Midterm 1 Flashcards
Light microscopy
Used to see live cells, colour, and whole tissues
Can target what you want to see
Whole tissues can be shown
Resolution limit = 0.2 nanometres (usually)
Transmitted Light Microscopy (TLM)
Bright Field Light Microscopy
Light passes through specimen and viewed
Optical techniques to increase the contrast of unstained living cells
Tissues must be cut into thin sections to see cells in them
types of Transmitted Light Microscopy (TLM)
Bright field
Dark field
Phase contrast: microscope shifts light and produces more contrast for more detail
Differential interference- contrast (DIC)
Emitted (Fluorescence) Light Microscopy (FLM)
Fluorescent molecules absorb light at specific wavelength (coloured), and emit light at a different wavelength which is viewed
Purpose is to visualize certain molecules or structures in cells
Molecules or structures are fluorescently labeled
Epifluorescence microscopes are used to illuminate the whole sample with a light source and the emitted light from the fluorescent label is detected
Fluorescence Light Microscopy (FLM) Structures inside can be label using:
- Fluorochromes: aka fluorophore, fluorescent chemical compounds
- Fluorochrome-linked antibodies: yellow fluorescent proteins (YPF), many different colours
- GFP and GFP variants (famous protein from jellyfish): green-fluorescent proteins (GFP)
types of Fluorescence LM
Immunofluorescence
Epifluorescence
Confocal
Direct Fluorescent Labeling: some fluorescent dyes can bind to structures and label:
membrane
Nucleus (binds DNA)
Mitochondria
Cytoskeleton
Indirect stain: immunofluorescence
Localizes proteins of interest in a cell using primary antibodies
Secondary Antibodies covalently linked to a fluorescent molecule recognize the primary antibody; provides signal amplification
Confocal fluorescence microscopy
use of lasers and optical sectioning removes out of focus light (increases resolution of light microscope).
Incoming light is focused on a single plane
Out-of-focus fluorescence form the specimen is excluded
Confocal microscopy cuts optical slices through sample
Advantages/disadvantages of Light Microscopy
Advantages: Can use color Can use live, whole cells Can track cells Cheap and easy to use
Disadvantages:
Can’t see smaller structures (organelles, ribosomes, etc)
Lower resolution
Electron Microscopy (EM)
Resolution limit = 0.2nm
higher resolution
Images are often black and white
Scanning Electron Microscopy (SEM)
The sample is coated with metal
Electron beam is focused on the specimen
Secondary electrons are knocked out of the specimen
A detector collects these shattered secondary electrons to build an image
Advantages/disadvantages of SEM
Advantages:
Can view surfaces (images appear 3D)
Disadvantages:
Cells must be dead
Complex specimen preparation; heavy metals- bit toxic
Microscope is expensive
Transmission Electron Microscopy (TEM) Advantages/disadvantages
Prepared specimens are sliced very thinly
Advantages: details of cytoplasm can be seen
Disadvantages: Cells must be dead Complex specimen preparation Difficult to know 3D shape of structures Plane of section: things look different from how you cut it High resolution
Features of Biological Membranes
- The membrane is a bilayer
- Biological membranes are made up of the phospholipid bilayer, and also have lipids, proteins, sterols, glycolipids, carbs, etc.
- All biological membranes are phospholipid bilayers but not all phospholipids are biological membranes - The membrane is organized but fluid- lipid
- The membrane has different permeability for different types of molecules
- The membrane is asymmetric
4 kinds of lipids:
Fatty acids, cholesterols/sterols,
Phospholipids,
Triaclyglycerols
Fatty acids
(micelle)
Fatty acids have a single tail & hydrophilic head group. They form micelles instead of bilayers or liposomes because of their shape. The hydrophilic heads face the aqueous environment.
cholesterols/sterols
Not a lot of opportunities for hydrogen bonding
Sterols are big and bulky carbon rings, with a little hydrophilic hydroxyl group.This means that they will form a layer on the water surface with the OH groups facing the water
Phospholipids
two fatty acids tails and hydrophilic head groups are necessary for formation of these structures in water. The polar heads face the water while the fatty acid tails form a hydrophobic core
Triaclyglycerols (triglycerides or triacylglycerols)
have three fatty acid tails, but do not form layers as they lack a polar head group
3 fatty acids esterified to a glycerol
Storage form of fatty acids
Neutral fats form oil droplets, not bilayers
Not strong hydrophilic group; not amphipathic
Thermodynamics of the hydrophobic effect
Minimum energy conformation (most stable) achieved by minimizing exposure of hydrophobic groups to water
Free energy of the system is minimized if the hydrophobic region (lipid tails) cluster together to limit contact with water, increasing the motional freedom of water
Water likes to form hydrogen bonds with other water molecules (energetically favorable- entropy of hydrophobic molecules decrease, but entropy of water increases)
These hydrogen bonds are continually breaking and re-forming; water molecules are constantly rotating as well
To form bilayers, lipids need to be _______ and the _________
amphipathic, right shape
Membrane fluidity
Lateral diffusion (2D movement) Both proteins and lipids can move within the 2D plane
How could a cell change its lipids to maintain appropriate fluidity?
