BIO3135 MIDTERM REVIEW - CELL BIO

Lecture 1 (intro):

- Characteristics of Eukaryotes
o Size: ~10-100μm (1*10^-6m)
o Separated from outside by a membrane
o Cells communicate with other cells
o High degree of organization and compartmentalization
o 70% water
o ATP required for many reactions
o Chemical composition: sugars, fatty acids, amino acids, nucleotides
- Plasma membrane
o Membrane formed from amphipathic molecules (hydro (phobic/philic))
o Must be permeable
o Allows the insertion of membrane proteins (ion channels) allows the passage or
exchange of important ions and molecules

Lecture 2 (Microscopy):

- Types of Microscopes
o Light microscope
 Conventional (e.g bright field, phase contrast)
 Fluorescence microscope
 Confocal microscope
 Two-photon microscope
o Electron microscope
- Light microscope
o Uses basic light path
o Used for live or fixed cells and tissues
o Tissues: Upright microscope (Light is directed through bottom of sample)
 Upright microscope is optimal for fixed samples
o Isolated cells: Inverted microscope (Light is directed through the top)
 Inverted microscope is optimal for live samples
- Four types of Light microscope
o Bright field
 Transmitted light
o Phase contrast
 Converts phase differences into changes in brightness
 As light travels through an unstained cell a phase shift occurs. The exploitation
of refractive indices allows for clearer images.
o Differential interference contrast
 Similar principle as with phase contrast but with more definition
o Dark field
 Lateral light source shows only scattered light
o Difference between Phase and Differential interference contrast
  DIC uses polarized light to visualizes the phase differences in an unstained cell

- Fluorescence Microscopy
o Used to detect the presence of certain ions and molecules
o Relies on atomic absorption of a photon and the following emission at a longer
wavelength to detect a light signal
o Requires the use of fluorescent dyes to stain cells, certain dyes are used for specific
wavelengths as their colour is only visible in that region (e.g DAPI used at 460nm, GFP at
500nm, FITC at 520nm)
o Similar in optical structure to LM
o High energy lamps (Hg) provide light, while light filters reduce unwanted background
noise.
o Once excited Chromophore (atom or group whose presence is responsible for the colour
of a compound) become visible at certain wavelengths

- Steps to Fluorescence microscopy

o Step 1: First barrier filter lets through only blue light with a wavelength between (450-
490) nm
o Step 2: Beam splitting mirror reflects light below 510nm but transmits light above
510nm
o Step 3: Second barrier filter cuts out unwanted fluorescent signals, passing the specific
green fluorescein emission between 520-560nm

- Tissue Preparation
o Cells must be histologically prepared in order to observe except for GFP (fluorescence)
o Fixation: cells are exposed to (aldehydes, acids, alcohols) in order to preserve and
stabilize them. This can produce side effects.
o Embedding: cells are embedded into plastic or polyethylene glycol
o Sectioning: Cutting of thin (1-10μm) tissue sections on a microtome
o Staining: Only if required, exposing cells to dyes

- Immunofluorescence
o Antibodies are produced in host animal and collected
o Fixed tissue is permeabilized and treated with primary antibody directed against a
specific antigen (primary antibody is programmed to attack antigen A)
o Antibody binds to antigen on or within cell
o Secondary antibody conjugated with fluorescent marker binds to primary antibody
(secondary antibody is programmed to attach onto primary antibody)
o Indirect immunohistochemistry labels cell structures (Fluorescence of secondary
antibody produces image of structure its bound to)
- Confocal Microscope
o Advantages:
 Technique that provides clear images with reduced “background” signal
  Particularly useful for applications involving thick sections or whole-mount
preparations
o Disadvantages:
 Costly
o Confocal refers to equidistance between light source and object, and object and
detector
o Utilizes fluorescence and high energy lasers (He-Ne, Ar)
o A pinhole focuses the light into a single point on the specimen producing an optical
section with low background noise
o Only light focused at the pinhole will enter the detector
o Precise targeting allows the confocal to provide clear X and Y axis images and potentially
Z axis

- Two Photon (non-linear) Microscopy

o Advantages:
 Non-linear technique that uses higher-order light-matter interactions from
multiple photons to generate contrast
 Allows for deep tissue imaging (up to 1mm depth possible)
 Light absorption occurs in the near IR region and NIR light penetrates deep into
tissue
o Disadvantages (very costly)
o Two-photon absorption involves “simultaneous” (~0.5fs) arrival at excitable molecule
o Excitation and emission occurs, similar to in fluorescence
o However, the signal is dependent upon photon density so absorption is spatially
confined
o In confocal, single photon absorption occurs throughout exc. Light cone
o Differs from confocal by excitation laser and detection pathway
o Rapid high-energy laser pulses (100fs; 100 MHz) are emitted
o Signal collected by detector depending on sample thickness
o No pinholes needed

- Things to Consider
o What are the primary differences between the different types of microscopy discussed
so far?
o Think about appropriate applications in which you would use regular fluorescence,
confocal and two-photon microscopy.

Lecture 3 (X-Ray microscopy):

- Electron Microscopy
o Two major types
 Transmission electron microscopy (TEM)
 Scanning electron microscopy (SEM)
o Bombards samples with electrons instead of light photons
 o Advantages:
 Resolution is ~2,000* that of a LM. 0.1nm=1.0 A (Angstroms). Lower resolution
in some biological preparations (2nm)

o Disadvantages:
 Very time-consuming preparation. But for some experiments, this is the only
appropriate technique

- Transmission Electron Microscope

o General configuration analogous to LM
o Electrons emitted at filament or cathode
o Accelerated by high voltage (105 V) in a vacuum
o Magnetic coils focus electron beam like a lens
o Sample may be stained producing “Electron dense” images
o Preparation for TEM
 Lengthy procedure that takes days to weeks to complete
 Tissue must be fixed in glutaraldehyde (Used to kills by crosslinking their
proteins)
 Addition of OsO4 increases electron density (used as a staining agent, the heavy
metal base contains many electrons allowing for good contrast)
 Dehydration and infiltration with a plastic resin gives extra support
 Ultrathin sections (50-100nm) must be cut with a diamond knife
 Sections cannot be handled directly
 Placed on copper grids
o TEM slides are prepared in slices, as such each image produced by TEM while be one
section of the sample. Once many slides are scanned a composite image of all TEM
images can produce a complete image of the sample

- Immunogold EM
o Similar to immunofluorescence, but a gold particle is used instead of dye to produce a
specific location of increased contrast. A gold particle is applied to the secondary
antibody to attack the primary antibody. Golds high electron density work well in EM as
the electrons fired at the gold scatter creating strong “dark spots” of contrast.

