Exam 4 Outline -1

8/22/2019 Exam 4 Outline -1

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cilia and flagella - structure and function

- three parts:- basal body (interchangeable with centriole), transitional zone, axoneme

- MT organization

- can be singlets, doublets, triplets; in doublets and triplets only the A filament is complete- basal body: 9 groups of fused triplets in cartwheel

- axoneme:- doublets are connected to one another by nexin protein

- nonmotile: 9+0 arrangement - 9 doublets in a circle around axoneme- may or may not have axonemal dynein

- motile: 9+2 arrangement - 9 doublets in a circle with two singlets in the center

- doublets connected to central pair via radial spokes as well as to one another with nexin; inner/outer dynein arms attached to doublets

- what cells?- only non-dividing- all vertebrate non-dividing cells have at least a primary cilium (nonmotile), many have beating cilia to induce

fluid flow (kidneys, trachea) and maintain proper functioning- functions of cilia/flagella

- single celled - often for motility; flagella and beating cilia = movement in single celled organisms and sperm- primary cilia - sensory (more below)- beating cilia - fluid flow that assists in tubule development and organ function

- primary/immotile cilia as signalling/sensory organelles- photoreceptors

- outer segment (discs w/ rhodopsin embedded) continually turns over, materials for construction andmaintenance must be transported from cell body

- connecting cilium is only link between cell body and outer segment; must be used as a highway

- motor based transport system called IFT transports all necessary lipids and proteins to outer segment- very fast - 2k opsin molecules/min through cilium in mammals

- olfactory neurons - similarities to photoreceptors

- OSN is main sensory cell containing components of olfactory signalling cascade- bipolar neurons with axons projecting through bony cribiform plate into oflactory bulb in brain

- dendritic knob on OSN is directly exposed to odors in nasal cavity- short dendrites containing multiple basal bodies - project into mucous of olfactory

epithelium- each OSN expresses only one type of odorant receptor

- all OSNs with a given receptor type connect to the same region in olfactory bulb

- sustentacular cells (SC) - support cells w/ microvilli- play roll in maintaining water balance and regulating mucosal ion content

- basal cells (BC) - replenish OSN and SC populations- olfactory signalling cascade:

- odorant binds receptor in PM of OSN cilia- activated receptor stimulates Golf, which activates adenylate cyclase type III (ACIII)- increased cAMP triggers opening of cyclic nucleotide gated Na+/Ca2+ channel (CNG)

- Na+/Ca2+ influx -> local membrane depolaraztion spreads from dendrites to entire plasmamembrane


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- voltage gated Na+ channels open in axon hillock, triggers influx of Na+, generates action

potential- AP triggers release of neurotransmitter (both excitatory and inhibitory), sensed as odor in

olfactory bulb

- cAMP activates PKA which regulates transcription and other signalling proteins- axonal/axonemal transport of cellular materials and organelles

- all MTs organized with - end towards centrosome- rate of axonal transport can be measured in vivo

- pulse-chase experiments - transport occurs bidirectionallyalong axons

- anterograde transport - cell body to synapse:

associated with axonal growth and delivery ofsynaptic vesicles

- retrograde transport - back towards cell body;brings old membranes from synapse to cell bodyto be degraded by lysosomes

- dorsal root ganglia extend from dorsal horn of spinal cordinto limbs

- inject radiolabelled amino acids into DRG axonnear cell body

- sacrifice mice at different time points, dissect out

injected axons- sciatic nerve is cut into small pieces and run out

on a gel to identify where radiolabelled proteinsare

- fastest moving material - vesicles at 3m/

s- slowest moving - tubulin subunits,

neurofilaments at 1mm/day- mitochondria move at intermediate rate

- meausre in giant axons of squid - squeeze cytoplasm from

axons so that organelles transported present as lumpsalong axon

- shows two organelles moving in opposite directions will slide past one another and continuealong axon

- MT motor proteins- kinesin - identified via purification of axonal extracts- fractionate axonal extracts into three components:

- purified organelles- organelle free cytoplasmic extract

- taxol-stabilized MTs- mix three components, add ATP, organelles move

along MTs

- no cytoplasmic extract = no organelle movement- ATP hydrolyzed to ADP = organelles fall off of MTs

- non hydrolyzable ATP (AMPNP) = organelles bindbut do not move

- so motor is bound tightly to MT in presence of ATP,

but requires hydrolysis in order to move

- suggested way of purifying motor protein- add cytoplasmic extract and organelles to

MTs in presence of AMPNP- allow organelles to bind, then precipitate

MTs- replace AMPNP with ADP (releases motor protein), run out on gel

- purification identified kinesin-1- dimer of two heavy chains, each associated with a light chain

- globular head domain, binds MTs and ATP - responsible for motor activity


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- short flexible linker region is

responsible for forward motility- stalk domain binds receptors on

membrane and cargo vesicles

- in vitro assays: kinesin-1 coatedbeads move towards + end of MT

- kinesin mediates anterogradeaxonal transport

- 45 kinesin family genes in human genome; 14classes based on sequence similarties anddifferences in motor domains and tail domains

- function of all kinesin molecules is not known, but all are + end directed motor proteins with only twoexceptions

