Biophysics of Molecules

Transcription

1 Biophysics of Molecules Cytoskeletal filaments, Actin polymerization and Actin Treadmilling Part 2 ( ) Dr. Carsten Grashoff MPI of Biochemistry cgrasho@biochem.mpg.de

2 Lecture Outline The Cytoskeleton 1. Actin dynamics and the f-actin network 2. Tubulin dynamics, the tubulin network and intermediate filaments 2

3 Why do you need to know this? Microtubules are essential for cell division (and therefore essential for life) Important diseases affect the tubulin network Most chemotherapies interfere with the tubulin network 3

4 The tubulin network f-actin tubulin 4

5 The tubulin subunits - the smallest subunits of the tubulin network are a-tubulin and b-tubulin - a-tubulin and b-tubulin form a heterodimer - both subunits have a GTPbinding site - the GTP on a-tubulin is trapped in the heterodimer and can never be hydrolyzed; b-tubulin can be in a GDP-or GTP-bound form 5

6 Microtubule formation - tubulin heterodimers form protofilaments, which have a polarity - 13 protofilaments form a microtubule, which resembles a hollow cylinder - due to multiple molecular interactions between subunits, microtubules are very stiff 6

7 Microtubules are very stiff - individual protofilaments are thermally unstable - assembled microtubule filaments are thermally stable 7

8 Assembly of microtubules Microtubule assembly involves three steps: - protofilament assembly - sheet assembly - elongation - shortly after subunit incorporation, GTP is hydrolyzed - if the rate of subunit addition is faster than hydrolysis a GTP-cap is formed 8

9 Tubulin polymerization - each subunit carries a GTP (T) before incorporation - hydrolysis reduces the binding affinity and GDPtubulin (D) is more likely to dissociate - on-rates are depending on the concentration of the free subunit - addition of GTP-tubulin to the (+) end is fast but slow for the (-) end 9

10 Tubulin treadmilling - the critical concentration of GTP-rich (+) end and GDPrich (-) end are different - at the appropriate subunit concentrations, when C C (T)<C<C C (D), tubulin filaments will undergo treadmilling 10

11 Dynamic instability of tubulin networks - if the GTP-cap is lost, a rapidly growing filament may quickly start shrinking, a process called catastrophe - if a GTP-cap is regained, a disassembling filament may start rapidly growing, a process called rescue - this inherently metastable behavior is known as dynamic instability 11

12 Dynamic instability of tubulin networks dynamic instability can be frequently observed in living cells 12

13 Dynamic instability in living cells 13

14 GTP-hydrolysis affects tubulin structure - GTP stabilizes a straight conformation - GTP hydrolysis leads to a structural change and protofilaments are bending - the GTP-cap structurally stabilizes the tip of growing microtubules 14

15 Growing and shrinking tubulin filaments 15

16 The tubulin network is highly dynamic 16

17 Regulation the tubulin network The problem Cells make a lot of a- and b-tubulin. How do cells prevent tubulin polymerization? 17

18 Stathmin-tubulin interactions - stathmin can interact with tubulin heterodimers - stathmin-bound tubulin heterodimers can not polymerize - stathmin is regulated during mitosis by phosphorylation - mutations in stathmin cause cancer (stathmin is also known as oncoprotein 18 ) 18

19 Regulation the tubulin network How is microtubule nucleation regulated? 19

20 g-tubulin-dependent tubulin nucleation - tubulin nucleation is facilitated by a third tubulin molecule: g-tubulin centrosome - g-tubulin concentrates in the microtubule organizing center (MTOC) called centrosome a-tubulin g-tubulin 20

21 The centrosome - microtubules are nucleated at the centrosome - microtubules (+) ends point outward towards the cell periphery 21

22 Centrosome architecture - each centrosome contains a pair of centrioles (a mother and a daugther centriole) - centrioles consist of a short cylinder of microtubules and are connected by accessory proteins 22

23 Centrosome replication during the cell cycle - centrosomes replicate during the cell cycle to generate two centrosome at the beginning of mitosis - centrosome replication is not fully understood, but many cancer cells have too many centrosomes 23

24 Regulation the tubulin network The problem Microtubules are very instable. (dynamic instability) How do cells stabilize microtubules? 24

25 Stabilizing microtubules at the (+) end - XMAP215-like proteins can be found in every eukaryotic organism studied so far - XMAP215 stabilizes the weakly bound tubulin dimer thereby decreasing dissociating rate 25

26 Stabilizing microtubules by MAPs - microtubule-binding proteins (MAPs) stabilize microtubules by direct binding - MAPs are highly expressed in the brain (i.e. in neurons) 26

