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PHYS5530M Physics of Biological Systems > Cytoskeleton Dynamics, Cell Motility and Cell Division > Flashcards

Cytoskeleton Dynamics, Cell Motility and Cell Division Flashcards

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1
Q

Crawling Cells

A

-cells migrate in response to specific external signals e.g. chemical and mechanical

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2
Q

Moving Tent Analogy

A
  • think of the cytoskeleton as a kind of structural framework like the poles and ropes holding up a tent (the membrane)
  • to move the tent in a storm you would want to make it crawl without losing contact with the ground
  • need to constantly relocate the anchoring points while rearranging poles and ropes in order to soften the wall you want to push forwards a simultaneously drag the back of the tent with you
  • cells use an array of proteins to build and deconstruct their own ‘poles and ropes’ to move the entire cell in response to signals
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3
Q

Cytoskeleton - Dynamic Scaffolds

A
  • the cytoskeleton provides the main structural and mechanical support for cell and cytoplasmic components
  • controls motion of cells, cell division and acts as a monorail for transporting organelles and materials
  • there are three major filament types; microtubules, intermediate filaments and actin filaments
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4
Q

Actin Filaments

A
  • extremely dynamic protein
  • formed by polymerisation of G-actin (small, cube-like, negatively charged ~5nm)
  • polymerise in presence of Mg2+ and ATP to form long spiral chains / filaments called F-actin
  • the rate limiting polymerisation step is getting n~3 polymers to form, from there polymerisation rapidly occurs
  • polymers have a positive and negatively charged end, polymerisation is preferential at the positive end
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5
Q

Factors Affecting Actin Polymerisation

A
  • ATP concentration
  • Mg2+ concentration
  • actin monomer concentration
  • Ca2+ concentration
  • actin binding proteins
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6
Q

Actin Filaments Steady State

A
  • known as ‘treadmilling’
  • monomer dissembly from the minus end is balanced by critical concentration of monomers in the cytosol meaning that the polymerisation rate at the plus end is equal
  • with one end dissembling at the same rate as the other end is polymerising the filament is essentially moving along
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7
Q

Actin Binding Proteins

A
  • huge toolbox of proteins that interact with actin monomers and filaments to control polymerisation, depolymerisation, network density, direction and mechanical properties
  • allows cell to remodel actin filament shape, move them and divide them
  • e.g. end-blocking proteins (capping), monomer sequestering proteins, cross-linking, bundling, filament severing, depolymerising, membrane-binding and myosin motors
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8
Q

Functions of Actin Filaments

A
  • networks of actin beneat the cell cortex which is a meshwork of membrane associated proteins that support and strengthen plasma membrane
  • allows cells to hold, move and support a variety of specialised shapes
  • involved in cell movement, division and muscle contraction
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9
Q

Cell Motility

A
  1. establish polarity - reorganisation of actin scaffold establishes actin dependent protrusion of cell’s leading edge which is composed of arm-like structures called lamelliopodia filopodia
  2. adhesion sites - during cellular are extension; plasma membrane sticks to surface at leading edge
  3. translocation of cell body (motility) - nucleus and cell body pushed forwards through intracellular contraction forces mediated by stress fibres (myosin)
  4. retraction - fibres pull rear of cell forward
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10
Q

Filopodia Formation

A
  • subset of uncapped actin filaments of ARP2/3 nucleated branch targeted for continued elongation by actin nucleating formin (Da2)
  • membrane curvature induced by pushing forces of the elongating filaments and recruit other components to site of filopodial initiation
  • incorporation of actin cross-linking protein fascin in shaft of filopodium generates stiff actin filament bundles
  • formin Da2 localised in the ‘tip complex’ and controls barbed end elongation of filaments
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11
Q

Adhesion Sites

A
  • membrane anchoring proteins link actin filaments to membrane which is important for the control of cell shape, maintenance of integrity, organisation of proteins into funcitonal dynamics
  • talin can link transmembrane integrins to actin directly or indirectly by interacting with other proteins
  • focal adhesions (once formed) act as molecular grips promoting protrusion of the leading edge whilst supressing membrane contraction
  • aid membrane protrusion by resisting actin retrograde flow hence indirectly promote the force produced by lamellipodial actin polymerisation
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12
Q

How is are new actin monomers continually provided for the extension of filopodia?

