VL 30 (Ralph Gräf) Flashcards
Molecular motors:
- dynein: MT; (+) → (-)
- kinesin: MT; (-) → (+)
- myosin: actin; (-)→(+)
Microtubuli end (+) at centromere and (-) at centrosomes
Mechanochemical cycle of myosin
Mechanochemical myosin cycle (left):
- strong affinity to F-actin in ADP-bound state
- low affinity to F-actin in ATP-bound state
- cocking of myosin head upon ATP hydrolysis
- power stroke upon Pi release
Mechanochemical kinesin cycle (right):
* high affinity to MTs in “0”, ATP and ADP+Pi state
* low affinity to MT in ADP-bound state
* power stroke upon ATP-binding
Dynein
- cilia, flagellae motor
- no structural similarity to myosin/kinesin
- motor head: 6+1 subdomains + dynacting-binding stem (N-term. tail) domain + MT-binding stalk domain
- 6 AAA domains (ATPases associated with various cellular Activities)
- many associated protein chains
- 2 dynein entities together with dynactin→processive motor
Movement in two phases:
1. Priming stroke upon ATP hydrolysis
2. Power stroke at Pi release
- complete dynein: two motor SU (NudE + LIS1 = regulators)
- cytoplasmic dynein always cooperates with dynactin (name, because it contains actin-like protein = Arp1)
Organelle and versicle transport along MTs:
Steps in cell migration
- Extension at leading edge
- Formation: new focal adhesions, pseudopods at leading edge
- Translocation of cell body (actin/myosin contractile mechanism at rear)
- Decomposition of old focal adhesion at rear end
- Integrin recycling via membrane vesicle transport from rear → front end
- Movement motor: actin (lamellipodia, filopodia, stress fibers)
- ECM-integrin interactions→integrin clustering at membrane
→ formation: focal contacts
→signal for focal contact formation - Central regulator: focal adhesion kinase (FAK; tyrosine kinase); bound by
paxillin; other kinases also involved (ILK, Src..)
Cell polarity in chemotaxis:
Polarization
* Differentiation between leading edge – uropod through
* Accumulation of specific proteins at both ends
→ cytoskeleton reorganisation *
Important molecular players
* G-protein-coupled receptors for signaling compound (evenly distributed) o phosphorylated phosphatidyl inositols
* intracellular signaling proteins
* actin
* myosin
Picture
* before signal: receptor molecules are evenly distributed around whole cell
* signal: bound to receptor molecule→signaling cascade
→cell alters shape
→uneven distribution of F-actin + lipid PIP3 (PIP2 + ATP)
–> front end: F-actin forms lamellipodium; PIP3
–> rear end: acto-myosin
signaling pathways in chemotaxis:
Steps in cell migration:
Front end
* branched actin filaments (nucleator: Arp2/3)
* Cofilin: actin depolymerase
* Profilin: actin polymerase
Rear end
* focal adhesion with contractile bundles/stress
fibers (F-actin, Myosin); bound to integrins
Signal-induced formation of actin structures
Left:
* On: free lipid modification; active; Rab hydrolyzes GTP over time
→phosphate dissociation; GTP-bound
* Off: lipid modification attached; GDP-dissociation from Rab over time
→ new GTP-binding; GDP-bound
* Regulators
–> GAP: GTPase-activation proteins → promote “off”-status (faster phosphate dissociation)
–> GEF: GTP/GDP-exchange factors → promote “on”-status (faster GDP-dissociation)
Right:
* RhoA → stress fibers
* Rac1 → lammelipodia
* Cdc42 → filopodia
Making of filopodia:
Convergent elongation model:
* Branched actin filaments (Arp2/3)
* Formin = starting point of filopodia; actin nucleator
Rac1 regulates lamellipodia:
Stress fibers: activation of myosin
- GGEF inactivation
- RhoA: off → on (Rho-GTP)
- activates Rho-kinase (substrates: myosin light chain phosphatase (MLC), CPI-17)
- Phosphorylated MLC→inactivated→promote contractility
- CPI-17-P = active
Stress fibers and filopodia: activation of formin
- Rho-type GTPase activates formin
- FH1 domain bind profilin-actin-ATP
- FH2 domain required for dimerization
Summary: Rho-GTPases in cell migration
Focal adhesions:
- Integrins: 2 chains (alpha + beta)
- Integrin/ECM interactions
- Integrins linked to actin cytoskeleton (linking-proteins: talin, vinculin, zyxin, pa