27 High MAP expression in the brain Distribution of two MAPs in a neuron (green: tau; red: MAP2) 27

28 The special case of the Tau protein - the tau protein binds to microtubules and stabilizes them - expression of tau in cells induces formation of microtubules - tau gets modified (phosphorylation, glycosylation, ubiquitinylation, cleavage, crosslinking, etc.) - tau also binds other proteins - the physiological role of tau is not totally clear 28

29 Tau aggregates cause Alzheimer s disease 29

30 Regulation the tubulin network The problem In preparation for cell division cells have to disassemble interphase microtubules quickly. How do cells break or disassemble microtubules? 30

31 Katanin, the cellular samurai hexameric AAA- ATPase complex - katanin is an AAA family ATPase - katanin forms a hexameric complex which uses ATP hydrolysis to break bonds between tubulin heterodimers - katanin is regulated by phosphorylation 31

32 Catastrophe factors - some kinesins such as Kinesin-8 and Kinesin-13 use ATP-hydrolysis to facilitate depolymerization - kinesins can walk on microtubules and induce depolymerization at the ends 32

33 Regulating tubulin dynamics 33

34 Fine tuning the tubulin network - many proteins are involved in the regulation of the tubulin network - spatiotemporal regulation (regulation in time space and time) within the same cell - there are many things we still need to learn 34

35 Let s summarize - the smallest subunits of the tubulin network is the tubulin heterodimer consisting of an a-tubulin and a b-tubulin - the b-tubulin subunit can be in the GDP or GTP-bound form - heterodimers form protofilaments; 13 protofilaments form a microtubule - tubulin networks are highly dynamic; they undergo treadmilling, rapid shrinking (catastrophe) and rescue; this phenomenon is called dynamic instability - nucleation of tubulin networks occurs at the centrosome using g-tubulin - cells express proteins which modulate the tubulin network: - stathmin to sequester heterodimers - microtubule-associated proteins (MAPs) to stabilize - katanin and catastrophe factors to break and depolymerize - these processes are regulated by complex signaling networks 35

36 Why is this important? Biological functions of tubulin networks 36

37 Microtubules essential role for cell division 37

38 Microtubules capture chromosomes during mitosis Growing microtubules Shrinking microtubules 38

39 Figure 16-85c Molecular Biology of the Cell ( Garland Science 2008) 39

40 Why this knowledge might save your life The problem with cancer is that cells division is not controlled. We need cancer cells to stop proliferating. 40

41 Tubulin drugs to fight cancer paclitaxel Taxus brevifolia (Eibe) - paclitaxel is a toxin of the taxane family - paclitaxel binds to b-tubulin and prevents depolymerization of microtubules - paclitaxel is used in chemotherapies against many cancers such as breast cancer, prostate cancer or lung cancer 41

42 Other toxins that affect tubulin Catharanthus roseus Vincristine Vincristine binds to tubulin heterodimers and inhibits filament assembly (for example used against lymphoma) Colchicum autumnale Cholchicine Colchicine inhibits microtubule filament assembly 42

43 Microtubules are also important for transport - microtubules are used for transport - in very long axons many microtubules are aligned - we distinguish: anterograde and retrograde axonal transport 43

44 Vesicle transport in cells video: Daniel v. Wangenheim 44

45 Microtubules build cilia and flagella - many bacteria but also mammalian cells such as sperm cells use flagella to move forward 45

46 Most of our cells have a primary cilium primary cilia in epithelial cells - most of our cells have a primary cilium ( i.e. fibroblast, epithelial cells, neurons, chondrocytes, etc.) - primary cilia sense mechanical flow and are connected to mechanosensitive calcium channels - loss of the primary cilium on kidney cells leads to polycystic kidney disease 46

47 Summary of biological importance cells critically depend on the tubulin network, because: - microtubuli are essential for cell division - microtubuli are the intracellular highways - the microtubular network supports cell shape and mechanical stability important diseases are connected to the tubulin network: - cancer - Alzheimer disease 47

48 The intermediate filament network 48

49 The intermediate filament network - not all organisms have intermediate filaments - the smallest subunit are elongated coiled-coil proteins (i.e. keratin) - parallel dimers assemble into tetramers - intermediate filaments do not have a polarity and do not bind nucleotides 49

50 Keratin networks in skin cells - the integrity of keratinocytes (skin cells) depends on keratin-based intermediate filaments - intermediate filaments fulfill important mechanical properties and influence the elasticity of cells 50

51 Keratin networks in skin cells - loss of the intermediate filament system in the skin leads to severe skin disorders such as epidermolysis bullosa 51

52 The three filament networks f-actin tubulin intermediate filaments thin (7 nm) thick (25 nm) intermediate (10 nm) ADP/ATP-binding GDP/GTP-binding no nucleotide binding polarity polarity no polarity decentralized centralized decentralized 52