Properties of a Cell

-cells are the fundamental unit of life and exhibit all characteristics of life
● Organelles do not; cannot reproduce themselves outside of host cell

Properties/characteristics of All Cells

-cells have 4 basic features:
● Plasma membrane
● Cytosol: semifluid substance
● Genetic material
● Ribosomes - make proteins
-all cells have metabolism - chemical pathways to convert energy into biochemical work

Cell Theory
-historical cell theory:
● All living things are composed of one or more cells
● Cell is basic unit of structure and organization in organisms
● Cells arise from preexisting cells
-added components to cell theory:
● Activity of organism depends on total activity of independent cells
● Energy flow (metabolism) occurs within cells
● Cells contain hereditary info in form of DNA/RNA
● Cells of similar organisms have similar composition

Prokaryotes
-prokaryotes vs eukaryotes:
Prokaryotes Eukaryotes

No nucleus or membrane bound organelles Presence of nucleus and membrane bound organelles

ribosomes ribosomes

Cell membrane Cell membrane

Split into bacteria/archaea Eukaryotes: protists, fungi, plants, animals

DNA in nucleoid and plasmids DNA packaged in chromatin (w/ histones) in

nucleus

Translation can begin before transcription finishes Transcription in nucleus; translation in cytoplasm

No mRNA processing mRNA processed before translation

1. prokaryote structure:
● Small (0.5-5µm)
● Lack membrane-bound organelles
● Nucleoid: single circular chromosome
○ Nucleoid is attached to cell
membrane; can’t move around freely
● Plasmid: small circular DNA molecule
(contain antibiotic resistance)
● Can have flagella or cilia
-divide by binary fission
2. Plasmid
-not all prokaryotes have a plasmid:
● Requires energy to maintain and reproduce
● If a cell is in an environment where it doesn't need antibiotic resistance, it can survive w/ out a plasmid
○ Reproduces by binary fission w/ out wasting energy/space on plasmid
● if no antibiotics are present, prokaryotes w/ no plasmid reproduce quicker, have selective advantage
-why have a plasmid?
● Plasmid may provide antibacterial resistance, which gives cells w/ plasmids a selective advantage
● Plasmids cause bacteria to take longer to reproduce, but help survival if there is selective pressure of
antibiotics
○ Prokaryotes w/ no plasmid are killed off, and prokaryotes w plasmid survive

-bacterial develop antibiotic resistance quickly b/c they can transfer plasmids b/w one another through a pilus
● Occurs during bacterial conjugation

3. Cell wall
-bacterial cell wall:
● Very strong and resists bursting - maintains shape
● Composed of peptidoglycan (protein+carb)
● Can be used to classify bacteria into 2 categories
-types of prokaryotic cell walls:
● GRAM positive:
○ Thick peptidoglycan layer
○ Plasma membrane
● GRAM negative:
○ Outer membrane
○ Thin peptidoglycan layer
○ Plasma membrane (inner)
-penicillin can kill GRAM-positive bacteria
● Interferes w/ peptidoglycan synthesis
● Has no effect on eukaryotic cells b/c they lack peptidoglycan cell walls
● GRAM-negative bacteria are more likely to be antibiotic resistant

Eukaryotes
1. Plants, animals, and fungal cells
-eukaryotic cells have similar organelles
● Minor differences (i.e. plants/photosynthetic eukaryotes have chloroplasts)
-all eukaryotes and prokaryotes have plasma membrane (double layer of phospholipids)
● Acts as selective barrier that allows sufficient passage of oxygen, nutrients, and waste through cell
-eukaryotes have more complex cytoskeleton than prokaryotes
-some eukaryotes have cell wall:
● I.e. plants and fungi, cell wall composition differs b/w them

-main breakdown of eukaryotic cells:

● Nucleus
● Cytoplasm: can be divided further into
○ Cytoplasmic inclusions: i.e. organelles, stored
nutrients, secretory products, cytoskeleton
○ Cytosol: liquid component of cytoplasm, aqueous saline
solution
Nucleus
-stores genetic material, DNA
-nuclear DNA is packaged into chromatin fibers
-Chromatin: eukaryotic DNA+proteins (twice as much protein as DNA)
● Majority of proteins are histones, but also contains non-histone proteins
○ Equal mass of histones and non-histone proteins
● Chromatin is usually uncondensed, but condenses to form chromosomes during cell division
● Components of chromatin:
○ Nucleosome: basic structural unit of chromatin
■ Nucleosome core particle: DNA wrapped around H2A, H2B, H3, H4 histones, and
sealed by H1 histone
■ Linker DNA: b/w nucleosome cores, contains
non-histone proteins
○ Histones: small proteins, contain high proportion of
basic amino acids (arginine and lysine)
■ +ve histone binds to -ve DNA

Nuclear Envelope
-defines boundary of nucleus
-is a double membrane: consists of inner and outer membrane
-has nuclear pores throughout membrane
● Allows nucleus to communicate w/ cytosol
-structure:
● Inner and outer nuclear membrane
○ Perinuclear space: b/w the membranes
● Nuclear lamina: fibrous network that provides structural support to
nucleus
● Nuclear pore complexes: channels for molecules travelling in and
out of nucleus

Nuclear pores
-form nuclear pore complexes (NPCs)
-what can travel through NPCs?
● Proteins:
○ DNA binding proteins (i.e. histones, non-histone proteins, activators/repressors)
○ mRNA-binding proteins
○ Lamins (components of nucleus)
○ Ribosomal proteins
● RNAs:
○ mRNAs, tRNAs, rRNA (component of 40S and 60S ribosomal subunits)

Nucleolus
-suborganelle of nucleus; mass of dense granules and fibers
-where ribosomes are assembled (40S and 60S subunits) and rRNA is synthesized