- kinesin-14 moves towards - end of MTs andfunctions in mitosis

- kinesin-13 destabilizes MTs

- binds and curves end of tubulinprotofilament to tubulin GDP

conformation- facilitates removal of tubulin dimers,

enhancing frequency of catastrophes

- require ATP hydrolysis to dissociatefrom -tubulin dimer

- defects in kinesin family proteins result in neurologicaldisorders

- kinesin-1 mutants = defective neurons

- axons are shorter- neurons accumulate cargoes (vesicles, synaptic membrane proteins, mitochondria and

prelysosomal particles; cytoskeletal elements) due to slow axonal transport- stores of kinesin decrease, axonal transport stalls causing progressive lose of neuronal

function

- kinesin-1 is required for neural function in flies- eventually results in distal paralysis

- neurons can no longer communicate with muscles- first exp model for human neurodegenerative diseases like ALS

- Charcot-Marie-Tooth peripheral neuropathy type 2A- autosomal dominant, caused by mutations in MFN2 (mitofusion) or KIF1B (kinesin-1B)- causes damage to peripheral nerves; characterized by loss of sensation in extremities

and postural tremor- onset typically in first or second decade of life

- some patients become dependent on crutches/wheelchairs, most do not- progressive degeneration


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- kinesin function

- kinesin heads bind to -subunits of tubulin in aprotofilament

- leading head binds ATP cases linker region to

zipper - point forward and dock in head- conf change throws ADP bound trailing head

forward to become new leading head- new leading head weakly binds MT binding site

16nm down- ADP exchanged for ATP, causing new leading

head to bind tightly to MT

- induces trailing head to hydrolyze ATP to ADPand Pi

- release of Pi from ADP bound trailing headcauses it to weakly associate with MT

- occurs in 16nm hand over hand steps

- kinesin is highly progressive- optical trap experiments and fluorescent

labeling experiments show that kinesin ishighly progressive, like myosin V

- 16nm hand over hand steps

- two heads work in coordination; one isalways contacting MT protofilament

- doesnt release, so progressive- despite lack of sequence homology

between myosin and kinesin, x ray

crystallography reveals that catalyticcores of each have same overal

structure- convergent evolution - making fold to hydrolyze ATP and generate work

- dyneins - retrograde transport

- predominantly - end directed motors- structure

- 2 large, 2 intermediate, and 2 smallsubunits

- two headed molecule built around twoidentical heavy chains- stem region - other dynein subunits bind

and associate with cargo via dynactincomplex

- linker - critical role in ATP dependentmotor activity

- head region - contains 6 AAA ATPase

repeats assembled into a flower likestructure

- stalk - embedded between 4th and 5thAAA ATPase domain; contains MTbinding domain

- major unanswered question: how MT

binding and ATP hydrolysis are linked,given they are physically separated

- the dynein power stroke- dynein head consists of planar ring with

7 domains - 6 AAA ATPase domainsand a heavy-chain C terminal domain

- 4 AAA domains retain ATP binding sequences- all four bind ATP, but only 1 hydrolyzes ATP (closest to tail domain)

- before power stroke - stem/tail attached to linker that lies across 1st and 3rd AAA repeat

- after hydrolysis of ATP, linker lies across 1st and 5th AAA domain; head rotates to bring stemand stalk closer together


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- 25nm stalk separates head from MT binding domain

- affect of ATP hydrolysis on dynein affinity for MT is poorly understood. ATP dynein has a lowaffinity, ADP dynein has a high affinity

- suggested that there is a sliding mechanism in recent literature

- power stroke model- AAA1 ATPase is free of nucleotide, dynein is bound tightly to MT and AAA5 domain is

latched to linker- ATP binding to AAA1 site induces conf change in head, rearranging so that linker

detaches from AAA3 and reattaches to AAA5- linker rearrangement moves stalk relative to rest of molecule and ATP is hydrolyzed- presence of ADP permits weak reattachment of dynein to MT, but at a location further

towards - end of MT- reattachment to MT triggers release of ADP + Pi, permitting strong binding to MT

- linker region straightens to post-powerstroke form, pulling cargo toward new bindingsite

- cytoplasmic dyneins do not interact directly with organelles

- dynactin links dynein to its cargo and regulates activity- dynactin - 11 subunits

- one domain built around Arp1, which assembles into short filament- + end of Arp1 filament is capped by CapZ - cargo binding region- second domain is long p150glued protein

- binds dynein and binds +TIP EB1- associates with + end of MT

- held in inactive state until + end reaches cortex- activated - pulls on MT that delivered it to cortex

- dynamitin holds two domains together

- when overexpressed, complex explodes- permits identification of processes dependent on dynein-dynactin

complex- activity of cytoplasmic dynein is controlled by associated protein

- NudE connects dynein intermediate and light chains to Lis1 protein

- Lis1 protein interacts with AAA ATPase domain, lengthening power stroke- increases processivity

- autosomal dominant mutations in Lis1 cause Miller-Dieker lissencephaly (smooth brain)due to defects in neuronal mitoses and migration of neurons to outer surface (cortical

plate) of cerebral cortex- activity of kinesin and dynein are tightly controlled in vitro- MT on glass slide with bound melanosome - changes direction in middle of sequence

- comparison of motor protein power strokes- myosin:

- myosin-ATP releases from actin- myosin-ADP-Pi binds weakly- apo-myosin binds tightly

- heads not coordinated in ATP hydrolysis and actin binding- fastest myosin moves at 60m/s - fastest of motor proteins

