Cytoskeleton: Microtubules Flashcards
What is tubulin
Gtpase
How are microtubules formed
From tubulin subunits
13 straight protofilaments - many flavours
Diameter microtubuels
250 angstrom diameter
Hollow tube
Polar filaments - + and neg diff properties
Are microtubules soft
NOO
GENerally rigid = 5200um persistant length
What do microtubules attach to
Typically attach to a central organizing centre - centrosome
What are microtubules the basis for
Larger structures = cilia, flagella
Describe mtoc
Where Mt grow and shrink from
Describe drosophila embryo synchronized division
Dynamics allow micortubule cytoskeleton to rapidly reorganize during mitosis
Why are microtubules essential
Broad range cell functions
Mitosis - chromosome segregation
Transport and organelle position - tracks for molecular motors
Cell shape and polarity -neuronal axon
Cell motility - cilia and flagella (sperm)
Signalling - primary cilia (in many places of bodu)
What makes up microtubule subunit
Heterodimer =
Made of
beta (exchangeable gtp site= hydrolysis happens here)
And
alpha tubulin (non exchange gtp site - locked in place, structural role - does not change)
Describe multiple isoforms of tubulin
7 alpha genes
7 beta genes
Super conserved bc have many important functions
Differences mainly in a the c term tail end
Mediate interactions with accessory proteins tailored to diff cell types
Describe microtubule assembly interactions
Alpha beta tubulin - assembly contacts = forms protofilaments that then interact
Lateral contacts = weaker
Longitudinal contacts = stronger
Describe microtubule assembly interactions Destabilization
Lattice destabilized by gtp hydrolysis - on beta subunit
When dynamic instability - mt shrinking and groaning
Describe microtubule assembly interactions- for final microtubule product
Alpha-alpha or beta-beta lateral contact except at the seam
Tube fold over itself
Why are weaker lateral interactions important
Allow for flexibility
Describe microtubule geometry
Typical = 13-3
Only geometry in which protofilaments are straight, has to be straight to make protofilament
Others are bendy bc of hydrolysis happening in microtubule when polymerizing
Do alll microtubule have exactly the same number of protofilaments
NOO can have diff numbers
But almost all are 13 protofilaments inside cells
What do actin and tubulin share
Polymerization characteristics
Describe polymer growth curve
Distinct s shape
Lag phase - nucleation limited, then growth phase, then eq phase
Dimers want to go back to monomers = rate constant high but then no elation= rate constant to make polymer increases
Proteins that help nucleation point are very important
Describe critical concentration
Balanced assembly and disassembly = critical concentration
Number of monomers adding to filament = number of monomers coming off
Steady state
Koff/Kon = Cc = 1 = no net polymer growths
Describe filament ends
Structurally distinct bc polarity
Minus end = slow growth
At plus end = faster growth
For generally polymers critical concentration same at both ends
Describe gtp binding and hydrolysis
Chemically different filament ends - depends on critical concentration
Cc(neg end) > Cc (pos end)
Bc micortubule cap usually at + end
What does ntp binding do
Drives polymerization
Increase affinity of subunit for polymer = increase kon(ntp)
Subunits add in ntp state
What does ntp hydrolysis do
Drive depolymerization by destabilizing lattice contacts = more likely to come out of polymers
Increase koff (ndp)
Describe dynamic instability - cap
Delay between subunit addition and gtp hydrolysis leads to a gtp cap - growing - means it’s stable and growing
Add at faster rate than hydrolysis
When subunit addition faster than ntp hydrolysis = polymer grows
Describe dynamic instability - disassembly
When hydrolysis catches up = ntp cap lost -= catastrophic disassembly
Constantly adding or losing subunits
Describe microtubule subunits in solution vs filament state
Straight = in Filament state
Curved in solution state, off axis
What must subunits do to incorporate into lattice
Subunits must straighten to incorporate into lattice
Add gtp tubulin to polymers = have to straighten out
Structural explanation for differences in on rates at + and - end
In gdp bound state = curved
Describe curved vs straight conformation
Generates lattice strain
Exchangeable gtp - added to straight protofilament - gtp hydrolysis changes subunit conf = weakens bond so now curved protofilament due to hydrolyzation gtp = microtubule polymerization - catastrophe event
Growing = gdp lattice under strain - wants to curved but locked straight
Shrinking = loss gtp cap allows gdp protofilaments to relax to curved state and disassemble
Describe microtubule catastrophe
Curved protofilaments associates with disassembly
