FINAL Flashcards
Cytological/Microscopic Observations of Cell Cycle
Cells at M Phase are easily identifiable
Cells have doubling times of about 20 hours. At any moment, 5% of cultured cells are at M phase suggesting that M Phase takes one hour to complete
What can the S Phase be labeled with? What does this tell us?
The S Phase can be labeled with Thymidine. The cells in S Phase can be identified now that Thymidine marks S Phases. The % of cells in S Phase (labeled) X length of one cell cycle. Determined that S Phase is 6 hours.
How is a Flow Cytometer measuring DNA cell contents?
DNA is labeled with fluorescence, like DAPI. Graph can be made to determine how many number of cells are in certain sets of DNA.
Cdc
Temperature sensitive mutants – can’t pass certain stages, like G1. Regardless, cells continue to grow.
Cell Fusion Experiment
Mitotic cell and G2 cell –> Induced condensation of chromosomes in G2 cells. Concluded that there is a factor in M Phase (MPF) cells’ cytosol that induces chromosome condensation and nuclear envelope breakdown.
Mitotic cyclins rise during ___ phase and drop during ___
Interphase; Anaphase
It oscillates
What is MPF composed of?
cdk1/cdc2
cyclin B
What is unique about yeast cell division
yeasts use cdk1 for their whole cycle
> > > Cyclins and CDKs of different cell cycle phases
G1 cyclin –> G1 CDK
G1/S cyclin –> G1/S-CDK
S cyclin –> S-CDK
M cyclin –> M-CDK/cdc2/CDK1
Regulating CDK Activity via Activating Phosphorylation
2-Step Process:
1) Cyclin partially activates CDK by moving T-loop
2) CAK phosphorylates CDK
Regulatory CDK Activity via Inhibitory Dephosphorylation
Wee1 Kinase: adds another phosphate, which inhibits CDK
CDC25 Phosphatase: removes second phosphate, activating CDK
Which AA are often phosphorylated in CDK
Serine and Threonine
Activating M-CDK
CDK1/CDC2 + M-cyclin –> inactive M CDK –Wee1 and CAK–> inactive M CDK (includes active and inactive phosphates) –CDC25+P–> Active M CDK (includes just active phosphate) –> Positive Feedback activates CDC25 (by phosphorylating it) and inhibits Wee1 (so that it can’t inactive phosphorylate )
CKI, and its example
CDK Inhibitor
P27 binds to CDK-cyclin (MPF) complex, inactivating it
Can act as a tumor suppressor
M Phase dependent events that are triggered by MPF
1) Chromatin condensation: Phosphorylation of condensin
2) Nuclear envelope breakdown: Phosphorylation of lamin
3) Golgi and ER Fragmentation: Phosphorylation of GM130
4) Spindle Formation: Phosphorylation of MTs, causing MT instability
How Phosphates affect Lamin
Phosphorylated Lamins: depolymerization (like in prometaphase)
Dephosphorylated Lamins: polymerization (like in telophase)
START
A point of no return for a cells to enter the cell cycles. The key regulatory point in the cell cycle before cells enter mitosis.
Exists in G1 is regulated by CDC2 (think back to temperature sensitive mutant)
LOOK AT SLIDE How is START regulated?
Cyclins regulate CDK deployed throughout cell cycle
RB (retinoblastoma) proteins
A tumor suppressor that prevents cells from entering the cell cycle.
Rb binds to E2F (a TF responsible for G1/S cyclin and S cyclin production), so that S CDK is not activated, and thus S phase can’t be entered.
Pathway:
Mitogen –> MAPK –> Myc –> G1-CDK –> Rb+2P –> activates E2F –> S cyclin/ G1/S cyclin –> S-CDK –> DNA synthesis
Ubiquitilation
E1, E2, and E3 are responsible for consecutively phosphorylating a protein. Upon polyubiquitlation, the compound will be targeted for degradation
What is CDC6?
In G1, it binds to origin of replication (ORC) and recruits needed pre-replicative complex and proteins, like helicase.
Causes S-CDK firing.
DNA replication needs the cdc6 to recruit but also cannot start replication until the cdc6 is removed.
In order to ensure only one cell cycle, CDC6 will be ubiquitlized for degradation.