- Scanning Electron Microscope

o Produces 3D images of surfaces structures.
o Used to study whole cells and tissues, rather than intracellular structures
o Principles of preparation and operation similar to TEM
o Cells/Tissues are coated in heavy metal
o Scattered electrons from the specimen’s surface are collected

- Ion Imaging
o Changes in intracellular ion concentrations (e.g Ca2+ and H+) are physiologically
important
 o Ion-selective indicators emit light depending on local ion concentrations
o These reveal rapid intracellular dynamics

- Ca2+ Imaging
o Intracellular Ca2+ is low
o Bioluminescent aequorin (Calcium activated photoprotein isolated from the hydrozoan
Aequorea Victoria) injected into a fish egg reveals Ca 2+ wave propagated during
fertilization.
o Other synthesized indicators molecules produce signal
 Fura-2: Ratiometric fluorescent dye which binds to free intracellular calcium,
excites at 340nm-380nm
 Fluo-4: Fluo-4 AM (acetoxymethyl ester) is injected into cells and is converted to
Fluo-4 Green-fluorescent calcium indicator, excites at 488nm
o Ratiometric imaging exploits differential wavelengths associated with ionic binding
(Fura-2)
o Dyes can be injected or “AM” analogues can be used to cross cell membrane
(acetoxymethyl ester)

- X-ray Crystallography
o Structure-function
o Macromolecules
o Atomic resolution
o X-rays (0.1nm)
o Crystallized proteins
o Bombardment and diffraction
o e.g. (interference patterns)

- X-ray crystallography (how it works)

o Fires beams of x-rays towards a crystalized protein structure.
o The diffraction of the x-ray is collected and produce an image of banding showing where
a structure is found.
o Multiple x-ray crystallography’s are assembled to form a better picture of the overall
shape
- Things to consider:
o Think about applications in Cell Biology in which specific techniques of microscopy are
most appropriate. Ex: would you use an EM to study intracellular Ca2+ dynamics?
o What are the major difference between each technique we’ve discussed?

Lecture 4 (Cytoskeleton filaments)

- Types of filaments
o Actin filaments (AFs, microfilaments)
o Microtubules (MTs)
o Intermediate filaments (IFs)
- Filament construction
o Small subunits form filaments
o Actin and tubulin, compact and globular
o Disassembly, diffusion, reassembly
o Protofilaments (A filament of polymerized tubulin in a cell, which becomes part of a
microtubule) (MTs)
o Weak noncovalent bonds

- Nucleation
o Process that occurs in the formation of a crystal from a solution, a small number of
subunits become arranged in a pattern characteristic of a crystalline solid, forming a site
upon which additional particles are deposited as the crystal grows.
o Growth of Actin filaments can be described in three phases (Slide 5 for image)
 First phase, Nucleation (lag phase): Small actin subunits bind together to form
larger oligomers
 Second phase, Elongation (growth phase): Oligomers begin to bind together to
form a rudimentary actin filament. During this phase oligomer addition> loss so
the strand lengthens.
 Third phase, Steady State (equilibrium phase): As the actin filament reaches its
final length the addition of oligomers begins to equal that of the loss. The
decrease in actin subunit concentration causes a plateau in growth as it no
longer has the required building blocks, or the Critical Concentration (Cc).
o Growth of Actin can be sped up by skipping the nucleation (lag phase) and adding
oligomers thus beginning the reaction at the elongation phase. Reducing the time
required for actin filament production

- Tubulin
o Heterodimer= 2 different proteins (Alpha-tubulin and Beta-tubulin) but is considered as
“1 subunit” of tubulin
o Each dimer binds to a GTP (Guanosine triphosphate); hydrolyzed at only 1 site
o Each Microtubule is formed from 13 tubulin protofilaments, which wrap around a
hollow core to form a cylinder
o Each strand contains two ends a “Plus (+)” and “Minus (-)” end.
 “Plus (+)” end contains only exposed Beta-tubulin, this end will elongate more
quickly
 “Minus (-)” end contains only exposed Alpha-tubulin, this end can elongate but
will substantially slower than the (+) end

- Actin
o Monomer of Actin Filaments
o Binds with ATP
o Contains both a “Plus (+)” and “Minus (-)” end
- Treadmilling and Dynamic Instability
o Describes the growth and shrinkage of filament proteins
o Actin and Tubulin catalyse hydrolysis of ATP (Actin) or GTP (tubulin)
o “T” or “D” form indicates if triphosphate or diphosphate for exists
o ATP (actin) or GTP (tubulin) Caps
o Critical concentration (Cc): where subunit addition equals subunit loss

- Treadmilling - Actin Filaments (slide 9 for image)

