BIS104 MT01

Microscopy, Protein Detection, Model Organisms, Nuclear Transport, Vesicular Transport

What does each part of a light microscope do?

Ocular lens: ?

Objective lens: ?

Condenser lens: ?

Ocular lens: captures image

Objective lens: forms image

Condenser lens: converges light on specimen

What is the resolution limit for a light microscope?

0.2µm

What is the equation for the resolution limit?

r=(0.61λ)/(nsinθ)

smaller value means better resolution

What is the numerical aperature?

NA = nsinθ

The manipulatable variables of resolution (n=refractive index, θ=half angle cone)

How would you decrease d (better resolution)?

Increasing the value of NA:

Increasing the refractive index (n). For example, by using immersion oil (max n=1.6)

Increasing the half cone angle (θ) by decreasing the working distance (ie. space between the objective lens and specimen).

What would increase d (worse resolution)?

A small NA due to a small n and small θ (from large working distance)

T or F?

Fluorophores can only be excited once

True, they fall back to a different ground state

If fluorophores always emit light of a lower energy than they absorbed, what does that mean about the wavelengths?

Fluorophores absorb a shorter wavelength of light than they emit.

Filters and mirrors of fluorescence microscopy …

First barrier filter: ?

Beam splitter: ?

Second barrier filter: ?

First barrier filter: selects wavelength of light allowed to enter (eg. blue)

Beam splitter: reflects light of a wavelength below a value towards the specimen (incoming light) and passes light of a higher wavelength (light reflected off the specimen)

eg. reflects blue but passes green

Second barrier filter: cuts out unwanted background fluorescent signals

Explain indirect immunofluorescence microscopy

A primary antibody is directed against the antigen of interest and a secondary antibody is directed against the primary antibody. The secondary antibody is coupled with a marker (eg. GFP) to be visualized. 

If multiple antigens are being observed, the secondary antibody cannot be from the same species as any of the primary antibodies or else cross reaction is possible. Best to have all different species.

What is the advantage of genetically modified fluorescent proteins derived from those isolated in nature (eg. GFP from jellyfish & DsRed from coral)?

Mutating their amino acids allowed for variants that offer a greater variety of colors and are brighter. They can even be turned ON/OFF 

How could you tag a protein?

You could insert a GFP gene before the stop codon (recombinant DNA). You must preform a functional assay to ensure the protein of interest maintains its function despite the presence of GFP (eg. make sure GFP is not blocking any binding sites)

What is the advantage of confocal microscopy? And what are methods?

Confocal microscopy increases resolution (ie. decreases the resolution limit) for fluorescent microscopy.

1. Pinholes: exclude fluorescent signals outside of the focal point, blocking interference

2. Super resolution: artificially combining images from different cycles of activation

3. Expansion microscopy: adhering the specimen to a gel that expands in aqueous solution to make the specimen up to 16x larger

Explain super resolution

Super resolution depends on the foundation that each fluorophore is excitable only once. By using cycles of low light to gradually excite just a few at a time, signals are less likely to interfere with each other. The closer to the center of the source, the brighter the signal. Thus an artificial center can be mathematically assigned. Tens of thousands of small successive groups can be combined to reconstruct a clearer complete image.

Electron Microscopy

• Uses an electron beam rather than a light source

• The electron beam has shorter wavelength and high frequency than visible light

• Better resolution

Transmission Electron Microscopy (TEM)

• The electron beam penetrates specimen

• Density informs image

• Use very thin sectioned sample embedded in Spurr’s plastic

• Thicker slices with higher voltage can be used for 3D reconstruction

  • Computer tomography (CT)

  • Cryo-EM (used for resolving protein structure)

• d=0.1nm (Å range)

Scanning Electron Microscopy (SEM)

• The electron beam reflects off whole samples

• Allows observation of surface and subcellular structure

• Black indicates a low or no signal (like no electrons hitting the detector)

• White indicates a high signal (a lot of electrons hitting the detector)

• d=0.5nm-10nm

How is an analog signal converted to a digital signal?

original signal → sample signal → reconstructed signal

• Original signal - visually convenient

• Sample signal - convenient for measurements

• Reconstructed signal - digital form of image (slightly removed from original signal)

How does pixel size affect spatial resolution?

Lower pixel size increases spatial resolution

What is bit depth?