- kinesin:- kinesin-ATP binds tightly to MT- kinesin-ADP-Pi binds weakly

- kinesin powerstroke is generated by exchange of ADP for ATP

- kinesin heads are coordinated in ATP hydrolysis and MT binding- fastest kinesins move at 2-3m/s - slowest of the motor proteins

- dynein:- dynein-ATP releases from MT

- dynein-ADP-Pi binds weakly- apo-dynein binds tightly

- dynein travels at a rate of 14m/s - intermediate


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- organelle transport via kinesin and dynein

- activity of both is tightly controlled in cells- melanosome transport

- melanophores are cells containing

melanin pigment in organelles calledmelanosomes that are bound to

dynein, myosin V, and kinesin- high cAMP - kinesin-2 disperses

melanosomes (darkens skin)- low cAMP - cytoplasmic dynein

aggregates melanosomes (lightens

skin)- fish and frogs can change

pigmentation states rapidly forcamoflage or social interaction

- controlled by neurotransmitters in fish

and hormones in frogs - activateGPCR and alters levels of cAMP

- hormone stimulation results inphosphorylation of kinesin light chain, inactivating motor

- dynein wins out and melanosomes move to center

- beating of cilia and flagella- power stroke begins at base and propagates along length of

flagellum- observation in flagellar axonemes from sea urchin sperm

- treat with detergent to remove membranes

- attach gold beads to flagellum - slide apart as wave movesthrough region

- suggests that MT filaments in axoneme slide past eachother during stroke

- axonemal dynein decorates A filaments

- heterodimers/-trimers; two or three heads- complex contains 9-12 polypeptides

- eukaryotic axonemal dynein has two globular heads; inprotozoa has three

- stem/tail binds A filament- globular heads have binding site for B filament- ATP present - axonemal dynein on A filament walks down

MTs on B filament- further analysis:

- treat isolated axonemes with detergent to removemembranes

- cleave nexin links with a light trypsin treatment

- add ATP - MTs slide apart- axonemal dynein molecules attached to A filament on one

doublet walks towards - end of B doublet on other filament- axonemal dynein is a - end directed motor protein

- cilia and flagella are dynamic structures

- MTs in cilia and flagella treadmill slowly

- new subunits must be transported to + end and then back to base- IFT B particles contain heterodimeric kinesin-2 and transport in

anterograde direction to tip at constant speed of 2.5m/s- IFT A particles contain cytoplasmic dynein and transport material

(including kinesin-2) to base at constant rate of 4m/s- particles travel along the outer doublets, underneath the plasma membrane

- cilia and flagella are responsible for fluid flow in organs- cilia in developing embryo responsible for creating fluid flow that causes left-right organization of developing

organs

- in lungs, reponsible for movement of mucous; removal of unwanted particles from lungs- in kidneys, responsible for fluid movement necessary for urination


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- in sperm, necessary for motility

- in photoreceptors, necessary for turnover of structures in outer segment- cilia function is disrupted by mutations in genes encoding IFT proteins - called ciliopathies

- Karagener s syndrome - recessive (1:32k live births):

- retinal degeneration- lack of odor sensation

- defects in organization of internal organs along left-right axis- male sterility

- respiratory infections- autosomal dominant polycystic kidney disease

- growth of cysts in kidneys

- affects 600k in US, 4th leading cause of kidney failure

epithelial tissues and cell junctions- epithelial sheets

- general structure

- polarized epithelial cells with apical and basal surfaces- basal lamina that is part of extracellular matrix

- connective tissue containing mesenchymal cells- types of epithelia:

- simple columnar epithelia

- elongated cells specialized for secretion or absorption- simple squamous epithelia

- composed of very thin cells, line blood vessels andmany body cavities

- transitional epithelia

- composed of several layers of cells with different shapes- line cavities subject to expansion and contraction

- stratified squamous epithelia (nonkeratinized)- line surfaces such as mouth or vagina

- resist abrasion but do not secrete/absorb materials into

cavity-junctions mediate cell adhesion - 3 types

- tight junctions- hold tissue together

- mediate flow of solutes through extracellular spaces betweencells forming and epithelial sheet

- maintain cell polarity between apical and basolateral regions by

preventing diffusion of membrane proteins and glycolipidsbetween two regions

- primarily in epithelial cells- anchoring junctions

- found in epithelial and non-epithelial

cells- three molecular parts:

- adhesive proteins in plasmamembrane that connect onecell to another on lateral

surfaces (CAMs) or to the

ECM on basal surface(adhesion receptors)

- adaptor proteins connectCAMs or adhesion receptors

to cytoskeletal filaments andsignalling molecules

- cytoskeleton- two types of anchoring junctions:

- adherens junctions

- form in apical region of cell underneath tight junctions and around entire circumference- desmosomes/hemi-desmosomes


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- form in lateral surface (desmosomes) or basal surface (hemidesmosomes)

- like spot welding- form spots of adhesion between two cells or between a cell and the ECM

- gap junctions

- found in epithelial and nonepithelial cells- form tiny holes in membranes, permitting rapid diffusion of small water soluble molecules and ions

bteween cytoplasm of adjacent cells- tight junctions form a seal between adjacent cells

- to illustrate:- use an electron dense tracer molecule (lanthanum hydroxide) added on luminal or basal side of

epithelium

- tight junctions in pancreas are impermeable to the large water soluble colloid lanthanum hydroxide- image epithelium after adding tracer molecule and can see that when added to apical side of epithelium,

it does not pass into spaces between lateral sides of cells; when added to basal side, shows that tracermolecule moves between cells but is stopped before reaching apical side