o Phase 1: Filaments added to ATP-actin.
o Phase 2: [ATP-actin] high, addition occurs at both ends.
o Phase 3: [ATP-actin] drops, addition becomes greater at plus end
o Phase 4: Steady state (Treadmilling)
o Treadmilling explanation
 Treadmilling is the process for which one end of an Actin filament or
Microtubule grows while the other end shrinks. This generates the treadmilling
action as the filament appears to be moving across the cytosol.
 Since both Actin filaments and Microtubules have (+) and (-) ends, shrinkage is
observed on the (-) end while grow is observed on the (+) end.
o Treadmilling is determined by the rate of hydrolysis and the Critical Concentration (Cc)
o The linear relationship of [subunits] vs elongation rate
 For treadmilling to occur the Cc(T)< C < Cc(D). (slide 11 for image)
- Dynamic Instability-MTs (slide 12 for image)
o [Tubulin] within critical values
o GTP cap
 A GTP cap is placed on the (-) end of the formed microtubule in order to prevent
shrinkage. The sudden loss of the cap causes rapid shrinkage and stalls any
further expansion. The recapping of GTP allows for the microtubules to regrow.
o Continuous transitions (catastrophe and rescue)
o Dynamic instability cycle or How hydrolysis depolymerizes MTs (slide 14 for image)
 Step 1: Stable protofilaments are made of GTP-Tubulin dimers formed in a
straight line.
 Step 2: GTP hydrolysis (GTP to GDP) changes the conformation of the straight
protofilament, this weakens the bond in the polymer
 Step 3: Once hydrolyzed the filament begins to depolymerize and break apart
 Step 4: The filament breaks into GDP-Tubulin bound dimers and is subsequently
phosphorylated becoming a GTP-Tubulin dimer returning back into the dimer
pool
o SEQUENCE OF EVENTS:
 Growth at + end
 GTP cap at end
 Loss of GTP cap (CATASTROPHE)
 Shrinkage
 Regain of GTP cap (RESCUE)
 Growth etc...
 o The kinetics of microtubule growth depends on [Tubulin]
o High [Tubulin] means "tapered end" and faster growth
o Low [Tubulin] means "Blunt end" and slower growth
- Treadmilling and Dynamic instability
o What is the purpose?
o Constant ATP consumption, worth it?
 Spatial and temporal flexibility with high turnover
 Fastest way to grow filaments without nucleation
 Exploration for attachment sites and remodelling

- Intermediate Filaments (slide 19 for image)

o (A,B) Dimer formation
 Two alpha-helical regions in monomer wrap together to form a coiled-coil dimer
o (C) Tetramer formation
 Two dimers are staggered together to form a tetramer
o (D) Tetramer-Tetramer association
 Two tetramers pack together
o Tetramers are packed into an array of 16 dimers in cross-section
o These have a rope-like appearance
o Formation by spontaneous interaction
o Disassembly likely regulated by phosphorylation

- Types of Intermediate Filaments

o Epithelial (Keratins)
 Most diverse family
 Type 1 and 2 keratin chains
 Gives strength in hair, nails, horns
o Axonal (neurofilaments)
 Found in central and peripheral axons of vertebrate neurons
 Type L, M or H
 Growth, increases axon diameter

- Things to consider
o Can you differentiate between the processes of treadmilling and dynamic instability?
o Think about the differences in assembly, growth and shrinkage between each of the 3
filament proteins discussed
o Where does ATP or GTP fit in?

Lecture 5 (Cytoskeleton Regulation)

- Accessory proteins
o Filaments (AFs, Mts, Ifs) are dynamic and under control of the cell
o Form higher-order structures (e.g. mitotic spindle)
o Accessory proteins modify these cytoskeletal dynamics
- Nucleation by γ-tubulin protein complex
o MTOC (the centrosome) in animal cells
o Spindle pole body in yeast
o γ -tubulin is highly conserved
o γTuRC (ring complex) accelerates MT formation
o Nucleation occurs at the (-) end.
o γ-tubulin ring complex acts as template & helps organize and bring all components
together
o Random way in which subunits join ring complex

- Nucleation by γ-Tubulin complex (slide 3 for image)

o MT initiation also occurs in cytosol
o E.g. plants
o MTs nucleate daughter MTs

- The centrioles and centrosome

o Centrosome contains 2 cylindrical centrioles
o 9 triplets of MTs
o Centrioles organize PCM (Pericentriolar Material)
o Centriole lumen and PCM contain γ-tubulin
o PCM initiates MT assembly
o Centrosome near nucleus
o (-) ends of MTs are anchored
o (+) ends of MTs emanate in astral configuration
o Centrioles act as MTOC

- Nucleation of AFs
o Formation
o Cell periphery (cortex), where density of AF proteins is highest
o Location related to AF function (cell shape, movement)
o Facilitated by ABPs (Actin Binding Proteins) including ARPs (Actin Related Protein)
o ARPs initiate nucleation of AFs

- Initiation of AFs [unbranched] (slide 8 for image)

o Initiation by formin, and ABP
o Correct alignment for polymerization
o E.g. filaments of muscle cells
o Brought together so that they all interact with each other (oligomer) maximize
interaction so that binding is stronger
o Arp2/3 complex acts to form a template

- Initiation of AF branches
o Cell periphery
o Arp 2/3 produces extensive branching
 o Complex contains 7 proteins
o Binds at (-) end
o 70o favourable angle
- Control of Subunit Pools
o Both actin and tubulin are maintained in the cytosol at high concentrations
o Concentration can exceed Cc
o Accessory proteins may sequester unused subunits (sequestering proteins)
o Sequestered proteins are not hydrolyzed
o Provide control or regulation of filament elongation

- Thymosin Sequesters Actin (slide 11 for image)

o Thymosin sequesters, but profilin recruits’ monomers
o Thymosin makes polymerization less favourable
o Profilin competes with thymosin and promotes assembly.
o Binds one actin monomer

- Stathmin Sequesters Tubulin (slide 12 for image)

o Stathmin binding prevents polymerization
o Decreases effective [tubulin]
o Promotes dynamic instability (catastrophe)
o Can bind 4 tubulin monomers at a time. 2 subunits at a time (alpha and beta)

- Mt-Associated Proteins (MAPs)

o Proteins that associates with tubulin, parts of proteins bind with microtubule & parts
that stick away. Allows for stuff to be transported across the cell and for space between
microtubules
o Several binding domains
o Length of projecting domain determines packing of MTs
o MAP2 (neuronal cell bodies)allows spacing of MTs; tau (axons)
o Tau and Alzheimer’s Disease (tau unable to bind to MTs)
o Poorly soluble (hyperphosphorylated) tau may induce neurodegeneration

