The Cytoskeleton (14-17) Flashcards
Why is organisation of movement required for things in cells?
For a e.g. protein a cell is a big space (similar to that of a country to a human)
→ networks of polymers enable efficient transport inside cells - gives organisation
What are the roles of the cytoskeleton?
Cellular → organisation of organelles, chromosome segregation, protein and RNA transport, cell division, cell motility and chemotaxis, maintaining cell integrity
Body → food mastication, digestion, blood circulation, communication, reproduction, body movement
What are the 3 types of cytoskeletal networks?
Made from protein polymers → coexist together, have different properties
Microfilaments → actin, 7-9nm
Microtubules → α,β-tubulin dimer, 25nm
Intermediate filaments → various subunits, 10nm
What are the properties of microtubules?
α,β-tubulin bind GTP (dynamic - energy requiring)
Rigid and not easily bent
Regulated assembly from a small number of locations
Highly dynamic, polarised
Tracks for kinesics and dyneins
Organisation and long-range transport of organelles
How are microtubules structured?
Polymers of a dimer containing 1 α-tublin and 1 β-tubulin
→ both subunits bind GTP - structure is dynamic which required a fuel source
→ barrel structure made up of protein filaments
→ repetitive arrays of α,β tubules dimers
→ each microtubule in the network grows out from a focus (the MTOC) at the nuclear periphery
→ subunits make side contacts with others in adjacent protofilaments to make sheets of usually 13 parallel protofilaments - zipper together to form a microtubule
Do microtubule protofilaments have polarity?
Yes → a plus end with a β-subunit, a minus end with an α-subunit
→ gives directionality to motor proteins
How do microtubules grow?
Microtubules preferentially grow at the +ve end
→ when soluble α,β (GTP bound) tubulin dimers are added (polymerisation) its followed by nucleotide hydrolysis GTP to GDP
→ +ve end addition is fast, hydrolysis lags behind - GTP bound β-tublin undergoes slow hydrolysis, gives +ve end a GTP cap
→ -ve end polymerisation is slower and hydrolysis catches up, so -ve end is always in the GDP form
What is the dynamic instability of microtubules?
Some microtubules rapidly growing, some rapidly shrinking
Rapid growth → with GTP-capped end
Accidental loss of GTP cap → lose ability to add β-tubulin-GTP - leaves GDP form at the +ve end triggers catastrophe
Catastrophe → change in growth - rapid depolymerisation of microtubule until a rescue event (new GTP bound β-tubulin added)
How are microtubules organised?
+ve ends of interphase microtubules → directed towards the cell cortex where they probe the inner face of the plasma membrane
→ microtubule tips pause here allowing proteins to interact - determine the length of the pause
→ microtubules can direct and maintain cell shape or determine where new sites of cell growth will occur
What is the different between growing and shrinking microtubule filaments?
Growing → straight edge, GTP cap
Shrinking → GTP cap lost, exposed GDP beta-tublin
→ causes rapid depolymerisation creating ram shaped ends, each protofilament separating fraying, curves break off to recharge with GTP
The cycle of microtubule polymerisation an depolymerisation is essential for their dynamic instability
→ cells use to transport things at end of growing microtubules - release upon depolymerisation
What is the difference between GTP- and GDP- bound alpha:beta-tublin dimers?
GTP-bound → straight heterdimer, 5° angle
→ microtubule assembly favoured
GDP-bound → bent heterdimer, 12° angle
→ hydrolysis of β-tublin-GTP to GDP causes conformational change in dimer - causes protfilamets to splay outwards - creates curved microtubules
What is end binding protein-1 (EB-1)?
Fusion protein that binds to GTP β-tublin cap at the end of growing microtubules
→ has binding sites that allows proteins to surf along microtubule to be deposited at plasma membrane upon disassembly
→ only has binding sites of growing end where α,β-GTP form, doesn’t bind α,β-GDP - marker for growing +ve end of microtubules
What are some drugs that cause microtubule instability?
Nocodazole & colcemid → structurally unrelated small molecules that bind to the α,β-tubulin dimer to prevent addition to microtubules
→ addition to cells prevents microtubule polymerisation and causes microtubules to disassemble
→ both are frequently used in biology research - useful in understanding microtubule biology
What are microtubule organising centres (MTOC)?
Microtubules grow from a MTOC → called centrosome in animal cells
→ the -ve ends are at the MTOC near the nucleus, +ve ends spread out towards the cell periphery
Centrosome → composed of two centrioles sounded by pericentriolar material (centrosome matrix) to which γ-tubulin ring complexes are associated
→ γ-tubulin - variants of α,β tubulin, unable to make long polymers but can make one ring - nucleate first addition of filaments
→ the γ-tublin ring is comprised of 13 subunits onto which α,β-tublin dimers bind
What is a part of the large tubulin gene family?
e.g. α/β → heterodimers form microtubules, found in all eukaryotes
γ tubulin → major component of ring complex, recruited to MTOCs - usually in centrosomes, found in all eukaryotes
δ and ε tubulin → components of centrioles and basal bodies, found in some eukaryotes and protozoa
What are microtubule used for?