- tight junctions found near apical side of cells

- nutrients are thus brought into the body by a transcellular pathway of specific membrane bound transportproteins

- some epithelia have selectively permeable tight junctions- permit certain small molecules and ions to cross via paracellular pathway

- extracellular loop of claudin is involved

- tight junction permeability can be regulated by signalling pathways, particularly GPCR and cAMP coupledpathways

- structure of tight junctions- ultrastructure

- when visualized by freeze fracture, tight junctions form a honeycomb pattern

- sealing strands are visible as ridges of intramembrane particles on cytoplasmic surface orcomplementary grooves on external face of membrane

- tight junctions do not connect to cytoskeleton- molecular structure

- claudin and occludin form sealing strand

for tight junction- both are 4 pass transmembrane

proteins with N and C terminalson cytoplasmic side

- occludin is less essential than claudin- tricellulin seals the membrane wherethree cells meet

- claudin- 24 members of claudin family in

humans- form paracellular pores, selective

channels that allow specific ions

to cross tight-junctional barrierfrom one extracellular space to

another- claudin-1-/- mice fail to make tight junctions and baby mice rapidly lose water and die

within a day of birth

- occludin

- c terminus of occludin binds to PDZ domains in large cytosolic adapter proteins- 80-90 aa domain found in 100 different proteins- serve as scaffolds to assemble larger complexes

- occludin recruits PDZ containing proteins to surface, where they interact with claudin

and stabilize tight junction- vibrio cholera alters composition of tight junctions by producing a protease that degrades the

extracellular part of occludin- results in massive loss of internal body ions and water into gut, leading to diarrhea


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- adherens junctions (type of anchoring junction) mediate reaggregation of dispersed tissue

- cells have ability to distinguish neighbors and who neighbors should be

- cells will migrate to be with correct neighbors- Holtfreter - cells sort by two mechanisms: differential adhesion and selective migration

- Steinberg - differential adhesion is enough to sort cells- differential adhesion hypothesis

- some cell types migrate peripherally when mixed with certain cell types, but migrate centrally when

mixed with other cell types- sorting follows certain rules:

- if final position of cell type A is internal to cell type B and B is internal to C, then A will always be

internal to C- hypothesis: cells rearrange themselves into most thermodynamically stable configuration with cells of

highest adhesitivity internal- Steinberg made movies of reaggregating cells to test hypothesis

- if morphogen model is correct, individual cells will move towards center/periphery of aggregate- if differential adhesion is correct, cells would form small clusters, then clusters would aggregate

to form larger and larger clusters - like hydrophobic effect

- results supported differential adhesion (shown above), but mechanism is still not understood- three components of adherens junctions

- CAM- E-cadhedrin in embryo, P-cadhedrin in

placenta, N-cadhedrin in neural plate, R-

cadhedrin in retina, B-cadhedrin in neuralcells

- cadhedrin is not the only CAM- cytoskeleton

- F-actin, unbranched

- adaptor proteins

- connect CAM to cytoskeleton

- a-catenin, b-catenin, p120-catenin- ZO1 adaptor protein interacts with several

different CAMs- cadhedrin

- function depends on Ca2+ in medium

- L-cells do not normally adhere to each other


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- if transfected with a cadherin transgene, cells adhere to each other if and only if Ca2+ is in

medium- cadhedrin type and expression levels control differential adhesion

- cells segregate according cadhedrin content/level

- cadhedrin is required for cell adhesion- inject a frog oocyte with antisense E-cadhedrin mRNA

- depletes E-cadhedrin protein and causes embryos to fall apart- targeted expression of dominant negative N-cadhedrin (extracellular domain, but no intracellular

domain) disrupts cell adhesion- E-cadhedrin activity is lost in cancer cells

- snail protein is transcription factor inhibiting expression of E cadhedrin

- absence of snail - cells express cadhedrin and adhere to each other- if forced to express snail, lose E cadhedrin expression and undergo epithelial to mesenchymal

transition- disassembly of all junctions

- cells become migratory

- desmosomal junctions- three components

- CAM- specialized cadhedrins called desmoglein and desmocollin have different cytoplasmic domain

than standard cadhedrins

- desmoglein discovered by studying people with autoimmune disorder, pemphigus vulgaris- patients produce antibodies targeting desmoglein, disrupts cell adhesion and induces

blisters (skin separates, space fills with fluid)- cytoskeleton

- intermediate filament proteins

- anchoring proteins- plakoglobin (related to -catenin), plakophilins, and desmoplakin

Intermediate filaments

- differences with other cytoskeletal elements

- form polymers of 10nm diameter- encoded in genome by 70 different genes

- 5 families by sequence homology- expressed in tissue specific manner

- not polarized- mechanism of assembly is not understood

- no known nucleating, sequestering, severing, or capping proteins

- structure of intermediate filaments- conserved rod domain flanked by nonhelical N and C terminal domains

- N and C terminal domains are different, dimer is polarized- rod domain is ~310 amino acids; -helical, coiled coil structure

- helix characterized by heptad repeat sequence with hydrophobid residues

usually at positions 1 and 4- helices of two proteins pack together, mediated by interactions

between these hydrophobic side chains- dimers associate with other dimers in anti-parallel fasion to form a non-

polarized, symmetric tetramer

- tetramers are offset so that C terminals at either end overhang

- tetramers are basic building block of IFs; assemble end to end to formprotofilament

