Microtubules

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Introduction
Microtubules Microfilaments Intermediate
filaments

Cytoskeleton

Kinesin Dynein Myosin
Motor proteins
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Scenario 3
Listeria move inside host cells by recruiting
actin
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Scenario 3
Listeria move inside host cells by recruiting
actin
Sheds light on crawling locomotion of animal cells
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Scenario 3
Listeria Human pathogen L. monocytogenes Risk
of infection from unpasteurized dairy
products Particularly dangerous in
pregnancy Grows at 30C, ie. in refrigerators Uses
an UTR as temperature sensor
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Scenario 3
Listeria Rod-shaped human pathogenic bacterium
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Scenario 3
Listeria Invades cells of host tissue, thus
evading immune response Moves inside and between
cells by recruiting a rocket-like tail of host
actin
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Scenario 3
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Scenario 3
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Scenario 3
Listeria moving in Xenopus extract Phase
contrast, and GFP-actin
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Scenario 3
Actin filaments
Moves inside and between cells by recruiting a
rocket-like tail of host actin
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Describe phenomenon of intracellular
locomotion of Listeria
Scenario 3
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Describe phenomenon of intracellular locomotion
of Listeria
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Scenario 3
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Scenario 3
Macrophage chasing and engulfing a
bacterium Shows protrusion is directed.
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Scenario 3
Crawling/gliding locomotion of animal tissue
cells Important component of mechanism is
protrusion of front. Typically a thin fan-like
structure, known as lamellipodium, or leading
lamella. Nerve growth cone is similar
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Describe phenomenon of intracellular
locomotion of Listeria
Scenario 3
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Describe crawling locomotion of animal cells
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Scenario 3
Crawling/gliding locomotion of animal tissue
cells Now good evidence that is driven by
polymerization of actin filaments at the
front Key host components of this mechanism are
recruited by Listeria
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Scenario 3
GFP actin at the leading edge
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Scenario 3
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Scenario 3
Speckle fluorescence - dilute GFP actin
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Scenario 3
Crawling locomotion of animal cells
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Describe phenomenon of intracellular
locomotion of Listeria
Scenario 3
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Explain role of actin polymerization in
protrusive phase of gliding locomotion
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Scenario 3
Actin is major component of virtually all
eukaryotic cells and is exceptionally highly
conserved
Top sequence fission yeast (S. pombe) Second
sequence human skeletal muscle Below is
consensus Red residues are identical
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Scenario 3

• Conservation of actin sequence
• is usually attributed to extremely large number
  of proteins (100) which interact with it.
• Among their functions
• Binding (sequestration) of G-actin monomers
• Self assembly
• Nucleation of filament assembly
• Scission (cutting) of filaments
• Cross-linking of filaments
• And many others

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Scenario 3
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List wide occurrence and discuss sequence
conservation of actin
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Scenario 3
G-(globular) actin is the protomer of actin
filaments molecular weight 43 kDa 4
sub-domains ATP-binding cleft
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Scenario 3
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F-(filamentous) actin Twin protofilaments
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?-carbon backbone of actin arranged as in F-actin
filament
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Scenario 3
Hydrophobic loop between protofilaments
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Scenario 3
Actin has common ancestor with prokaryote
(bacterial) MreB Bacterial protein MreB forms
large fibrous spirals underlying membrane of
rod-shaped cells. Has role in determining cell
shape. Mre murein cluster e. (Murein is the
bacterial cell wall peptidoglycan). MreB has 3D
structure extremely similar to G-actin, although
overall sequence identity is only 15. MreB
forms filaments very similar to single actin
protofilament
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Scenario 3
MreB bacterial actin-like molecule. Same fold,
but not sequence
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Scenario 3
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Discuss resemblance between actin and a bacterial
protein
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Scenario 3

• Polymerization of actin in vitro
• Experimentally controlled by ionic strength
• LOW, approx Water
• (mM ATP mM Mg2) solubilises G-actin monomers.
  
• (Low salt promotes electrostatic repulsion)
• 0.1 M NaCl as inside cells
• drives filament assembly, forming F-actin
  filaments
• (favours hydrophobic interactions)

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Scenario 3
Actin filaments microfilaments Diameter 7
nms Compare microtubules Diameter 25 nms
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Scenario 3
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Describe in vitro polymerization of actin,
referring to significance of ionic strength and
draw simple diagram of actin filament, with
dimensions.
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Scenario 3
Polymerization induced by adding salt. Has lag
phase which can be abolished by adding nuclei
seeds
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Scenario 3
Why F-actin grows more easily than it starts
Catch 22
Need structure like this to start filament
growth Green squares hydrophobic loop which
moves to stabilise twin protofilaments
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Scenario 3
Hydrophobic loop between protofilaments
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Scenario 3
How to tell which end is which
Pointed minus end
Barbed plus end
Decoration of an F-actin filament with the motor
domain of myosin ( myosin S1)
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Scenario 3
Pointed end
Barbed end
Myosin-decorated filament used as seed
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Scenario 3
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Discuss co-operativity of assembly, and
requirement for nucleation, referring to
experimental evidence
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Scenario 3
Arp2/3 complex - filament initiator
A signal-regulated cellular device for
initiating actin polymerization Complex of seven
subunits including Actin-related proteins Arp2
and Arp3. Discovered in Acanthamoeba by Laura
Machesky but is conserved from yeast to man
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Scenario 3
Arp Actin-Related Protein Arp 2/3 complex, with
5 other proteins Activation may move arp2 and
arp3 into spatial relation as if actin monomers
in the actin filament
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Scenario 3
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Scenario 1

?-tubulin
Recall involvement of ?-tubulin in MTOCs
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Scenario 3
Listeria recruits host cell Arp2-3 complex via
single bacterial protein (Act A) and generates
own propulsive actin tail
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Scenario 3
Great experiment! beads coated with Listeria
protein Act A moving in a cell extract (GFP-actin)
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Scenario 3
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Describe role of arp2/3 complex in nucleation,
and its recruitment by Listeria ActA
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Scenario 3
Arp2/3 complex - filament initiator
A signal-regulated cellular device for
initiating actin polymerization Localises at
motile regions of animal cells Generates new
actin filaments in response to signals, by
starting new branches (dendritic polymerization)
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Scenario 3
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Cartoon of lamellipodium from Vic Smalls website
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Scenario 3
Speckle fluorescence - dilute GFP actin
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Scenario 3
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Draw diagram of dendritic polymerization, includin
g Arp 2/3 complex, and showing actin polarity
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Scenario 3
Arp2/3 complex - filament initiator
Activation by Listeria requires just Act A
protein. Activation in mammalian cells depends
on signalling proteins which join the arp2/3
complex e.g WASP WASP Wiskott-Aldrich
Syndrome Protein Syndrome is X-linked recessive
defect in leucocyte chemotaxis.
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Scenario 3
Macrophage chasing and engulfing a bacterium
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Scenario 3
END
Listeria move inside host cells by recruiting
actin