BIS104 MT1

Dinesh-Kumar Cell Bio UCD SQ2025

How are coronaviruses (CoV) named?

Named for the crown-like spikes on the surface and belong to the family Coronaviridae within the order Nidovirales

What do coronaviruses infect?

vertebrates such as humans, birds, bats, etc

SARS-CoV-2 (COVID) Genome

single stranded; positive sense (can be directly translated into proteins)

Flu/Influenza virus

ss negative sense RNA virus (needs RdRp, cannot be directly translated)

What did Robert Hooke discover?

Termed pores inside of cork as cells bc they reminded him of the cells inhabited by monks. Were actually empty cell walls made of dead plant tissue

What did Antonie van Leeuwenhoek discover?

• Examined pond water and observed microscopic “animalcules”

• Saw bacteria from peppercorn water and dental plaque

• Credit for discovering living cells, first to see bacteria

Cell Theory; who was it articulated by and what was added later

• Articulated in mid-1800s by Matthias Schleiden, Theodor Schwann, Rudolf Virchow

  1. All organisms are composed of one or more cells

  2. The cell is the structural unit of life

  3. Cells arise only by division from a pre-existing cell

• Added since:

  4. Cells contain genetic information (DNA) passed to next cell generation

Prokaryotic vs Eukaryotic Cell Differences

• Prokaryotic

  • bacteria

  • Genetic material in nucleoid

  • single circular chromosome

  • cytoplasm is devoid of membranous structures

• Eukaryotic

  • Plants, animals, protists, fungi

  • Genetic material is membrane bound (nucleus)

  • single linear molecule of DNA

  • More complex (structurally and functionally)

Prokaryotic vs Eukaryotic Cell Similarities

• Share an identical genetic language, a common set of metabolic pathways, and many common structural features

• Both may be surrounded by a rigid cell wall that protects the cell

• Both bounded by plasma membranes of similar construction, serving as selectively permeable barrier

Lecture 3 04/04

What are the six model organisms for eukaryotic cells

a. Escherichia coli (E. coli/ bacteria)

b. Saccharomyces cerevisiae (Brewer’s yeast)

c. Arabidopsis thaliana (thale cress/ flowering plant)

d. Caenorhabditis elegans (roundworm/ nematode)

e. Drosophila melanogaster (fruit fly)

f. Mus musculus (mouse)

Light microscope

uses refraction of light rays to magnify an object

Light Microscope: Condenser lens

Directs light toward the specimen

(more technical description - gathers the diffuse rays from the light source and illuminates the specimen with a small cone of bright light that allows very small parts of the specimen to be seen after magnification)

Light Microscope: Objective lens

collects light from the specimen

Light Microscope: Ocular Lens

Forms an enlarged, virtual image

Light microscope: Resolution

Ability to distinguish two points:

• Numerical aperture measures the lens light-gathering qualities

• Limit of resolution depends on the wavelength of light

Light microscope: Visibility

• requires specimen and background to have different refractive indexes

• Stains add color and contrast

• Suitable for tissue slices and non-living cells

3 ways to study cells

1. microscope

2. Genetics: Break the function of genes

   • difficult to conclude sufficiency

   • redundancy

3. Biochemistry

What does it mean when a gene is necessary for a function?

The function cannot happen without that gene. If you knock it out and the function is lost, it’s necessary

How do you experimentally test if a gene is necessary?

Use a knockout experiment by removing or disabling the gene and observe if the function is lost

What does it mean when a gene is sufficient for a function?

the gene alone can cause the function to happen, even in a system where it usually doesn’t occur

How do you experimentally test if a gene is sufficient?

Add the gene or protein to a new system that normally doesn't do the function and it now does, the gene is sufficient

Limit of Resolution Equation

D = (0.61*λ) / (n*sinø)

• n*sinø = numerical aperture (N.A)

• sinø = maximum amount of light collected (1.0)

• λ = wavelength

• n = refractive index (R.I)

  • air =1.0

  • oil = 1.5

  • H₂O = 1.3

Limit of Resolution for:

1. Naked Eye

2. Light Microscopy

3. TEM

4. Fluorescence Microscopy

1. Naked eye = ~200 µm

2. Light microscopy = 0.2 µm

3. TEM = 2nm

4. FM = 0.2 µm (still a light microscopy)

Lecture 4 04/07

Transmission Electron Microscopy (TEM)

