Term
|
Definition
| a network of interconnected filaments and tubules that extends throughout the cytosol, from the nucleus to the inner surface of the PM |
|
|
Term
| cytoskeleton structural components |
|
Definition
Microtubules (MT)
microfilaments (actin filaments)
intermediate filaments (IF) |
|
|
Term
|
Definition
Structure and support
Intracellular transport
Contractibility and motility
Spatial organization |
|
|
Term
| techniques for studying the cytoskeleton |
|
Definition
Microscopy:
Immunofluorescence microscopy
Live cell imaging
Fluorescent monomers
Time-lapse videos
EM
Inhibitors (Table 15-3)
microtubules formation: Colchicine, Nocodazole,
Microtubule dissembly: Taxol
microfilament formation: Cytochalasin D |
|
|
Term
|
Definition
Tubulin subunit - alpha/beta dimer
highly conserved protein across all eukaryotic species
the alpha -tubulin - GTP bound permanently
the beta -subunit – has GTPase activity and hydrolyzes GTP after the dimer is incorporated into the MT |
|
|
Term
|
Definition
|
|
Term
| microtubule assembly in vitro: initiation |
|
Definition
tubulin dimers associate end-to-end to form short protofilaments
Aggregation of dimers to form oligomers, known as nucleation 1
Joining of oligomers to form protofilaments 2
protofilaments associate side-by-side to form sheets 3
sheet closes to form tubular structure 4 |
|
|
Term
| microtubule assembly in vitro: initiation |
|
Definition
|
|
Term
| Microtubule Assembly In Vitro: MT elongation |
|
Definition
tubulin dimers (both GTP bound) associate and dissociate at each end 5
tubulin dimer in wall of MT (b-subunit) hydrolyzes GTP to GDP+Pi |
|
|
Term
|
Definition
|
|
Term
| The Kinetics of Microtubule Assembly In Vitro |
|
Definition
In vitro assembly of MT is temperature dependent: polymerization (assembly) at 37C and depolymerization (disassembly) at 4C or 0C
Critical concentration Cc of tubulin dimer: MT forms/grows when the dimer
concentration is above Cc and MT shrinks when it is below Cc . |
|
|
Term
| Treadmilling of Microtubules |
|
Definition
Different Cc at the two ends of the MT assembly
-Lower Cc at the plus end
The free tubulin concentration can thus be higher than Cc at the plus end but lower than Cc at the minus end
When that happens, polymerization will take place at the plus end while depolymerization takes place at the minus end
Tubulin dimers added to the plus end are lost after getting to the minus end |
|
|
Term
| The GTP Cap and Its Role in the Dynamic Instability of Microtubules |
|
Definition
When tubulin concentration is high, a GTP cap is formed at the tip of the MT
-The speed of adding tubulin-GTP onto the MT exceeds that of the GTP hydrolysis in the wall of the MT
A MT tip with a GTP cap is stable and allows more tubulin dimers to be added. |
|
|
Term
| The GTP Cap and Its Role in the Dynamic Instability of Microtubules |
|
Definition
When tubulin concentration is low, no GTP cap is formed at the tip of the MT
-The speed of GTP hydrolysis in the wall of the MT exceeds that of the addition of tubulin-GTP onto the MT.
A MT tip without a GTP cap is unstable and allows rapid depolymerization |
|
|
Term
| Microtubule dynamics in cells: dynamic instability |
|
Definition
Dynamic Instability -
-individual MTs oscillate between phases of elongation and ‘catastrophic’ shortening
-intrinsic property of cytoplasmic MTs in cells |
|
|
Term
|
Definition
|
|
Term
| MTs originate from Microtubule-Organizing Centers in cells (mtoc): |
|
Definition
MTOC:
serves as the nucleation and anchoring site for MTs in the cells
basal bodies and centrosomes
Basal body located from the base of cilia serves as a MTOC
The MT will “grow out” from the basal body because tubulin dimers are added to the basal body
EM examination reveals that MTs grow much faster from one end (plus end) than the other (minus end) |
|
|
Term
| MTs originate from Microtubule-Organizing Centers in vivo |
|
Definition
Centrosome: two centrioles and associated pericentriolar materials
Centrioles
-9 sets of MT triplets
-Oriented perpendicular to each other |
|
|
Term
|
Definition
Cell locomotion
Muscle contraction
Maintaining cell shape
-Cell cortex beneath plasma membrane
Use actin as building blocks (G-actin and F-actins)
-Two stranded helix |
|
|
Term
| Microfilament Assembly In Vitro |
|
Definition
Many similarities to MT assembly
Reversible assembly: critical concentration
Polarity of assembly
Plus/Minus ends and treadmilling
Basic units is ATP-bound, hydrolyzed once in filaments
Kinetics
Lag (nucleation) phase
Rapid (elongation) phase
Plateau phase: gain=lost |
|
|
Term
| microfilament assembly in vitro |
|
Definition
|
|
Term
| Intermediate Filaments Contrast with MT and MF |
|
Definition
10-12 nm in diameter
Less organized appearance, but more stable and less soluble
No polarity: antiparallel tetramer subunits
Protein subunits fibrous
Protein sequences are tissue specific- Medical diagnostic use
No motor proteins, mainly structural |
|
|
Term
| classes of intermediate filaments |
|
Definition
|
|
Term
|
Definition
Motility at the subcellular level