• X bit = 2^x steps of color

  • eg. 8 bits = 2^8 + 256 steps

• Greater bit depth allows greater intensity resolution

• Bit depths of different colors can be combined (RGB)

  • 24 bit = 8 bit (red) + 8 bit (green) + 8 bit (blue)

Bit depths to know …

Naked eye limit: ?

Typical commercial use: ?

Lab use: ?

Naked eye limit: 5 bits

Typical commercial use: 8 bits

Lab use: 16 bits

Methods of detecting intracellular proteins with probes …

1. Antibody probes

2. Separating proteins

3. Detection of protein-protein interactions

Antibodies aka. immunoglobulins (Igs)

• Heterotetramer (150kDa)

  • 2x light chains (25kDa)

  • 2x heavy chains (50kDa)

  • associated via disulfide bonds

• The variable region contains bivalent antigen antigen-binding domains

• Produced by effector B-cell clones

  • Each clone only produces one specificity (recognizes one epitope)

• NOT suitable for SDS-PAGE because they are denatured into heavy and light chain bands

Different Ig classes

IgG, IgM, IgA, IgD, IgE

• IgG - highest levels of opsonizationa. ndneutralization

• IgM - produced first, pentameric form

• IgA - expressed in mucosal tissues as dimer

• IgD - unknown

• IgE - involved in allergy response

How do you produce antibodies from a host?

Injection/Exposure → host’s immune response generates antibodies → collected via blood plasma (or chicken’s eggs)

• Repeat exposure until response is strong enough

• More alien the antibody, the more likeley antibody generation will be successful

Hybridoma

Effector B-cell fused to tumor cell to combine Ig production activity with infinite cell division

Why do antibodies need to be humanized for immunotherapy?

Antibody of foreign species would be attacked by immune system

How do you produce a chimeric antibody for immunotherapy?

1. Generation of highly specific and potent monoclonal antibody in host

2. Clone cDNA encoding chains of corresponding antibody

3. Make recombinant DNA by replacing gene encoding the constant region with human counterpart

4. Mass production of humanized chimeric antibodies in hosts like yeast, bacteria, or plants

Herceptin

• Monoclonal antibody immunotherapy example

• Ig interupts dimerization of receptors in order to prevent trigger of tumor cell proliferation and tags for destruction

• Anti PD-L1

  • Blocks T-cell PG-1 and tumor cell PD-L1 interaction so that T-cell kills tumor cell

Methods of protein separation …

• Differential centrifugation

• Column chromatography

• SDS-PAGE

• 2D SDS-PAGE

Explain differential centrifugation

Allows for a fractionation of cell contents via density. Each time test tube is centrifuged, supernatant is recollected and re-centriguged at a higher gravity.

Cell contents that are too close in density can be centrifuged in a sucrose gradient for finer separation

In what can cell contents be collected from density dependent centrifugation?

1. Nuclei

2. Mitochondria, lysosomes, peroxisomes

3. Plasma membrane, ER

4. Ribosomes

5. Cytosol

Ion-exhange column chromatography

• Separation based on charge

• Glass beads have positive charge that trap negative molecules

• Salt wash at different concentrations used to gradually elute trapped molecules

  • Stronger association elutes last

Afinity column chromatography

• Separation based on a specific ligand association with a specialized matrix

Gel-Filtration column chromatography

• Separation based on size

• smaller molecules get trapped in a “maze”

• Larger molecules elute first

SDS Polyacrylimide-Gel Electrophoresis (SDS-PAGE)

• Separates proteins based on molecular weight

• Smaller molecules migrate to the anode first

• Proteins coated with SDS (ionic detergent) give them a negative charge, causing proteins to migrate towards the anode (+ charge)

• Can also be treated with reducing agents (DTT, B-mercaptoethanol) to denature

2D SDS-PAGE

• Two proteins have similar molecular weight are very unlikely to also have the same charge weights

• 1. Isoelectric focusing (IEF) - separates based on charge

  • Proteins migrate laterally along a stable pH gradient (basic/high pH → acidic/low pH

  • Protein stops migrating at a pH that matches Pi

• 2. SDS-PAGE - separates based on size

  • Done second because SDS-PAGE denatures the protein and thus the Pi would no longer be accurate

What does the purification of protein complexes by affinity tags and their antibodies provide evidence for?