Ribosomes in Eukaryotes
-both in eukaryotes and prokaryotes
-composed of rRNA and proteins
-function: protein synthesis, assemble amino acids into polypeptides
-location: free in cytosol or bound to ER membrane (only in eukaryotes)
● Proteins synthesized by free ribosomes vs ribosomes attached to ER have diff fates
Endomembrane System
-membrane system inside of cell - forms multiple compartments
-components: nuclear envelope, ER, Golgi apparatus, Lysosomes, Vacuoles, plasma membrane
● Components are continuous (membrane bridges) or connected via transfer by vesicles

-nuclear envelope: outer nuclear membrane is continuous w/ rough ER, and perinuclear space is continuous w/
ER lumen

-ER and Golgi apparatus sort/transport proteins destined for:

● Secretion out of cell, incorporation into plasma membrane, or incorporation
into lysosomes
-proteins transported from ER, through Golgi apparatus, and to plasma membrane or
lysosome

1. Endoplasmic Reticulum
-network of interconnected internal membranes
● Largest organelle (10% of cell volume, 50% of cell membrane)
● Extends from nuclear membrane throughout cytoplasms
● Contains single internal space - ER lumen
-3 main regions:
● Rough ER: has ribosomes on membrane surface
○ Synthesizes membrane components and secretory proteins
● Smooth ER: no ribosomes
○ Lipid synthesis, carb metabolism, Ca2+ storage, detoxification of drugs
● Transitional ER: region where secretory vesicles (w/ protein or lipids) exit en route to Golgi apparatus
○ Process: vesicles exit transitional ER, go to ER-Golgi Intermediate Compartment (ERGIC),
and then to Golgi apparatus
-ER structure:
● Rough ER: forms stacks of cisternae, and ribosomes are attached to cytosolic surface of rough ER
● Smooth ER: connected to cisternae and forms networks of tubules

2. Golgi Apparatus
-structure: not physically continuous w/ ER, series of flattened membrane
sacs (cisternae)
● Has 2 faces: entry/cis face and exit/trans face
○ Cis face: adjacent to ER
○ Trans face: points towards plasma membrane
-process:
● Vacuoles (w/ proteins/lipids) arrive to cis Golgi network from ER
● go through cis, medial, and trans cisternae (where they’re
processed/modified/packaged)
● Contents sorted/distributed by trans Golgi network to go to
destination
-function:
● Further modifies lipids/proteins produced by ER
○ I.e. proteins are further glycosylated
● Acts as sorting station
○ Proteins are sorted fro transport to destination (i.e. lysosomes, plasma membrane, secretion)
● Synthesizes sugars for glycoproteins
● Some lipids (glycolipids and sphingomyelin) are synthesized within Golgi complex
● In plant cells, Golgi apparatus produces polysaccharides of cell wall
3. Lysosomes
-found in animal cells, specialized vesicles derived from Golgi body
-structure:
● Full of hydrolytic enzymes that digest/degrade macromolecules and organelles
○ Contains 50 types of degradative enzymes: nucleases, proteases, lipases, glycosidases
● Membrane-bound proton pumps: keep internal environment at pH=5
○ b/c lysosomal enzymes are acid hydrolases, only active at pH=5, not at neutral pH=7
● H+ ATPase: in lysosomal membrane, maintains lysosomal lumen at acidic pH
-why don’t lysosomes digest themselves?
● Enzymes only act upon specific substrates
● Proteins can be tagged for disposal, allowing degradative enzymes to
digest them
● Glycocalyx is physical barrier b/w degradative enzymes and lysosome
membrane
-types of lysosomes:
● Primary lysosome: spherical, don’t contain particulate debris
● Secondary lysosome: larger/irregular shape, results from fusion of
primary lysosome w/ aged/defective organelles
-how do materials get to lysosomes (3 pathways):
● Endocytosis: molecule taken up from outside of cell
● Phagocytosis: large particle (i.e. bacteria) taken up from outside of cell
● Autophagy: digestion of aged/defective organelles (cell’s own components)
-mutations in genes encoding lysosomal acid hydrolases cause 30 diff human disease (lysosomal storage
diseases - under degraded material accumulates within lysosomes)

4. Vacuoles
-large vesicles from ER and Golgi apparatus
-functions:
● Nutrient storage
● Food vacuole - digestive function
● Contractile vacuole - expels excess liquid upon contraction
● Central vacuole (in plants) - regulates turgor pressure

Mitochondria
-functions: site of Krebs cycle, ETC, oxidative phosphorylation, ATP production
-structure:
● Surrounded by inner and outer membranes, b/w which is intermembrane space
● Inner membrane:
○ Folded into cristae (increases SA of membrane)
○ Contains ETC components (principal site of ATP synthesis)
○ Contains Krebs cycle enzyme - succinate dehydrogenase
● Outer membrane: converts lipid substrates into forms metabolized in matrix
● Mitochondrial matrix: interior, contains enzyme for metabolic processes
○ Enzymes responsible for oxidative breakdown of carbs/lipids via CAC
○ Contains circular DNA molecules (mitochondrial genome) and mitochondrial ribosomes
-reproduces through binary fission (like bacteria)
-vary in shape
Chloroplasts
-contained in plants and photosynthetic eukaryotic cells
-structure:
● Surrounded by double membrane
● Thylakoids: interconnected sacs flattened to form disks
○ Thylakoids grouped into grana, and embedded in stroma (matrix of chloroplast)
● 3 internal compartments:
○ Thylakoid lumen: inside thylakoid
○ Stroma: inside chloroplast but outside thylakoid membrane
○ Intermembrane space: b/w inner and outer membranes
-function:
● Photosynthesis: light rxns performed in thylakoid membrane during
daylight, produce ATP and NADH
○ Dark rxns in stroma use ATP/NADH to convert atmospheric CO2
into sugars (CO2 fixation)

Peroxisomes
-surrounded by single membrane
-produces hydrogen peroxide (H2O2), converting it to water using catalase
-functions:
● Fatty acid oxidation: produce H2O2 to break down fatty acids
○ Unlike mitochondria, peroxisomal fatty acid oxidation doesn’t lead
to ATP formation
● Decomposition of H2O2
● Plasmalogens biosynthesis: most abundant lipid in nervous tissue
● Convert stored fatty acids to carbs in germinating seeds