Proteolysis of regulatory proteins to control cell cycle
1) SCF: has an F-box, which ubiquitizes CKIs, in order to initiate S-Phase
2) APC/C binds to CDC20, activating APC/C, which ubiquitilizes M Cyclin. Degradation of cyclin, causes cell to enter Anapahase
Timeline of cyclin and CDKs
MPF/M‐Cdk is only activated at the end of G2 phase, and is inactivated in anaphase.
Cyclin-B/M‐cyclin accumulates during interphase, and is degraded in anaphase.
Anaphase‐promoting complex(APC)/cyclosome marks cyclin-B for destruction.
How anaphase occurs?
M-CDK and CDC20 activate APC/C
APC/C causes the ubiquitylation and destruction of securing, which frees separase.
Separase is activated.
Separase destroys cohesin (which normally keeps chromosomes bound together.
Anaphase begins
What are cell checkpoints and what activates them?
Points of the cell cycle where things can be halted until conditions are better.
Activated by:
Incomplete cell division (Inhibits M CDK)
Chromosomal DNA is damaged (inhibits G1/S/M CDK)
Replicated chromatids are not aligned at the metaphase plate (inhibits APC/C)
Chromosomes are not properly attached (inhibits APC/C)
Endoreplication
Repeated S phase without M phase
Checkpoint Mechanisms for DNA Damage
Sensory: DNA detects damage??
Signaling: Kinase cascade resulting in the phosphorylation of P53, which transcribes P21
Effector: P21 is expressed as CKI, which inactivates S CDK, inhibiting S Phase
Cell Cycle Arrests
Anaphase wont start until….
ALL kinetochores are captured by MTs in the spindle.
Monitored by proteins at the kinetochore
Mitotic Checkpoint Complex (MCC)
When SAC is activated, it inhibits APC/C, allowing cyclin B and Securin to remain active
MAD2
Part of the MCC.
If chromosomes are not attached to the microtubules at prometaphase, MAD2 will be become present. Thus MCC will inhibit APC/C
Types of mitotic microtubules
Kinetochore microtubules: attaching kinetochore/chromosome to the spindle
Astral microtubules: separating spindle poles
Interpolar microtubules: elongating spindles
What forms two poles
Duplicated centrosomes
Acentrosomal Spindle
lacks centrosomes, therefore dynein and kinesin-14 are used to maintain spindle polarity
Kinesin-5 (BimC)
Maintains bipolarity of spindle; antiparallel crosslinking
Kinesin-14
Engaging both spindles
Kinesin-4/10
Bring chromosomes to the metaphase plate; outward push
Dynein
Further separate spindle poles
How do Kinetochores remain attached to kinetochore microtubule fiber?
Depolymerizes MT from the plus end; the force pulls the kinetochore MT to the poles
Stages of chromosomal separation during anaphase
AnaphaseA: sister chromatid/chromosome separation; kinesins promote depolymerization
AnaphaseB: spindle elongation
o Plus end‐directed kinesin motors act at interpolar microtubules to slide them apart; o Minus end‐directed dynein motors walk along astral microtubules to further separate the spindle poles.
Cytokinese in Animals vs Plants
Animals = cleavage furrow, in which actin and myosin II constricts to cleave cells: contractile ring; but the final fission depends on MT Plants = phragmoplasts
Nondisjunction and example
Uneven chromosome splitting.
Trisomy (Down Syndrome)
Asymmetric cell division of a neuroblastoma
Neuroblastoma asymmetrically divides into a neuroblastoma and GMC (ganglion mother cell).
GMC will divide into two differentiated neurons, while the neuroblastoma will continue to asymmetrically divide
Cell division plane
Determined by the spindle position, in which cells will be split between centrosomes.
