Cell bio7

• 1.Advanced Cell Biology 2014 1nd Semester Department of Animal Science Chungbuk National University 7th Lecture

2. 1st week : Introduction 3rd week :Research Strategies For Cell Biology 5nd week : Nucleus, Transcription and Splicing 7nd week : Membrane and Channel 9nd week : Membrane Trafficking 11nd week : Cell Signaling 13nd week : Cytoskeleton 15nd week : Cell Cycle 3. Cytoskeleton : Scaffolds in the Cells Architectural scaffolds : Supports constructions 4. Three major cytoskeleton components Microfilaments Microtubule Intermediate filaments (Actin) (Tubulin) (Keratin, vimentin..) Microfilaments 5. Cytoskeletons as Road in the Cell Highway Interstate Highway = Microtubule? Local Roads Microfilaments (Actin) 6. Actin Monomeric Actin (42kDa) (G-Actin) Filamentous Actin (F-Actin) Actin is the second abundant protein in the world Polymerization Depolymerization 7. Actin Filament: the main building block of cytoskeleton Borish & Svitskina 8. Roles of Actin Filament Remodeling in various cellular process CytokinesisCell Motility PhagocytosisVesicle Transport Asymmetric spindle Positioning in Oocyte 9. Actin Filament is dynamic Formation of Actin filaments When? How long? Where? How looks like? 10. New Filament Generations (Actin Nucleators:Arp2/3, Formins, Spire) Capping (CapZ, Tropomodulin) Depolymerizations/Severing (Cofillin, Gelsolin) Monomeric Actin Binding Protein (Profilin, Thymosin beta-4) 11. Actin Nucleators Determine type of Filament Structure Arp2/3 complex :59 Annual Reviews AR131-19 REGULATION OF ACTIN FILAMENT NETWORK 651 ure 1 Electron micrographs of branched actin laments. (a) Electron micrograph of leading edge of a migrating Xenopus keratocyte prepared by detergent extraction and ry shadowing. (b) Detail of boxed region from (a), with a branched lament network ed in blue. (c) Gold-labeled antibody to ARPC2 localized to a branch point in the ment network at the leading edge. (d ) Branch formed in vitro by pure actin laments Acanthamoeba Arp2/3 complex. (ac) modied from 14. (d ) Modied from 13. p2/3 complex (hereafter referred to as nucleation promoting factors), are being Formins Straight actin bundles In Stress fibers and filopodia Branched actin filaments in Lamellipodia Spire Straight Fiber Oocyte development Vesicle Transport 12. Arp2/3 complex: 6 Protein complex makes branched networks Arp2 Arp3 ARPC3ARPC1 ARPC5 ARPC4 ARPC2 Dominguez and Namgoong, Comphrensive Biophysics 2012 Arp2 Arp3 ARPC3ARPC1 ARPC5 ARPC4 ARPC2mother filament barbed end barbedend Nucleation Promoting Factor (NPF) 13. 18 85 161 200 245 436 498 N-WASP Pro-rich 26 136 200 270 400 501 WH1 GBDB AW CW WAVE2 Pro-richhelix B AW C JMY MA spectrin-like AW CW WPro-rich 40 300 620 790 855 983 profilin-actinregulation/localization Prototypical NPF AW CWWPro-rich actin Arp2/3 167 202 290 303 360 465 WASH Pro-richWHD1 AW CWHD2 Arp2/3 complex WCA + actin transition complex activated complex + branch 70 2 4 5 1 Arp2 Arp3 3 Nucleation Promoting Factor (NPF) are essential for Arp2/3 mediated filament nucleations 14. Formins Rho actin Pro-rich (FH1) FH2CCDD DADDIDGBD 73 130 377 452 570 736 1175 1200 autoinhibition profilin-actin DD DID DAD Complex of DID and DAD DID GTP-Rho DD GBD Binding of Rho GTPase linker lasso knobcoiled coil post FH2 Domain 3 1 4 21 2 4 barbed end barbed end 3 1 4 2 1 2 4 3 Responsible for the straight bundles of filaments (Stress fiber, Fillopodia) 15 formins (8 subclass) in human and mouse Caps fast growing ends (barbed end) of actin filament and enhance elongation rates Regulated by autoinhibition and small GTPase (Rho) 15. Signaling activating Actin nucleators Rho-family GTPases (Rac, RhoA, cdc42) play crucial roles in activation of Actin Nucleators Rac -> WAVE -> Arp2/3 Cdc42 -> N-WASP -> Arp2/3 RhoA -> Formin 16. 