- AF binding proteins
o Cofilin destabilizes AFs
 Binds to side of proteins
 Binding induces mechanical stress
 Treadmilling, turnover
 Cell locomotion
o Tropomyosin stabilizes AFs
 Binds to side of proteins
 Muscle contractions
 Vital part of muscle spindles
- Modifications at AF ends
o Capping (increases critical concentration)
o Recall elongation slower at (-) end
o E.g. CapZ (+), elongation occurs only at (-) end
o E.g. tropomodulin (-) in muscle contraction

- Modification at MT ends (slide 17 for image)

o Capping in MTs has a dramatic effect on dynamic instability
o Important during mitosis
o Dismantle spindle so cell can go through cytokinesis

- Cross-Linking Proteins and AFs (slide 19 for image)

o Formation of higher-order structures
o Spacing of 2 actin binding domains of cross-linking protein determines type of structure
o 2 major groups: Bundling and Gel-forming
o Actin bundling proteins (slide 20 for image)
 Alpha-actin important part of muscle spindle
 Allows for proper spacing (contractile bundle)
 Fimbrin allows for close packing
o Gel-forming proteins (slide 21 and 22 for image)
 Forms network/mesh/cortex below plasma membrane
 Ex. Filamen. Lamelapodia (flat foot)
 Spectrin (flexibility of red blood cell core)
- Changes in cell shape during embryonic development (slide 23 for image)
o Vertebrate embryo
o Formation of neural tube during nervous system development
o Neural plate must thicken then fold to form a tube
o Cell height- MTs
o Folding into tube- AFs

- Induction by extracellular signals

o Example: crawling neutrophil
o Chemical activates WASP (Wiskott-aldrich syndrome protein)
o Polymerizing filaments push membrane
o Sequence of steps
 Extracellular signal
 Activation of WASP
 Nucleation and branching by Arp2/3
 Promotion of assembly by profilin
 Elongation reduced by capping proteins
 Destabilized by cofilin and return of subunits to pool

- Things to consider
o Think about how each accessory protein affects the stability of MTs and AFs
 o Many of the dynamic mechanisms that we discussed do not apply to Ifs, why?

Lecture 6 (Cytoskeleton III: Molecular Motors)

- Motor Proteins
o Use energy derived from hydrolysis of ATP to produce mechanical force
o Binds to cytoskeleton (AFs, MTs)
o Produces net movement of proteins or “cargo”
o Divided into 3 families
 Myosins: Move along AFs
 Kinesins: Move along MTs (+) - towards growing end (anterograde)
 Dyneins: move along MTs (-) - towards nucleus (retrograde)
- Myosins
o Diverse family of motor proteins
 Most eukaryotes (I, II, V)
 Plants (XI, XIII)
 Protozoa (XIV)
 Head region conserved (catalytic regions)
o Function
 “Conventional” myosins
 E.g. type II: Muscle contractions, cytokinesis
 “Unconventional” myosins
 E.g. type V: organelle transport

- Myosin II (see slide 4 for image)

o Structure
 Heavy chains (1*)
 Heads and tails in helix form
 Light chains (2*)
 Essential and regulatory chains
 Allow for different protein changes
 Arrangement and movement
 Heads are catalytic, provide force (only one used due to so many myosins)
 Tails mediate dimerization with other myosins
 Light chains amplify conformational change

- The myosin cycle (see slide 5 for image)

o Attached: No ATP, locked. Rigor complex. Myosin bound firmly to AF
o Release: ATP bound, conformational change (away from AF)
o Cocked: Hydrolysis, conformational change toward (+) end of AF
o Force-generating: Weak binding, Pi release, power stroke, ADP lost.
- Thick Filament Formation
o Tail-tail interactions
o Heads oriented in opposite direction
o Formation of bundles in sarcomere of muscle cells

- Sarcomere (see slide 7 for image)

o Structural unit of a myofibril (elongated contractile threads) in striated muscle (muscle
tissue)
o Dark bands = myosin bundles
o Light bands = no myosin, just AF
o Z disc = inbetween the light and dark bands. Made of alpha-actin (crosslinking protein).
Spaces out accessory proteins & cap Z which stabilizes growth of AF

- Ca2+ dependence of Muscle Contraction

o No Ca2+, tropomyosin blocks myosin-AF binding
o Ca2+ important for muscle contraction
o Ca2+ released from sarcomere and binds to troponin C
o Conformational change in troponin I
o Tropomyosin (bound to troponin I) moves and exposes binding sites
o Myosin-AF binding
o Muscle contraction

- Myosin II S1 Fragment
o Addition of protease enzyme
o Myosin cleaved between neck and tail
o S1 contains catalytic site
o Can be immobilized on glass surface
o Slide AFs in vitro
- Contractile Ring
o Important during cytokinesis
o Myosin II and actin filaments
o Redistribution of filaments
o Thickness of ring never changes due to AFs breaking off

- Myosin V (see slide 12 for image)

o Long neck region
o “Hand over hand” steps
o Processivity
o Carries cargo (organelles)
o Ex. AFs on the outer part of cells. Pigment granules can be brought to AFs by MTS &
change the colour of the cell

- Kinesins
o 1960s: Proteins involved in axonal transport were unidentified
 o Radioactive 3H leucine moved along sciatic nerve in cat
o 410 mm/day
o Discovery
 MTs adhered to glass coverslips
 Axoplasmic extract from squid giant axon+ ATP induced organelle movement
 ATP but no axoplasm, no movement
 With non-hydrolysable form of ATP (AMP-PNP), vesicles bound to MTs but no
movement
 Kinesin isolated and identified as motor protein
o Kinesin structure (see slide 17 for image)
 Similar basic structure as myosins
 Head region conserved
 Tail regions are diverse
 C-terminal domain attached to cargo
 Most travel on MTs toward (+) end
 KIFC2 travels toward (-) end
 ATP-binding sites (yellow, image) similar
 Mt vs. AF binding sites differ
 Linker region interacts with catalytic core, thus swinging arm around.
o Kinesin Cycle
 Processive steps along MTs
 Step 1: ATP binding of leading head induces conformational change in
its linker region; trailing head advances
 Step 2: ADP induces weak binding of leading head
 Step 3: Hydrolysis of trailing head induces detachment; ADP dissociates
from leading head (Repeat...)