• Uses electrons instead of light → very short wavelength → higher resolution (2nm)

• Can see membranes and very fine structures

TEM limitations

• requires extensive sample prep

• not commonly used unless necessary

• needs training; typically done through collaboration

Transmission Electron Microscopy:

1. Limit of resolution

2. Magnification

3. What helps provide contrast

4. What kind of images may be taken

1. Limit of resolution = 10-15 Å

2. Magnification enhanced 10⁵ times due to increased resolution

3. heavy metals stains provide contrast

4. photographic emulsion or digital images may be taken

Fluorescence Microscopy

• Useful for in live-cell imaging

• Uses fluorophores that absorb light (excitation) and emit longer wavelength light (emission)

• Works best with flat or thin cells/tissues

• Limitation: blurred images in thick staples due to multiple focal planes and out-of-focus light

Fluorescence Microscopy 3 parts to setup

1. First barrier filter

2. Beam-splitting mirror

3. Second barrier filter

Fluorescence Microscopy: 1) First Barrier Filter (light & wavelength)

lets through only blue light with a wavelength between 450-490nm

Fluorescence Microscopy: 2) Beam-Splitting Mirror

Reflects light below 510 nm but transmits light above 510nm

Fluorescence Microscopy: 3) Second Barrier Filter

Cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520- 560 nm

Fluorophores

Molecule that absorb photons of a specific wavelength and release a portion of the energy in longer wavelengths

Fluorescence Components

• Fluorophores: Used to visualize otherwise transparent cells/structures

  • Fluorescein: excite w/blue light (450-490 nm) → emit green (520-580 nm)

  • Rhodamine: excite w/green light (535 nm)→ emit red (610 nm)

Fluorescence Microscopy: Common Dyes

• DAPI: stains chromatin → nucleus appears blue

• Mitotracker Red: stains mitochondria

• Other dyes based on target and desired emission color

Immunofluorescence Microscopy

• Used when specific dyes don’t exist for certain proteins

• A technique that uses fluorescently labeled antibodies to visualize specific molecules. Relies on the principle that antibodies bind specifically to their target antigens

• Fluorochrome-conjugated antibodies are used to locate specific cellular structures

Direct Immunofluorescence

• Antibody binds target protein

• Antibody is directly conjugated to fluorophore

• Simple but weaker signal

Green Fluorescent Protein (GFP)

• Can be recombined with genes of interest in model organisms

• Expressed with the host gene of interest

• Used to follow a gene of interest

Indirect Immunofluorescence

• Primary antibody binds proteins

• Secondary antibody (w/fluorophore) binds primary antibody and carries fluorescent dye

• Signal Amplification: multiple secondaries can bind one primary

Immunofluorescence Microscopy Primary/Secondary Antibody Example

• Labeling Golgi with an antibody to a known Golgi protein

• Use DAPi (nucleus), rhodamine (Golgi), fluorescein (tubulin) for multi-color labeling

• Primary antibody has to be very specific to mitochondria

Lecture 5 04/09

Laser Scanning Confocal Microscopy

• Produces an image of a thin plane located within a much thicker specimen

• Solves thickness/image clarity issue in fluorescence microscopy

• a laser beam is used to examine planes at different depth in a specimen

• computer can compile images for 3-D modeling

Key advantages and disadvantage of Confocal Laser Scanning Microscopy

• Advantages:

  • pinpoint illumination using lasers

  • better control of light wavelength via illuminating aperture

  • pinhole instead of barrier filter to precisely collect light

  • allows optical sectioning (taking images at multiple focal planes

  • Uses photomultiplier to combine signals into crisp 3D images

• Disadvantage:

  • Very expensive ($600k-1M), usually available through core facilities

Live Cell Imaging

• A technique that allows researchers to observe living cells (cell activity) over time

• Avoids traditional dyes (which kills cells)

• Uses YFP, RFP, GFP

  • Proteins are fused genetically (gene A + GFP gene → fusion protein)

GFP Excitation and Emission Wavelengths in Live Cell Imaging

• Excitation

  • 375nm - 480nm

• Emission

  • 510 nm

Yellow Fluorescent Protein (YFP) Excitation and Emission Wavelengths in Live Cell Imaging

• Excitation

  • 513 nm

• Emission

  • 527 nm

Red Fluorescent Protein (RFP) Excitation and Emission Wavelengths in Live Cell Imaging

• Excitation

  • 558 nm

• Emission

  • 583 nm

Fluorescent Protein Spectra Problems and Solutions

• Problem: Spectral overlap; too similar to be used together (GFP and YFP)

• Solution: Use fluorophores with non-overlapping emission spectra (GFP and RFP)

GFP may affect…

• localization

• function of the protein

How would you confirm GFP fusion is accurately representing the original protein?