Separation of chromosomes during cell division
Intracellular movement of vesicles and organelles
Motility at the cellular level
Cilia/flagella-based movement
Amoeboid movement
Tumor metastasis
Chemotaxis
Motility at the tissue level
Muscle contraction: the combined effect of many muscle cells moving simultaneously |
|
|
Term
| microtubule-based motility |
|
Definition
ATP dependent
Motor proteins walk on MT track
Motor proteins carry cargos |
|
|
Term
| microtubule-based motility |
|
Definition
|
|
Term
| interaction Between Motor Proteins And MT/MF Produce Motion |
|
Definition
|
|
Term
|
Definition
|
|
Term
|
Definition
two globular heads and one coiled-helical tail
heads
MT binding domain
ATPase site
generate force on MTs through ATP hydrolysis
tail - binds cargo |
|
|
Term
|
Definition
MT stationary
8 nm/step
1 ATP/step
Walking motion
One head/”foot” detaches and binds a new - tubulin
Trailing head/”foot” detaches, walks over the other head/”foot” and binds another new - tubulin
Microtubule stays stationary
tail - carries cargo |
|
|
Term
| Cytoplasmic Dynein/Dynactin Complex |
|
Definition
|
|
Term
| Cytoplasmic Dynein/Dynactin Complex |
|
Definition
heavy chains, intermediate chains and light chains
Heavy chains bind to MT and engage in “walking” toward the minus end
Light chains interact with a Dynactin complex, which in turn, binds to membrane of the cargo |
|
|
Term
| MT serves as “Tracks” for vesicle transport inside the cells |
|
Definition
Transport of secretory vesicles
-Vesicles from ER delivered to Golgi (near centrosome)--Dynein
-Retrograde transport--Kinesin
-To the plasma membrane--kinesin |
|
|
Term
| Movement At The Cellular Level: Cilia And Flagella |
|
Definition
Motile appendages of eukaryotic cells
-specialized projections of cell cytoplasm - inside Plasma Membrane
-basal body (MTOC) at base of structure
Common structure base but different size and number
Cilia: short and numerous
Flagella: general long and limited to one or few per cell |
|
|
Term
| The Structure of Cilium/Flagellum: |
|
Definition
| an Axoneme connected to a Basal body by a Transition zone |
|
|
Term
| Cross-section of an Axoneme |
|
Definition
Central Pair (2)
-13 protofilaments each
Outer doublets (9)
-doublet MTs
-A tubule: 13 protofilament;
-B tubule: 10 or 11 protofilament, shares with A tubule
-dyneins attached to A tubule can interact with B tubule on adjacent doublet |
|
|
Term
| Dynein “Walking” On MT Generates Sliding Force |
|
Definition
dynein arms (on A tubules) walks toward (-)end of adjacent B tubule - pushes adjacent tubule toward (+)end direction. This process is ATP dependent.
dynein is ‘(-)end-directed motor’
this “sliding” is normally restricted by nexin links:
-Axoneme treated with protease that breaks nexin links
-Addition of ATP - dynein arms generate force on adjacent doublet - telescopes axoneme to 9x original length |
|
|
Term
| Summary: MT and Cilia/Flagellum |
|
Definition
Growth at MT plus end contributes to the formation of cilia/flagellum
Dynein-mediated MT sliding cause bending and motion of cilia and flagellum
Kinesin and Dynein-mediated vesicle transport delivers/retrieves materials to/from the tip (plus end) of cilia/flagellum. This is called Intraflagellar Transport. |
|
|
Term
| Actin-Base Cell Motility The Myosins |
|
Definition
Head domain (2 for myosin II)
actin-binding site
ATPase site
Neck region
binds light chains
4 for myosin II (calmodulin related)
Different ‘tail’ domains
specific cell functions
some forms dimerizes through coiled-coil domain interactions (e.g. myosin II). |
|
|
Term
| Organization of Skeletal Muscle Tissue |
|
Definition
Muscle - tissue
bundles of myofibers
Myofiber - muscle ‘cell’
fusion of many cells into multinucleated cell with large interior cytoplasmic space filled with myofibrils
Myofibril
end-to-end linkage of sarcomeres
Sarcomeres
contractile unit |
|
|
Term
|
Definition
Skeletal and Cardiac muscle
-Striated: alternating dark and light bands
-A band and I band
Sarcomere
-One A band, two half I band on each side of A band
-A band: thick or thick+thin
H zone: thick only
-I band: thin only
-boundaries:
center of I band: Z line
A sarcomere runs the lengths between two Z lines |
|
|
Term
| structure of a sarcomere: thick and thin filaments |
|
Definition
Thin filaments
-Actin filaments attached to each Z-disk by + end
-Decorated by Tropomyosin and Troponin
-capped at the (+)end by Cap Z and at the minus end by Tropomodulin (actin filament capping proteins)
-long nebulin molecule lies along actin filament - length of nebulin = length of thin filament
Thick filaments
-Myosin bipolar filaments in center of sarcomere bundled together by Myomesin
-In skeletal and cardiac muscle cells - ~300 myosins/bipolar filament (thick filament) |
|
|
Term
| Table 16-2 Major Protein Compounds of Vertebrate Skeletal Muscle |
|
Definition
|
|
Term
| The Slide-filament Model For Muscle Contraction |
|
Definition
Thick filaments (A band) and thin filaments remain constant length but slide with respect to each other, increasing overlap.