- 4 protofilaments form protofibril

- four protofibrils assemble into 10nm IF

- so 16 interlocked protofilaments- IFs are more stable, but still dynamic

- add biotin-labeled type I keratin to fibroblasts- inorporated into existing cytoskeleton within 2-4 hours

- five classes of IFs

- class I - acidic keratins- in epithelial cells


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- propsed to be involved in tissue strength and integrity

- class II - basic keratins- in epithelial cells

- proposed to be involved in tissue strength and integrity

- class III - desmin, GFAP, vimentin- muscle cells, glial cells, mesenchymal cells

- proposed to be involved in sarcomere organization and integrity- class IV - neurofilaments (NFL, NFM, NFH)

- neurons- proposed to be involved in axon organization

- class V - lamins

- nucleus- proposed to be involved in nuclear structure and organization

- IF diseases- epidermolysis bullosa simplex

- normally keratin 14 forms heterodimer with keratin 4 in basal cells of epidermis and assemble into

protofilaments- as cells mature, keratin 4 and 14 are replaced by keratin 1 and 10

- K14 lacking N or C terminal domain will form heterodimers with K4, but dimers cannot form tetramersand assemble into protofilaments

- basal cells become fragile and unable to withstand abrasion

- causes epidermis to separate from dermis, causing blisters- disruption of desmosomes at CAM or cytoskeleton results in weak cell-cell adhesion

- Hutchinson-Guilford progeria- caused by mutations in LMNA

- rare spontaneous dominant mutation in lamin A/C gene (LMNA)

- 2003 - mutation is single base change that activates cryptic splice site and produces truncated lamin Aprotein (progerin)

- premature aging disorder- normal appearance at birth

- as they grow, develop characteristic features

- baldness- pinched nose

- aged skin- dwarfism

- craniofacial defects resulting in a small face and jaw- life expectancy is 13-14 years

- death usually due to cardiovascular disease

- nuclear instability can lead to widespread death in tissues that undergo high levels of stress -e.g., heart muscle

- nuclear membranes show abnormal morphology- the mutant LMNA is processed incorrectly

- normally synthesized as large precursor protein, modified by addition of farnesyl group (lipid)

- farnesyl modification is common in proteins localized to membranes (like ras)- ras converting enzyme cleaves precursor at site of farnesylation

- last step is blocked in HGPS- farnesylated intermediate accumulates in nuclei, causing deformities

- farnesyl transferase inhibitors identified as part of screens to treat cancer

- can blocking farnesylation of precursor protein prevent symptoms?

- mouse model experiments promising, clinical trials begun in 2007


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mammalian cell cycle

- divided into 3 stages- G0 - cells are quiescent; can be reversed

- interphase

- G1 - first gap phase; period between birth of daughter celland onset of DNA synthesis

- cells grow in size and make critical decisionsabout wether to divide, become quiescent, or

differentiate- S - DNA replication and centrosome duplication

- cultured cells - typically 6-8 hrs - determined by

size of genome- G2 - second gap phase; lasts 3-5 hours

- cell prepares for mitosis, but not fully understoodhow

- M - mitosis - 5 stages

- prophase- breakdown of interphase microtubule displace and

replacement by mitotic asters- mitotic aster separation

- chromosome condensation

- kinetochore assembly- metaphase

- nuclear envelope breaks down- chromosomes are captured, bi-oriented and brought to spindle equator

- chromosomes line up at metaphase plate

- anaphase- APC/C activated, cohesins degraded

- anaphase A: chromosome movement to poles- anaphase B: spindle pole separation

- telophase

- nuclear envelope reassembly- assembly of contractile ring (actin)

- cytokinesis- reformation of interphase microtubule array

- contractile ring forms cleavage furrow- embryonic cell cycle lacks G1 and G2 to allow for more rapid division

- centrosome duplication

- semi-conservative- two halves separate and serve as templates

for construction of new half- occurs once and only once per cell cycle

- incorrect number of centrosomes leads to

defects in spindle assembly and errors inchromosome segregation

- results in aneuploidy- two centrosomes move apart along nuclear envelope

and amount of g-TuRC increases dramatically

- increases nucleation of MTs - centrosome maturation

- cohesins- structure

- 4 subunits - 2 are Smc1 and Smc3 (structuralmainenance of chromosomes) - coiled coil proteins with ATPase domain at one end

- subunits linked together by kleisin- cohesins link sister chromatids after S phase

- cohesin complex is disrupted at metaphase-anaphase transition- deplete cohesins from xenopus egg extracts via treatment with anti-cohesin antibody

- DNA replicated, but sister chromatids did not associate properly

- unkown if single cohesin complex spans both sister chromatids, or if several cohesins link up like achain


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- cohesins are loaded during G1 phase, but do not have cohesive properties

- activated during DNA replication and hold sisters together- conversion requires help from cohesin loading factors

- during G2, sister chromatids are connected along entire length by cohesins

- Mei-S332/Sgo proteins recruit PP2A to centromere- PP2A protects cohesins located at centromere from removal during metaphase

- aurora kinase and polo kinase phosphorylate cohesins to release them from chromatin- spindle formation

