MCB Topic 19 Regulation of Actin Assembly- Prof. David Rivier

Transcription

1 MCB 252 -Topic 19 Regulation of Actin Assembly- Prof. David Rivier MCB 252 Spring 2017

2 MCB 252 Cell Biology Topic 19 Regulation of Actin Assembly Reading: Lodish , 17.7

3 MCB 252 Actin Cytoskeleton A. Properties of Actin B. Role of Actin in Cell Movement C. Regulation of Actin Assembly in Cells D. Stable Actin Structures E. Muscle Cells and Myosin Motors

4 Actin Cytoskeleton Properties of Actin Actin assembly Role of ATP Treadmilling Role of actin in cell movement Actin is required for movement Actin assembly can drive movement Regulation of actin assembly in cells Proteins involved in actin assembly/disassembly Turning the protein involved in assembly and disassembly ON and OFF Building stable actin structures

5 How Does a Cell Regulate Where Assembly Occurs?

6 What are the properties of Actin polymerization in cells? 1. Nucleation is regulated so that polymer formation only occurs is specific regions of the cell. 2. Dissociation of monomers from the polymer is regulated so that dissociation primarily occurs at the minus end. 3. Association of monomers with the polymer is regulated so that growth occurs almost exclusively at the plus end. 4. Regulation is mediated by proteins that interact with G- actin or with the ends of F-actin.

7 How is assembly of actin restricted to particular regions of the cell (of the cytoplasm)? 1. Regulation is mediated by proteins that interact with G- actin or with the ends of F-actin. 2. Signals are received by one side of a cell but not another. Signaling regulates the function of the proteins involved in actin assembly. First we will look at the mechanisms by which proteins modulate F-actin assembly/disassembly. Then we will look at regulation- how the proteins that modulate assembly are themselves turned ON or OFF in response to signaling.

8 Proteins that Facilitate Various aspects of F-actin assembly: A Parts List STEP Sequestering G-actin: Nucleation: Elongation: Disassembly: PROTEIN(S) Thymosin β4 Formin, Arp2/3 complex Formin, Profilin Cofilin, Gelsolin PREVENTING ASSEMBLY AND DISSASEMBLY (Capping Proteins) Plus end: CapZ Minus end: Tropomodulin

9 Cells assemble actin polymers and move faster than they can produce actin. How do they do this? Answer: The concentration of G-actin in the cell is 100 x greater than the critical concentration BUT G-actin in sequestered in a pool that is not in a form that can be polymerized. Binding of Thymosin β4 to G-actin holds G-actin in a form that does not polymerize. To form a polymer, G-actin must be released from Thymosin β4.

10 Thymosin β-4 Binding to Actin

11 How is Sequestration of ATP-actin by Thymosin β4 Regulated? Sequestered Free Lodish 8 th edition Fig Answer: We do not know. Possibility 1: signaling Possibility 2: equilibrium/ buffering

12 Nucleotide exchange factor Profilin: Role 1

13 Profilin Profilin binds at hinge Profilin binds G-actin so that the surface that binds the plus end is exposed and the surface that binds the minus end is blocked

14 Actin + Profilin in Pymol T19M1 Actin_and_Profilin_Pymol

15 Cofilin Facilitates disassembly of ADP-actin (minus end)

16 Cofilin induces torsional stress to ADP-actin polymers Cofilin is thought to sever the filament at junction Part of Lodish 17-11

17 Severs actin filaments Gelsolin Family Binds plus end at point of severing, creates new minus end -> prevents grow at new plus end, allows disassembly from new minus end Gelsolin regulated by Ca++

18 Facilitation of Treadmilling

19 Proteins that Facilitate Various aspects of F-actin assembly: A Parts List STEP Sequestering G-actin: Nucleation: Elongation: Disassembly: PROTEIN(S) Thymosin β4 Formin, Arp2/3 complex Formin, Profilin Cofilin, Gelsolin PREVENTING ASSEMBLY AND DISSASEMBLY (Capping Proteins) Plus end: CapZ Minus end: Tropomodulin