Cell Evolution
-prokaryotes were alone for 2.1 billion years before endosymbiogenesis occurred
-endosymbiogenesis: eukaryotic cells came from prokaryotic cells through symbiosis
● Form of horizontal gene transfer b/w prokaryote and proto-eukaryote
○ Bacterial genome incorporated into proto-eukaryote
-Process of endosymbiogenesis:
● 1st endosymbiogenesis event: cell swallows bacteria cell capable of aerobic respiration
○ Produced mitochondria
● 2nd endosymbiogenesis event: cell swallows bacteria capable of photosynthesis
○ Produced plastids (i.e. chloroplasts)

-endomembrane system arose through infolding of plasma membrane

● Allowed compartmentalization and increased SA to perform various cells
● Results in nuclear envelope, ER
● Independent of endosymbiogenesis (mitochondria/chloroplasts not part of endomembrane system)

-evidence of endosymbiogenesis (mitochondria/chloroplasts arose from bacteria)

● Size: similar in size to bacteria (1-10 microns)
● Structure: both have double phospholipid bilayers (inner/outer membranes)
○ Arose b/c primitive mitochondria/chloroplasts entered eukaryotic cells via endocytosis
● Function:
○ Mitochondria: similar characteristics to aerobic bacteria (use O2 in ATP production)
○ Chloroplasts: similar characteristics to photosynthetic bacteria (both have chlorophyll)
● Gene expression: similar to bacteria, both have circular DNA, RNA, ribosomes (70S)
● Reproduction: both divide independently of cell through binary fission (like bacteria)
Homology vs Analogy
-homology: genetically related
● Divergent evolution: 2 species w/ common ancestor experience adaptive radiation b/c of different
selective pressures
-analogy: not related genetically
● Convergent evolution: 2 species w/ no common ancestor experience convergent evolution b/c of similar
selective pressures
Simple and Compound Microscopes
-simple microscope: has one lens
-compound microscope: multiple lenses in parallel
-potential issues: light refracted by lenses doesn’t converge on single
focal point, resulting in blur/color on edges
● Chromatic aberration: b/c light of diff wavelengths has diff
refractive indices and leads to focus issues
● Spherical aberration: loss of definition in image due to
imperfections in lens surface geometry
● Solution: multiple lenses stacked together, forcing light to interact at same focal plane
○ Aka achromatic lenses

Light Microscope
-advantage: can see live cells
-disadvantage: individual cells are usually transparent, so difficult to see fine components

Visualization in Light Microscopes

1. Magnification
● Light source provides light from below, passing through sample and lenses, causing
magnification
● Occurs b/c lenses distort light and magnify the object
-lenses in a light compound microscope:
● Ocular lens (aka eyepiece): close to eye, always 10x magnification
● Objective lens (aka scanning objective): close to sample, magnification varies
-total magnification in compound microscope:
● Ocular magnification x objective magnification
● I.e. 10 (eyepiece)*40(objective)=400 times magnification

2. Contrast: make different things look different from each other

-can increase visualization by increasing contrast

Methods of increasing contrast:

-Staining w/ dyes - usually involves killing the cell
● Some stains keep cells alive (i.e. methylene blue)
● Commonly used dyes: haemotoxylin and eosin (most common), Giemsa stain, fluorescent dyes
● Stains interact w/ cellular structures differently, b/c each stain has different properties (i.e. chemical
charge, basic/acidic)
○ Haemotoxylin w/ Al salts stains acidic structures dark purple (basophilic dye)
■ Used to stain nucleus
○ Haemotoxylin counterstained w/ eosin, which stains basic structure light pink (eosinophilic
dye)
● When staining, use buffers to maintain pH
○ If pH changes, this changes the structure of the stain (i.e. H and E)
○ I.e. increasing pH: adds protons to eosin
○ I.e. decreasing pH: removes protons from haemotoxylin
-increasing contrast using properties of light (i.e. refraction, reflection)
● Brightfield: light passes from source directly through object and is
captured by objective lens
○ Normal microscope
● Darkfield: light blocked from passing directly below object to objective
lens
○ Light comes from around sample and bounces of edges
○ Scattered light captured by objective lens
○ Produces image w/ increased contrast
● Phase contrast: uses phase differences in travelling light wave and
converts it to differences in contrast
○ Caused by differences in refractive index in different components
of transparent sample

3. Resolution: minimum distance at which 2 points are distinguishable

● Higher resolving power=can see small things
○ Allows us to see objects as separate rather than single blob
● Human eye resolution: 100µ
● Theoretical light microscope resolution: 0.2µm
○ Objects close than these distances cannot be distinguished
-equation:

● d=minimum distance at which you can distinguish 2 objects

● λ=wavelength
● NA=numerical aperture (measuring of light gathering power of lens)
○ NA=nsinθ
-improving resolution:
● Decrease wavelength of light
○ Using electrons, which have smaller wavelength than photons
■ Resolution is theoretically unlimited but practically limited to 0.1 nm b/c of lens
● Increase NA (increasing θ or n)
○ Oil immersion: increases refractive index (n) by causing light to travel more slowly
■ Oil has larger refractive index than air
○ w/ oil immersion, resolution becomes:
Fluorescence Microscopy
-advantage: easier to distinguish and identify specific parts of cell and visualize specific molecules
by using dyes or fluorescent probes
● Unlike light microscopy, where live cells are often transparent
● Also, can be used on live cells
-mechanism behind fluorescence:
● Fluorescent molecules absorb light at one wavelength (excitation wavelength) and emit
light (fluoresce) at another, longer wavelength (emission wavelength)
○ Always emits longer wavelength b/c photons will have lower energy
○ Energy of photon inversely proportional to wavelength (E=hc/λ)
● 3 stages of fluorescence:
○ Excitation: photon supplied by external source (laser) and is absorbed by
fluorescent molecule, creating excited state (S1’)
○ Energy dissipation: energy of excited state is partially dissipated, yielding a
relaxed state (S1)
○ Fluorescence emission: photon is emitted, returning fluorescent molecule to
ground state (S0)