In plants, it is determined by the preprophase band (PPB) orinetation
(Revise) Different kinds of P ;)
P27 – inhibits M phase
P53 – DNA damage; activated by UV damage; activates P21
P21 – DNA damage; inhibits S-Phase
How does APC/C affect M CDK
APC/C inhibits Cyclin B, which stops M CDK and M Phase
Major Steps of Apoptosis
1) Cytoplasm shrinks/ chromosomes condense
2) Nuclear fragmentation and cleavage of DNA
3) Fragmented cytoplasm and bleb formation
4) Formation of apoptotic bodies
5) Phagocytes, like macrophages, engulf apoptotic bodies
Ced3/4 Mutations
Encodes for caspases (suicide proteins); mutants lack the gene, thus cells wont die; used worm for example
Caspase binding behavior
cysteine is in the enzyme’s active site
aspartic acid is at the cleavage site of target proteins
Target proteins include lamins (breaks them down), raf kinases (prevents development/growth), and CAD (chops DNA)
Cleaved DNA can be detected
Phosphatidyl Serine
Normally in the inner leaflet of cells;
Rich concentration on the outside of apoptotic cells
Known as the “corpse marker” because macrophages identify apoptotic cells via PS
How is a caspase activated
Inactive initiator caspase (monomer) –Apoptotic signals (cleaves and dimerizes)–> Active caspase (dimerized) –> activates executioner caspase (via dimerization and cleavage) –> Apoptosis
CAD pathway
CAD-iCAD –Executioner Caspase–> iCAD cleaved –> CAD is activated –> DNA fragmentation
Process can be visualized via staining or gel filtration
BCL2
Sits on Mitochondria to control cytochrome exiting. Multiple BCL2 work together to form channels for it.
They are a family of proteins that trigger apoptosis, ONLY if BH4 domains are :
Anti-apoptotic: BCL2 and BCLXL; has BH4
Pro-apoptotic proteins: BH3-only and effector BCL2 family (BAX and BAK)
Cleavage furrow ends with ____
Interpolar Microtubules
How do activated BH3-only proteins affect cytochrome activation
1) Inactivates anti-apoptopic BCL2 proteins
2) Aggregates effector BCL2 family proteins
Ways cells survive/ avoid apoptosis
1) Increased production of anti-apoptotic BCL2 family proteins
2) Inactivation of pro-apoptotic BH3-only proteins
3) Inactivation of anti-IAPs (inhibitors of apoptosis)
Refer to graph
Assembly of Apoptosome
1) Apoptotic stimulus
2) BCL2 proteins aggregate and release cytochrome c
3) Arpf1 (w/ CARD) is recruited
4) cytochrome c and Apa-f1 arrange into an apoptosome
5) Apoptosome recruits procaspase/caspase-9 (w/ CARD)
6) Caspase-9 is activated at apoptosome
7) Executioner caspase is activated
8) Cascade cascade leads to apoptosis
Apaf-1 and Melanoma
Malignant melanomas have inactive Apaf-1.
Apaf-1-negative melanomas are chemoresistant, thus they can’t respond to P53 signals from UV damage
HID
Lecture 23/24
Induction of apoptosis via extrinsic stimuli
Tc/NK cells kill apoptotic cells
1) Tc/NK have fas ligad
2) Fas ligand binds to infected cell’s Fas Death Receptor
3) Triggers the recruitment of the FADD (Fas-associated death domain) protein
4) Consequently, free caspases will be recruited to form the DISC (death-inducing signaling complex)
5) Caspase-8 is activated
6) Caspase-8 activates executioner caspase (caspase-3)
> > > How does a lymphocyte kill tumor/infected cells?
Secretory granules are transported along MTs towards the centrosome (near PM and target cell)
How do polycyclic aromatic hydrocarbons (PAH) in smokes induce apoptosis
These potent air pollutants, also found in oil, smoke, tar, and coal, are recognized by Ahr (aromatic hydrocarbon receptor), which induces the expression of pro-apoptotic proteins, like Bax, causing apoptosis of oocytes in the ovary
How are cells organized into tissues
1) Adhesion, which anchors cells together and anchors them to the basal lamina (cell-matrix)
2) ECM (which makes up most of our daily products and food)
What composes different plant cell shapes
Cell wall polysaccharides allow plant cells to acquire their distinct morphologies during differentiation
Beta-1,4-linked glucose units
How microtubules affect cellulose shape
Phenomenon: nascent cellulose microfibrils are arranged in parallel to cortical microtubules beneath the plasma membrane.
Hypothesis: cortical microtubules govern the orientation of newly deposited cellulose microfibrils.
Microfibrils expand in the horizontal direction, thus so does cellulose
How are polysaccharides connected to the PM?