20 Formin facilitates nucleation and elongation Note: this is only part of the Formin protein: the FH2 domain Formin facilitates assembly of unbranched (linear) polymers

21 Formin: Nanotechnology T19M2Formin.mov Formin accelerate the rate at which steady state is reached

22 Formin is a Machine!!! Profilin Role 2: increase the rate of actin assembly at + end Formin arms bind profilin to further facilitate assembly at + end

23 Proteins that Facilitate Various aspects of F-actin assembly: A Parts List STEP Sequestering G-actin: Nucleation: Elongation: Disassembly: PROTEIN(S) Thymosin β4 Formin, Arp2/3 complex Formin, Profilin Cofilin, Gelsolin PREVENTING ASSEMBLY AND DISSASEMBLY (Capping Proteins) Plus end: CapZ Minus end: Tropomodulin

24 Actin Related Proteins = Arps Arp2 and Arp3 are Actin Family Members Specialized for a Role in Nucleation

25 Arp2/3 Complex a Second Way to Nucleate Arp2/3 complex nucleates branched actin polymers

26 Another view of nucleation by the Arp2/3 complex

27 Repeated Rounds of Arp2/3 nucleation results in branched actin network

28 Another view of nucleation by the Arp2/3 complex

29 Proteins that Facillitate Various Aspects of F-actin Assembly: A Parts List STEP Sequestering G-actin: Nucleation: Elongation: Disassembly: PROTEIN(S) Thymosin β4 Formin, Arp2/3 complex Formin, Profilin Cofilin, Gelsolin PREVENTING ASSEMBLY AND DISSASEMBLY (Capping Proteins) Plus end: CapZ Minus end: Tropomodulin

30 Capping Proteins Four possible consequences of capping: 1. Prevent further growth 2. Restrict growth or shrinkage to one end 3. Form a stable filament (not growing, shrinking or t-milling) 4. Stop polymerization in regions where you don t want it

31 Proteins work in coordination How does polymerization generate force on the membrane? We will come back to this question later

32 Properties of Actin Actin assembly Role of ATP Treadmilling Actin Cytoskeleton Role of acting in cell movement Actin is required for movement Actin assembly can drive movement Regulation of actin assembly in cells Proteins involved in actin assembly/disassembly Turning the protein involved in assembly and disassembly ON and OFF Building stable actin structures

33 How does a cell regulate where assembly occurs?

34 How does a cell regulate where assembly occurs?

35 Logic of Actin Regulation In Movement Cells can respond quickly to signals. Cells can rearrange their actin cytoskeleton quickly. To achieve these quick responses cells have all the components and machinery synthesized but the most of the components are in an inactive form. Signaling results in activation of the components.

36 Wound Healing: One Example of Signaling in Cell Migration In a wound, blood platelets are exposed to extra cellular matrix (ECM) which leads to activation of the platelets. Activated blood platelets contribute to blood clotting. Activated platelets also send signals that result in the movement of fibroblasts and epithelial cell to move into the damaged area to effect healing.

37 3 main types of actin filaments involved in this type of motion Filopodia Filopodia and the Lamellopod are at leading edge (branched) Stress fibers will pull the tail end forward (talk about later) How does signaling set up and maintain cell polarity?

38 RAS superfamily Alberts Table 15-5 Families often named for the first member identified

39 Activation of Ras Family Members is Similar to RAS Signaling triggers activation GAP resets the signal making the response transient

40 Activated Rho Triggers Formin Activation Rho inactive until a signal is received

41 Activated Cdc42 triggers WASp activation Activated WASp activates Arp2/3 complex and therefore triggers branched polymer formation

42 Coordinated Regulation of Cdc42, Rac and Rho 3 G-proteins and 3 Responses

43 Coordinated Regulation of Cdc42, Rac and Rho

44 Signaling Coordinates Multiple Aspects of Actin Dynamics Activate nucleation and branching Inhibit capping and stress fiber formation

45 Rho promotes several aspects of stress fiber function

46 How Do We Know Which Rho Family Members do What? Recall that Ras is mutated in ~ 50% of Human Cancers How Does Ras Contribute to Cancer?