I band in each half sarcomere shortens
Z-lines are pulled together all along the length of myofibril
sliding driven by “walking” of myosin head toward the plus end of thin filament
-The amount of force/tension the muscle can generate during contraction depends on the number of myosin heads than can make contact with the thin filament |
|
|
Term
| Cycle Of Cross-bridge Formation And Muscle Contraction |
|
Definition
|
|
Term
| Cycle Of Cross-bridge Formation And Muscle Contraction |
|
Definition
Transient cross-bridges formed by interaction between the myosin head and F-actin in the thin filament holds the thick and thin filaments together loosely
1-Pi release cause pivot of head on neck and tighter binding
2- release of ADP is accompanied by a powerstroke that pulls the thin filament towards the ____ of the Sarcomere
3-ATP binding dissociates head from actin filament
4-Hydrolysis of ATP into ADP+Pi cocks myosin head toward actin (+)end (spring-loading)
head rebinds to actin filament upstream (towards the + end) of original position
Energy: 1 ATP/powerstroke |
|
|
Term
Actin-myosin alone
Tropomyosin |
|
Definition
unregulated
myosin head crossbridges free to interact continuously with actin filaments
Muscle would contract continuously
lies along both sides of actin filament
2 positions
“normal” state - blocks myosin head interaction with actin filament
“sliding-off state” - allows myosin head interaction with actin filament |
|
|
Term
| Troponin - 3 protein complex |
|
Definition
TN-T -
binds complex to Tropomyosin
TN-I -
required to position TM in ‘normal’ state
TN-C -
related to calmodulin
binds Ca2+ with KD ~ 10-3 mM |
|
|
Term
|
Definition
forces Tropomyosin into “sliding-off” state position on thin filament (removes inhibition)
allows force production |
|
|
Term
| Ca2+ regulation of skeletal muscle contraction |
|
Definition
Rise in Ca2+ concentration to ~10-3 mM
Ca2+ binds to TN-C
Tropomyosin slides out of the way
Contraction
Decrease in Ca2+ concentration , <10-4 mM
Ca2+ removed from TN-C
Tropomyosin blocks Myosin binding
Relaxation |
|
|
Term
| Skeletal muscle regulation of Ca2+ concentration |
|
Definition
Sarcoplasmic reticulum (muscle smooth ER)
membranous system wrapping around myofibrils/sarcomeres
T-tubules
invaginations of plasma membrane penetrate deep into myofibril cytosol
close proximity to SR |
|
|
Term
| Sarcoplasmic reticulum (muscle smooth ER) |
|
Definition
membranous system surrounds myofibrils
SR membrane Ca2+-ATPase pumps
pump Ca2+ from cytoplasm into SR lumen
actively establish resting level of Ca2+ in cytosol (<10-4 mM)
voltage-gated Ca2+-release channel
Can be activated by membrane depolarization
opens to release Ca2+ down concentration gradient |
|
|
Term
| Turning on: nervous stimulation – muscle contraction |
|
Definition
Nerve cell stimulation initiates voltage depolarization in myofibril
T-tubules transmit voltage depolarization that opens SR voltage-gated Ca2+ channels
Ca2+ concentration in myofibrils rises - Ca2+ binds to TN-C and allows actin-myosin force production |
|
|
Term
| Turning off - nervous stimulation stops - relaxation |
|
Definition
Ca2+ channels close
Ca2+ ATPases re-establish low Ca2+ concentration (<10-4 mM) in muscle cell cytosol
Ca2+ is lost from TN - TM moves to inhibitory ‘off’ position on thin filament |
|
|
Term
| in common and differences |
|
Definition
Common:
Ca2+ , Myosin binds actin, muscle contraction
Ca2+ , Myosin can not bind, muscle relaxation
Difference:
Ca2+ come from outside of the cell
Calmodulin and phosphorylation is involved
No Troponin based regulation |
|
|
Term
| Actin-based motility in non-muscle cells: Cell crawling through Lamellipodia and filapodia |
|
Definition
Lamellipodia: protrusion of a sheet of cytoplasm
Filapodia: protrusion of thin, pointed structure
Focal adhesion: local contact with surface to provide traction.