- mitotic spindle is MT based machine dedicated toseparating sister chromatids

- contains three classes of MTs

- astral MTs extend from spindle pole to cellcortex

- kinetochore MTs extend from spindle pole tochromosome

- polar MTs extend from one pole towards

metaphase plate, interact with MTs from otherpole in anti-parallel manner

- responsible for pushing duplicatedcentrosomes during prophase,maintaining spindle structure, and

pusing spindle poles apart inanaphase B

- function- use early embryonic cells of frogs to observe

parts of cell cycle - large (easily manipulated) and

rapidly divide (~30 min; only S and M phases)- can make pure cytoplasmic extracts to

allow manipulation of cell cycle undersimplified conditions

- egg cytoplasm contains tubulin, ATP and

cytoplasmic proteins necessary formitosis

- sperm nucleus contributes centrosomes,DNA, and nuclear proteins

- MT instability increases in M phase cell extracts- entry into mitosis signals abrupt change in cell MTs

- interphase - cell contains a few long MTs

- M phase - MTs converted into a larger number of shorter, more dynamic MTs aroundcentrosome

- during prophase (and prometaphase),half life of MTs decreases dramatically

- increase in MT instability coupled with

increased ability of centrosome tonucleate MTs results in very dense,

dynamic array of MTs- add centrosomes and FL-tubulin

to xenopus egg extracts; MTs

nucleate from centrosomes

- MTs in mitotic extracts differ frominterphase extracts because ofincreased frequency of catastrophes

- arises from decrease in XMAP 215

(stabilizes MTs)- kinesin 13 (catastrophe factor) destabilizes MTs, levels remain constant


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- spindles and kinetochores

- kinetochores- specialized protein complex associated

with centromere

- centromere - specialized sequenceof DNA; varies greatly in size

- can visualize proteins using antibodiesfrom patients with lupus

- lupus is autoimmune disease(1.5-2mil in US; over 90% women15-45 yrs)

- symptoms include achy joints,fever, prolonged/extreme fatigue,

rashes, anemia, light involvement,and hair loss

- immune system loses ability to

distinguish self from other, will oftenmake antibodies against DNA, kinetochores, etc

- kinetochore proteins act to capture + ends of spindle MTs- varies in number of MTs attached - yeast is 1 MT, humans ~30, plants by hundreds

- constructed in back to back orientation, prevenging MTs from same pole attaching to

kinetochores on both sister chromatids- divided into inner and outer layer

- MT + ends are embedded in specialized MT attachment sites in outer kinetochore- MTs are selectively stabilized when captured by kinetochores, decreasing the number of catastrophes

- selective stabilization is caused by a gradient of ran GTPase activity

- ras-related nuclear antigen (ran) cycles in and out of nucleus as part of nuclear import/export machinery

- ran-GEF is bound to DNA and activates ran in nucleus- ran-GAP is distributed evenly throughout cytoplasm after disassembly of nuclear

envelope

- so, high levels of ran-GTP near chromosomes (high ran-GEF beats out activity of ran-GAP)

- ran-GTP activates unknown proteins that induce the release of MT stabilizing factorsfrom cytosolic protein complexes

- so, stabilzation of MTs near chromosome, biasing growth of MTs towardkinetochore

- attachment is unstable until chromatid is bi-oriented

- finding kinetochores- spindle MTs use search and capture mechanism to find and bind to kinetochores

- kinetochore binds to MT, laterally or at the end- if lateral, MT slides toward attachment site and converts to end on orientation,

stabilizing MT

- at the same time, MTs from the other spindle are attaching to kinetochore on sister chromatid,ensuring that they will eventually migrate in opposite directions


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- movement of chromosomes to

midpoint during metaphase- chromosomes oscillate in

position during early

metaphase and attach to theend or side of MT

- dynein/dynactin providesstrongest force pulling

chromosomes towards pole (-end)

- kinesin 13 facilitates this by

depolymerizing + end of MT- eventually MT from opposite

pole finds free kinetochore onopposite sister chromatid -now bi-oriented

- once some chromatid pairs arebi-oriented with others only

attached to one pole, CENP-E/kinesin7 from free kinetochorebinds to side of neighboring polar MT and walks to + end, helping orient kinetochore to attach to

MT from opposite pole- kinesin 4 is located on chromosome arms, with head domain oriented away from chromosome

- binds polar MTs and pulls arms towards center of spindle- congression involves bidirectional oscillation of chromatids

- one set of kinetochore MTs shortens, while those on the other side lengthen

- on shortening side, kinesin 13 aids in MT dissembly- dynein-dynactin complex moves to - end

- lengthening side - kinesin 7 holes on to growing MT- bi-orientation stabilizes MT

attachments

- intial MT attachment tokinetochore is weak,

permitting errors inattachment to be corrected

- attachments remain weakuntil bi-orientation occurs

- creates tension

across chromosome;increased tension

stabilizes MTattachments

- kinetochores assemble at

chromosome region markedby H3 histone variant, CENP-

A- complex process involving over 40 different proteins

- highly conserved from yeast to humans

- Ndc-80 complex is long and flexible; links + end of MT to inner kinetochore

- function controlled by proteins in chromosomal passenger complex (CPC)- early mitosis - CPC located at inner centromeric region of chromosomes- CPC contains aurora-B (kinase)

- phosphorylates several kinetochore components in centromeric region, including

Ndc-80 - pNdc-80 has weak association with MT- PP1 is associated with outer kinetochore and continually removes phosphates from Ndc-80 and

other proteins- so Ndc-80 switches back and forth constantly

- tension created by bi-orientation pulls both kinetochores away from CPC

- also pulls Ndc-80 to increase spacing between inner/outer kinetochore plates


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- result: aurora B can no longer phosphorylate Ndc-80 and other kinetochore

components- unphosphorylated Ndc-80 has strong attachment to + end of MT

- separation of chromatids during anaphase A (spindle poles

do not separate)- kinetochore MTs are under tension; remove cohesin

attachments between sister chromatids - freeschromosomes to move

- shortening of kinetochore MTs drives movement ofchromosomes during anaphase A

- kinesin 13 depolymerizes both + and - ends, and

loss of GTP cap provides energy for movement- process recapitulated in vitro - add

metaphase chromosomes to purifed MTs- chromosomes bind + end.