The Cellulose Synthase (CESA) Complex is linked to cortical microtubules through interacting/connector proteins.
____is considered to be a source of renewable bioenergy.
Cellulose
What happens when ECM proteins are damaged/destroyed by matrix proteases
Osteoarthritis and Cancer Metastasis, destroying ECMs near it
What is the most abundant ECM protein? What are its various forms?
Collagen; Types consist of: Fibrils (made of glycine and 3x chains X many strands), Nonfibrils, Network‐forming, and Transmembrane
Composed of many triple-stranded collagen fibrils
How are collagen fibrils made?
Procollagen is secreted from a vesicle. Proteinase cleaves procollagen ends, liberating the collagen molecule
How does a cell move along the basal lamina?
1) Actin polymerizes at the plus-end, to protrude a lamellipodium
2) Focal contacts contain integrins, which keep the anterior edge locked
3) The posterior end is released/contracts
4) Cell moves along the basal lamina
What fills the space of the ECM?
Glycoproteins and proteoglycans
- Hyaluronic Acid serves as a central strand for proteoglycans (composed of GAGs/gycosaminoglycans linked to core proteins)
- Proteoglycans are bound to the hyaluronic acid via link proteins
Bone marrow and eye jelly are composed of ___
Polysaccharide ECM
Properties of cancer cells
1) Reduced dependence on neighboring cells for survival
2) Less prone to kill themselves via apoptosis
3) Tends to proliferate indefinitely; no contact inhibition
4) Cancer cells exhibit high genetic instability; increased mutation rate
5) Abnormally invasive: may miss specific cell-adhesion molecules, like cadherins
6) Often survive and proliferate in foreign tissue; metastasize
Contact inhibition
Cells stop other cells from continuous division; like how a single epithelial is maintained;
Cancer cells don’t demonstrate contact inhibition, thus they can form large mounds
What is metastasis? What are its steps?
The spread/survival of cancer cells at a new site
1) Cancer cells break down basal lamina
2) Cancer cells enter capillaries
3) Cancer cells travel through blood stream
4) Cancer cells escape from the blood stream, then spreads into a new region of the body
Microevolutionary process that causes cancer
1) Increased cell division
2) Decreased apoptosis
How do pap smears determine cervical cancer?
Normal cells have a smaller nucleus than precancerous or invasive carcinomas; increased number of chromosomes in cancer cells
What are carcinogens? Examples
Cancer-causing agents Include: 1) Sun exposure/ UV light 2) Smoke/ Benzopyrene 3) Deamination 4) Food contamination, like AflatoxinB1 These all can induce mutations. Generally mutations occur in unimportant regions of chromosomes, but if they occur in important regions then cancer can occur
Ames Test
Identifies potential carcinogens
Assay that tests carcinogen initiatives
Testing potency of agent which causes mutation
1. Takes bacteria strain which requires the AA histidine for growth and mutates at one point so that it can no longer make their own histidine
2. Substance mixed with liver homogenate, which can modify the substance (cyt p450 enzyme causes modification to activate the carcinogens) and control, incubate and grow an identical amount of bacteria
3. Surviving ones have the initial mutation corrected (correcting the defects) or two mutations balance each other out
• Whenever a colony is formed, a new mutation has been introduced to correct the original one
• More colonies=more likely it can cause tumor
Microtubules dictate how cellulose is laid down
In plant cells
Plectin/Distan vs Viniculin/Talin
Both use integrin:
Plectin and Distalin bing to keratin in hemidesmosomes;
Vinculin and Talin bind to actin for actin in desmosomes
CDC25, Wee1, and CAK all occur during which phase
Strictly M Phase
What composes FADD and DISC
FADD: Death domain and Death Effector Domain
DISC: FADD + Death Receptor + Death + Initiator Caspase -8
Types of Adhesive Junctions
1) Desmosome – connect intermediate filaments (keratin) between cells
2) Adherin Junctions – connects to actin filaments between cells; involved in neuralation
3) Hemidesmosomes – attaches epithelial cells to basal lamina
How were induced plutipotent stem (iPS) cells made?
