

Formaldehyde

forms methylene bridges between reactive groups and nucleic acids (cross-links)
reaction functional groups:

• amido

• guanidino

• thiol

• phenol

• imidazole

• indolyl

not a good preservative for microtubules

Why use light microscopy in cell biology?

to study live samples with good resolutions

Resolution

the minimum distance at which two distinct points of a specimen can still be seen

Resolution of objective lens

d = 0.612(wavelength)/NA
NA = nsin(alpha)

Optical lens

bends light, focuses/defocuses light, forms an image

Optical filter

selectively transmits light having certain properties (wavelengths) while blocking the remainder

Total magnification

multiply the eyepiece power by the objective power

Cover slip

protects objective lens from contacting the specimen and creates an even thickness for viewing

Limitations of Light Microscopy

wavelengths of light vary from 400-700nm but light diffraction limits resolution to 200-250nm —> magnification limited to 1500x

Phase Contrast Microscopy

produces high-contrast images of transparent, living specimens by translating variations in phase into corresponding changes in amplitude

Refractive index

n = c/v

n = refractive index
c = phase velocity of a wave phenomenon (light or sound) in a reference medium
v = phase velocity in medium itself

Diffraction

the tendency of light to bend around objects

Constructive interference

waves that combine in phase add up to a relatively high irradiance, coherent

Destructive interference

waves that 180degrees out of phase cancel out and yield zero irradiance, coherent

Incoherent addition

waves that combine with lots of different phases nearly cancel out and yield very low irradiance

Irradiance

power of EM radiation at a surface per unit area (Wm^-2)

Negative phase contrast

surround wave travels through more material and is retarded in phase, materials with larger optical path length (OPL) appear darker

Positive phase contrast

surround wave travels through less material and is advanced in phase, materials with larger optical path length (OPL) appear brighter

What role does the condenser annulus play in phase contrast?

it generates a hollow cone (ring shaped) illumination so only a ring of light enter the condenser —> ensures the specimen is illuminated in a way that produces direct and diffracted light

What role does the phase plate play in phase contrast?

it selectively retards or advances the ring-shaped light relative to the diffracted light coming from the specimen —> converts invisible phase differences into amplitude intensities, creating contrast

Confocal microscopy

generates high resolution images and 3D reconstructions

types:

• laser scanning

• spinning disk

• slit scanning

Why can confocal microscope provide high resolution images?

two confocal pinholes allow for high resolution in xyz, pinhole 1 focuses on a small area of the specimen and pinhole 2 selects the in focus signal

Lateral resolution

0.61*(wavelength)/sqrt(2)NA

Axial resolution

2(wavelength)n/sqrt(2)NA²

optimal pinhole size

pinhole = 2.5(wavelength)M/piNA
M = magnification

Why can confocal microscope provide a 3D reconstruction of a specimen?

• sectioning

• scanning

• reconstruction from 2D to 3D via computer

optical sectioning by confocal detection

the confocal pinhole is in the conjugated plane of the focal plane

types of confocal microscopes

laser scanning
spinning disk
slit scanning

Point scanning

high spatial resolution, slow speed, fixed samples

Spinning disk

fast due to parallelized detection, live cell imaging

Slit scanning

intermediate speed, balance between resolution and photodamage

Super-resolution microscopy

allows images to have resolutions higher than those imposed by the diffraction limit

Single molecule biology

one molecule —> one signal
versus ensemble of molecules gives one wide signal

single molecule fluorescence techniques

allow for manipulation and measurement of single biological molecules within a cell/culture —> reveals action on molecular level

fluorescent probes

organic dyes (fluorophone)

fluorescent proteins

quantum dots

dyed polymer particles

GFP (green fluorescence protein)

unique protein that emits green color in blue/UV light (comes from jellyfish)

Single molecule localization principle

image the fluorophones individually over multiple cycles and combine the images to get an accurate representation

STORM and PALM

stochastic optical reconstruction microscopy and photoactivated localization microscopy

—> utilize functions to localize centroids of individual fluorophones and reconstitute these centroids to form super resolution images

resolutions of microscopes

conventional light microscope ~200nm
phase contrast microscope ~200nm
laser scanning confocal microscope ~140-180nm
super resolution light microscopy ~1-100nm

properties of water

universal solvent (dissolves polar molecules and ions)
high specific heat
high latent heat of vaporization
cohesion (between water molecules) and adhesion (between water and other polar molecule)
density

surface tension

a measure of the force necessary to stretch or break the surface of a liquid (hydrogen bonds)

protein function

structural
signaling
catalysts
immunity
gene regulation
transport
poisons

peptide bond

amino acids are joined together in a polypeptide chain through the formation of a peptide bond to make proteins

amide character in peptide bond

the peptide bond is also an amide and undergoes resonance —> therefore rigid and have some double bond character

ampholyte

amino acids can act as an acid or base —> can have both acidic and basic functional groups

amino acids classification

polar charged
polar uncharged
nonpolar
side chains with unique properties

Polar charged amino acids

aspartic acid
glutamic acid
lysine
arginine
histidine

have hydrophilic side chains that can act as acids or bases that tend to be fully charged (+ or -)

polar uncharged amino acids

serine
threonine
glutamine
asparagine
tyrosine

have hydrophobic side chains that have partial charge (+ or -)

nonpolar amino acids

alanine
valine
leucine
isoleucine
methionine
phenylalanine
tryptophan

have hydrophobic side chains (almost entirely C and H), associate with lipid bilayer

unique amino acids

glycine
cysteine
proline

glycine

has one H atom side chain —> can fit in either hydrophilic or hydrophobic environment
resides where two polypeptides are close together

cysteine

side chain has polar, uncharged character
forms covalent bonds with another cysteine to form a disulfide bond