• Knockout gene A, reintroduce A-GFP, check if function is restored (valid)

• Change GFP position (N-terminus vs. C-terminus), if only one works then position matters

• Biochemical methods to confirm location (subcellular fractionation)

Differential Centrifugation

• Allows the isolation of particular organelles in bulk quantity

• Isolated organelles can be used in cell-free systems to study cellular activities

Pellet

Heavier components settle to the bottom depending on speed

Supernatant

Lighter materials or liquid above the pellet

Subcellular Fractionation (Differential Centrifugation) Spin Speed (pellet and supernatant)

• 600g

  • Pellets: Nucleus

  • Supernatant: Everything else

• 15,000g

  • Pellet: mitochondria, lysosomes, peroxisomes

  • Supernatant: ER, Golgi, Plasma Membrane, Cytosol

• 100,000g

  • Pellet: membranes (ER, Golgi, plasma membrane)

  • Supernatant: Cytosol

Subcellular Fractionation Process

1. cell homogenization fragments cytoplasmic membranes

2. Vesicles derived from the end-membrane system from similar sized vesicles (microsomes)

3. microsomal fraction can be fractionated into smooth and rough fractions

4. Once isolated, the biochemical composition of various lipid and protein fractions can be determined

Sucrose Density Centrifugation

Organelles separate into bands at their equilibrium density in sucrose gradient

Sucrose Density Gradient Centrifugation Involves

• solubilizing membranes using mild detergents

• layering on a 0.5-5M sucrose gradient

• Spinning at high speed (~65,000)

Sucrose Density Gradient Centrifugation Bands

• ER - Upper Layers

• Golgi - Middle

• Plasma Membrane - lower

• Mitochondria - Even lower

• Lysosomes - Around same as mitochondria

How to detect proteins in Isolated Fractions

• SDS-PAGE (sodium dodecyl sulfate, polyacrylamide gel electrophoresis)

  • SDS denatures and gives proteins uniform negative charge (unfolds into linear protein)

  • Proteins separated by size only

SDS-PAGE Steps ( Part of Western Blotting Process)

1. Run SDS-PAGE

2. transfer proteins to a membrane

3. Incubate with primary antibody (specific to target protein)

4. Detect with secondary antibody conjugated to HRP (enzyme)

5. Add substrate → produces color/light at protein band

Where in the cell does protein C reside?

A. Cytosol

B. Golgi

C. ER

D. Nucleus

E. Mitochondria

E. Mitochondria

Where in the cell does protein A reside?

A. Cytosol

B. Golgi

C. None of the above

D. Nucleus

E. Mitochondria

B. Golgi

Where in the cell does protein B reside?

A. Cytoplasm

B. Golgi

C. None of the above

D. Nucleus

E. Mitochondria

D. Nucleus

Positive and Negative Control for Protein localization

• Positive Control:

  • Known nuclear protein detected to confirm successful nuclear isolation

• Negative Control:

  • Known cytoplasmic protein should not be detected in nuclear fraction

  • If detected → contamination of nucleus w/cytoplasmic material

Lecture 7 04/14

Plasma Membrane

• the outer boundary of the cell that separates it from the world

• is a thin, fragile structure about 5-10nm thick (which is why we wouldn’t use light microscopy-0.2um)

• Need electron microscope to examine

• All membranes examined closely from plants, animals or microorganisms have the same ultrastructure

Functions of Cell Membranes in Plant Cell: Compartmentalization Example

Vacuoles/lysosomes contain acid hydrolases functioning at low pH (sequestered)

Functions of Cell Membranes in Plant Cell: Enzyme Housing (localization)

CO2 fixation catalyzed by enzyme associated with outer surface of thylakoid membranes of the chloroplasts (energy production)

Functions of Cell Membranes in Plant Cell: Selective Permeability/Barrier

Water molecules are able to penetrate rapidly through plasma membrane, causing pressure against cell wall