Taking a fibroblast from an adult skin fibroblast, then using a retrovirus to create an iPS cell (which could then make any embryonic cell)
Types of Cell-Cell Junctions; the size of the gap between the junctions
1) Tight Junction – seal off cells, polarizing cells and affecting permeability
2) Gap Junction – only found in animal cells; allows <1kDa molecules to pass; composed of 6 connexins, making one connexon; two connexons can be joined to allow molecule mobility; creates a hydrophilic channel; dopamine blocks these junctions in neurons; 1.5nm diameter, with 2-4nm gap
3) Plasmodesmata – in plant cells; allows big breaks between cell walls, enabling ER and other organelles to be cross-linked/communicate between cells; 20-200nm channels
4) Adhesive Junctions – consist of desmosomes, hemidesmosomes, and adherent junctions; 20-30nm gaps
Cadherin
A transmembrane protein used in adhesive junctions;
are hompohilic and depend on Ca2+ ;
link embryos and tissues together;
like pairing with same type of cadherin molecules;
bacteria` phages can use cadherins to infect cells
Active Transport Pathways
- coupled transporters
- ATP-driven
- Light or redox-driven
Na+ and Glucose transport
Symport
What does the Na+/K+ pump maintain
Osmotic Pressure
Omeprazole
Inhibits H+/K+ Atpase pump, inhibiting the release of HCl; stomach ulcer medication
Presynaptic vs Postsynpatic membrane
At presynaptic membrane:
1) Open the voltage-gated Ca2+channel
2) Trigger vesicle fusion (exocytosis)
3) Release neurotransmitter: electrical signal to chemical signal
At postsynaptic membrane
1) Open transmitter-gated channel: ion flow
2) Change membrane potential
3) Fire action potential: chemical signal to electrical signal
Where does gated transport occur in the cell?
Nucleus
What does the nucleus do?
Keeps ribosomes (and thus protein translation) out of the nucleus, thus requiring splicing and processing to occur first
Nuclear localization signal
Lys-Lys-Lys-Arg-Lys
Bind to import receptor
Can be in any position in the polypeptide (even split up) Key: basic residues
Can artificially lead to nuclear localization
Nuclear Pore Complex (NPC)
Also known as nucleoporins
Regulates entrance/exit of the nucleus.
Up to 60kDa will be allowed to enter nucleus, but the larger the molecule, the longer it will take.
Proteins entering and RNA leaving are energy-dependent transports
Ran-GTP in NES vs NLS
Ran-GEF produces Ran-GTP;
In NLS, Ran-GTP releases cargo in nucleus;
In NES, Ran-GTP binds cargo in nucleus, allowing it to leave nucleus
Mitochondrial peptide signal
alpha helix with positive charges/ hydrophilic region and hydrophobic region
TOM and TIM
TOC and TIM
Subsequent mitochondrial/chloroplast import receptors;
In animals and plants, respectively
Transport pwathways
1) Secretory: ER –> Golgi –> PM, lysosome, etc.; biosynthetic
2) Endocytic: Inward from PM; Uptake
3) Retrieval: Returns molecules; Recycling
SEC12 and SEC17
Encodes vesicle budding from the ER and vesicle fusion and targeting to the Golgi vesicles, respectively
Free ribosome vs. ER-bound ribosome translation
Free ribosome is post translational; protein is made before heading to its location
ER-bound ribosome is co-translational; Protein is incorporated into the ER while translation is occurring
Start vs Stop-transfer signals; and directionality
Both hydrophobic, transmembrane binding sites in the ER;
Start starts the region, while stop ends it;
Positive end will always face the cytosol
Ribosome –> ER transmembrane pathway
Ribosome–ER signal sequence binds to SRP;
SRP binds to SRP receptor;
Protein goes through translocator;
Signal peptidase removes translocator and cleaves start sequence
FRET components
Blue: Donor/emission
Yellow: Excitation/acceptor
Blood Type Enzymes
B A
Golgi/ER/MTOC relationship
ER (+) MTOC (-) Golgi (+)
2 mutations and 3 diseases associated with intermediate filaments
2 mutations:
1) Null – no keratin; skin ruptures easily
2) Autosomal Dominant Mutation – large agregrates of keratin; little damage will destroy the cell