• Co-localization

  • Co-localization is NOT concrete evidence of direct interaction (ie. the proteins could be indirectly interacting)

  • Can inform the hypothesis of direct interaction

How to perform purification of protein complexes by affinity tags and their antibodies …

Uses a known protein as bait to find out what it forms a complex with

1. Bait protein is attached to a tag (eg. GFP)

2. Complex forms on bait in affinity column

3. SDS-PAGE

4. Excise bands and digest with trypsin

5. Analyze with mass spectrometry

Mass spectrometry

• separates peptides by their mass to charge ration (m/z)

• Different m/z causes different speed of migration which leads to unique peaks

• For tandem mass spectrometry, proteins are fragmented

How can you confirm direct protein-protein interaction?

1. Yeast two-hybrid assay

2. FRET

Explain a yeast two-hybrid assay

This approach is based on the foundation that yeast Gal4 TF is split into a binding domain (BD) and active domain (AD). The BD and AD must indirectly interact through intermediate proteins to activate transcription of the lacZ reporter gene.

Proteins X & Y of interest are fused to the BD & AD (X-BD & Y-AD). If X & Y directly interact the Gal4 TF will regain activity resulting in expression of the lacZ reporter gene visible through the production of a purple color.

Explain fluorescence/Forster resonance energy transfer (FRET)

This approach is based on the foundation that fluorophores emit a longer wavelength of light than they absorb. This means that different fluorophores can have overlapping excitation and emission wavelengths.

This approach is based on the foundation that yeast Gal4 TF is split into a binding domain (BD) and an active domain (AD). The BD and AD must indirectly interact through intermediate proteins to activate transcription of the lacZ reporter gene.

Proteins X & Y of interest are fused to paired fluorophores. If X & Y interact directly, they will be close enough that the emission from one fluorophore would excite the other. Thus, when exciting the system with only the excitation wavelength of the first fluorophore (and not the second), you would observe the emission wavelength of the second fluorophore instead of the first fluorophore’s.

eg. Excite a blue fluorophore with violet light → blue emission used to excite a green fluorophore → observer only sees green emission

Requirements of model organisms …

• Easy to grow

• Inexpensive

• Simple genetic make up

• Short lifetime

• Easy to manipulate

• Ethical to work with

Model organism applications …

• Discovering new genes

• Proving functionality of genes of interest

• Vaccine & antibody manufacturing

Zmapp

Polyclonal antibody cocktail produced in tobacco plants that combats Ebola

Protein Roadmap - Gated transport …

• Nucleus ←→ Cytosol

Protein Roadmap - Engulfment …

• Cytosol → Nucleus

• Cytosol → Lysosome

Protein Roadmap - Protein translocation …

• Cytosol → ER

• Cytosol → Mitochondria

• Cytosol → Peroxisomes

• Cytosol → Chloroplast & Plastids (in plants)

Protein Roadmap - Vesicular Transport …

• ER ←→ Golgi

• ER → Peroxisomes

• Golgi ←→ Early Endosome

• Golgi ←→ Late Endosome

• Golgi ←→ Secratory Vessicle

• Golgi → Plasma Membrane & ECF

• Early Endosome → Late Endosome

• Early Endosome ←→ Plasma Membrane & ECF

• Late Endosome → Lysosome

What organelles’ lumens are topologically equivalent to (or continuous with) the ECF?

• ER lumen

• Golgi lumen

• Transport vessicles

• Lysosomes

T or F? Cargoes of the secretory pathway will never touch the cytosol.

True

Nuclear lamina:

filamentous structural proteins of the nuclear envelope

Nuclear Pore Complex (NPC):

• collection of nucleoporins (nups)

• around 125 MDa (million Daltons)

• Cytosolic fibrils form a nuclear basket facing the ICF

• FG-nups with phenylalanine-glycine repeats form a gel mesh that acts as seive

Nucleus ←→ Cytosol through NPC traffic size limits

• <5000Da freely permeable

  • the larger, the slower

• >60kDa requires active transport

Why is spatial regulation of protein synthesis necessary for eukaryotes?