- Degree of unsaturation in lipids; fatty acid saturation
- Higher number of saturated lipids; more tightly packed; more Van der Waals interactions; less fluid
- Higher number of unsaturated lipids; more kinks in the fatty acid tails due to double bonded structure; more fluid - Fatty acid tail length
- Shorter tails (<18) are more fluid
- Phospholipids with shorter fatty acid chains have less surface area & therefore fewer van der waals interactions - Amount of sterol in the membrane
How does the amount of sterols change membrane fluidity?
- At low temperatures, sterols can increase fluidity by preventing tight packing of fatty acids (fewer Van der Waals interaction)
Sterols will make spaces in the membrane and increase fluidity - At high temperatures, the ring structures of sterols act to ‘stiffen’ the cell membrane. This is because the sterols provide more surface area to form more van der waals interactions with the fatty acid tails
Lipid rafts
Microdomains in the plasma membrane rich in specific types of lipids (sphingomyelin & cholesterol)
Lipid rafts are thicker than other regions of the cell membrane- much more ordered
How does attachment to structures inside/outside the cell/membrane protein adhesion to other molecules affect membrane fluidity?
The important take-away is that the attachment of membrane proteins to the cell cortex, ECM, etc will result in these becoming anchored proteins. As such, they are no longer going to be able to move laterally in the membrane bilayer
Integrins
proteins embedded in the membrane attached to fibronectin which make sure integrins stay stabilized
Adhesion to protein outside the cell (ECM = extracellular matrix)
cadherins
Adhesion to neighboring cells- attach together and stay together
Cell to cell adhesion molecules (cadherins) linking the plasma membranes of neuronal cells
Tight junctions
Barriers to diffusion: Tight junctions are adhesions between neighboring epithelial cells that form ‘kissing points’ between the two cells so nothing leaks in between the cells
Membrane permeability is dependent on the properties of the molecules
Gases and hydrophobic molecules diffuse freely across lipid bilayer- no problem interacting with hydrophobic core Small uncharged polar molecules diffuse well across lipid bilayers Diffusion of large uncharged polar molecules across lipid bilayers is negligible Charged substances (ions) cannot diffuse across the lipid bilayer
Primary Structure - sequence of amino acids (peptide bonds)
Start with N-terminal
end with C terminal
Peptide bonds (covalent bonds between amino acids)
Read in groups of 10
Amino Acid Residue - amino acid has been incorporated into a primary structure
Secondary Structure- backbone interactions (H-bonds): alpha-helix
H-bonds are:
Are repetitive all the way along the backbone of the alpha-helix
H- bonds from one amino (n) group and carbonyl (n+4) that is 4 positions away
These interactions do not involve side chains/R-groups; many sequences can adopt helical structure
Parallel to the long axis of the helix
R- groups project outwards
Nothing can travel through alpha-helix “pore”
Too tight and no space for molecules to travel through
Secondary Structure Backbone interactions (H-bonds): beta-sheets
R groups point up & down alternating away from the peptide backbone
R groups are above or below the plane of the sheet; thereby have different properties from one side to the other
Beta sheets are usually twisted and not completely flat
Can be parallel and antiparallel on the same sheet
Protein domains: how proteins are functionally organized
Secondary structure elements fold into domains within a tertiary structure
Examples of protein domains:
Transmembrane domain- part that functions to be part of a membrane DNA binding domain- helps enzymes bind to DNA with domains Catalytic domain (which carries out enzymatic activity) cAMP(cyclic-AMP) binding domain- specific site or domain cAMP an important signal molecule
Tertiary & quaternary structure depend on….
and types of interactions include……….
side chain interactions (R-groups) Types of interactions: Hydrogen bond Ionic bonds Van der waals interactions Disulfide bonds
Bilayer and protein structure formation is similar as both are driven by thermodynamics in terms of
Non-covalent & covalent interactions ensure the most stable final conformational state
Increase the stability of the system
R-groups in the right position to facilitate H-bonds
Integrated proteins
proteins directly attached to the membrane; amphipathic. Can monomeric or multimeric
Asymmetry: the orientation of transmembrane proteins matter; the leaflet of attachment matters
Transmembrane protein - through membrane
Monolayer- associated with one layer of the membrane
Lipid-linked - covalently attached to lipid and the lipids are directly attached to the bilayer
Peripheral proteins
bound to membrane surfaces through non-covalent association with other membrane proteins
Asymmetry: different proteins attach to different sides
Attached to membrane indirectly
Transmembrane domain
part of a membrane protein that passes through the lipid bilayer
Most transmembrane domains are alpha helices, though some are beta barrels (larger pore)
To make pore, need multiple alpha- helices or a beta-barrel
The inner core of the beta barrel:
Hydrophilic and polar
In this environment facing inwards, there’s water. They need to interact with water and form H-bonds
The R-groups have to interact with the aqueous environment. The ability to form H-bonds with water molecules will stabilize this protein, and hydrophilic/polar R-groups will facilitate formation of these H-bonds.
The outside of the beta barrel:
Hydrophobic and nonpolar
They are interacting with fatty acids and lipid tails
The R-groups have to interact with the strongly hydrophobic environment made up of the fatty acid tails. The ability to form Van der Waals interactions will stabilize this protein, and hydrophobic/nonpolar R groups will facilitate this
At the top and bottom of the barrel
Hydrophilic and polar
The R groups have to interact with the aqueous environment. The ability to form H-bonds with water molecules will stabilize this protein, and hydrophilic/polar R-groups will facilitate formation of these H-bonds. Plus, there are some hydrophilic head groups from the phospholipids that will play into this too