o Myosin and Kinesin compared

Myosin II Kinesins
Attaches to AFs Attaches to MTs
Contract muscle Transport organelles
Rapid Power stroke Longer attachment
No head contraction Processive steps
5% of time attached 50% attached
ATP binding ->release ATP hydrolysis -> release
2-60 μm/s <2 μm/s
<8 pN theoretical force <5 pN

- Dyneins
o (-) end directed
o MT motor proteins
o 2 major divisions
 Cytoplasmic, e.g. retrograde vesicular transport
 Axonemal, e.g. beating of cilia and flagella
 o Cytoplasmic Dynein (see slide 21 for image)
 Dynein cannot bind directly to cargo
 Attachment to MT mediated by dynactin complex
 Named because of the inclusion of actin (red)
 Other accessory proteins

- Axonal transport (see slide 22 for image)

- Role of AF vs. MT-based motors (see slide 24 for image)

- Things to consider
o Structure is related to function. Think about the differences between types of motors at
the molecular level that lead to differences in function
o Why isn’t there a single motor proteins that performs all of the roles discussed?

Lecture 7 (Cell Cycle 1: Intracellular Control)

- Why control the cell cycle?

o The cell cycle is a complex system of coordinated processes that must occur in a specific
sequence
o If performed incorrectly or out of sequence, results may be catastrophic
o Regulatory proteins and biochemical switches control progression through the cell cycle
o This system monitors intracellular and extracellular environments

- Cdks control the cell cycle

o Cdk= ” Cyclin-dependent kinase ”
o Activity of cyclin (and thus Cdks) rises and falls with cell cycle
o Molecular switches that regulate important events:
 DNA replication
 Mitosis
 Chromosome segregation
 Cell proliferation

- Classification of Cyclins and Cdks (see slide 5 for image)

o Cyclins in all eukaryotes (4 major classes):
 G1/S cyclins: Bind Cdk near end of G1 and lead cell into DNA replication
 S-cyclins: bind Cdk during S phase and are required for DNA replication, control
early mitotic events
 M-cyclins: promote the events of mitosis (Rise of M-cyclins = initiate mitosis &
destroy of M-cyclins = end of mitosis)
 G1-cyclins (in most cells): promote passage though restriction point in late G1
- Cdks are protein kinases (see slide 6 for image)
o Protein kinases are proteins that take phosphate from ATP & attaches it to another
protein, i.e phosphorylation
o Like all protein kinases full activation of Cdk requires phosphorylation, ATP is hydrolyzed
to add a phosphate group to a serine side chain found on the Cdk. Phosphatase removes
the phosphate group to deactivate the Cdk

- Cdk activity is regulated (see slide 7 for image)

o Cyclical regulation
o As Cdk enters different stages of mitotic development, different cyclin types bind to it.
 Cdk enters the G1 phase empty. Near the end of G1, S-cyclin is bound to it. This
triggers DNA replication machinery, once the S-cyclin has fulfilled it job it is
degraded and the Cdk enters the G2 phase.
 As Cdk leaves the G2 phase, M-Cyclin is bound to it to trigger Mitosis machinery.
Once the task is fulfilled, M-cyclin is degraded, the Cdk leaves the M phase and
enters the G1 phase, thus repeating the cycle.

- Activation of Cdk-cyclin (see slide 8 for image)

o Stage of activation of Cdk
 Inactive: In the inactive stage Cdk is not bound to cyclin, without cyclin the
Activation loop or T-loop blocks the entrance of ATP into the active site.
Preventing activation of the Cdk enzyme
 Partially Active: Once Cyclin has bound to the Cdk, the T-loop’s conformation is
changed to no longer block the active site, ATP can now be bound.
 Fully Active: Once the active site becomes open, CAK (Cdk-Activating Kinase)
hydrolyzes ATP to phosphorylate the Cdk activating it.

- Inhibition of Cdk (see slide 9 for image)

o Two ways to inhibit Cdk
 Wee1 kinase phosphorylates the Cdk-Cyclin complex inhibiting it, this can be
reversed by Cdc25 phosphatase removing a phosphate group. E.g.
phosphorylating M-Cdk to prevent triggering of Mitosis machinery
 p27 is a CKI (Cyclin-dependent Kinase Inhibitor) which wraps around the Cdk-
cyclin complex to inhibit activity. p27 prevents the cell cycle from progressing to
the G1 phase.

- Ubiquitin and Protein degradation (see slide 10 for image)

o Ubiquitin causes ubiquitination, marks protein for degradation (done by proteasome)
o A ubiquitin ligase is required to catalyze addition of a polyubiquitin chain to a protein
o Polyubiquitin chains are produced by the addition of one subunit to a desired protein.
The addition of one protein causes the binding of many more.
o Polyubiquitin chains are used to tag proteins that as destined for degradation, in order
for the proteasome to degrade a protein a polyubiquitin chain must be present.
- SCF and APC are Ubiquitin Ligases (see slide 11 for image)
o SCF (Skp, Cullin, F-box containing complex)
o SCF can lead to destruction of G1/S cyclins (see slide 5)
o SCF can lead to destruction of CKI (Cdk Inhibitor Protein),
o Explanation of image (control of proteolysis by SCF)
 Step 1: CKI is phosphorylated by a kinase, in-order to be recognized by the SCF
complex.
 Step 2: The CKI is ubiquitinated by the SCF complex and sent to the proteasome
for degradation
 Step 3: S-Cdk is not deactivated; the cell proceeds to S phase
 Step 4: Positive effect on cell cycle (i.e. the continuation of the cell cycle)

- SCF and APC are Ubiquitin Ligases (II) (see slide 12 for image)
o APC (Anaphase Promoting Complex)
o APC can lead to destruction of securin, leading to chromatid separation
o APC can lead to destruction of M-cyclin
o Explanation of image (control of proteolysis by APC)
 Step 1: APC is activated by Cdc20, active APC ubiquitinates the Cdk-Cyclin
complex (bound to M-cyclin).
 Step 2: The ubiquitinated M-cyclin is sent to the proteasome for degradation.
 Step 3: Cell permitted to leave M-phase (destruction of the M-cyclin by
proteasome)
 Step 4: Positive effect on cell cycle (cell cycle continues)