A
  • to get new actin to the front of filopodia, the other end is constantly recyced so equilibrium is never reached
  • filament half-life is ~1min due to the constant depolymerisation and polymerisation
  • actin sequestering proteins catalyse exchange of ADP/ATP and increase polymerisation rate at the barbed end
  • actin cross-linkers and binding proteins link two actin filaments at specific angles (70’)
  • capping proteins bind to plus or minus end to control access
  • actin severing proteins cause depolymerisation at minus end via ADP
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13
Q

Force Generation in the Cytoskeleton

A
  1. polymerisation, Fmax = 5-10pN

2. molecular motors, Fmax = 1-10pN

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14
Q

Myosin Motors

A
  • proteins present in virtually all eukaryotic cells
  • different classes for different functions e.g. filopodia growth, movement of cargo, cell division and molecular contraction
  • convert chemical free energy of ATP to mechanical force and movement
  • move along actin filaments in defined directions (either towards plus or minus end) depending on myosin class
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15
Q

Myosin Powerstroke

A
  1. myosin head lacks a bound ATP and is attached to the actin filament
  2. ATP binding to head induces small conformational shift reducing affinity for actin so the head releases
  3. ATP binding also causes a large conformational shift in the myosin lever arm moving the myosin head moving further along the filament, ATP is hydrolysed
  4. myosin motor head makes weak contact with the actin filament and a slight conformational change occurs on the myosin promoting the relase of an inorganic phosphate
  5. release of phosphate reinforces binding of actin and myosin and triggers the powerstroke, forces are generated on the actin filament
  6. myosin regains original conformation, ADP is released but the myosin head is still tightly bound (back to the start)
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16
Q

Myosin Walking Speed

A
  • one discrete step is tightly coupled to ATP hydrolysis
  • rate limiting steps:
  • -ADP release
  • -ATP binding (in low ATP concentrations)
  • other chemical steps are much faster
17
Q

Myosin Average Translocation Velocity

A

v = ds / (1/k1[ATP] + 1/k2)

  • where ds is the step size
  • and k1 is the 2nd order ATP binding rate constant
  • and k2 is the 1st order ADP dissociatoin rate constant
18
Q

Myosin Maximum Translocation Velocity

A

vmax = v ( k2/k1[ATP] + 1)

19
Q

Testing Myosin Activity

A
  • can watch movement of fluorescently labelled actin filaments on a myosin coated surface
  • high speed AFM now allows direct visualisation of myosin v stepping along an actin filament
20
Q

AFM vs. High Speed AFM

A
  • traditional AFM takes ~5mins to scan surface
  • an nm tip is oscillated and scanned across surface, changes in oscillation are used to detect topography whilst feedback loop maintains minimal contact with the surface
  • the speed limit of AFM is set by the slowes component of the system so to increase the speed every elemnt of the process has to be optimised
21
Q

Bacterial Cell Motility

A

-bacterial cell flagella generate corkscrew type motino with a continuously applied force for movement through a relatively high viscous world

22
Q

Cytoskeleton - Dynamic Scaffolds

A
  • microtubules organise the cytoplasm, position the nucleus and organelles, and provide tracks for cargo transport
  • in cell division, microtubules function to physically segregate chromosomes and orient plane of cleavage
23
Q

Microtubules

Structure

A
  • formed via assembly of tubulin dimers of alpha and beta tubulin which string together to form long protofilaments
  • 13 protofilaments form a hollow straw shaped filaments of microtubules
  • polymerisation occurs by binding GTP which hydrolyses to GDP
  • microtubules have polarity with + and - ends
24
Q

Microtubules

Dynamic Instability

A
  • individual microtubules switch randomly between growing and shrinking states
  • sometimes they switch several times in their lifetime
  • catastrophe = rapid depolymerisatoin
  • rescue = polymerisation
25
Q

Microtubules

Dynamics

A
  • minus ends of microtubules anchored to microtubule organisation centres (MTOCs)
  • primary MTOC in the cell is the centrosome, usually located adjacent to the nucleus
  • microtubules tend to grow out from centrosome to plasma membrane
26
Q

Microtubules

Motor Proteins

A
  • as with myosin motors and actin, kinesin and dynein motors move along microtubules using ATP
  • kinesin moves towards the positve end, dynein moves towards to negative end
  • functions: intracellular transport, spindle formation and chromosome separation in cell division and applying force
  • e.g. kinesin bound to two anti parallel microtubules can cause them to move adjacent to each other
27
Q

Mitosis

A
  • cell division
    1) prophase
    2) prometaphase
    3) metaphase
    4) anaphase
    5) cytokinesis
28
Q