47 How Does Ras Contribute to Cancer? What is the mutant form of Ras that contributes to cancer?

48 Thinking about Genetics

49 Thinking about Genetics Lodish 5-2

50 Thinking about Genetics Genotype RAS-WT RAS-WT RAS-CA RAS-WT RAS-CA RAS-CA rasδ RAS-WT rasδ rasδ Phenotype Divides when signaled Divides without signal Divides without signal Divides when signaled Cannot Divide RAS-WT = Wild-type RAS-CA = Constitutively active (also referred to in textbook as Ras D ) rasδ= completely inactive gene (null phenotype)

51 Thinking about Genetics Genotype RAS-WT RAS-WT RAS-CA RAS-WT RAS-CA RAS-CA rasδ RAS-WT rasδ rasδ Phenotype Divides when signaled Divides without signal Divides without signal Divides when signaled Cannot Divide Dominant mutations are often Gain-of-Function Mutations; Here Ras has gained the ability to signal in the absence of the signaling molecule. Recessive mutations are typically Loss-of-Function Mutations; Here Ras has lost the ability to signal in the presence of the signaling molecule.

52 How Does Ras Contribute to Cancer? What is the mutant form of Ras that contributes to cancer? How can you use this info to test whether Rho, Rac and Cdc42 play a role in regulating the actin cytoskeleton?

53 Activated Mutants of Rho Family Members Stress Fibers Everywhere Lamellipodia Everywhere Filopodia Everywhere

54 How do we know which Rho Family Members Regulate which Processes? Transfect Activated Mutant Forms

55 Role of Rho Family Members in Wound Healing Loss of Rac, Cdc42 or Rho activity leads to defects in wound healing

56 Let s return to the mechanics of membrane extension How does polymerization generate force on the membrane?

57 How Does Actin Polymerization Drive Movement? It s not yet clear, 3 models 1. Actin flexing 2. Pushing on membrane-bound proteins 3. Rectified diffusion (the Brownian Ratchet)

58 Actin Flexing Model Lodish5th edition, 19-27

59 Formin: Nanotechnology T19M1Formin.mov Formin accelerates the rate at which steady state is reached

60 Pushing Model of Membrane Extension Problem with this Model: Formin not at leading edge

61 Does Cdc42 (or Rac) Travel with the Plus end? Is a Formin Family Member Associated with Leading Edge?

62 How Does Listeria Nucleate Actin Polymerization?

63 Listeria ActA protein Actin assembly drives movement of Listeria

64 Nucleation of Branching by Infectious Agents

65 Proteins act in coordinated fashion Capping Occurs Once the Filament is No Longer Pushing on the Membrane

66 Logic and Design Rationale Roles of CapZ To insure that actin polymerization only happens at the appropriate sites: 1. Nucleation proteins are activated 2. Random nucleation events are made unproductive CapZ is located throughout the cytoplasm CapZ binds the ends of polymers and blocks further polymerization Randomly nucleated polymers are quickly capped Formin blocks capping by CapZ (physically) Leading edge membrane inhibits CapZ Branched polymer no longer contacting memb. gets capped

67 Logic and Design Rationale Roles of CapZ Properly nucleated polymers are protected from capping and are therefore free to elongate Any polymers that are nucleated as a result of the random collision of 3 actin monomers are quickly capped by CapZ and removed by disassembly from the minus end.

68 Role of Actin in Endocytosis

69 Role of Actin in Endocytosis

70 Role of Actin in Endocytosis

71 Role of Actin in Phagocytosis