- SCF and APC are active during different stages (see slide 13 for image)
o APC is active only during G1 phase and at the beginning of Anaphase
o SCF is active during the start of S-phase (through S, G2, M) until the start on anaphase
o APC-Cdc20 (activation) leads to reduction in M-cyclin, M-Cdk is no longer active which
increases APC-Cdh1 keeping M-cyclin levels low and the cell leaves M-phase
o APC-Cdc20 activation leads separase activation and anaphase

- Activation of M-Cdk triggers mitosis (see slide 14 for image)

o Once M-cyclin binds to Cdk, CAK and Wee1 can phosphorylate the complex. If both
Wee1 and CAK phosphorylate the M-Cdk it becomes inactive, Cdc25 can
dephosphorylate the complex and return it to its active state, the active state of M-Cdk
positively increase the activation of Cdc25 (positive feedback). Activation of M-Cdk also
enhances the feedback of Wee1 increasing the amount of inactive M-Cdk.
o S-Cdk phosphorylates (activates) Cdc25 during M-phase

- “Checkpoints”
o There are three checkpoints to be covered
 1. DNA replication checkpoint
 2. Spindle attachment checkpoint
 3. DNA damage checkpoints (several)
 - DNA replication checkpoint
 Step 1: Detection of un-replicated DNA
 Step 2: Cdc 25 not activated; M-Cdk activation is blocked (slide 14)
 Step 3: Cell does not progress into mitosis

- Spindle attachment checkpoint

 Step 1: Unattached kinetochore
 Step 2: Mad2 (Mitotic Arrest Deficient) binds
 Step 3: Cdc20-APC activation blocked
 Step 4: No chromatid separation

- Chromatid Separation (see slide 17 for image)

o M-Cdk activates APC, APC ubiquitinates the securin which is inhibiting the separase, the
securin is sent for degradation.
o The active separase causes the chromatids to split (Metaphase to Anaphase)

- DNA damage checkpoint (G1) (see slide 19 for image)

o Explanation of image
 Step 1: Damaged DNA causes protein kinase activation and phosphorylation of
p53 (p53 is a gene regulatory protein)
 Step 2: If Mdm2 is bound to an unstable or broken p53, p53 is sent to the
proteasome for degradation.
 Step 3: Stable active p53 binds to regulatory protein of p21 gene, and produces
p21 mRNA
 Step 4: p21 mRNA is translated into a protein, this being p21 CKI, this inhibition
of the G1/S Cdk and S-Cdk prevents the cell from entering S-phase (negative
effect on cell)

Lecture 8 (Cell Cycle II: Extracellular Control)

- Total Cell Mass

o Dependent on extracellular factors
 1) Growth Factors increase synthesis and decrease the degradation of proteins
 2) Mitogens: Increases cell division
 3) Survival factors: decreases apoptosis
 Note: Some growth factors also have mitogenic and survival effects.

- Growth Factors
o Needed for growth of animal cells
o Increase synthesis and decreases protein degradation
o E.g. platelet derived growth factor (PDGF), epidermal growth factor (EGF), nerve growth
factor (NGF)
o Stepwise path
 Step 1: Extracellular signalling proteins (GFs)
 Step 2: Cell surface receptors
  Step 3: Intracellular pathways

- Growth factors (II) (see slide 5 for image)

o Act through receptor tyrosine kinases (“enzyme-linked receptors”)
o Extracellular binding, transmembrane domain, and intrinsic enzyme activity
(“autophosphorylation”)
o Ligand (dimer) binding leads to receptor dimerization and transfer of P1 from ATP by
tyrosine kinase (TK domain)
o “Autophosphorylation” initiates intracellular pathways.
o Explanation of image
 Step 1: receptor tyrosine kinase is made of two separate structures, a signal
molecule binds in both of the inactive receptor tyrosine kinase, activating it.
 Step 2: Tyrosine kinase cross phosphorylates, this draws signalling proteins to
bind to phosphorylated sites creating a signal transduction

- Growth factors (III) (see slide 6 for image)

o Phosphorylated receptor activates PI-3 kinase
o Phosphorylation of ribosomal protein S6 and increased translation of many important
proteins
o E.g. NGF and TrkA receptors in neurons.
o Explanation of image
 Step 1: Activated growth factor receptor binds an activated PI-3 kinase which
phosphorylates a PI(4.5)P2 into a PI(3,4,5)P3.
 Step 2: PI(3,4,5)P3 activates TOR (protein kinase), which activates S6K (protein
kinase) that then activates S6 (ribosomal protein) which causes protein
synthesis and cell growth. An intrinsic pathway

- Mitogens
o Extracellular signals necessary for cell proliferation
o E.g. PDGF, EGF, erythropoietin (EPO)
o Increases cyclin synthesis, and thus Cdk activation
o Promotes entry into S-phase from G1
o G1 restriction point
 Also, a checkpoint but requires an extracellular signal (mitogen)
 Most vertebrate cells are in G0 (G0 is state in which the cell cycle is inactive)
 No mitogen present causes cell to enter G0 (e.g. neurons, muscle)
 If mitogens are present causes cell to re-enter G1 and continues (e.g. fibroblasts,
PDGF, wound healing)
- Mitogens stimulate Cell Division (see slide 10 for image)
o Mitogen binding
o Ras (G-protein “switch”)
o MAP kinase activation
o Increase in Myc gene expression
o E.g. PDGF, EGF
- Mitogens stimulate Cell Division (II) (see slide 11 for image)
o Myc is a gene regulatory protein
o Explanation of image (two pathways)
 First pathway
 Step 1: Myc activates cyclin gene transcribes more cyclin
 Step 2: Increased cyclin causes G1-Cdk activation
 Step 3: G1-Cdk activation causes Rb phosphorylation (inactivation)
causing cell to enter S-phase
 Second Pathway
 Step 1: Myc activates SCF subunit gene transcribes more SCF
 Step 2: SCF increases p27 degradation (a CKI)
 Step 3: Less p27 causes S-Cdk activation which causes Rb
phosphorylation (inactivation), cell then enter S-phase.