• So that introns are not translated

• Accomplished by the nuclear envelope keeping ribosomes out and immature mRNA in

Nucleus → Cytosol through NPC

• Mature mRNA

• tRNA

• ribosomal subunits (rRNA incorporated)

Cytosol → Nucleus through NPC

• Proteins needed for chromosome replication

• Proteins needed for transcription

• Ribosomal proteins (to build subunits)

Transmembrane protein translocation:

uses specific receptors that recognize signal peptides and translocators

Signal peptides:

amino acid sequences that are recognized by sorting receptors and inform protein destination

• On N-terminus if needed for co-translational transport

• C-terminus is more accessible and open to modifications

• Sequences can be spaced out in primary structure but revealed in 3D folded arrangement

Signal peptides on N-terminus …

• ER import signal

• Mitochondria import signal

• Plastid import signal

Signal peptides on C-terminus …

• Peroxisomes import signal

• ER retention signal

ER import signal

• On N-terminus

• Includes a long hydrophobic amino acid sequence

ER retention signal

KDEL

Nuclear localization signal (NLS)

• Nuclear import signal

• Key basic residues

• Positive amino acids

• Experiment with frog eggs showed NLS was in the tail region of the protein of interest

G-Proteins:

• Capable of binding guanine in GTP/GDP form

• Function as molecular switched (involved in regulation)

Ran Proteins:

• Nuclear G-protein small enough to difuse through NPC when NOT loaded

• Also a GTPase

  • Can hydrolyze its bound GTP to cause dissociation from effector

• Active state = Ran-GTP

  • Can bind to effectors

• Inactive state = Ran-GDP

  • Cannot bind to effectors

3 classes of regulation of Ran-GTP/GDP

1. GAP

2. GEF

3. GDI

cytosolic GTPase Activating Protein (GAP)

• Accelerates GTP hydrolysis

• Facilitates Ran-GTP → Ran-GDP

• GAP in cytosol

• If there is more GAP present, then Ran is more likely in its inactive state

nuclear Guanine Nucleotide Exchange Factor (GEF)

• Promotes the exchange of GDP for GTP

• Facilitates Ran-GDP → Ran-GTP

• GEF in the nucleus

• If there is more GEF present, then Ran is more likely in its active state

GDP Dissociation Inhibitor (GDI)

• Sequesters bound GDP to lock the inactive state when cell is at rest

• Counteracts GEF

Importin:

• Recognizes NLS

• The binding of Ran-GTP in the nucleus causes the importin to release its cargo

• Dissociation of Ran-GDP in the cytosol allows the importin to pick up cargo

Exportin:

• Recognizes nuclear export signal (NES)

• The binding of Ran-GTP in the nucleus causes the exportin to bind its cargo

• Dissociation of Ran-GDP in the cytosol allows the exportin to release the cargo

Cholesterol biosynthesis is regulated by spatial sequestration of regulatory nuclear localization

Negative feedback loop

• High cholesterol inhibits cholesterol synthesis

  • Cholesterol binds INSIG+SCAP, which acts as a lock, sequestering the TF SREBP to the ER

• Low cholesterol favors cholesterol synthesis

  • When unlocked, a protease cleaves SREBP so that it is free to enter the nucleus, where it activates transcription of cholesterol genes

  • The interaction of SCAP+SREBP is also necessary

Ran GTPases impose directionality on nuclear transport through NPCs

• Due to enzyme localization …

  • Cytosol contains mostly Ran-GDP

  • The nucleus contains mostly Ran-GTP

• The gradient of the two forms drives nuclear transport in the correct direction

Proteins shuffling back and forth between the nucleus can have both import and export signals. How is this regulated?

• Calcium, calcineurin, and phosphorylation

• In cytosol, dephosphorylation causes calcineurin to block export signal

• In nucleus, phosphorylation causes calcineurin to release and phosphates mask the import signal

Protein import into the mitochondria and chloroplast …

• Positive aa on top of 3D structure

• Requires assembly of translocators on membrane

  • One acts as receptor of signal peptide

  • TOM & TIM for mitochondria

  • TOC & TIC for chloroplast

• These are double-membraned organelles with 3 intracellular spaces

• Active transport

• Import signals get cleaved → mature proteins are smaller than their cytosolic precursors

T or F? 90% of the proteasome is made in the cytosol.

True, therefore post-translational translocation is necessary.

T or F? Translocated proteins are smaller than their cytosolic precursors.

True, because their import signals get cleaved by peptidase

What does vesicular transport offer?