3 disease:
1) Epidermolysis Bullosa (EB) – Connective tissue disease resulting in blisters
2) ALS – Abnormal assembly of neurofilaments blocking axonal transport
3) Laminopathy – Accelerated aging and muscular dystrophy
Regions composed of keratin
1) Keratin – epithelial cells
2) Neurofilaments – neuron shape
3) Lamins – maintains nuclear shape
4) Vimentin – other cells
Microfilament proteins
1) Profilin – recruits actin monomers; promotes polymerization
2) Thymosin – promotes depolymerization
3) Formin – contains lots of proline to recruit profilin, thushelping in actin polymerization
Microfilament ends
Barbed end = positive end; needs less monomers, thus has a smaller Cc
Pointed end = minus end; has a higher Cc because it require more actin monomers
Phases of microfilament, microtubule, and intermediate filament growth
1) Lag Phase/ Nucleation – three monomers join together
2) Growth Phase/ Elongation
3) Equilibrium Phase/ Steady State – basically treadmilling; monomers from minus end fall off, then are used for polymerization on positive end
Pase of microfilaments and microtubules
Microfilaments = ATPase Microtubules = GTPase
Microtubule composition and function
13 protofilaments, composed of alpha and beta-tubulin dimers; beta-tubilins can bind to GTP or GDP; upon GDP, tubulins fall off
Used in mitosis, maintaining cell and organelle shape,
Proteins Associated with Microtubules
1) EB1 – plus end polymerization
2) XMAP215 – plus end polymerization
3) Kinesin-13 – plus end depolymerization
4) MAP2 – larger protein spacing
5) Tau – smaller protein spacing
6) Stathmin – inhibits depolymerization by keeping MT bound together
7) Katanin – chops MTs; minus end depolymerization
8) Dynein – ATPase minus end motor; lissencephaly; moves from ER/PM to MTOC/Golgi
9) Kinesin – ATPase dimer; plus end motor
Adhesive Junctions/ Cadherins are used to composed ____
Embryos and tissues; compose cell
Hemidesmosome Structure
Kertain –> Dystonin/Plectin –> Integrin/Collagen –> Laminin –> Collagen
Intermediate Filament
Desmosome Structure
Keratin –> Dense Plate –> Cadherin
Intermediate Filament
Adherin Junctions
Actin –> Catanin –> Cadherin
Microfilament
What is fibronectin’s purpose?
Connects ECM to cell (integrin);
has an RGD sequence which binds to integrin
How can HER2 genes become cancerous?
Gene amplification –> Overproduction
Oncogene
How does c-myc result in overexpression?
Rearrangement causes c-myc –> IgH/c-myc
Now has a stronger promoter –> Overproduction
Oncogene
RasV12
A proto-oncogene that can become an oncogene via point mutation: Gly –> Val
Can no longer hydrolize GTP, thus remains active
Can be stopped via drug that inhibits MAP Kinase
Tel/PDF
Fusion protein Tel/PDF oncogene –> constant kinase
Activation of RTK constitutively by gene fusion inducing dimerization
Oncogene
Virus-induced cancer
HPV
Virus inserts into genome, thus allowing replication, and spreading
Why doesn’t a normal + tumor fused-cell produce a tumor cell
Normal cells have tumor-suppressor genes
Rb gene
Tumor suppressor gene
Related to the eye
Cancer from somatic mutation via recombination –> loss of healthy form
Regulates E2F –> inhibiting S phase
Oncogene vs. Tumor Suppressor
Oncogene: Few mutations can hyperactivate proto-oncogenes –> oncogenes; dominant
Suppressors: Many mutations can inactivate tumor suppressor genes; recessive
APC
Frequently mutated protein in colon
Tumor suppressor
Truncated gene
> > > Philadelphia Chromosome
Chromosome translocation forms Ber-Abl, which activates Map kinase
Oncogene
X’some 22 and 9
Gleevec Drugs
Attaches to polypeptide junction of fused proteins/genes, inactivating them
effector BCL2 family
Bax and Bak
Cc
The lower Cc, the less monomers required for polymerization
Because Cc requires less monomers, it polymerizes faster
Treadmilling occurs when Cc(+) < C < Cc(-) and
Cc(T) < C < Cc(D)
C > Cs (-) then polymerization occurs at both ends
C < Cc (+) then depolymerization occurs at both ends