- Abnormal mitogen stimulation

o Mutations can sometimes lead to abnormal mitogenic stimulation
o Excessive increase in Ras or Myc mimics stimulation
o E.g. cancer, inappropriate entry into S-phase
o Normal cells detect these changes and prevent further division

- Prevention of Abnormal mitogenic stimulation (see slide 13 for image)

o Recall, p53 is normally degraded and cell cycle normal
o But if excessive Myc is produced, p19ARF will bind Mdm2 and stabilize (release) p53
o In cancer, this mechanism may not work because of p19 and p53
o Explanation of image
 Step 1: Excessive Myc production, p19ARF binds to Mdm2, allowing freed p53 to
become stable and active
 Step 2: Stable and active p53 causes either cell cycle arrest or apoptosis

- Survival factors
o Extracellular signals required for cells to survive
o Secreted by cells in surrounding tissues
o Without it, cells undergo apoptosis
o Some growth factors are survival factors
o E.g. Nervous system development

- Survival factors (II) (see slide 15 for image)

o Extracellular signal
o Explanation of image
 Step 1: Survival factor enters receptor, PKB is activated and begins two
pathways.
 Pathway 1: PKB phosphorylates Bad/Bcl-2 complex splitting inactive Bcl-
2 from Bad. The split Bcl-2 is now active and causes apoptosis.
  Pathway 2: PKB phosphorylates active gene regulatory proteins (GRP)
inactivating it. Expression of cell death promoting genes leads to
apoptosis
o Bcl2 family of proteins promote or inhibit apoptosis by regulating cytochrome c release
from the mitochondrion

- Things to consider
o There are many details that you’ve learned in these two lectures. Try to piece
everything together and think about where in the cell cycle everything fit, i.e.
chronological order
o What roles do feedback mechanisms play in cell cycle control?
o Remember the difference between extracellular and intracellular mechanisms of cell
cycle control.

Lecture 9 (Cell Cycle III: Apoptosis)

- Apoptosis
o Programmed cell death
o Number of cells is, in part, controlled by regulation of cell death
o Differs from necrosis: trauma or cytotoxicity leading to decrease in ATP and Na+/K+-
ATPase activity, followed by lysis.
o General characteristics
 Cell must undergo significant biochemical and morphological change
 Initiation by intracellular or extracellular signal
 Activation of a series of proteins involved in promoting apoptosis (or inhibition
of those that prevent it)
 Important intracellular proteins necessary for survival are cleaved during
apoptosis
 Orderly disposal of dead cell.
o Usually more cells are produced than necessary, initially those that make important
synaptic connections are kept, the rest die off
o Structural changes during apoptosis
 1) Chromatin condenses; shrinkage of cytoplasm
 2) Nucleus fragmented; DNA “laddering”; blebbing, cell fragmentation
 3) Phagocytosis of apoptotic debris
o Phagocytosis
 Asymmetric distribution of plasma membrane is lost
 Negatively charged phosphatidylserine then becomes exposed on the outside of
cell
 The cell is then marked for phagocytosis by a macrophage
- Caspases
o Identification of ced-3 gene led to the discovery of homologous family of proteins
“caspases”
o Caspases are proteases that cleave essential proteins
 Cleaves protein kinases (FAK (Focal Adhesion Kinase), disrupts cell adhesion)
  Cleaves lamins (disassembly of nuclear lamina)
 Cleaves Cytoskeleton (changes in cell shape)
 Activates CAD (Caspase-Activated DNAse), (DNA fragmentation)
o Involved in most changes observed in cell death
o This enzyme has a Cysteine residue at its catalytic site and cleaves other proteins at an
ASPartate site (CASPase=caspase)
o Caspases may also cleave each other, leading to their activation.

- Procaspase activation (see slide 11 for image)

o Initiator or “pro” caspases are inactive
o An apoptotic signal triggers assembly of adaptor proteins that activate caspases
o These caspases go on to activate executioner caspases by proteolytic interactions
(cleavage)
o Explanation of image
 Step 1: Apoptotic signal connects two inactive caspase monomers in the
adaptor binding domain.
 Step 2: Protease domain cleaves the cleavage site and creates the active
caspase
 Step 3: Active caspase cleaves an executioner caspase creating more active
caspases, which makes more… then apoptosis

- Caspase cascade (see slide 12 for image)

o An active caspase activates another caspase
o Irreversible, “all-or-none”
o Amplification of signal
o Result: intracellular proteins are cleaved

- 2 major apoptotic pathways

o Extrinsic
 Procaspase activation triggered from outside the cell
 “Cell death” receptors
o Intrinsic
 Procaspase activation triggered from inside the cell
 E.g. intracellular damage
 E.g. via Bcl-2 family of proteins

- Extrinsic pathway (continued) (see slide 14 for image)

o Extracellular signal (Fas ligand) activates death receptor (Fas protein)
o Recruitment of adaptor proteins and procaspase activation
o Caspase cascade
o Explanation of image
 Step 1: Killer lymphocyte binds its Fas ligand into Fas-death receptor.
  Step 2: FADD adaptor protein (composed of two domains (death domain and
death effector domain)) binds its death domain to the activated Fas death
receptor.
 Step 3: Caspase B death effector domain binds to FADD’s death effector
domain, creating the DISC (death inducing signalling complex). DISC activates
and cleaves caspase B creating apoptosis.

- Intrinsic pathway
o An intracellular death signal initiates caspase cascade and cell death
o Initiated by e.g. DNA damage or loss of survival factor
o Bcl-2 protein family
 Bcl-2 inhibits apoptosis
 Bax and Bak act on mitochondria and release cytochrome c
o Balance of these determines fate (i.e. life or death) of cell.

- Intrinsic pathway (II) (see slide 16 for image)

o Apoptotic signal activates Bcl2 family proteins (e.g. Bax) to form aggregates in outer
mitochondrial membrane
o Induces cytochrome c release from intermebrane space.