• Neuronal activities (NT exocytosis)

• Blood vessel health (endocytosis of pathogens + debris)

• B-cell exocytosis for secretion of antibodies

• Secretion of digestive juices via exocytosis

• Healthy eyes (tears + moisture)

T or F? Pathogens often hack membrane traffic machinery.

True!

Secretory pathway:

• aka. Biosynthetic

• Outwards from ER → Golgi → Early/Late Endosomes (→ Lysosomes), Secratory Vessicles, PM, ECF

Endocytic Pathway:

• aka. Uptake

• Inwards from PM

Retrieval Pathway:

• aka. Recycling

• Returning molecules to their residing organelles

Pulse-Chase experiment

• Performed in pancreatic acinar cells (make digestive enzymes)

• Pulse phase: cells fed with radioactive aa (3H-leucine)

• Chase phase: cells fed with regular aa

• Alternation of phases allows to track batches of synthesized proteins

• Shorter pulse phase = better acuity

  • But trade off with visibility

• Discovery of secretory pathway: ER → Golgi → Secretory granules

How did we find out sec12 encoded GEF protein

Because enlarged ER in sec12 mutants indicated vesicles couldn’t leave ER

How did we find out sec17 encoded SNARE protein

Because sec17 mutants accumulated vesicles in their cytoplasm, indicating they couldn’t enter the Golgi

How do we determine the hierarchal relationship among genes?

Examining double mutants to study up/down stream effects

Endoplasmic Reticulum (ER)

• Continuation of the nuclear envelope

• Starting point of the secretory pathway

• Constantly being remodeled in living cells

• Dynamic tubular structures

• Sheet-like structures

  • Large ER sheets are a sign of ER stress

How do proteins enter the ER?

• A. Co-translational translocation

  • more common

  • mRNA brought to ER and transcribed by ER-bound ribosome directly into the ER lumen through a channel

• B. Post-translational translocation

  • Protein translated by free ribosome in cytosol

  • Guided by chaperons

Microsome experiments

• ER near impossible to isolate so broke down ER and homogenized

• ER derived lipids spontaneously reformed into microsomes (smaller compartments)

• Centrifugation in sucrose gradient separated smooth and rough ER

  • Rough ER denser bc ribosomes

• Able to study ER

Discovery of ER import signals

• labeled protein of interest

  • If it remained in the microsome, then it occurred via co-translational translocation and thus required an ER import signal

  • If it was found in the supernatant, then it occurred via post-translational translocation

• ER import signal always cleaved thus mature protein is smaller than one made at free ribsosome

Co-translational translocation process

1. Signal Recognition Particle (SRP) recognizes and binds to the signal sequence of a growing peptide

2. SRP targets translation machinery to ER by binding to SRP-receptor on ER membrane coupled with the protein translocator (how ribosomes end up docked on rough ER)

3. Protein translated through channel of protein translocator

4. Peptidase cleaves the signal sequence and the protein is released into ER lumen

5. SRP is released and recycled

Insertion of transmembrane proteins into the ER membrane

• Modulated by start-stop signals determined by switch of aa charges (cytosolic side is + and ER lumen is -)

  • START = + → -

  • STOP = - → +

• Hydrophobic domains left in membrane

• Sequence between start-stop is transmembrane and inside ER

Protein maturation inside the ER lumen

• Signal peptidase cleaves signal peptide at N-terminus

• Glycosylation starts in ER

  • N-linked glycosylation assists with protein folding (Glycosylation site is NXT or NXS)

  • Lipid linked oligosaccharide anchored in ER membrane is transferred to the asparagine (N) residue of translocating protein

• Folding

  • Disulfide bonds form between cysteine (C) residues

  • Chaperons ensure proper folding and assembly (ie. BIP)

• ER performs quality check before shipping out proteins

Binding Protein (BiP)

• Chaperon in ER that retains incompletely assembled antibodies in the ER

Golgi apparatus

• Sorting of secretory vesicle

• Very dynamic

• Polarized

  • Entry face = Cis

    • Vesicles added to this face

    • Only retrograde vesicles leave this face

  • Exit face = Trans

    • Vesicles bud off from this face

• Each cisternae is biochemically specialized

Cis Golgi Network (CGN):

• anterograde or retrograde transport

Trans Golgi Network (TGN)

• Protein sorting station

• Anterograde transport (onwards only)

Golgins

• Proteins that highlight the membrane of the Golgi

• Necessary for interaction with vesicles