- dilute mixture - concentration of free

tubulin drops below Cc;chromosomes move toward - end of

MT- at anaphase, CPC remains at midzone as

chromosomes are pulled apart - associates with

polar MTs- centralspindlin complex joins CPC

- contains a + end directed kinesin motor protein- during anaphase B, centralspindlin recruits Cyk4, an exchange factor for RhoA

- RhoA-GTP activates formin, which nucleates assembly of actin filaments into

contractile ring- separation of chromatids during anaphase B

(spindle poles separate)- kinesin 5 is bipolar kinesin molecule

associated with overlapping polar MTs -

walk to + end of MTs- run in place, pushing polar MTs

apart with - end leading (like actinfilaments when myosin heads are

attached to glass coverslip)- spindles move apart, polar MTs mustgrow to accomodate increased distance,

even as kinetochore MTs continue toshrink

- dynein/dynactin complex is attached toplasma membrane

- cannot move

- aster MTs project + ends towardscell cortex

- dynein walks towards - end,generating pulling force onspindle and moving it towards cell

cortex

- cytokinesis- final step of cell cycle- cleavage furrow is first visible manifestation of cytokinesis

- occurs at same time as telophase (nuclear evenlope reassembly, chromatin decondensation)

- furrow deepens rapidly and spreads around cell until cell is divided in two- underlying structure is contractile ring

- contractile ring - actin based- as cells enter mitosis, stress fibers dissemble and actin reorganizes

- actin and myosin II accumlate at midpoint as sister chromatids separate in anaphase, forming

contractile ring- assembly of filaments is initiated by formin


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- contraction of ring exerts enough force to bend a glass fiber

- ring maintains same thickness as it contracts, suggesting total volume/number of filaments decreasessteadily

- actin filaments are highly dynamic

- polar MTs have furrow inducing capability- proteins that induce furrow formation are localized to overlapping interpolar MTs

- RhoA small G protein (regulates actin polymeriazaion by controlling formin activity; regulates myosin IIactivity by activation of kinases that phosphorylate myosin light chain)

- Cyk4 is a rhoA regulatory protein- cross section - rhoA forms ring in cell cortex, but cyk4 is localized to equatorial plane of cell, associated

with MT bundles

- regulation of the cell cycle- fuse cells with ethylene glycol

- fuse M-phase cell with G1 cell- nuclear envelopes of G1 phase cells retract and DNA condenses

- diffusible factor in M phase cells that can induce G1 to enter M phase

- MPF - M phase factor- fuse S phase cell with G1 cell; add radiolabeled thymidine

- thymidine incorporated into G1 phase DNA- indicates initiation of new round of DNA synthesis

- diffusible factor initiates S phase (SPF)

- if fuse G2 with S phase cell; no DNA synthesis occurs- SPF does not act on G2 phase nuclei

- use of xenopus embryos in studying cell cycle regulation- advantages:

- abundant embryos that can be obtained at will

- embryogenesis stimulated by addition of hCG - was once used as pregnancy test- rapid embryogenesis

- large, easy to manipulate- good fate map

- gene knock down technology

- good source of material for biochemistry- disadvantages

- tetraploid - no genetics- poor optics

- inefficient transgenesis- cell cycle naturally regulated in all animals

- oocytes arrested in G2; meiosis induced by steroid hormone

- egg arrests in metaphase of meiosis II until fertilization- induces completion of meiosis, pronuclear fusion, and 12 synchronous mitotic

cleavages

- polar bodies are waste products of meiosis (DNA that is not kept in oocyte)- meiotic cell cycle

- 4 haploid daughters created by two rounds of division

- one S phase followed by two rounds of cell division- first division - meiosis I - homologous chromosomes are pulled to opposite poles

- sister chromatids move together - kinetochores of each sister chromatid are attached toMTs from single pole (co-orientation)


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- second division - meiosis II - sister chromatids are pulled apart

- bi-orientation of MTs attached to kinetochores- no interphase/prophase between meiosis I and II

- during oogenesis, one daughter cell of meiosis I is discarded and becomes first polar body; one

daugher cell from meiosis II is discarded to become second polar body

-

- back to xenopus oocytes- Mitchison and Kirschner - biochemical studies of cell cycle regulation

- cytoplasm from oocytes arrested in meiosis II induces immature oocytes arrested in G2 to enter

into meiosis- therefore, a soluble factor (MPF - maturation promoting factor) can induce entry into meiosis as

well as mitosis- MPF activity found in mitotic cells from all species assayed and can work across species

boundaries- mitotically arrest mammalian cells by treatment with colchicine- treatment with cytoplasm from frog oocytes induces entry into mitosis

- factor purified by column chromatography, acts just like cytoplasm- MPF activity changes throughout cell cycle