-optical path of fluorescent microscope:

● 1st barrier filter: lets through only blue light w/ wavelength b/w
450-490 nm
● Fluorescent dye, fluorescein, in specimen is excited by blue light
● Fluorescein emits light (fluoresces) at a specific and longer
wavelength (green light)
● Beam-splitting mirror: reflects light below 510 nm but transmits
light above 510 nm
○ Stops blue light, lets green light go through
● 2nd barrier filter: cuts out unwanted fluorescent signals, only
passing specific green fluorescein emission b/w 520-560 nm

-labelling structures w/ fluorescence microscopy:

*remember, some things naturally fluoresce (autofluorescence)
● I.e. chlorophyll autofluoresces under UV light

1. Structure-specific dye: some fluorescent dyes stain specific structures (i.e. DAPI)
● DAPI binds strongly to AT-rich regions in DNA and used to stain nuclei
● DAPI can pass through intact cell membrane, so can be used to stain live and
fixed (dead) cells

2. Immunohistochemistry: reveals specific proteins in fixed cells (dead)

-Uses antibodies to label specific/structures/molecules
● b/c antibodies are highly specific to an antigen
● Antibodies: Y-shaped proteins used by immune system to identify/neutralize
foreign objects
-process: aka immunofluorescence microscopy
● One primary antibody binds to target structure (antigen A) and another secondary antibody (w/
fluorescent probe attached) binds to primary antibody
○ Use of secondary antibodies results in amplification of fluorescent signal
○ Increases sensitivity of
immunofluorescence (easier to
detect target)
-structure of primary and secondary antibodies:

-different fluorescent probes can be used in the same cell

● Multiple lasers required to stimulate each fluorescent dye

3. Fluorescent reporter genes - i.e. GFP

-green fluorescent protein (GFP) of Aequorea victoria can be used as fluorescent label - reveals specific
proteins in living cells
● Naturally fluorescent protein: stimulated by blue light, emits green fluorescence
● Small in size - 27 kDa (small size ensures it doesn’t affect protein function significantly)
● Structure:
○ Barrel made of 11 beta strands and 1 alpha helix in middle
○ Fluoresces b/c of motif of 3 amino acids in alpha helix in center of barrel
■ Serine, tyrosine, and glycine so close together that their chemical groups
spontaneously bind together into GFP chromophore
● Why does GFP fluoresce: when excited by light, GFP goes from linear chain to cyclized backbone
w/ 5 atom ring

-how does this method work?

● Gene for GFP can be fused w/ other genes to study
expression and localization of specific proteins
● Fluorescent microscopy of GFP-tagged proteins follows the
localization and movement of protein of interest within
living cell
■ Wherever protein-of-interest is located,
attached GFP glows green

-what if you want to mark more than one protein?

● Need more than one color
● Achieved by mutating key amino acids around GFP
chromophore motif to produce changes in fluorescent color
○ Yellow GFP version - citrine
○ Cyan GFP version - mCFP
● Discovered another fluorescent protein, dsRED, from choral species Discosoma
○ Issue w/ dsRED: formers tetramer when you stick it on a protein
○ Through some mutations, scientists turned dsRED into monomer, mFRP (monomeric red
fluorescent protein)
■ Further mutated into mORANGE, mCHERRY, mPLUM
4. Ion Sensitive Indicators
-light emitting indicators can measure rapidly changing intracellular ion concentrations
-ion-sensitive indicators used to measure dynamic and temporal changes of:
● Intracellular ions (i.e. Ca2+, Mg2+, Na2+, H+)
● Cyclic AMP
● Cyclic GMP
● Kinase activity
-ion-sensitive indicators change in presence of these compounds
-example:
● Aequorin is luminescent protein from Aequorea victoria that emits blue light in
presence of Ca2+
● Aequorin injected into fish egg
● When egg fertilized by sperm, rapid rise in intracellular Ca2+ occurs as a wave
from site of entry by sperm
○ Shown by Aequorin

5. Confocal Microscope
-uses laser (1 pure wavelength of light)
-confocal microscope removes out-of-focus light from parts of cell above/below focal
point
● Generates a single, thin optical section
○ Gets rid of fuzziness/blurriness
-multiple optical sections can be digitally combined to create 3D image

6. Fluorescence Resonance Energy Transfer (FRET)

-looks for protein interactions in living cells
-intermolecular FRET: shows 2 specific proteins in cell are interacting
● Blue protein has BFP attached, and green protein has GFP
attached
○ When BFP shined w/ violet light, emits blue
○ When BFP shined w/ blue light, emits green
● When blue protein is on its own:
○ Shine violet light and produce blue light
○ Green protein isn’t excited
■ Shows no interactions
● When both proteins are interacting (coupled) in a living cell:
○ Shine violet light and emits blue light, which excites the fluorescent tag on green protein and
green light is emitted
■ Shows protein interaction
-requirement for intramolecular FRET: both proteins must be expressed at the same time
-intermolecular FRET: protein fused w/ 2 fluorescent proteins (i.e. BFP and GFP)
● Protein can either by inactive or active (requires change in structure)
-Process of intermolecular FRET:
● RFP and YFP fused on 2 diff parts of same protein
● When protein is inactive, RFP and YFP are too far apart. RFP activated and only fluorescence from
RFP is visible
● When protein is active, structure changes and RFP and YFP are brought close together
○ Activated RFP passess energy to YFP, emitting different color light
-proteins in intermolecular FRET are biosensors
● Show when show when cell is active or inactive in response to different stimuli

Electron Microscopy
-always involves fixation of cells - kills the cell and pauses it in single state
● Thin sections produced by microtome
○ Results in series of “cuts” through the cell
○ Must try to imagine 2D slices in 3D conformation
-an electron in an electron microscope w/ accelerating voltage of 100,000: λ=0.004nm
● In theory, resolution of electron microscope is 100,000 times greater than light microscope