proline

side chain has hydrophobic character
creates kinks in polypeptide chains and disrupts secondary structure

essential amino acids (need through diet)

histidine
isoleucine
leucine
lysine
methionine
phenylalanine
threonine
tryptophan
valinen

nonessential amino acids (synthesized in body)

alanine
arginine
asparagine
aspartate
cystine
glutamic acid
glycine
ornithine
proline
serine
tyrosine

polypeptides

40-50 amino acids
too small to form functional domains, not yet a protein

primary structure

simply sequence of amino acids in polypeptide chain

secondary structure

the folded structure that forms within a polypeptide due to interactions between atoms
alpha helix or beta pleated sheet (parallel or anti parallel) —> held together by H-bonds between the carbonyl-O and amino-H of amino acids

tertiary structure

due to interactions between the R groups of amino acids including hydrophobic interactions, disulfide bonds

disulfide bond

pairs of cysteines form disulfide bonds between different parts of the main chain; adds stability and is common in extracellular proteins

quaternary structure

when multiple polypeptide chain subunits come together
folded proteins bind together to form dimers, trimers, and high order structures

Non-covalent bonds

required to maintain secondary, tertiary, quaternary structure (H bonds, electrostatic/salt bridges, van der Waals)

Salt bridges

electrostatic bonds between oppositely charged groups
strength usually 4-7 kcal/mol

Hydrogen bonds

formed by sharing of proton between donor and acceptor groups
strength around 2-5 kcal/mol, ideal distant 2.8-3 angstrom

van der Waals

formed between dipoles of atoms

Hydrophobic interactions

results from inability of water to form hydrogen bonds with certain side chains

X ray crystallography

produces high resolution protein structures, based on scattering of X rays from electron density

Crystallization

if crystals are successfully formed, they can diffract to a high enough resolution

Post translational modifications of amino acids

Phosphorylation (typically Ser, Thr, Tyr)

• gain of charge, binding

Glycosylation (typically Asn, Ser, Thr)

• solubility, stability, binding

Acetylation/Acylation/Methylation (N-term, Lys, Arg)

• loss of charge, stability

Lipidation/phenylation (typically Cys)

• membrane anchoring

Ubiquitination/Sumoylation (Lys)

• degradation, signaling

Protein domains

functional regions within a protein that can perform specific roles

• SH2

• PTB

• SH3

• PH

• WW

• DD

• DED

• BH

Antibodies

highly specific

recognizes 8-10 amino acids, both sequence and conformation

terminal deoxynycleotidyl transferase (TdT)

adds diversity to antibodies by adding nucleotides between the V (variable), D (diversity), and J (joining) regions

activation-induced cytidine deaminase (AID)

makes random mutations (somatic hypermutation) in antibody variable regions, can make antibody more strongly bind to their target

gel electrophoresis

DNA fragments separated through agarose or polyacrylamide gels

DNA is negatively charged, migrates towards (+) electrode

gels are porous and separate based on size

Southern blotting

DNA isolated and resolved on gel
Nucleic acids are blotted by capillary action or by second electrophoresis
membrane is probed with complementary DNA sequence
probe binds to its complementary sequence

Restriction Fragment Length Polymorphism (RFLP)

result from base changes in nucleotide sequences and upon cleavage with restriction enzyme

used in direct detection of disease causing mutation, DNA fingerprinting, and linkage of polymorphism with gene mutation

polymorphism is the existence of two or more variants at significant frequencies in population

isolating RNA

cells broken with detergents
RNAase must be inactivated
RNA purified

Northern blotting

RNA —> gel electrophoreisis —> transfer to membrane —> probed with complimentary cDNA —> autoradiography

PCR

denaturation —> annealing of primers —> DNA synthesis
requires DNA polymerase, nucleotides, oligonucleotide primer, DNA template, cycling machineW

Western blot

protein —> gel electrophoresis —> transfer to membrane —> probed with antibody —> chromogenic reaction

EMSA (electrophoretic mobility shift assay)

probe : DNA —> variation can be RNA

SouthWestern assay

protein resolved —> transferred to membrane —> probed with DNA probe

use of antibodies

immunohistochemistry
protein localization
protein trafficking
blocking reagents

principles of centrifugation

separates particles based on size, shape, density, viscosity, and rotor speed; more dense particles sink

F = Mw²r

• M = mass

• r = radius of rotation

• w = avg angular velocity

• F = force

w = 2pi(rev)(min^-1)/60

differential centrifugation

based on differences in sedimentation rate of biological particles of different size, shape, density

DNA cloning

cut DNA at precise locations
attach DNA of interest to a cloning vector producing a recombinant DNA molecule
introduce recombinant DNA into a host cell
screen host cells containing recombinant DNA

cloning vectors

must have replication origin, selectable markers, and cloning sites

vector uses

propagation of cloned DNA fragment for further analysis and/or manipulation
construction/screening of genomic and cDNA libraries
shuttle vector —> shuttling cloned genes between organisms
expression vector —> expressing a cloned protein coding gene at relatively high levels for purification of the protein

insertion inactivation

selection strategy for distinguishing bacterial colonies harboring recombinant molecules from the colonies only containing the parent vector

DNA library

genomic —> contain introns, exons, promoters, enhancers, etc
complementary DNA (cDNA) —> only contains coding sequences for protein, relative abundance of sequences in a cDNA library will relate to the abundance of the original mRNA in the tissue of origin

screening a library

molecular probes:

• probes for DNA sequences

• probes for expressed proteins

overexpression

introduction of plasmid DNA, mRNA, proteins via microinjection, electroporation, viral mediated, genetic-based

Loss of function

gene silencing or knock-down (removing/inactivating a specific gene)