L02: Cytoskeleton
• 3 different types of filaments : actin filaments, microtubules and intermediate
filaments
• Actin is located in the outside of the cell
• String Actin filaments- F actin
• Actin filaments have some polarity due to the turnover
• The turnover is due to the acting molecule having ATP bound
• Actin hydrolyzed ATP to ADP forming old actin
• ATP end= new end, ADP end = old end – process called treadmilling
• Helper protein – small GTP binding proteins ( proteins from the RAS family)
• Rac protein is regulated by nucleotide (active vs inactive)
• GEFs bring new GTP to activate RAC
• GAPs activate the intrinsic GTP activity
• Depending of the GTP protein, the F acting structure differs
• Branch vs unbranched – branched are simulated by ARP2/3 Complex, Linear
are promoted by Formins
• ARP 2/3 speeds up acting filament formation
o 1. ARP2/3 complex associated with the acting filament (mother filament)
when activated by a NPF
o 2. When binding occurs new actin filaments are brought in
o 3. This process is made at 70 degree angle, and in close proximity to the
membrane
• There are actin proteins that cut actin filament – Cofilin, Sevelin (chop actin
filaments back to smaller units
• Profilin= actin in a storage complex (G actin is in a reserved)
• Proteins prevent the addition and removal of actin units- capping proteins
• Arp2/3 is able to do it’s jobs o well as it is very similar in structure with actin
• Formins- for linear actin, whole family of proteins
• Formins have to have at least these 2 domains to be classified as Formins –
FH1 AND FH2
• FH1- binds to profilin (profilin is a protein that binds to G actin
• FH2- business site. (Actin polymerization)
• In order to activate formins, they need to be bonded to a small GTP binding
protein (Roh or Rac) to end terminal
• Formin activity is all over the cell for different function
• F actin can be in tight parallel bundles, anti parallel bundles/contractile bundles
or gel like networks/mesh work
• Mesh work arrangement –
• spectrin (tetramer) has an actin binding site and it cross links actin. – crucial for
red blood cells
• Alpha actinin which functions as a dimer- crucial for cross linking F-actin in
muscle cells
• Alpha actinin forms contractile bundles and fibrin forms parallel bundles
• There are 6 different types of actin type
• Actin on its own works but it's NOT efficient
,• Actin is 42kDa
• Microtubules are hollow tubes - Filamental structures are former of tubulin
dimmer which are then arranged into profilaments which are the arranged into
microtubules
• The stabilization of the tubulin dimmer is controlled by a nucleotide (GTP)
• The hydrolyzation of GTP to GDP weakens the microtubules
• Tubulin is 55 kDa, 52 kDa
• Microtubules also have polarity generated by the position of the centrosome,
which is located at the microtubule organizing center
• There are many tubulin associated proteins which help regulate it
o Centrosome- multiprotein complex which organizes tubulin
o Stathmin- prevents assembly (similar to cofilin in actin filaments)
o Kinesin 13, Katanin- Fragment microtubules
o MAP(microtubules associated proteins)- stabilize
o Tau – important crosslinker (important in Alzheimer's)
• Tubulins have a C terminal tail which allows for regulation (by addition or
removal of amino acids)- post transcriptional modification.
• Post transcriptional modification affects the function and interaction of the
molecule therefore controlling the role of microtubules
• Intermediate filaments are not polar, formed by coiled-coiled dimers
• Due to the lack of polarity, intermediate filaments can’t be used for certain
functions, but they are used for structure stabilization
• NLS – nuclear localization site- sequence of amino acid that targets a protein to
the nucleus – used for stabilization of nucleus lamina
• Intermediate filaments very depending on the tissue
o Keratins- in epithelial tissues
o Vimentin- in fibroblast and endothelial cells
o Neurofilament proteins- nerve cells
o Nuclear Lamins- contain an NLS – in nuclear Lamins
• Intermediate filament can be used as disease markers
• Lamins interact with chromatin
• LAD= lamina associated domain- determines where chromatin is read out
• If LAD is present genes are usually not read at a high level, as it’s covered, and
therefore no access to transcriptional machinery
• Therefore, genes that need to be transcribed a lot are usually located near the
nucleus
• There’s crosstalk between the different cytoskeleton filament systems
For exam:
- Don’t need to know all actin protein, but need to know how actin is regulated, and
be able to give some examples that do different things
- Know some names of drugs in each cytoskeleton filaments
L03: Cells - Culture Imaging
• The color of the medium can tell you how well the cells are doing due to the pH
meter
• HeLa cells double every 24h –HeLa cells are immortalized
• In cell culture, you either study cells that proliferate or cells that differentiate
(central dogma of cell culture)
2
,• To change from proliferation to differentiation- grow cells at high density, add
differentiation factors, withdraw growth factors
• To change from differentiation to proliferation- add serum
• Trypsin is a protease that chops the connections the cells make with substrate
• Freezing medium - contains a protective agent (10% DMSO) that prevents
crystal formation
• Cells are kept liquid nitrogen
• Yellow medium- yeast growth – not sterile growth
• Mycoplasma- bad contamination as it slows down growth of cells, affects the cell
identity and very infections
• 2D to 3D growth makes cell cultures more realistic
• Immunofluorescence- Use antibodies that recognize a particular protein. Use a
counter stain for the DNA using a reagent such as dapi that intercalates into the
DNA and allows the visualization of nuclear in blue
• Fluorochromes are excited by a short wavelength and then they emit a longer
wavelength. If something is green in the microscope it’s excited by blue light
o This is because in the fluorochromes in the excited state fall into a ground
state, therefore there’s energy lost allows for emission of fluorescent
• The tail in fluorescent dyes allows for the linking, in immunofluorescence the tails
allows for the linking to the antibody
• FRET- for FRET, the fluorescence protein used need to have overlapping
absorption and emission spectrum, in order to have an energy transfer.