Electron Microscope Structure

-larger than light microscope
-upside down:
● In light microscope, light source is below sample
● In electron microscope, light source is above sample
-uses beam of electrons, instead of beam of light
-uses magnetic coils to focus electron beam and create magnified
image of specimen, instead of glass lenses
-entire column is maintained under ultrahigh vacuum

Optical Path in TEM

-electrons are emitted by a cathode when its electrically heated
● Electric potential of cathode is kept at 50,000-100,000V
-electric potential of anode is zero
● Voltage drop causes electrons to accelerate as they move toward anode
-path details:
1. Electromagnetic condenser lens: focus electron beam onto specimen plane
● Doesn’t create magnified image of specimen
● Specimen must be thin - 50-100 nm thick
2. Electromagnetic objective lenses: YES magnification
● Pick up electrons passed through specimen
● Focus electrons on focal plane of objective lenses
● Create magnified image of specimen on focal plane
3. Electromagnetic projector lens: equivalent to ocular lens in light microscope, YES magnification
● Pick up electrons focused on focal plane of objective lens
● Focus electrons on viewing screen or photographic film
● Create magnified image of specimen on viewing screen or photographic film

Optical Path in Compound Light Microscope

1. Condenser lens: focuses light from bright source onto specimen
● Doesn’t create magnified image of specimen
2. Objective lens: pick up light transmitted by specimen and focus it onto
focal plane of objective lens
● Creates magnified image of specimen
3. Ocular lens (aka eyepiece): magnifies image on objective focal plane
and focuses it on objective focal plane (line of vision)

TEM vs Light Microscope Path

-TEM: resolution of 0.2 nm
● Electromagnetic condenser lens: NO magnification (focuses
electrons on sample)
● Electromagnetic objective lens: YES magnification
● Electromagnetic projector lens: YES magnification
-light microscope: resolution of 200 nm
● Condenser lens: NO magnification (focuses light on sample)
● Objective lens: YES magnification
● Ocular lens: YES magnification

Focal Plane in Electron Microscope

-in microscope, focal plane can be moved/adjusted
● Would result in a different view by going through diff slices of the cell
○ Focus on foreground, background is blurry

Staining for Electron Microscopy

-contrast in electron microscope depends on differences in electron density of organic molecules in cell
-efficiency of stain is determined by atomic weight of stain
-most stains used in electron microscope are heavy metals:
● I.e. uranium (uranyl acetate), lead (lead citrate), osmium (osmium tetroxide)

Immunogold electron microscopy

-antibodies attached to electron-dense colloidal gold particles (5-20 mm in
diameter)
● Antibodies interact only w/ specific antigen (i.e. catalase)
-thin sections of glutaraldehyde-fixed cells (or tissues) treated w/
gold-labeled anti-catalase antibodies
● Allows you to determine subcellular location of catalase in
electron microscope
● Gold particles indicate presence of catalase in peroxisomes (image
to right)
○ Gold particles show up as black dots b/c electrons don’t
pass through gold
TEM vs SEM
-transmission electron microscope (TEM)
● Good for visualizing internal features of cell
● Preparation of specimen:
○ fixed
○ Stained w/ electron dense material (i.e. heavy metal)
○ Embedded in resin
○ Thinly sliced
● Electrons pass through specimen and electron stained areas appear dark
● Resolution: up to 0.3 nm
● Magnification: 2,000,000
-scanning electron microscope (SEM):
● Good for visualizing external features (i.e. immunogold method wouldn’t be good for SEM)
● Heavy metals added to outside of specimen
○ Cells often coated w/ thin layer of gold
● Uses scattered electrons to produce 3D image
● Resolution: 3-20 nm
○ Lacks the resolution and magnification of TEM

-disadvantage of electron microscopy: only dead cells can be observed

Basic Components and General Functions of Plasma Membrane
-plasma membrane is boundary separating living cell from its surroundings
● In eukaryotes, cell membranes also form internal compartments
-functions of plasma membrane:
● Important/export of molecules
● Receiving info
○ Signaling b/w cells, sensing/responding to environmental changes
● Capacity for movement and expansion (i.e. amoeba)
○ Allows deformation w/ out tearing
● Internal membranes enclose intracellular compartments
○ Act as selective barriers b/w cytosol and organelle interior
○ Distinct internal chemistry of each organelle is b/c of subtle differences in organellar
membrane composition (i.e. diff membrane proteins)

Fluid Mosaic Model

1. Membrane composed primarily of phospholipids
2. Cell membrane are fluid - flexible and move
3. Cell membranes are a mosaic - other components are embedded within phospholipid bilayer
● Phospholipid bilayer associated w/ lipids and proteins
○ I.e. cholesterol, proteins (i.e. channel receptors), carbohydrates (i.e. glycolipids/glycoproteins)

Phospholipid Bilayer
-phospholipids: most abundant membrane lipids
● Amphipathic, have hydrophilic head and hydrophobic tail
● Structure:
○ 2 fatty acids linked to polar head group (w/ phosphate)

-formation of phospholipid bilayer:

● When phospholipids are added to aqueous solution, spontaneously form
sealed bilayer w/ hydrophilic heads on outside interacting w/ water, and
fatty acid tails interacting w/ each other
○ Results in most energetically favorable arrangement, b/c it avoids
exposure of hydrophobic hydrocarbon tails to water

Micelles, Liposomes, and Sheets

-phospholipids spontaneously form into bilayer sheets, liposomes, and micelles

-Cholesterol is transported in blood in natural micelles:

● LDL - low density lipoprotein: lipids+proteins
● HDL - high density lipoprotein
● Chylomicrons
-Natural micelles are produced from ER
● Lipid droplet builds up inside ER
membrane and buds off as a micelle

-transport of natural micelles throughout body:

● Cells produce HDL, which removes excess cholesterol from cell
● HDL is sent to liver, which converts HDL into bile salts
● Liver releases VLDL (very-low density lipoprotein), turns it into LDL
● LDL delivers cholesterol to cells
-micelles can be used to deliver drugs by targeting them to specific tissues
● Phospholipids added to solution containing drug, spontaneously forming micelle/liposome w/ drug
encapsulated inside
● Add target molecules onto outside of micelle, targeting it to a specific tissue/cell
○ Allows you to direct the drug to a specific tissue
● Liposome fuses w/ membrane of specific cell, releasing contents into cell.