All contain actin filaments |
|
|
Term
| Extracellular Matrix of Animal Cells |
|
Definition
ECM: structure immediately outside the PM, consists of long fibers embedded in a hydrated gel-like network
The protein components of the ECM are produced by and secreted from the cells
Collagens: structural fibers
Proteoglycans: major components of the matrix/gel (“goo”)
Fibronectins and laminins: adhesive molecules
bone, cartilage and connective tissue |
|
|
Term
| connecting cells with ECM |
|
Definition
Proteoglycans: a special class of heavily glycosylated proteins (made in the rER, glycosylated in Golgi, and secreted to the outside of the cells via vesicles)
-Core protein
-glycoaminoglycans (GAG)
Hyaluronate
Chondroitin
keratan
Fibronectins/Laminins (from ECM)
-bind Collagen and Integrin
Integrins (from PM) |
|
|
Term
| The Integrins: The Fibronectin/laminin Receptor |
|
Definition
Integrins: Integrating cytoskeleton with ECM
A heterodimer of / subunits
a5b1: fibronectin receptor
a6b1: laminin receptor
Transmembrane protein
Extracellular domain bind fibronectin and laminin
Cytoplasmic domain indirectly interact with cytoskeleton
-Focal adhesion |
|
|
Term
| function of the integrins |
|
Definition
Cell attachment
Cell movement: provide anchorage and traction for actin-based motility
Signal Transduction
-Normal cells-anchorage-dependent growth: cells need to be attached to ECM/base to grow, otherwise die.
Integrin clustering activates growth
-Cancer cells: anchorage-independent growth: cells grow fine without attaching to ECM/base.
The growth pathway is constitutively activated. No need for adhesion/integrin clustering |
|
|
Term
| Cell-Cell Adhesion Proteins |
|
Definition
Cell to cell adhesion mediated by TM proteins (adhesion receptors) on the PM of the neighboring cells
-identical proteins on other cells - homophilic interactions
-different types of proteins on cells or in the extracellular matrix (ECM) - heterophilic interactions
Most adhesion receptors are connected to cytoskeleton with their intracellular domain
Some serve as receptors for pathogens |
|
|
Term
| Human Pathogens and Cell Adhesion Proteins |
|
Definition
Yersinia Pestis: Plague; category A bioterrorism agent
Other category A agents include:
Anthrax; smallpox, Ebola virus..
can be easily disseminated or transmitted from person to person;
result in high mortality rates and have the potential for major public health impact;
might cause public panic and social disruption;
and require special action for public health preparedness. |
|
|
Term
| cell junctions in animal cells |
|
Definition
Adhesive junctions
-Cadherin-mediated
-Cell to cell, cell to ECM connections
-Mostly localized adhesions
Tight junctions
-Permeability barrier
Gap junctions
-Direct, open connection |
|
|
Term
|
Definition
Mediated by TJ proteins:
-Claudin, occludin, JAM (junctional adhesion molecule)
Forms tight seals between the membranes of the neighboring cells
Establishes cell polarity |
|
|
Term
| tight junctions and cell polarity |
|
Definition
Prevent lateral diffusion of membrane protein and lipid to maintain polarity
Prevent movement of material through space between cells |
|
|
Term
| tight junctions and cell polarity |
|
Definition
|
|
Term
| cell polarity and absorption |
|
Definition
|
|
Term
| cell polarity and absorption |
|
Definition
|
|
Term
|
Definition
Direct connections of cytoplasms of neighboring cells for communication
Areas of multiple tunnel-like connections between cells
-Units of connection: connexons |
|
|
Term
| gap junction communication experiment |
|
Definition
Experiment (W. Lowenstein. problem 17-9, pp505)
injected 2 fluorescently-labeled probes into one cell with gap junctions with neighboring cell
1200 dalton MW compound passed to neighboring cell
2000 dalton MW compound remained in injected cell |
|
|
Term
| gap junction communications |
|
Definition
small molecules (Ca2+ and cAMP) can pass from cell to cell
Elevated levels of Ca2+ close gap junctions
-prevents death of cell if neighboring cell dies and loses control of Ca2+ concentration |
|
|
Term
| flow of information in cells |
|
Definition
|
|
Term
|
Definition
The Nuclear Envelope
-Inner membrane
--The nuclear lamina
-Outer membrane
--Rough ER
-Nuclear Pore Complex
The Nucleoplasm
-The Nuclear Matrix
-Chromatin
The Nucleolus |
|
|
Term
| nuclear matrix and nuclear lamine |
|
Definition
Nuclear Matrix (nucleoskeleton)
-Insoluble, 3-D fibrous network inside the nucleus after extraction of the chromatin
Nuclear Lamina
-Thin IF network immediately underneath the nuclear envelope
-Lamin A, B, C |
|
|
Term
|
Definition
| A large subnuclear organelle with a spherical structure but no membrane |
|
|
Term
| nucleolus as a Site of rRNA Synthesis |
|
Definition
A nucleolus is formed around the ribosomal DNA (rDNA) repeats, which are called nucleolar organizing regions (NORs).