Less stable Region of micortubule contains gdp tubulin dimers
Once cap gone = splay at end and shrink
Describe microtubule rescue
Critical concentration gets High enough so then can state adding back on gtp subunits = growth
Regain of gtp cap = recscue
What is an inherent property of tubulin
Individual filaments cycle between catastrophe and rescue
Name all microtubule interacting proteins
Nucleation and anchoring
Sequestration
Destabilization
Cross linking
Stabilization
Growth acceleration
Adaptors
Name the 2 classes of microtubule interacting proteins
Structural maps - microtubule associated proteins
Motor proteins - kinesin and dyenin
Describe maps
Co purify with tubulin during cyclic assembly/disassembly
Binds and stabilizes mts - promotes assembly
Ex = map-2, tau (Alzheimer’s) in neurons
Describe motor proteins
Convert energy of atp hydrolysis into direction motion along microtubules
Kinesin = mostly + end directed
Dyenins = minus end directed
Describe mtocs
Centrosome = mtoc neg end
Nucleating site for mts
Ex = animal centrosome = organized by amorphous pericentriolar material - cloud of protein surrounds centrosome
Ex = yeast spindle pole body = organized in paracrystalline plaques embedded in nuclear envelope
VERY DIFF CELLUALR STRUCTURES - SAME MECHANISM FOR NUCLEATING MTS
Describe what nucleates microtubules
Y-TUBULIN complexes
HOMOlog of alpha/beta tubulin
Y tubulin - small complex 300kda, yTuSC
Y tubulin ring complex - yTuRC = 2.2 mda
Circulates in peri centriolar space - like lock washers
ESSENTIAL FOR NUCLEATION IN ALL EUKARYOTUES
Describe nucleation mechanism
YTURC = micortubule template
Serves as first ring of tubulin in mt
First circle of mt - creates seam - started in ring complex
What do yturcs do
Cap and stabilize mt minus end
After ncuelation - y turcs stay attached
=why Little or no dynamics at - end, and all the activity at + end
What will bind + end mt
Gtp cap - on plus end - proteins bind to it to. Stabilize it so can keep growing or destabilize it
Describe factors that stabilize + end
Map
Suppress catastrophe and growth rate enhanced
=long less dynamic mts - improve growth state
Tips = tack growing end, enhance growth rate and mediate attachment and stabilization
Protein - if attached to growing end and will walk along faster
Describe factors that destabilize + end s
Kinesin 13 = catastrophe factor
Peels away gtp cap
Result = shorter, more dynamic microtubuels
Name side binding maps
Katanin and spastin
Map2 and tau
Describe Katanin and spastin
Sever microtubule in middle = 2 neg ends
No gtp in middle = expose 2 gdp ends = depolymerizes fast
Bind sides mts and severs them
Describe map2 and tau
Bind and cross link mts in neurons
Describe motor proteins - what are they
Binding domains attach to cargo
Motor domains move along mts
Kinesin and dyenin hydrolyze atp to generate energy for movement
Direction of movement depends on which motors are bound and active
Which direction does dyenin move in
Moves towards neg end - cell body
Which direction does kinesin move in
Twoards plus end - axon Terminal (more Like myosin’s)
Describe kinesin structure
Cargo binding domains
Mt binding and catalytic core (atpase), Motor - feet walking along
Describe dyenin Structure
Cargo binding
Catalytic core - atpase (AAA atpase), changes positions of feet = walks
Mt binding = where feet are and walks along
Name and describe families of kinesins
Classified by domain organization
Motor head and atp hydrolysis one end, then cargo binding other = kinesin 1 (+ end directed)
Kinesin 3 = short (+ end directed)
Mt sliding = kinesin 5, binds itself and makes bipolar motor (+ end directed)
Kinesin 13 = non motor, binds mt ends and increases dynamic stability
Kinesin 14 = neg end directed, wants to go towards that, creates chroms
Describe mechanism of kinesin movement
2 feet bind mt
Linker - adp unstructured but with atp = thrust forwards
Cargo binding domains doesn’t interfere with feet
Lagging head = tightly bound, atp bound,
Leading head = loosely bound, to adp
Hydrolysis on lagging head weakens attachment - trailing foot hydrolysis = becomes weak = neck region flips over and steps right
Detachment of lagging head - exchange at leading head thrusts linker arm forwards
Lagging head becomes leading head - step then swing then keep walking along
Neck region responsible for whipping action of brining adp motor head off microtubule
How does kinesin move
Cooridnateion of atpase cycles between heads -causes head over head walking
Bc one head is slays bound - motion is very processive - kinesin takes long trip along mt
- doesn’t wrap around but walks along
Describe dyenin structure - specific s
Huge protein = 500kda
Cargo binding domain near tail
Mt binding stalk = between 2 aaa domains
Completely unrelated to kinesin part of aaa atpase fam of enzymes