Types of Phospholipids
1. Phosphoglycerides:
-Main structural features of glycerol phospholipid:
● 3 carbon glycerol backbone
● 2 long-chain fatty acids linked through ester bond to glycerol
● 3rd carbon in glycerol linked to phosphate
● Phosphate attached to different head groups

-phosphatidylcholine (0 net charge): most common type of phospholipid in cell membrane

● Has 2 fatty acids bound to glycerol
● Glycerol bound to phosphate group, which is attached to another small polar molecule (choline)
● Function: important structurally and used to make acetylcholine

-differences in chemistry (i.e. charge of phospholipid) results in differences in function (i.e interaction w/
enzymes)
-other glycerol phospholipids:
● Phosphatidylserine
● Phosphatidylinositol (-1 charge): important in cell signalling
● Phosphatidylethanolamine

2. Sphingolipids: can also be phospholipids, but non-glycerol phospholipid

-structure of sphingolipids:
● Contain sphingosine backbone (1 amino group and 2 hydroxyl groups) instead
of glycerol
● Structure of sphingomyelin:
○ 2 fatty acid chains bound to serine (not glycerol)
○ Serine is bound to phosphate group, which is attached to polar choline
● Function of sphingomyelin: sphingomyelin is important in myelin sheath
surrounding nerve cell axons

-4 major phospholipids in mammalian plasma membranes shown below:

Phospholipids in Archaea
-archaea: have branched isoprene fatty acid chains, ether linkage b/w fatty acid
and glycerol, and L-glycerol
-eukaryotes: have unbranched fatty acid chains, ester linkage b/w fatty acid and
glycerol, and D-glycerol

Cholesterol
-cell membranes contain up to 1 cholesterol for every phospholipid molecule
-structure:
● Amphipathic: hydrophilic head and one hydrophobic tail
● Consists of 4 hydrocarbon rings and single hydroxyl group
-cholesterol is a type of sterol related to testosterone
-unlike eukaryotes, fungi and protists use ergosterol in cell membranes instead of
cholesterol
● Ergosterol is a good target for antifungal drugs
○ Antifungal drugs only attack ergosterol, not cholesterol used in
eukaryotic organisms

Carbohydrates in Cell Membrane

1. Glycolipids: lipid w/ attached carbohydrate
-structure:
● 2 hydrocarbon chains of fatty acids linked to polar head group, which is attached to
a polar carbohydrate (i.e. glucose or galactose)
● Can have glycerol or sphingosine backbone
-location: found on non-cytosolic side of eukaryotic plasma membrane, the surface
● Makes them good for cell-cell recognition

-glycolipids are diverse in structure/function

● Need to be specific in function; identify different molecules
● Glycolipids contain variety of head groups
-function:
● Protect membrane from harsh conditions
● Alter electrical field across membrane due to charged sugars
● Help concentrate Ca2+ on outer membrane
● Cell recognition
● Provide entry point for bacterial toxins and viruses
● Bases for blood types ABO (sugars on outside of RBC membrane)

2. Glycoproteins
-cells recognize other cells by binding to molecules often containing carbs, on the extracellular surface of
plasma membrane
● Glycoproteins: carbohydrates covalently bound to proteins
-ABO blood groups based on cell surface glycoprotein markers
Membrane Fluidity
-3 types of phospholipid movements in bilayer
● Lateral movements: membrane lipids can diffuse laterally,
occurs 107 times per second
● Rotation: individual lipid molecules can rotate quickly around
axis
● Flexion: lipids can contract and expand
● Flip-flopping: transverse movement of lipid from one
monolayer to another
○ Rare, requires enzyme activity and doesn’t occur spontaneously
○ Catalyzed by flippase or scramblase

-factors influencing membrane fluidity

1. Length of hydrocarbon tail
● Shorter chain length increases fluidity of bilayer - reduces
hydrophobic interactions b/w tails

2. Level of saturation of hydrocarbon tails:

● Lipid bilayers w/ large proportion of unsaturated hydrocarbon
tails are more fluid than those w/ lower proportions
● Kinks caused by cis-double bonds prevent tight packing of
hydrocarbon tails
○ Kink produced b/c carbons in double bond cannot
rotate freely

3. Cholesterol Presence
-Cholesterol stiffens bilayer and makes it less fluid
-Process:
● Cholesterol inserts into membrane
● Polar hydroxyl group is close to polar head groups of phospholipids
● Rigid hydrocarbon tails of cholesterol interact w/ and partially immobilize regions of
fatty acid chains that are adjacent to phospholipid head groups
● More cholesterol=less fluid membrane

-how do cells regulate membrane fluidity?

● Many organisms/cells can respond to temperature changes by adjusting levels of
unsaturated fatty acids/cholesterol/short fatty acid in membrane
● Often used in organisms that do not control their body temp (i.e. bacteria)
FRAP Experiment (Fluorescence Recovery After Photobleaching)
-FRAP measures membrane fluidity
● If membrane proteins aren’t anchored and can diffuse, rate of fluorescence recovery is high and
quickly fill back bleached area
● If proteins are bound tightly, will not experience fluorescence recovery after photobleaching

-membrane proteins fused w/ GFP

-select area of membrane is bleached w/ strong laser
● Produces bleached region w/ no fluorescence -
electrons of fluorescent molecule are lost, so no more
fluorescence can be emitted
-after some time, fluorescence in previously bleached region is
recovered
● Caused by fluid motion of phospholipids, so
fluorescent phospholipids diffuse into bleached region
and bleached phospholipids diffuse away

Asymmetry of Membrane
-cell membranes are asymmetrical
-lipid compositions of 2 monolayers of lipid bilayer are different
-asymmetry arises b/c glycolipids and sphingomyelin are produced by enzymes exposed to Golgi lumen and
are not substrates for lipid translocators (aka flippases)
● Thus, glycolipids and sphingomyelin are only on outside of cell membrane
● Flipasses are unable to move them from one leaflet to another