It is the factory in which 28S, 18S and 5.8S ribosomal RNAs (rRNAs) are transcribed, processed and assembled into ribosomal subunits |
|
|
Term
|
Definition
Numerous openings at the NE that connects the cytosol and the nucleoplasm
-3000-4000/nucleus
-Channels with120 nm in diameter
-Large protein complex (~120 mDa) consists of dozens of different polypeptides (Nups)
Functions to allow nucleo-cytoplasmic exchange of macromolecules |
|
|
Term
| the structure of the nuclear pore |
|
Definition
Inner and outer membrane fuse together to provide opening
Lined by a multi-protein structure called nuclear pore complex (NPC)
-NPC arranged in a octagonal pattern
-Two rings (eight subunits each) surrounding a central transporter which can open to allow passage of molecules
-Rings and the transporter are connected by eight spokes
A basket of fibers at the nucleoplasmic side |
|
|
Term
| the structure of the nuclear pore |
|
Definition
|
|
Term
| Macromolecular Transport into and out of the Nucleus |
|
Definition
|
|
Term
| Macromolecular Transport into and out of the Nucleus |
|
Definition
Nuclear Import of proteins:
-Transcription factors
-Histones
100 histones/pore/minute
-Ribosomal proteins
Nuclear Export of RNA and proteins
-All FOUR types of cellular RNA
-Partially assembled ribosome subunits
4-5 subunits/pore/minute |
|
|
Term
| Transport Through the Nuclear Pore Complex Nuclear Import |
|
Definition
|
|
Term
| Transport Through the Nuclear Pore Complex Nuclear Import |
|
Definition
Active transport mediated by NLS
-Nuclear Localization Signal containing proteins (cargo) are destined for the nucleus
Stretches of positively charged amino acids (e.g. PKKKRKV)
-NLS interacts with its receptor (importin)
-Importin docks the complex at the NPC
-Translocation mediated by the central transporter
RanGTP terminates the import
process by binding to import
receptor and dissociating the cargo |
|
|
Term
| Transport Through the Nuclear Pore Complex Nuclear Export |
|
Definition
Active transport mediated by NES
-Nuclear Export Signal containing proteins (cargo) are transported back to the cytoplasm after their nuclear experience
Leucine-rich signal (LR-NES): eg. LPPLERLTLD
-NES interacts with its receptor (exportin), which is in complex with RanGTP
-Exportin mediate the transport of NES-bearing cargo through the NPC
GTP hydrolysis by Ran terminates the export process and dissociate the cargo |
|
|
Term
| cytoplasm- rangdp, nucleus- rangtp |
|
Definition
RanGEF exclusively nuclear
Turns RanGDP into RanGTP
RanGAP exclusively cytoplasmic
Turns RanGTP into RanGDP
RanGTP terminates the import process by binding to import receptor and dissociating the cargo
RanGTP bound for export, Hydrolysis stops the export
process by dissociating the cargo
The recycle of the RanGDP
not clearly understood, maybe
simple diffusion |
|
|
Term
| Interspecies heterokaryon analysis |
|
Definition
Some proteins shuttle between the cytoplasm and the nucleus
gffp- fusion gene, transfection in a human cell for 24 hours
then add a mouse cell and protein synthesis inhibitor fusion for one hour |
|
|
Term
| organization of DNA in genomes |
|
Definition
A copy of an organism’s complete genetic information
-Mostly DNA
--“Naked” DNA:
Circular
Linear
--Chromatin/Chromosomes
-RNA genome for many viruses |
|
|
Term
| Interconversion of Relaxed and Supercoiled DNA |
|
Definition
cccDNA (covalently closed circular DNA):
Supercoiled circle
oc DNA:
Open/Relaxed circle
Linear DNA |
|
|
Term
| Reactions Catalyzed by Topoisomerases I and II |
|
Definition
Topoisomerases introduce transient breaks into the DNA strand to release tension, thus relaxing the supercoiled DNA
single and double stranded breaks |
|
|
Term
| Thermal Denaturation of DNA |
|
Definition
Temperature increases , Hydrogen bonds break, DNA strands separate
ssDNA absorbs more UV light than dsDNA
Tm: the melting temperature
50% increase in A260
Proportional to GC content |
|
|
Term
| DNA Denaturation and Renaturation |
|
Definition
After denaturation:
Temperature decreases, Hydrogen bonds re-from, DNA strands re-anneal together
The ssDNA will find its partner though base-pairing
The basis for nucleic acid hybridization |
|
|
Term
| Gel Electrophoresis of DNA |
|
Definition
| DNA (RNA) molecules carry inherent negative charges due to its phosphate backbone |
|
|
Term
| cleavage of dna by restrictive enzymes |
|
Definition
| RE: endonucleases from bacteria that cleave DNA at specific sequences (sites), generating fragments called restriction fragments. |
|
|
Term
|
Definition
| Restriction mapping: using RE to probe the sequence organization of DNA. |
|
|
Term
| DNA Sequencing- sanger method- Chain termination |
|
Definition
|
|
Term
| Examples of Some Sequenced Genomes |
|
Definition
| Pyrosequencing and Illumina Deep Sequencing |
|
|
Term
| The Eukaryotic Cell Cycle-Overview |
|
Definition
Cell growth accompanies cell division
Genetic information must be faithfully passed down to new generation of cells
Cell division goes through discrete stages known as cell cycle phases |
|
|
Term
| the eukaryotic cell cycle |
|
Definition
|
|
Term
|
Definition
M-phase = Mitosis: during which the cell physically divides
Interphase
-G1 - ‘Gap phase 1’
growth and preparation for another round of cell division
-S - Synthesis phase DNA replication
2N to 4N
-G2 - ‘Gap phase 2’
checkpoints to ensure that chromosome duplication is complete
Doubling time (the generation time): the time to go from one cell to two cells.
