Lecture 08 (Mbc C103)

Front 1

Phagocytes

Back 1

* specialized cells that carry out the process of phagocytosis

- phagocytosis = cellular eating

Ex. neutrophils, macrophages

Front 2

Chemotaxis

Back 2

* process by which a cell can undergo directional movement controlled by a chemical gradient

* used by neutrophils to look for bacteria

Front 3

Chemoattractants

Back 3

1) chemokine, cytokines: secrete by immune cells, chemicals given off during inflammation

2) bacterial proteins like fMLP

3) complement components like c5a

Front 4

Chemoattractant Receptors

Back 4

* have a seven transmembrane domain

* coupled to a signalling cascade that consists of a heterotrimeric GTP binding protein

Front 5

Heterotrimeric GTP Binding Protein

Back 5

* consists of three subunits:
- α (binds to GTP)
- β, γ (no binding to GTP)

Front 6

Gs protein (stimulatory G protein)

Back 6

receptor → Gs → activates adenylate cyclase (ATP → cAMP + PPi → → → downstream responses)

* target of cholera toxin: locks Gs into GTP bound form = constant production of cAMP

Front 7

Gi protein (inhibitory G protein)

Back 7

receptor → Gi → Rho family

* leads to the ability for neutrophils to target bacteria via chemotaxis

* target of pertusin toxin: inhibits Gi
= prevents activation of pathway
= inhibits chemotaxis

Front 8

Rho Family of GTPases

Back 8

* consists of monomeric GTPases

* mediates cell migration

* exists in two forms:
- GDP (inactive)
- GTP (active)

* GDP → GTP via GEF
* GTP → GDP via GAP

Front 9

Steps in Cell Migration

Back 9

1) polarization (Cdc42)
- cell must have front and rear
- front faces high [ ] of chemoattractant

2) protrusion (Rac)
- pseudopodium extends membrane forward

3) adhesion (Rho)
- pseudopodium attaches itself to surface via focal adhesion

4) translocation (Rho)
- cell squeezes itself at read to move cell forward

5) de-adhesion
- cell leaves old focal adhesion behind

Front 10

Actin

Back 10

* cell migration mediated by actin cytoskeleton

* exists in two forms:
- monomer form
- polymer form: non-covalent attachment of monomers into filament-like structure (major fxnal unit)

Front 11

Actin Polymerization (monomers → polymers)

Back 11

1) nucleation: monomers come together to form a seed; filaments created

2) elongation: more monomers are added to the ends

* nucleation and elongation drive the process of protrusion: generate the force to push a cell fwd as it moves

3) steady state: constant polymerization and depolymerization

Front 12

Rac

Back 12

* responsible for the forward movement of the front of the cell

* Rac GTPase → WASP → Arp 2/3 complex → nucleation and branching of actin filaments

Front 13

WASP

Back 13

* Wiskott-Aldrich Syndrome Protein

* effector protein

* syndrome patients get increased infections because immune cells can't migrate or phagocytose

Front 14

Integrins

Back 14

* contained inside the focal adhesions

* activated by Rho during adhesion in a bidirectional manner

* transmembrane receptor proteins
- outside te cell they interact with ECM (cells are sitting on them)
- inside the cell they interact with actin

Front 15

Phalloidin

Back 15

* drug that binds actin filaments

* prevents depolymerization

Front 16

Cytochalasin

Back 16

* inhibits actin polymerization and depolymerization

* if its addition inhibits bacteial entry, then entry of the bacterim is an actin-dependent process

Front 17

Stages of Phagocytosis

Back 17

1) Recognition of Pathogen and Binding

2) Internalization

3) Phagosome Maturation

4) Killng and Degradation of Pathogen

Front 18

Recognition of Pathogen and Binding

Back 18

* opsonization: bacterial surface opsonized (coated) with molecules that allow it to be recognized

* opsonins: molecules that coat the surface of bacteria and allow them to be phagocytosed
- rcognized by receptors

Ex. complement proteins (C3b) and antibodies

Front 19

Internalization

Back 19

* phagocyte grabs on and internalizes the particles

activation of receptors that recognize the bacterium
↓
Rho family
↓
Cdc42 and Rac activation
↓
WASP protein
↓
Arp 2/3 complex
↓
actin polymerizes beneath phagocyte and extends the surface to grab onto bacterium and pull it into cell

Front 20

Zipper Mechanism

Back 20

* non-phagocytic cells (ex. epithelial cells) req that pathogens induce their own phagocytosis

* bacterium only polymerizes actin in the immediate area

Front 21

Trigger (Splash) Mechanism

Back 21

* non-phagocytic cells (ex. epithelial cells) req that pathogens induce their own phagocytosis

* bacterium polymerizes actin all over the cell

Front 22

Maturation of Phagosome

Back 22

* once it gets into the cell, bacterium is in phagosome

* phagosome undergoes maturation, which involves acquisition of bactericidal components

* not a linear process: involves constant budding and fusion

early endosome
↓
multivesicular body
↓
late endosome
↓
lysosome

* maturation accompanied by increasing acidification
- vacuolar ATPase is an ATP-hydrolyzing enzyme that pumps protons into the lumen of compartment

Front 23

Bafilomycin

Back 23

* inhibits vacuolar ATPase
= inhibits acidification

Front 24

Antimicrobial Components of Lysosome

Back 24

* low pH

* ROIs

* toxic nitric acid

* enzymes that degrade bacterial proteins
- nucleases, proteases, lipases, glycosidases

* iron competiors (lactoferrin)

* antimicrobial peptides (defensins)

Front 25

Strategies for Surviving Phagocytosis

Back 25

* relevant to intracellular pathogens

1) escape: get out of phagosome ASAP
ex. Listeria escapes to cytosol and lives there

2) prevent maturation of endosomal compartment into a lysosomal compartment
- manipulate the transport of cargo into endosomal compartment

3) survive in the lysosome

Front 26

Autophagy Pathway

Back 26

* bacteria that have escaped into the cell may be subjected to an internal phagocytic pathway

* intracellula pathway normally involved in degrading intracellular components
ex. organelles in need of degradation

* involves sequestration and maturation