• As cAMP increases, FRET decreases
• FRAP- used to see protein dynamics
L04: Apoptosis 1
• Cell death is necessary for: development, fight infectious agents, get rid of
malfunctioning cells, necessary for physiological processes, allows immune
system to develop
• Necrosis- severe or sudden injury
o Plama membrane main site of damage
o Leakage of intracellular contents
o Potent inflammatory response
• During necrosis, there’s swelling, water leaking in through the plasma membrane
as plasma membrane is damaged and the cells swell up, eventually leading to
disintegration or bursting of the cell, leading to splurging out of its intracellular
contents and leading to inflamation
• Apoptosis
o No leakage of intracellular contents
o No inflammatory response
o Rapid clearance of apoptotic cells
• Apoptosis is triggered by intracellular signals leading to changes in nucleus,
cytoplasm and changes in mitochondria, changes to the cortex of the cell that
cause the chromatin and organelles to condense, the cellular membrane to bleb
and break off into apoptotic bodies.
• Apoptotic bodies are eaten by phagocytes
• Apoptosis is regulated in two ways: lack of trophic signals (growth factors, etc…)
or trigger of specific signals
• During apoptosis :
3
, o Changes in plasma membrane- externalization of membrane
phosphatidylserine (PtdSer)- lipid usually only found in the inner leaflets of
the cell
o Changes in plasma membrane- Membrane undergoes a morphological
change to form blebs- big bobbles of membrane that bulge out into the
extracellular world- no breakage of membrane therefore internal contains
stay inside the cell
o Changes in plasma membrane- The breakage is prevented by class of
enzymes called transglutaminases that are triggered upon apoptotic onset
that form an impermeable mesh work which stops cellular contents leaking
out
o Changes in nucleus- shrinkage and condensation of chromatin
o Changes in nucleus- Endonuclease activation- cleaves DNA into smaller
fragments
• PtdSer is a very acidic polar phospholipid
• In apoptotic cells PtdSer is externalized because normal homeostatic
machineries act to maintain this asymmetric distribution of this lipid are
compromised when cells enter apoptosis
• 2 enzymes responsible for keeping PtdSer inside healthy cell: phospholipid
scramblase, flippase
• Flippase- ATP- dependent enzyme responsible for keeping PtdSer inside
• Scramblase- ADD FAQ INFO- it's activated by calcium and calcium levels rise in Commented [BM1]: faq asked on function
apoptosis when mitochondria start to fragment. The activity of scramblase is Commented [BM2R1]: response: Scramblase effectively
countered by flippase, an ATP requiring enzyme that takes the PtdSer from the scrambles the phosphatidylserine across inner and outer
outer leaflet and moves it onto the inner leaflet leaflets of the plasma membrane. It is active in healthy cells
and apoptotic cells; in apoptotic cells the other enzyme,
• When apoptosis is triggered, as well as enhancing the activity of scramblase, flippase (that usually restores PtdSer to the inner leaflet of
caspase proteins that are effectors of apoptosis act to inhibit flippase the PM), is inactivated, allowing PtdSer to accumulate on
• In apoptosis scramblase increases levels of functions and flippase is inhibit and the outside face.
is not able to undo scramblases work- leads to then symmetric distribution of
PtdSer in the outer leaflet of plasma membrane
• PtdSer in the outer leaflet is a recognition signal for phagocytes and
macrophages that have PtdSer receptors and therefore bind to it and act as a
“eat me” signal
• Membrane blebbing- actin filaments in the periphery are connected by the motor
protein myosin. If myosin is activated, it will walk on the actin and contract and
bring the actin together this enhances the contractility of the cell. By enhancing
the contractility of the cortical actin network, the plasma membrane is able to be
pulled away.
• Myosin is the motor protein involved in membrane blebbing =
• Myosin is activated when its phosphorylated by a protein called ROCK
o Myosin light chain( gets phosphorylated by ROCK and therefore activated
and allowing for contraction
o This happens in down stream of the signaling pathway activated by
GTPases called RhoA
▪ RhoA activates ROCK1
▪ ROCK phosphorylates myosin light chain, activates it and causes
contractility
▪ ROCK1 also phosphorylates myosin light chain phosphatase (MLC
phosphatase) and inhibits it
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