Mitosis

Prophase

A

1) prophase
- duplicated nuclear DNA condenses from random polymer to discrete chromosomes
- microtubules nucleate at two centrosomes located on the outside of the nucleus
- polymerisation of overlapping microtubules between centrosomes pushes them apart forming mitotic spindle

29
Q

Mitosis

Prometaphase and Metaphase

A

2) prometaphase
- nuclear membrane disintegrates
- microtubules emanating from the spindle poles attach to the centromere of each chromosome
3) metaphase
- pulling forces from each pole cause chromosomes to align equi-distant from each spindle pole

30
Q

Mitosis

Anaphase and Cytokinesis

A

4) anaphase
- two sets of chromosomes are pulled apart towards each spindle pole
5) cytokinesis
- contractile ring of actin and myosin becomes smaller and smaller until two nuclei are pinched off into separate cells

31
Q

Mitosis

Early Stages

A
  • chromosome condensation is driven partly by protein complexes, condensins, which are thought to act by modifying cross-linking and supercoiling properties of DNA
  • dynamic instability used to explore cellular compartment rapidly to find and capture chromosomes, and bring them to the centre of the cell
  • random growth and shrinkage is an efficient method for exploring the volume within the cell allowing quick chromosome attachment to spindle poles
32
Q

Mitosis

Miotic Spindle

A
  • self-assembly from nucleated microtubules
  • responsible for separating chromosomes correctly into daughter cells
  • bundles of parallel microtubules from each spindle pole attach to the protein complexes (kinetochores) on each chromosome arranging DNA into a metaphase ‘plate’ in centre of cell
33
Q

Mitosis

Pulling Chromosomes Apart by Motors

A
  • plus ends from opposite poles overlap a the centre of the spindle, antiparallel
  • miotic spindle uses combination of microtubule polymerisation and moecular motors to push centrosomes and, pull chromosomes apart
  • kinesin 5 is critical with 4 motor domains allowing simultaneous movement at ~20nm/s to the plus ends of each of two microtubules it crosslinks
34
Q

Mitosis

Control of Spindle Length

A

-depolymerisation at spindle poles (where minus ends are nucleated) acts to decrease spindle length, S at rate Vdepol
-antiparallel, overlapping regions of microtubules at the centre of the spindle, length L, are pushed apart by motor complexes at rate Vsliding
-the length of overlap, L, is also increased by polymerisation at rate Vpoly:
dS\dt = 2 [ Vsliding - Vdepol ]
dL\dt = 2 [ Vpoly - Vsliding ]
-L is heavily dependent on kinesin activity

35
Q

Mitosis

Actin Contractile Ring

A
  • generates constricting force to separate cell into two
  • composed of actin filaments and motor proteins, myosin-2
  • ring forms under surface of membrane and is linked to membrane such that when it constricts it creates cleavage furrow that partitions the cell in two
36
Q

Super Resolution Microscopy

A
  • super resolution microscopy has revolutionised fluorescence techniques
  • spatial resolution is improved from ~400nm to ~20nm
  • conventional fluorescence microscopy resolution is diffraction limited
  • there are two main categories of super resolution:
    1) structrued illuinated microscopy (e.g. STED)
    2) single molecule localisation microscopy (e.g. STORM)
37
Q

Stochastic Optical Reconstruction Microscopy (STORM)

A
  • in each imaging cycle, only a fraction of fluorophores turned on allowing their position to be determined with nm accuracy
  • this is repeated over several cycles with fluorescence in different places far enough apart that the signals don’t overlap
  • the signals are localised to the centre
  • these frames are added together to form a high resolution image, ~20nm resloution
38
Q

Stimulated Emmision Depletion Microscopy (STED)

A
  • fluorescence probe first excited, by light, from ground state to singlet state
  • the de-excitation by light via stimulated emmision or spontaneously by fluorescence emission
  • fluorescent confinement obtained by co-aligning gaussian excitation beam (described above) with secon doughnut shaped beam with ‘zero’ -intensity point in the centre
  • the combined beam has a very narrow effective PSF
39
Q

Bacterial Cell Division

A
  • reproduce very fast, >30x speed of mammalian cells
  • E coli. uses MinCDE system (MinC, MinD & MinE) which oscillates from cell-pole to cell-pole measuring the length of the cell unti it is correct for cell division
  • using ATP, MinC oscillates to create an inhibitory gradient to localise division machinary, FtsZ ring, at the centre of the cell

PHYS5530M Physics of Biological Systems (12 decks)

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