-breakdown of asymmetry:
● Phosphatidylcholine: mostly in extracellular leaflet
● Phosphatidylinositol: mostly in intracellular leaflet
○ Involved in intracellular signalling
● Glycerol phospholipids w/ terminal primary amino group: phosphatidylethanolamine and
phosphatidylserine are mostly on intracellular leaflet
● Cholesterol: distributed equally in both leaflets
○ This lipid spontaneously shuttles (flip-flops) b/w leaflets

-function of membrane asymmetry:

● Creates difference in charge b/w 2 leaflets - important for nerves/muscle cells
● Cytosolic proteins require specific lipid head groups to function
○ Example: enzyme protein kinase C binds to areas of membrane rich in phosphatidylserine
and requires its negative-charged head for activity
● Phospholipids needed for cell signalling
○ Phospholipase enzymes cleave phospholipids; resulting fragments act as short-lived
intracellular messenger molecules
 ● In animals, asymmetry used to distinguish b/w live and dead cells
○ When cell undergo apoptosis, phosphatidylserine rapidly translocates from cytosolic to
extracellular leaflet
○ Signals nearby cells (i.e. macrophages) to phagocytose dead cell

Lipid Rafts
-lipid rafts: domains of different compositions known formed by lipid bilayer
● Facilitated by protein interactions w/ lipids and other proteins
-lipid rafts are temporary, dynamic, and vary in size
-structure:
● Lipid rafts are regions where lipid, protein, and cholesterol concentrations are higher
● Contain specific proteins and lipid
-function:
● Used for cell recognition, enzyme function
● Adjusted according to needs of cell

Membrane Composition
-varies by organism, cell, organelle , regions of the same membrane
-is complex and dynamic (fluid mosaic)
-is essential to function

Membrane Contact Sites (MCS)

-membrane contact sites: b/w organelles, important regulated routes for inter-organelle communication
essential for cell homeostasis
● Organelles either touch, or are very close
-ER forms MCSs w/ mitochondria, Golgi, endosomes, peroxisomes, lipid droplets, and plasma membrane
● Results in closely opposed (10-20 nm) but not fused membranes containing molecular machinery
● Mediate essential cellular processes like lipid and ion exchange, organelle positioning, organelle
biogenesis

-examples of MCSs in cell:

1. ER-Mitochondria MCSs:
● Ion exchange: Ca2+ released from ER at MCS and funneled into mitochondria
○ Ca2+ involved in cell signaling (in muscle/nerve cells)
● Required to make new mitochondria: ER-mitochondria MCSs define sites of mitochondrial division
○ 1. ER tubules wrap around mitochondria to form MCSs defining fission position
○ 2. Constriction and division machineries are recruited (i.e. actin and myosin constrict
mitochondria)
■ Mitochondria is split, binary fission occurs
2. Endosome-ER MCS: endosomes (transport vesicles originating from trans-Golgi network) are bound to ER
network
● Pull ER tubules w/ them as they traffic along microtubules
3. ER-Plasma membrane MCSs: transfer phospholipids to plasma membrane
● Ion channels at these sites replenish ER Ca2+ stores

-summary: MCSs provide highly integrated communication b/w organelles essential for cell physiology
● High proportion of proteins identified that specifically regulate MCS function are mutated in variety of
diseases (i.e. ALS, retinal dystrophy, etc.)
Overview of Protein Structure
-R-groups: side chains, what makes an amino acid unique
● Determines protein property
-protein backbone: N-C-C-N-C-C
-N-terminus: end w/ amino group
-C-terminus: end w/ carboxyl group
-4 orders of protein structure:
● Primary: amino acid sequence
● Secondary: caused by HB b/w amino and
carboxyl
○ R groups interact
○ Forms alpha-helices or beta-pleated
sheets
● Quaternary: multiple proteins interacting
-amino and carboxyl undergo condensation rxn and form peptide bond, w/ water side product

Membrane Proteins
-membrane proteins carry out most of the functions of membrane (diversity in functions)
● 50% of membrane weight
● Much larger in weight than lipids, so 1 protein per 50-100 lipid molecules
● 30% of animal genome codes for membrane proteins
-2 types of protein’s associated w/ cell membrane
● Peripheral membrane protein: next to membrane
○ Function in transmitting info b/c they’re not stuck to membrane
● Integral membrane proteins: in membrane

Peripheral Membrane Proteins (aka membrane associated proteins)

-peripherally associated w/ membrane, not actually inserted into membrane
● Easily separated from membrane

-how do peripheral membrane proteins associate w/ membrane?

1. Some directly associated w/ membrane by covalently attached
lipids/glycolipids
2. Some indirectly associated w/ membrane by electrostatic interactions w/
integral membrane protein

-membrane proteins can be associated w/ only one side of bilayer:

● 4. Anchored to cytoplasmic face of lipid membrane by amphiphilic alpha-helix
● 5. Anchored to cytoplasmic face by covalently bound fatty acid chain or prenyl group
● 6. Anchored to extracellular face by oligosaccharide linker attached to phosphatidylinositol
(phospholipid) - glycosylphosphatidylinositol (GPI)
anchor
-membrane-associated proteins can be non-covalently attached to membrane proteins
● Intracellular or extracellular proteins may be anchored noncovalently to
transmembrane proteins

-functions: involved in signalling b/c of transient associations (can dissociate and go

somewhere else)
● Roles in intracellular signalling
● Roles in transmitting info received from external signals

Dissociating Peripheral Membrane Proteins

-peripheral membrane proteins dissociate from membrane following treatments w/ polar reagents (i.e. extreme
pH, high salt concentrations) that do not disrupt lipid bilayer
-extreme pH or high salt disrupts electrostatic interactions (b/w proteins) and separates peripheral protein from
integral protein