How does dyenin move along micorubuels
When phosphoryltaed = twists and turns = causes foot to move up during power stroke
Rotation fo atpase ring change in linker ring. Contacts
Dimerization required for processivity = middle of molecule spins to moev foot
When spin = put one foot in front of other
Describe large mt assemblies
Related structures with similar organizations
Cilia- mt related cores
Flagella - long mt in sperm
Centrioles - mtoc
Basal bodies -derived from centrioles
Describe centriole
Core fo centrosome
Describe basal body
Anchors cilia/flagella
Derived from centriole
Describe flagella
Long, whip like motion
Sperm motility
Not like bacterial flagella
Describe cilia
Shorter, wave like motion
Primary cilium - signal receptor
How do cilia and flagella form
Form bundles of mt doublets
9 + 2 organization
Complete a microtubule, b microtubule shares protofilaments from a
2 cargo binding domains
Other dyenin arm - can bind cargo portion microtubule
Mt binding domain - grabs onto next double and bind them - but cannot move bc linked by nexin so they bend
Describe organization of cilia and flagella
Dyenin wants to walk towards minus end
Causes mt bending - when power stroke
How do centrioles and basal bodies from
Bundles of mt triplets
Centrioles - have 3 mts = a (complete), b and c (share - incomplete)
Describe mitotic spindle
Mother of all mt assemblies
Entire mt network is completely reorganized once per cell cycle
Enormous machine for accurate segregation of genome
Microtubules excluded from nucleus dispersed throughout cytoplasm in g2
Name the 3 classes of microtubules of mitotic spindle
Astral
Kinetochore
Inter polar
Describe astral microtubule - gen
Position within the cell
Look for outside of cell - look for poles of cells, grab cortex at poles diving cell - dyenin here
Describe kinetochore microtubule - gen
Neg end centrosome
Grow and shrinks - finds super chromatids and binds at kinetochore
Attachment to chromosomes
Describe interpolar microtubule - gen
Growing and skinning
Spindle stability
Look for other mts - and bind then grab each other - kinesin between them
Describe how mitotic spindle sculpted
Motor proteins sculpt spindle
Spindle organization results from exquisite balance of forces
Forces from both molecular motels and intrinsic mt dynamics
What does dyenin do - for mitotic spindle
Pulls poles twoards cortex
Astral mt - dyenin grabs cell wall and pulls centrosome back twoards cell
What does kinesin14 do - for mitotic spindle
Cross link - pulls poles together
What does kinesin5 do - for mitotic spindle
Sliding - push pole apart - want to go to neg end
Polar mt
= will push a way and slide microtubules apart = pulls centrosome away
What does kinesin4,10 do - for mitotic spindle
Move chromosomes to metaphase plate
What do kinetochore microtubules do for mitotic spindle
While rest going on = kinetochore micortubules grab dna and pull apart
Describe Dynamic tension
Balance of forces to align chromosomes
Cut sister chromatids with laser - other arm pushes, arm without kinetochore = more away from pole
Arm with kinetochore moves towards pole
Push = inter polar or astral micortubule
Pull = kinetochore mt
Small perturbations to system allow large rapid responses
Check point - need all dna to line up at metaphase plate
Constant push and pull = to bring dna back towards centrosome, centrosome move back and away from each other
Describe attachment of mts to chromosomes
Mts stabilized by interactions with kinetochore
Mechanism of kinetochore attachment = inside kinetochore= proteins that like to bind polymers, hook and pull and separate chroms - monomers come off
Kinetochore mts grab kinetochore proteins and start to depolymerize - pull dna back = proteins phosphorylated so micortubule depolymerizes and little arms grab to next point
Depolymerization slower eand regulated - prevent catastrophe
Phopshatase to undo it
Describe chromosome separation components
2 components = anaphase a, and anaphase b
Describe mt depolarization driven chromosome separation
Anaphase a = movement of chromosomes towards poles
Shortening kinetochore microtubuels - movement of daughter chroms to poles, forces generated mainly at kinetochore
Polymer short
Chroms come apart
Describe motor driven chromosome separation
Movement of poles away from each other
Walk towards neg end - dyenin mechanism
Pulls poles back twoards edge -away from dna
Astral mts
Sliding force generated between inter polar microtubules from opposite poles to push poels apart, inter polar micortubules also elongate, pulling force acts directly on the poels to move them apart
what does brain have
Lots of microtubules = need to be cold, fall apart at 40 degrees - disassemble
What does taxol do
Binds lattice micotubuels and stops growth