Functions of Cell Membranes in Plant Cell: Transport

Membrane transporters import/export ions, solutes (ex. H+) into extracellular space

Functions of Cell Membranes in Plant Cells: Signal Transduction

Receptors (ex. growth hormones) on membranes initiate intracellular responses, as a result of response to external stimuli

Functions of Cell Membranes in Plant Cells: Cell-cell communication

• Plants: Plasmodesmata

  • materials move directly from cytoplasm of one cell to its neighbors

• Animals: gap junctions

Ernest Overton - 1895

• Showed that membrane is semipermeable

• More soluble the dye, the better it enters the cell

  • Tested on root hair cells where the more lipid-soluble a solute was, the more rapidly it entered

• Membrane is made of of lipids

Gorter and Grendel - 1925

• Used RBCs to extract lipids

• Found lipid bilayer via monolayer spreading experiments through modified Langmuir trough.

  • Calculated surface area of RBC membranes, found 2:1 ratio of lipid to cell surface area

Membrane lipids are ____ which contain both hydrophilic and hydrophobic regions. Name three main lipid types.

• amphipathic

• Phospholipids (names based on polar head)

• Sphingolipids

• Cholesterol

What is membrane fluidity dependent on?

• Saturated vs. unsaturated fatty acids

  • The more unsaturated fatty acid you have, membrane will be more fluid

  • The less unsaturated fatty acid you have, membrane will be less fluid

• Other factors include temperature, cholesterol

How would lipids move between bilayers

• Flippase - energetically impossible without it

• Cell membrane would likely be in liquid, crystalline state at warmer temps around 37ºC and rotate axis/move laterally through plane

Danielli A Davison 1935

• discovered membranes also contain proteins

• Experimented by observing oil droplets from fish eggs absorbing proteins

Frye and Edinin 1970

Discovered proteins are mobile within the bilayer

• Experimented by fusing human and mouse cell

Lecture 8 04/16

Singer & Nicolas (1972)

Discovered Fluid Mosaic Model

• Membrane is fluid: both lipids and proteins can move (mobile)

• Membrane is a mosaic: contains different lipids and proteins interspersed

FRAP (Fluorescence Recovery After Photobleaching)

• Used when you want to know whether protein is static or mobile on membrane

• Study lateral movement of proteins within the membrane

FRAP Steps

1. Fuse protein to GFP

2. Bleach a membrane region with laser (confocal microscopy)

3. If fluorescence recovers → protein is mobile

4. If it doesn’t recover → protein is static

List the Membrane Proteins

• Multipass Membrane

  • Extracellular peripheral membrane protein

• Singlepass Membrane

  • Intracellular peripheral membrane protein

• Lipid-Anchored Proteins-

  • Acetyl

  • Prenylated

  • GPI

• Beta Barrel Proteins

Integral Membrane Proteins (Transmembrane)

• Typically contain one or more transmembrane helices

• Function as receptors that bind ligands, channels, or transporters to move ions/solutes across the membrane

• Amphipathic

  • Single pass: One hydrophobic region → crosses membrane once

  • Multi-pass: Multiple hydrophobic regions → crosses multiple times (beta barrel proteins)

Hydrophobicity Plot

Used to identify hydrophobic alpa-helices (15-20 amino acids) in integral proteins

Peripheral Membrane Proteins

• Noncovalently (loosely/weak electrostatic) bonded to polar head of lipid bilayer and/or integral membrane protein

• Dynamic relationship with membrane, being recruited or released as needed

Lipid Anchored Proteins: What they do and lipid names

• Covalently bonded to a lipid group that resides within the membrane

  1. Myristoylation (or Acetylation)

  2. Prenylation

  3. GPI Anchored Protein

Lipid Anchored Protein: Myristoylation Example

• Modification at glycine/methionine

• Anchors protein to cytoplasmic side

• GFP must be fused to C-terminus to avoid blocking anchoring

Lipid Anchored Protein: Prenylated Example

• Modification at cysteine near the C-terminus (CAAX motif)

• Anchors protein to cytoplasmic side

• GFP must be fused to N-terminus

Lipid Anchored Protein: GPI Example

• Added in Golgi

• Anchors protein to Outer (extracellular) leaflet of membrane

• No transmembrane domain required

Functional Importance of Membrane Proteins

• Approx. 30% of human proteins are membrane-associated

• Difficult to study due to solubility and extraction issues

• Critical for signaling, transport, structure