Photobleaching fluorescent actin in a fibroblast

1
Photobleaching fluorescent actin in a fibroblast
Experiment 1

• The fluorescent mark moves backward with respect
  to the front cell edge (and with respect to the
  substratum).

Alberts Fig. 16.56
Slow moving cell
Conclusion 1. Actin meshwork is flowing
backward 2 New actin is polymerized at the edge
Y-L Wang et al., 1985. JCB 101597-602
2
Models of actin filament growth during
protrusion Treadmilling versus nucleation-release
Vic Small
Tim Mitchison
3
Photoactivation of fluorescence in a moving
keratocyte

• The fluorescent mark moves backward with respect
  to the front cell edge (but not with respect to
  the substratum).

Fast moving cell
Theriot and Mitchison, 1991 Nature 352 352-131
4
Treadmilling vs. nucleation-release
This is figure 4, from Theriot and Mitchison,
1991. It is posted under papers on
HuskyCT Image quality is lost when converting to
PDF
5
Rate of actin turnover is consistent with
nucleation-release model

• Photobleaching experiments show rate of actin
  filament turnover to be greater than expected if
  treadmilling of long filaments occurs
• BUT Small criticized interpretation of
  photobleaching experiments
• Depolymerization of actin filaments makes
  estimate of filament turnover artificially high

• (Small) EM studies show long actin filaments in
  lamellae of slow moving fibroblasts
• (Others) EM studies in rapidly moving cells show
  a dense meshwork of actin filaments of different
  lengths

6
Current view

• Dendritic nucleation model (Mullins, 1998)
• Confirmed by the discovery of Arp2/3 at leading
  edge of motile cells
• Varible lengths of F-actin in lamellae of motile
  cells
• Treadmilling occurs in individual filaments that
  are nucleated and released from these sites (if
  uncapped at both ends).
• Treadmilling occurs in the whole mass of actin
  filament meshwork

Question If the rearward movement of bleached
marks is due to treadmilling, then why do we
observe faster rearward movement (w.r.t.
substratum) in cells with slow rates of actin
turnover?
7
When actin polymerization is inhibited the actin
meshwork continues to move rearward
Mechanical inhibition of retrograde flow (see
movie)
8
Adhesions allow mechanical coupling between a
contractile cytoskeleton and the substratum

• In fast moving cells contractile forces at the
  front edge are low compared to the strength of
  adhesions
• No rearward actin flow so that most newly
  polymerized actin contributes to protrusion
  bleached marks are stationary w.r.t. substratum
• In slow moving cells contractile forces at the
  front are high (but not higher) compared to the
  strength of adhesions
• Actin flows rearward because most newly
  polymerized actin feeds the flow protrusion
  is limited - bleached marks move back w.r.t.
  substratum

9
The tail of Listeria monocytogenes Lessons
learned from a bacterial pathogen

• Found in soil, on plants animals
• Associated with eating contaminated dairy
  products, plants
• Infects intestinal cells and spreads from cell to
  cell
• Intracellular motility essential for spread
• Can cause, meningitis, septicemia, abortions
• Old, very young, and immunocompromized people at
  risk

10
Movie

• Listeria rocketing in infected cell

11
Listeria highjacks host cell functions

• Listeria express surface proteins internalins
  (e.g. InlA, InlB)
• InlA binds to E-cadherin
• A cell-cell adhesion molecule
• Recruitment of adhesion proteins links bacterium
  to cytoskeleton triggers phagocytosis
• Listeria secrete listeriolysin O (LLO) and
  escapes into cytoplasm
• Optimal activity pH 5.5 same as inside phag.
  vacuole
• Bacterium recruits host cells actin and ABPs to
  move intracellularly
• Induce membrane extension phagocytosed by
  neighboring cell and infects it

12
Advantages of studying Listeria

• 1. Doesnt have the drawbacks of other whole cell
  systems
• e.g. Some cytoskeletal mutations are lethal
• Functions of many ABPs are redundant
• Difficult to reconstitute cell motility it
  requires a plasma membrane
• 2. No plasma membrane
• 3. Motility can be reconstituted in vitro
• 4. The tail of Listeria is analogous to a
  lamellipodium of a moving cell

13
How does actin polymerization drive the movement
of Listeria?

• 1. Insertional actin polymerization occurs at
  back edge of bacterium
• Polymerization fluorescently labeled actin shows
  brighter regions at back edge
• 2. Photobleaching experiments show that the tail
  remains stationary as bacterium moves forward
• 3. Depolymerization occurs at the same rate
  throughout the tail
• tail length is usually constant
• a decreasing gradient of filament density exists
  from the front to rear of the tail
• F-actin half life 30 sec

loss
addition
Filament density
Distance um from back
14
How does actin polymerization become localized at
one end of the bacterium?

• 1.Identification of nucleation factors
• 2. Symmetry breaking (later)

• In the early 90s used a genetic screen in mutant
  Listeria that could not form tails, and normal
  ones
• Found a single gene actA - encodes a bacterial
  surface protein ActA
• Can induce tail formation in
• Immotile Listeria, other bacteria, polystyrene
  beads

15
Act A is required for tail formation
Bact. Memb. anchor sequence

• Does not bind directly to actin
• Looked for proteins that localized to the back
  edge of bacterium that are not seen in the tail.
• 1. Found VASP (vasodilator-stimulated
  phosphoprotein)
• discovered by immunofluorescence studies
• Binds to proline rich region of Act-A
• Known to be associated with F-actin and focal
  adhesions in lamellipodia
• 2. Profilin binds VASP
• VASP and profilin accelerate filament elongation
  but are not nucleators
• Evidence Actin clouds form in profilin depleted
  cytoplasmic extracts
• VASP-actin complexes have no nucleating activity
• How is elongation accelerated?
• Poly proline regions bind multiple VASP molecules
• Evidence Bacterial speed is proportional to
  number of proline-rich repeats in ActA
• GFP-profilin concentration at back edge is
  proportional to speed

16
ABPs in Listeria tails are the same as in
lamellipodia

• Arp2/3 (Welch et al., 1997)
• isolated by column chromatography from host cell
  (platelet) cytoplasm - required for
  polymerization is activated by ActA
• Capping proteins e.g. gelsolin - found throughout
  tail
• Is enriched at bacterial surface but ActA thought
  to suppress capping here
• ADF/Cofilin - found throughout tail
• important for increasing actin filament turnover
  by 10-100 times compared with in vitro
• Immunodepletion leads to formation of very long
  tails
• Addition of excess decreases tail length but
  increases speed
• Crosslinking proteins -eg. Fimbrin, ?-actinin -
  found throughout tail, structural role
• introduction of dom. negative fragment stops
  bacteria movement

17
Organization of actin filaments in Listeria is
similar to that of lamellipodia

• Y shaped cross-links containing ARP2/3 are
  present
• Evidence of other kinds of crosslinking exists