Organisation of organelles
Chromosome segregation
Protein and RNA transport
What are kinesin and dynein motors?
Motor proteins responsible for numerous microtubule-dependant transport events in eukaryotic cells
→ organisation of the ER and Golgi depends on the orientation of microtubules and motor activity
Kinesin motors → almost exclusively +ve-end directed motors, 14 structurally related classed, use ATP to generate force
→ generally direct organelles (e.g. ER) and vesicles towards the plasma membrane
→ e.g. early endoscopes and secretory vesicles for exocytosis
Dynein motors → exclusively -ve end directed motor, part of very large dynein-dynactin family, structurally unrelated to kinesis, use ATP to generate force
→ direct organelles (e.g. Golgi) and vesicles away from the plasma membrane
→ e.g. last endoscopes for endocytosis, lysosomes and ER-Golgi intermediate compartment
Some organelles use both kinesin and dynein
How are kinesin-1 motor proteins structured?
Dimer at head bind microtubule - walking along
→ other end tail bound to cargo e.g. vesicle
→ connected via stalk
How does kinesin generate movement?
Moves processively along microtubule - action of the two heads are co-ordinated, one of them is always bound
Two identical motor heads → each consists of a catalytic core and neck linker
→ both contain ADP which move randomly - when one finds microtubule it binds tightly causing ADP to release
→ ATP then rapidly enters the empty nuceltodide binding site - triggers neck linker to zipper onto catalytic core which throws the second head forward neat the next binding site on microtubule
→ backward head hydrolyse ATP and release Pi
Every step uses one molecule of ATP
What is involved in dynein-based organelle transport?
Has associated proteins → second microtubule binding site, allows attachment of cargo - 11 subunit complex called the dynactin complex
→ goes 100x faster than kinesin
→ moves exclusively towards then end of microtubules
Where is bi-directional organelle or vesicle transport seen?
e.g. in neurones - moving up and down axons
→ vesicles containing neuropeptide - retrograde and anterograde movement
→ organelle, depending on the signal, where its got to on the neuron can move towards the synaptic termini and deliver its neuropeptide - then move backwards to pick up more in the cell body
What are the properties of microfilaments?
Actin binds ATP → fuel source
Form rigid gels, networks and linear bundles
Nucleated from a large number of places → primarily under cell cortex
Highly dynamic
Polarised → important for motor proteins directionality, tracks for myosins
Contractile machinery and network at the cell cortex
Where are microfilaments (actin) found in gut epithelial cells?
Microvilli → have straight actin, extend plasma membrane area
Cell cortex → has branched network of actin filament - important for processes like endocytosis
Adherens belt → has straight actin, contractile belt using myosin motor proteins to respond to force from adjacent cells - maintains integrity of the lining
Where are microfilaments (actin) found in fibroblast cells?
Migrating cells have actin filament structures - fibroblast moves to source of wound
Filopodia, stress fibres and lamellipodium → act co-ordinately to move cell forwards
→ filopodia & stress fibres - straight actin
→ lamellipodium/leading edge - branched actin
Cell cortex → matrix of branched actin
Where are microfilaments (actin) found during cytokinesis?
Contractile ring → cells undergoing devision stop what they’re doing and undergo cytokinesis
→ ring constricts two cells apart after nuclear division to enable cellular division
What is actin?
Actin is a globular (G-actin) protein → divided by a central cleft at top that binds ATP, gives polarity
Actin filament (F-actin) → appears as two strand of subunits, 14 actin molecules in each strand, covering a distance of 72nm in length
→ strands fit together as a clockwise helical twist - symmetry every 36nm
ATP binding cleft always binds to the opposite side of the adjoining actin molecule - gives filament polarity
→ - end has actin binding cleft exposed, + end has no cleft
What end does actin polymerisation occur at?
Rate of assembly at + end is faster (10X) than the - end, rate of dissociation similar
→ in the filament ATP hydrolyses to ADP-Pi and Pi is released slowly giving rise to a filament containing ATP-actin, ADP-Pi actin and ADP-actin
→ ATP-actin is added preferentially to the + end while ADP-actin disassembles at the - end - giving rise to tread milling subunits
What are some compounds that bind actin to stabilise/destabilise actin filaments?
Cytochalasin D & Latrunculin A → binds actin monomers and prevents actin polymerisation
→ interfere with ATP binding cleft
Phalloidin → binds and stabilised actin filaments, this can be labeled with florescent dye for staining actin filaments in cells - prevents disassembly