Integral Membrane Proteins

-major functions:
1. Transport: ion channels
2. Enzymatic activity: i.e. succinate dehydrogenase is
embedded in membrane
3. Signal transduction: hormone receptors
4. Cell-cell recognition: glycoproteins
5. Intercellular joining: multiple cells attached together (i.e.
tight junction)
6. Attachment: to other surfaces (i.e. focal adhesions)

-transmembrane proteins: span entire membrane

● Have both hydrophobic and hydrophilic regions
● Hydrophobic region: interacts w/ fatty acid chains
○ Have amino acids w/ uncharged and nonpolar
side chains
● 2 hydrophilic regions: interact w/ aqueous solution
○ Have amino acids w/ charged or polar side
chains
Dissociating Integral Membrane Proteins
-to study transmembrane proteins, they must be solubilized by disrupting lipid bilayer
● Done so by reagents that disrupt hydrophobic interactions, like
detergents
-detergents: small, amphipathic, lipid-like molecules containing both
hydrophobic and hydrophilic groups

-2 commonly used detergents:

● SDS (sodium dodecyl sulfate): strong, ionic detergent
○ Has ionized group at hydrophilic end
● Triton X-100: mild, non-ionic detergent
○ Has non-ionized but polar group at hydrophilic end

-process - when detergents are mixed in great excess w/ membranes:

● Hydrophobic ends of detergent molecules interact w/ hydrophobic region of transmembrane proteins
and phospholipids
● Hydrophilic ends of detergent bind to hydrophilic portion of transmembrane proteins, creating
protein-detergent complexes
-summary: detergents surround membrane and break
it into pieces, removing transmembrane proteins

Structure of IMP
-transmembrane proteins are commonly composed of alpha-helix domains
● Single polypeptide chain turns around itself to form a cylinder
● NH group of peptide linked linked by HB to C=O group of neighboring peptide bond
○ Located 4 peptide binds away in same chain

-possible IMP structures:

1. alpha-helix type 1: all amino acid side chains are hydrophobic
● Hydrophobic amino acid side chains are exposed on outside of alpha-helix, in contact
w/ hydrophobic tails of lipid molecules
-type 1 form single, uniformly hydrophobic transmembrane alpha-helix

-function: receptors for extracellular signals

● Ligand binds to ligand-binding domain of transmembrane protein
● Signal causes conformational change in regulatory domain
2. Alpha-helix type 2:
-structure:
● Hydrophobic amino acid side chains lie on one side of alpha-helix, and
hydrophilic side chains lie on other side
● Hydrophobic side chains - exposed to lipids of membrane’
● Hydrophilic side chains: form hydrophilic pore (water-filled pore) by
packing several helices side-by-side in a ring within hydrophobic
environment of lipid bilayer
○ Produces a pore

-example: water-filled channels

● Hydrophobic amino acid side chains one one side of each helix contact hydrophobic tails of lipid
molecules
● Hydrophilic side chains on opposite side of helices form water-filled pore
-image to the right: 5 transmembrane alpha-helices form water-filled channel across lipid
bilayer

-function of water-filled pores

● Involved in selective transport of large polar molecules and small charged
molecules across membranes
○ I.e. amino acids, nucleotides, sugars, ions (Na+, K+)
● Ion channels

3. Beta barrel
-formed by beta-pleated sheets
● Individual polypeptide chains in beta-pleated sheet are held together by HB
b/w peptide bonds in different polypeptide chains
● Adjacent polypeptide chains can run either in same direction (parallel
beta-sheet) or in different direction (antiparallel beta-sheet)
-structure:
● Hydrophobic amino acid side chains contact hydrophobic core of lipid bilayer
● Hydrophilic side chains face inside of barrel and line aqueous channel
-function:
● Water-filled pores inside beta-barrel function in selective transport of large
polar molecules and small charged molecules across
membrane
○ I.e. amino acids, nucleotides, sugars, ions
● Form porins
Glycosylated Membrane Proteins
-many transmembrane proteins are glycosylated (have sugar attached)
● Sugar residues (oligosaccharides) are added onto extracellular section of protein

-glycoprotein: mostly protein (w/ oligosaccharide chain)

-proteoglycan: larger carbohydrate chains w/ some peptide attached

Cytoskeletal Connections
-connections w/ cytoskeleton provide mechanical strength and restrict diffusion of some
proteins
-there are many connections b/w cytoskeleton and integral membrane proteins (act as anchors)
● IMPs connect to extracellular matrix, can be used to send signals b/w cells
● Cytoskeletal connections w/ membrane proteins gives cell/membranes their
structure
● Anchors different proteins on membrane in correct position
-example: bacteria stick genome to cell membrane

Protein Distribution in Membrane

-proteins aren’t distributed the same in a single cell, expressed in different parts:
● A. Proteins self-assemble into large aggregates
○ Function together
● B. Proteins are tethered on outside of cell
○ Attached to fibers in extracellular matrix
● C. Proteins are tethered on inside of cell (i.e. cytoskeleton)
● D. Proteins interact w/ proteins on surface of another cell
○ Connects cells together (i.e. sheet of epithelial cells in skin)
○ Example: proteins anchor cell to basal lamina

-shown to the right: cells have different surfaces, each part has different
proteins w/ different functions
● apical surface: cell membrane close to lumin
● Basal surface: faces basal lamina
● Lateral membrane: on side of cell

How do proteins influence membrane shape?

-membrane-bending proteins deform bilayers
-membrane assumes different shapes w/ help of proteins
● Protein interacts w/ membrane in different ways and fold it into
different shapes
● structure=function
-possible shapes:
● Flat sheets, narrow tubules, round vesicles, fenestrated sheets, pancake shaped
● Golgi apparatus
What can you predict about these proteins based on these figures?
-hydropathy index: how hydrophobic are these proteins
-1st protein (Glycophorin): integral membrane protein that passes through membrane once
● Very hydrophobic component surrounded by 2 hydrophilic components
-2nd protein (bacteriorhodopsin): has 7 very hydrophobic regions
● Corresponds to 7 alpha helices passing through hydrophobic portion of cell membrane