- Intrinsic pathway (III) (see slide 17 for image)

o Formation of “apoptosome”
o Explanation of image
 Step 1: Apoptotic stimulus causes mitochondrion to release cytochrome c,
cytochrome c activates Apaf1.
 Step 2: CARD domain of Apaf1 assemble into an apoptosome, the CARD domain
of caspase-9 binds to Apaf1. (7 needed to make apoptosome)
 Step 3: Activation of capase-9, which cleaves and thereby activates executioner
caspases, leads to caspase cascade.
- Intrinsic pathway (IV) (see slide 18 for image)
o Explanation of image
 Activation of PKB by survival factor creates active Bcl-2, which prevents the
release of cytochrome c from mitochondrion creating caspase cascade, cleavage
of intracellular proteins.
 Survival factors prevents the synthesis of Bax. If DNA damage occurs and there
is no survival factors than activated p53 creates Bax, which releases cytochrome
c form the mitochondrion, caspase cascade, cleavage of intracellular proteins.

- Apoptosis in the developing nervous system

o NGF is a survival factor and a growth factor
o Released by target cells
o Bax is an important mediator
o Mutation in genes encoding important proteins in the apoptotic pathway have dramatic
consequences.
 - Things to consider
o How do extrinsic and intrinsic pathways of apoptosis differ? How is the caspase cascade
started in each case?

Lecture 10 (Mitotic spindle)

- Major events of mitosis

o Centrosome duplication (G2)
o Mitotic spindle formation (prophase)
o Chromosome condensation (prophase)
o Breakdown of nuclear envelope (prometaphase)
o Chromosome attachment to MTs begins (prometaphase)
o Metaphase plate
o Anaphase

- Increased Dynamic changes (see slide 3 for image)

o Of cytoskeleton and MTS
o Decreased activity of MAPs by phosphorylation & increased kinesin activity
(catastrophins) and vice versa.
o Want MTs to change and grow in order to attach - facilitates formation of mitotic
spindle
o Image explanation
 Step 1: M-Cdk (by phosphorylation) leads to reduced MAP activity and increased
Kinesin activity.
 Step 2: Those changes causes increases in dynamic changes of MTs

- Components of Mitotic Spindle

o 1. Microtubules
 Astral (diff directions)
 Kinetochore (bind to chromosome pairs)
 Overlap (interaction with each other, crosslinked with molecular motors)
o 2. Motor Proteins
 Kinesin-related (+)
 Dynein (-)
o 3. Chromosomes (chromatids)
o 4. Centrosomes
 Centrioles
 PCM
- Motor proteins contribute to spindle assembly (see slide 5 for image)
o Multimeric motor proteins “cross-link” antiparallel MTs
o (+) end-directed kinesin-related proteins (KRPs, e.g. BimC or kinesin-5)
o KPRs self associate with each other at their tail domains
o Slide overlap MTs past each other
o Promotes elongation of mitotic spindle, increase area as centrosomes divide from each
other
- Motor proteins contribute to spindle assembly (see slide 6 for image)
o Motor proteins cross-link adjacent MTs
o Multimeric (-) end-directed dyneins and kinesin-14 arrange minus ends
o Creation of foci at spindle poles

- Spindle Pole separation (see slide 7 for image)

o Prophase
o KRPs push overlap MTs
o Dyneins pull astral MTs

- Chromosome Condensation (see slide 8 for image)

o Occurs at prophase
o Cohesin and condensin have similar ATP and DNA-binding domains
o Cross-linking
o Most cohesin is replaced by condensin during prophase
o Condensation dependent on ATP hydrolysis and phosphorylation of condensin by M-Cdk
o Condensin limited at kinetochore which holds it together

- MT-Kinetochore attachment (see slide 9 for image)

o Prometaphase
o Astral MTs “capture” kinetochores
o Increases MT stability
o Astral MTs become Kinetochore MTs
o Bipolar attachment (from both sides)

- MT-Kinetochore attachment (II)

o (+) end attached “end-on” to kinetochore
o Metaphase
o Formation of metaphase plate
o (+) and (-) end-directed motor proteins provide stability of chromosome along equator
of spindle. Translates into alignment on metaphase plate

- MT-Kinetochore attachment (III) (see slide 11 for image)

o Motor proteins: balance at the MT-kinetochore interface
 KRP (i.e. depolymerase)
 CENP-E (CENtromere-associated Protein E)
 Dynein
o Motor proteins: balance by astral ejection force (see slide 12 for image)
 Push chromosomes away from the poles, toward the equator

- Consequence of missing a motor protein (see slide 13 for image)

o Chromosomes are trapped near (-) end of MT (poles)
o Kinesin protein (KID) is lacking so chromosomes don't make it to metaphase plate
- Poleward flux (see slide 14 for image)
o If a chromosome pair is not centred, there is a change in rate of addition & loss of
subunits on both sides
o Addition at the shorter side and loss at the longer side
o Balance at metaphase plate, 1. Motor proteins, 2. Poleward flux

- Anaphase A separation (see slide 16 for image)

o Poleward (-) movement of chromatids by shortening of kinetochore MTs.
 1. MT depolymerization at (+) ends by KRPs
 2. Continual loss of tubulin at (-) ends (i.e. as in poleward flux) without addition
at (+) ends.
- Two Separation forces (see slide 17 for image)
o MT disassembly drives chromatid movement
o Poleward (or microtubule) flux at onset of anaphase

- Combined model for Anaphase A (see slide 18 for image)

o Kinesin-13 depolymerizes at both ends.
o Dam1 ring maintains attachment between MT and chromatid in yeast
o Depolymerization powers movement

- Anaphase B separation
o Pulling by motor proteins at poles
o Pushing by motor proteins at central spindle (i.e. at overlap MTs)
o Further elongation of spindle

- Action of motor proteins in Anaphase B

o KRPs cross-link and push overlap MTs apart
o Dyneins anchored to cell cortex pull themselves toward (-) end of astral MTs

- Things to consider
o Are you familiar with the roles of each different type of MT during mitosis?
o Motor proteins?
o Think how chromosomes are balanced at metaphase and how chromatids separate
during anaphase.