18-24 hours for typical cultured mammalian cells
G1+S+G2+M
Interphase
G1 – highly variable, typically 8-10 hours
S – 6-8 hours
G2 – 4-6 hours
M-phase
30-45 minutes |
|
|
Term
|
Definition
G0 phase cells
-Permanent - ‘terminally differentiated’ cells such as neurons that no longer divide unless transformed to become cancerous
-Temporary - stem cells waiting for signals to re-enter cell cycle and make new cells for normal replenishment, e.g. wound healing |
|
|
Term
|
Definition
G1 to G0
differentiation factors signal cell to exit cell cycle and become a specialized cell (e.g. neuron or hepatocyte)
G0 to G1
Normal - Growth Factors, e.g. EGF signals cell to re-enter cell cycle and proliferate
Abnormal - Cancerous transformation |
|
|
Term
|
Definition
G1 is the cell growth phase
-Cell volume (size) generally increases to ensure a daughter cell will not be only half the size
-Prepares for DNA replication
-Length highly variable
Fast dividing cells have very short or even no G1 phase
Embryonic cells have large enough volume to be divided many times without growth (no need for G1). |
|
|
Term
|
Definition
S: DNA replication phase
-Amount of DNA doubles after the completion of S phase
-Length depends on the number of active DNA replicons
Fast dividing cells can have very short S phase
Total number of replicons increases and all the replicons are activated (firing) at once
G2: another growth phase
Prepares for mitosis |
|
|
Term
| Multiple Replicons in Eukaryotic DNA |
|
Definition
|
|
Term
| the directionality of dna synthesis |
|
Definition
DNA synthesis: addition of new nucleotides
-Mediated by DNA polymerase
-Can only happen at the 3’ end of the DNA strand
-Base-pairing determines what the next nucleotide is
-Phosphoester bond formed |
|
|
Term
| Discontinuous DNA Synthesis of the lagging strand at a Replication Fork |
|
Definition
Leading strand: new strand grows the same direction the replication fork moves
Lagging strand: new strands grows the opposite direction the replication fork moves |
|
|
Term
| Proofreading by 3’to 5’ Exonuclease |
|
Definition
DNA polymerase
5’-3’ DNA polymerase activity
3’-5’ exonuclease activity |
|
|
Term
| The Role of RNA Primers in DNA Replication |
|
Definition
DNA synthesis: addition of new nucleotides. DNA polymerase can only add nucleotides to an existing chain
-The need for a primer
-The primer is an RNA of 3-10 nts
-Primase initiate primer synthesis from scratch using DNA as a template
-The primers are later removed |
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Term
| Proteins Involved in Unwinding the DNA at the Replication Fork |
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Definition
DNA helicase: unwinds the double-stranded DNA
Topoisomerase: removes the tension generated ahead of the fork during unwinding/replication
ssDNA binding proteins: protect the separated DNA strands from degradation |
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Term
| DNA Replication: Problem at the end |
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Definition
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Term
| The Extension of Telomeres by Telomerase- replication of the lagging strand of a linear chromosome encounters a problem at the 3' end |
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Definition
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Term
| DNA Amplification Using the Polymerase Chain Reaction- kary b mullis |
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Definition
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Term
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Definition
M-phase: separation of the two copies of the genomic DNA which was created in S phase by DNA replication
Mitosis: nuclear division
Cytokinesis: cytoplasmic division |
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Term
| DNA packaging: Nucleosomes |
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Definition
Nucleosome core:
146bp of DNA wrapping around 8 histones
H2A, H2B, H3, H4
Histone H1 binds to the linker region |
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Term
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Definition
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Term
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Definition
Prophase
nucleus
chromatin---chromosome
Cytoplasm
centrosomes (duplicated during S phase) move apart
Mitotic spindle formation
Interpolar MT
Astral MT |
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Term
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Definition
nuclear envelope breakdown, allowing MT to enter nuclear area to make contact with chromosome
Kinetochore formation and attachment to MT
Kinetochore MT
Interpolar (polar) MT
Astral MT |
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Term
| centromere and kinetochore |
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Definition
Centromere
Constriction at chromatid
Contains simple, repetitive DNA called CEN sequences
Two sister chromatids are attached at the centromeric region
Kinetochore
layered plate-like protein structure associated with centromere DNA in sister chromatids
One for each sister chromatid and facing the opposite way
In contact with the + end of kinetochore MT (30-40 MTs for a mammalian kinetochore) |
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Term
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Definition
Metaphase
chromosome maximally condensed
sister chromatids aligned in spindle with each kinetochore connected to one or the other pole by ‘kinetochore’ MTs
Metaphase plate
Ready for separation
Karyotype
Metaphase arrest allow visualization of individual chromosomes
Metaphase - 3 sets of spindle MTs
‘kinetochore’ MTs - Kinetochore to pole