1253 1128 158 445 507 Sra1 Nap1 90 172 202 498 559 W C Ahelix meander Pro-rich region WAVE1 Abi2 75 HSPC300 L697 Y704 L841 F844 W845E434 F626 M632R190C179 basic Rac1-binding SH3 Nap1 HSPC300 Sra1 Abi2 WAVE1 WAVE1 Polybasic region (proposed membrane-binding site) proposed Rac1-binding site W C C-terminal part polybasic region and Pro-rich sequence of WAVE1 substituted by a short linker (disordered in structure) Pro-rich region and SH3 domain removed for crystallization disordered 17. Actin Depolymerization - ADF/Cofillin : Bind to Actin Filaments (ADP-Actin) - Favor Depolymerization of Actin - ADF/Cofillin signaling RhoA->ROCK-> LIM Kinase -> Cofillin Phosphorylation (Inactivation) Slingshot Phosphatase Dephosphorylated Cofilin (Active) 18. Actin Capping Proteins - Barbed End (Fast-Growing End) Capper CapZ : Stop Elongation of Actin Filaments - Pointed End Capper Tropomodulin : Protect Actin filaments from Depolymerization 19. Capping Proteins : inhibit elongation and stabilize actin filaments CapZ : Capping fast growing (Barbed) end Gelsolin : Severing and Capping Proteins 20. Tropomyosin + Tropomodulin : Guardian of actin filament Tropomodulin Tropomyosin 21. Dendritic Nucleation Model 22. Myosin : Molecular Motor on Microfilaments 23. Myosin Actin in Skeletal Muscle 24. Myosin walk on actin filaments - Except Myosin VI, every myosin move from Slow growing end (pointed end) - Hydrolysis of ATP provides energy for the movement 25. Regulation of Myosin Activities Skeletal Muscle : Calcium mediated Troponin-Tropomyosin Smooth Muscle : Myosin Light Chain Kinase Myosin Light Chain Phosphatase 26. Cell Motility Actin Polymerization is the main driving force for cell migration 27. Actin filaments in Lamellipodia is mainly formed as branched filaments by Arp2/3 complex Actin filaments in Fillopodia is mainly formed as straight bundle by Formins and Arp2/3 complex 28. Cytokinesis 29. http://bement.molbio.wisc.edu/node/13 30. Endocytosis 31. Asymmetric division Actin Cap 4N GV GVBD Spindle Formations Spindle Migration MII Arrest 2N 2N 1N 1N Fertilization Polar Body Adapted from Yi and RI, Cytoskeleton (2012) 32. Actin Nucleators in Asymmetric Spindle Migrations Gene Phenotype Method Referenc es Formin (FMN2) Failure of Spindle Migration Symmetric division Knockout mouse fmn2 (-/-) Spire Failture of Spindle Migration Failure in Cytokinesis siRNA knockdown How about Arp2/3 and Nucleation Promoting Factors? 33. Microtubule Consists with alpha and beta Tubulin (Binds GTP or GDP) Alpha and Beta Tubulin 34. Mitotic spindle 35. Centrosome 36. Microtubule-binding proteins 37. Microtubule-based Motors - Kinesin : Minus Plus End Directed Motor Proteins - Dynein : Plus -> Minus Directed Motor Proteins 38. Kinesin walking on microtubule 39. Moving vesicle along with microtubule