- donor cytoplasm from oocytes at different stages of cell cycle goes into recipient

oocytes arrested at G2 phase- progesterone induces MPF in donor cytoplasm; levels are high as cells pass through

meiosis I- fertilization triggers a rapid decrease in MPF activity; remains low until embryonic

mitosis begins


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- treat embryos with cyclohexamide to block increase in MPF - shows MPF activity

depends on new translation- MPF activity is independent of nucleus and transcription

- components required for cell cycle progression are stored in cytoplasm of

xenopus oocytes- sea urchin embryos as source for cell cycle material

- divide synchronously, like frog embryos- many can be obtained and fertilized in vitro

- large cells, easily staged in cell cycle with a microscope- Timothy Hunt

- extracts of embryos at different

times postfertilization, ran totalproteins on gel

- most bands increased in intensity,reflecting new protein synthesisas cell number increases

- however, some oscillated inintensity

- intensity of these bandsincreases duringinterphase and peaks

early in mitosis, followedby an abrupt crash just

before cleavage- protein - cyclin B

- cyclin B is continually synthesized during embryonic cell cycles, but abruptly

degraded following each anaphase- cyclin B controls entry into mitosis

- make extract from unfertilized eggs (arrested in metaphase of meiosis II); add spermnucleus

- nucleus progresses through cell cycle, as judged by morphology

- DNA de-condenses and replicates one time- replicated chromosomes condense and nuclear envelope dissembles, as in

intact cells- spindle forms, separates chromosomes, and nuclear envelope reassembles

- MPF activity phosphorylates histone H1; compare MPF levels to cyclin B levels byexamining H1 kinase activity in vitro

- MPF activity rises and falls in synchrony with cyclin B concentration

- cyclin B also controls exit frommitosis

- chromosome decondensationand nuclear envelopereassembly take place with

MPF/cyclin B levels decrease- test if decrease in cyclin B

levels is essential for exitfrom mitosis

- deplete RNA

transcripts from

extract- add mRNA encoding

a mutant cyclin Bprotein that is non-

degradable- MPF activity remains

elevated in these extracts- early mitotic events occur, but not late events

- so: rise of MPF activity is necessary to enter mitosis, fall of MPF activity and

exit from mitosis depends on degradation of cyclin B protein- vertebrates contain 3 B type cyclin proteins: cyclin B1, B2, and A


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- factors that control cell cycle were identified by genetic experiments in budding yeast (S. cerevisiae)

and fission yeast (S. pombe)- can use cell morphology to find

location in cell cycle

- if you mutate a gene required forcell division, mutant cells will not

divide to form a colony - how doyou isolate mutants?

- use conditional loss of functionmutants - easy in yeast

- candidate gene for MPF in yeast

- S. pombemutants thatpass through cell cycle

normally at permissivetemperatures but arrest atspecific stage at non-

permissive temperature

- mutant (cdc2) found

- prevents entry into mitosis - G2 arrest leads to larger than normal cells

- dominant mutation in same gene - cdcD - produce cells that are too small

- S. cerevisiaecdc15- mutants

- cdc15- mutants fail to exit mitosis

- complete anaphase, but not cytokinesis

- cdc28mutants arrest in G1 phase, do not enter S phase (candidate for SPF)

- mutants in cell cycle control genes arrest all cells at sam stage of cell cycle

- mutants essential for life of cell, but not for cell cycle arrest at random positions in cell cycle

- genetic screen for cell cycle arrest mutants

- S. pombe- cell cycle control exerted primarily at G2 to M transition

- cdc2ts mutants, enough MPF activity remains at non-permissive temperature topermit cells to enter S phase, but not enough to enter mitosis

- in cultures of mutant cells, some arrest in G1, but most arrest in G2- cdc2acts during both G2 to M and G1 to S transitions, but the G2 to M transition

is more sensitive to reductions in MPF activity

- S. cerevisiae- cell cycle control primarily at G1 to S transition

- partially defective cdc28mutants arrested in G1, but completely defective

mutants arrest in G1 or G2, depending on where in cell cycle the cells were atthe time of temperature shift to non-permissive levels

- cdc28acts during both G1 to S and G2 to M transitions, but G1 to S transition is

more sensitive to reductions in SPF activity

- cdc2 and cdc28 encode cyclin dependent kinases - CDK

- small proteins with kinase activity

- human CDK2 is 63% icentical with S. pombecdc2 at amino acid level, and can rescue

S. pombecdc2-mutants

- human protein can perform all essential functions of yeast protein

- cdc13 encodes a yeast cyclin B orthologue - cdc2/cdc13 complex is S. pombeMPF

activity

- cyclins are regulatory subunits of CDKs

- T-loop blocks access of protein substrates to phosphate of ATP in free CDK

- cyclin A binding induces conformational changes that pull T loop from active

site; 1 helix moves into catalytic cleft at the same time- phosphorylation of T160 induces further conformational changes, increasing

affinity of CDK for substrate proteins

- identification of CDK regulatory proteins

- cdc25- loss of function mutants cannot enter mitosis at non-permissive temperature -like cdc2- mutants

- Cdc25 overexpression decreases cell size, reflecting shorter G2- indicates Cdc25+ activates Cdc2/Cdc13 complex

- wee1- loss of function mutants enter M phase too soon (shortened G2) and are small

- when Wee1+ is overexpressed, cells are larger - prolonged G2- demonstrates Wee1+ activity inhibits Cdc2/Cdc13 function

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