interpolar MTs - form poles into central spindle where they overlap with IP MTs from other pole
astral MTs projecting from pole toward cell surface
Aligned chromosomes are being pulled by balanced forces from the opposite poles |
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Term
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Definition
Separation of sister chromatids
Anaphase A
Chromosome to pole
Anaphase B
Pole-pole separation
Fast process |
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Term
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Definition
Kinetochore MTs shorten as chromosomes move toward poles
Motor protein in the kinetochore promote MT depolymerization at the (+) end, moving the chromosomes towards pole
Motor protein in the spindle pole promote MT depolymerization at the (-) end), reeling in the kinetochore MT and the chromosomes
Taxol inhibits chromosome movement by inhibiting MT depolymerization |
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Term
| The Use of Laser Photobleaching to Study Chromosome Movement During Mitosis |
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Definition
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Term
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Definition
Anaphase B requires ATP
Kinesin-like (+) end-directed motors slide interpolar MTs and push poles apart
Dynein-like (-) end -directed motors associated with cell membrane may pull on astral MTs |
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Term
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Definition
Telophase
cleavage furrow forms on cell surface at position of original metaphase plate
Contractile ring formed just beneath the PM
nuclei reform in daughter cells
chromosomes de-condense |
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Term
| cytokinesis: cleavage furrow |
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Definition
Contractile ring formed just beneath the PM
Ring of actin and myosin II filaments produces ‘purse-string’ contractile force that cleaves cell into two cells
Ring always in plane of metaphase plate |
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Term
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Definition
Contractile ring formed just beneath the PM
Ring of actin and myosin II filaments produces ‘purse-string’ contractile force that cleaves cell into two cells
Ring always in plane of metaphase plate |
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Term
| Cell Cycle Regulation-overview |
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Definition
Progression through cell cycle is regulated at multiple transition points
-Ensure the completion of a previous phase before commencing the next
-Can respond to signal for growth or arrest
-G1/S transition
-G2-M transition
-Metaphase-Anaphase transition
G2-M transition
Ready for cell division? Accumulate more cytoplasm
Need for cell division?
Metaphase-anaphase transition
Chromosomes properly attached to the mitotic spindle?
Chromosomes properly aligned at the metaphase plate? |
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Term
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Definition
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Term
| Molecules Regulating Cell Cycle |
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Definition
Cdks: cyclin-dependent kinases
-Inactive unless bound to cyclins
-Phosphorylate key proteins involved in cell cycle progression
-Called cdc (cell division cycle) genes in yeast
Cyclins: activator proteins for CDKS
-Expression level oscillates up and down through out the cell cycle
G1 cyclins
Mitotic cyclins |
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Term
| M-phase signal: Maturation Promoting Factor (MPF) |
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Definition
Frog oocyte
-stimulated to mature by female hormone
-cytoplasm extracted from oocyte in M-phase - microinjected into a G2-arrested oocyte - stimulates injected cell to enter M-phase without hormone
demonstrates that a ‘Maturation-Promoting Factor’ or M-phase-Promoting Factor (MPF) exists in the cytoplasm of M-phase cells |
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Term
| MPF is a cyclin-Cdk complex |
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Definition
Mitotic Cyclin (Cyclin B)
-synthesized continuously in cells to maximum amount at M-phase -
-during M-phase cyclin B is destroyed by proteolysis
-MPF is the complex of mitotic cyclin and mitotic CDK (CDK1) |
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Term
| Phosphorylation and Dephosphorylation in the Activation of a Cdk-Cyclin Complex |
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Definition
Protein kinase: add phosphate groups to target protein
Phosphatase: removes phosphate groups from target protein |
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Term
| The Mitotic Cdk Initiates M-phase |
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Definition
Nuclear envelope breakdown
Phosphorylation of lamins cause them to depolymerize, lamina dissembles
Chromosome condensation
Phosphorylation of condensin
Phosphorylation of histones
Mitotic spindle formation
Phosphorylation of MAPs
Activate Anaphase promoting complex (APC) |
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Term
| The APC and the Separation of Sister Chromatids |
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Definition
Anaphase-Promoting Complex :
targets Securin for breakdown
-Cohesin holds sister chromatids together
-Separase can breakdown cohesin
-Separase is normally bound and secluded by Securin
-APC degrades Securin
-relieves inhibition of sister chromatid separation
-starts anaphase |
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Term
| The APC and The Mitotic Cyclin Breakdown |
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Definition
Anaphase-Promoting Complex
-activates Mitotic Cyclin breakdown mechanism
negative feedback on MPF activity
cyclin B contains ‘destruction box’ protein sequence motif that is recognized by APC
Ubiquitination: ubiquitin-mediated proteolysis
mutation of destruction box prevents cyclin B degradation - prevents M-G1 transition
-Breakdown of mitotic cyclin necessary for cytokinesis |
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Term
| The Mitotic cyclin breakdown and cytokinesis |
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Definition
Cleavage furrow myosin II
-MPF phosphorylates an inhibitory site on cleavage furrow myosin II - inhibits myosin activity and prevents cleavage until MPF activity falls
-drop in MPF activity - phosphatase dephosphorylates myosin - allows force production for cytokinesis |
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Term
| Cell Cycle Regulation: G1/S transition |
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Definition
Mammalian cells that no longer progress through cell cycle are most commonly stopped (“arrested”) at late G1
Restriction point (“start” for yeast) prevent the cells that are “not ready” from going into DNA replication
-No nutrients
-No space
-Protein synthesis halted
-Defective DNA
Passing through the restriction point (“start” for yeast) is the main steps that commits a cell to proceed through the rest of the cycle
Positive mitogenic/ proliferation signals required for cell to pass G1 restriction point to enter S phase
Controlled by G1 cyclin/Cdks
G1/S control is often lost in cancer cells |
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Term
| Cell Cycle Regulation: Retinoblastoma |
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Definition
No external mitogenic signal - Retinoblastoma (Rb) inhibits G1-S transition -
-sequesters E2F transcription factor that stimulates expression of proteins required for DNA replication
Mitogenic signal (e.g. growth factor) - stimulates synthesis of Cyclin D - activates Cdk4/6 kinase activity
Cyclin D-Cdk4/6 phosphorylates Rb - releases E2F
E2F stimulates expression of proteins (early response genes) required for DNA replication - cell enters S phase |
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Term
| Cell Cycle Regulation: Growth factors |
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Definition
Positive mitogenic/ proliferation signals required for cell to pass G1 restriction point to enter S phase
-activation of RTK-Ras-MAPK pathway
Positive mitogenic/ proliferation signals required for cell to pass G1 restriction point to enter S phase
-Activated MAPK translocates (NLS-based nuclear import !!) into the nucleus and activate transcription factors
-Cyclin D made and Cdk activated
-Cyclin D-Cdk4/6 phosphorylates Rb - releases E2F |
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Term
| Retinoblastoma: a tumor suppressor |
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Definition
Tumor suppressor - 'loss of function' mutation leads to uncontrolled cell division
Mutated (loss of function) Rb fails to bind E2F - no inhibition of G1-S transition even when cyclin D-Cdk4/6 is not activated
Individuals with mutated Rb genes develop eye tumor
Human Papilloma Viruses (HPV) infection can cause cervical cancer
Once inside the cells, HPV make several viral proteins, one of them called E7
E7 binds to Rb, pulling it away from E2F.
E7 is called an oncogene |
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Term
| Cell cycle regulation: damage checkpoints |
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Definition
G1/S transition (Rb): brake of cell cycle, responds to growth signal.
In addition, the progression through cell cycle is constantly monitored
cycle is also blocked when there is
-DNA damage (G1 , S, G2)
-Incomplete DNA replication (G2)
-improper spindle function (M) |
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Term
| Cell cycle regulation: DNA damage checkpoint |
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Definition
p53 protein - transcription factor normally very unstable - stabilized by presence of damaged DNA
-presence of p53 stimulates expression of p21
-p21 inhibits cyclin-Cdks - arrests cell cycle until DNA damage is repaired
When DNA is beyond repair, p53 turns on the cell death pathway and cell commits suicide |
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Term
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Definition
p53 mutation inactivates p53 (loss of function) -
allows cell to replicate damaged DNA
Replication of damaged DNA can lead to replication and expression of mutated genes that cause cancerous transformation
p53 mutated in majority of cancers |
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Term
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Definition
First: you know the following from intro bio or genetics so we can get to Meiosis……
Sexual reproduction
Gametes and zygotes
Diploid and haploid cells |
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Term
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Definition
Reduce the number of chromosomes by half in the gametes
Starts with one diploid cells, ends up with four haploid cells
One round of DNA replication followed by two successive cell divisions
Meiosis I and Meiosis II
-Meiosis I: reduction division
-Meiosis II: a mitosis |
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Term
| Meiosis II: a mitosis of the two haploid cells |
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Definition
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Term
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Definition
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