ACTIN BINDING PROTEINS pathways to sculpt functionally polymorph actin structures Beáta Bugyi Muscle Biophysics University of Pécs – Medical School PhD Summer School Budapest, August 28 – 30, 2018 Department of Biophysics UNIVERSITY OF PÉCS – MEDICAL SCHOOL – DEPARTMENT OF BIOPHYSICS CYTOSKELETAL DYNAMICS LAB http://cytoskeletaldynamics.wix.com/mysite
OVERVIEW CONCEPTS BEHIND ACTIN’S DIVERSIFICATION Contrasting the functional polymorphism of cellular actin networks and the inherent actin dynamics What are the molecular origins of sculpting functionally polymorph actin structures and providing them with spatio-temporal regulation? Evolutionary perspective Prokaryotic vs. Eukaryotic diversification Basic inventory of actin binding proteins (ABPs) How ABPs recognize ‚their own actin’? STRUCTURAL-FUNCTIONAL ASPECTS OF ABPs IN BUILDING FUNCTIONAL ACTIN NETWORKS The classic models of thin filament regulation: Tropomyosin Assembly and maintenance of thin filaments Co-factors of the actin monomer pool: T b 4/WH2, Profilin Dynamics at the filament barbed end: Capping protein, Formin Dynamics at the filament pointed end: Tropomodulin, Leiomodin OUTLOOK Dynamic regulation of non-muscle actin networks: Arp2/3 complex machinery Master regulator concept in actin’s diversificaiton Alternative ways to self-organize functionally diverse actin networks
FUNCTIONAL POLYMORPHISM OF ACTIN NETWORKS – MANIFESTATION EUKARYOTIC CYTOSKELETON 1942, ~ 1970 Straub FB. Studies 1942 Ishikawa H. et al. J. Cell Biology1969 Beating of neonatal cardiomyocyte ( α -actinin – Movement of B16 melanoma cell (EGFP-actin). AcGFP in Z discs). Klemens Rottner Institute of Genetics, University of Bonn, Shintani SA. et al. Journal of General Physiology 2014 Germany
FUNCTIONAL POLYMORPHISM OF ACTIN NETWORKS – MANIFESTATION EUKARYOTIC CYTOSKELETON 1942, ~ 1970 Straub FB. Studies 1942 Ishikawa H. et al. J. Cell Biology1969 PROKARYOTIC CYTOSKELETON ~ 1990s Fink G. et al. Cell 2016 NUCLEOSKELETON ~ 2000s Viita T. et al. Handb Exp Pharmacol. 2017 Bajusz C. et al. Histochem Cell Biol. 2018 Beating of neonatal cardiomyocyte ( α -actinin – Movement of B16 melanoma cell (EGFP-actin). Klemens Rottner Institute of Genetics, University of AcGFP in Z discs). Bonn, Germany Shintani S. A. The Journal of General Physiology 2014 3D electrical connections nanofabricated biocomputers ENGENEERING Galland R. et al. Nature Materials 2013 actomyosin machinery ~ 2010s Nicolau D. et al. PNAS 2016 metallized actin
SPONTANEOUS ASSEMBLY PATHWAYS OF ACTIN STRUCTURES SPONTANEOUS DE NOVO ASSEMBLY OF INDIVIDUAL SUBUNITS INTO POLYMERS ACCOPANIED BY AN INCREASE IN ACTIN’S ATPaseACTIVITY + end + end length (bar = 1 m m) a - end - end time (bar = 10 s) KYMOGRAPH 𝑤 𝑓𝑚𝑝𝑜𝑏𝑢𝑗𝑝𝑜 = 𝑢𝑏𝑜𝛽 = = ∆𝑚𝑓𝑜𝑢ℎ(𝜈𝑛) = ∆𝑚𝑓𝑜𝑢ℎ ∗ 370(𝑡𝑣) ∆𝑢𝑗𝑛𝑓 (𝑡) ∆𝑢𝑗𝑛𝑓 (𝑡) 𝑤 𝑓𝑚𝑝𝑜𝑏𝑢𝑗𝑝𝑜 = 𝑙 𝑃𝑂 𝐻 − 𝑙 𝑃𝐺𝐺 = 𝑙 𝑃𝑂 𝐻 − 𝑑 𝑑 note 𝑂 = 1 ! 𝑙 𝑃𝑂 𝑐𝑏𝑠𝑐𝑓𝑒 𝑓𝑜𝑒, 𝐵𝑈𝑄 = 11.6 μ𝑁 −1 𝑡 −1 𝑙 𝑃𝑂 𝑞𝑝𝑗𝑜𝑢𝑓𝑒 𝑓𝑜𝑒, 𝐵𝐸𝑄 = 1.3 μ𝑁 −1 𝑡 −1 TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY (TIRFM) OBSERVATION OF THE ASSEMBLY OF INDIVIDUAL ACTIN POLYMERS Bugyi B. Muscle Contraction - A Hungarian Perspective 2018
SPONTANEOUS ASSEMBLY PATHWAYS OF ACTIN STRUCTURES SPONTANEOUS ASSOCIATION OF INDIVIDUAL POLYMERS INTO HIGHER ORDER STRUCTURES SIDEWISE ASSOCIATION ENDWISE ASSOCIATION cross-linking/bundling annealing radial thickening lateral growth Bugyi B. Muscle Contraction a Hungarian Perspective 2018
SPONTANEOUS DISASSEMBLY PATHWAYS OF ACTIN STRUCTURES SPONTANEOUS DEPOLYMERIZATION DISSOCIATION OF INDIVIDUAL SUBUNITS FROM POLYMERS + end - end MICROFLUIDICS-ASSISTED TIRFM OBSERVATION OF THE DISASSEMBLY OF INDIVIDUAL ACTIN POLYMERS 𝑙 𝑃𝐺𝐺 𝑐𝑏𝑠𝑐𝑓𝑒 𝑓𝑜𝑒, 𝐵𝐸𝑄 = 5.4 𝑡 −1 𝑙 𝑃𝐺𝐺 𝑞𝑝𝑗𝑜𝑢𝑓𝑒 𝑓𝑜𝑒, 𝐵𝐸𝑄 = 0.25 𝑡 −1 Shekar S. et al. Current Biology 2017, Carlier MF. et al. Methods in Enzymology 2012
SPONTANEOUS MECHANICAL FORCE GENERATION OF ACTIN STRUCTURES Limulus acrosomal bundle microfabricated wall 𝑀 𝐹𝐽 = 𝑀 𝑞 𝑛𝑏𝑦 = 𝑙 𝐶 𝑈 ∆ ln(𝑑 𝑙 𝑃𝑂 𝐺 = 𝜌 2 𝐹𝐽 𝐺 ) 𝑀 2 𝑙 𝐶 𝑈 𝑙 𝑃𝐺𝐺 ∆= 2.7 𝑜𝑛 𝐺 = −𝑙𝑒 Apparent elongation (nm) FORCE (pN) 𝑀 𝑞 𝐹𝐽 𝐺 (10 −26 𝑂𝑛 2 ) (𝑞𝑂) (𝜈𝑛) F-actin 9 3.6 0.25 - 0.56 Ph-F-actin 18 7.2 1.3 Time (s) Footer MJ. et al. PNAS 2007, Kovar DR. et al. PNAS 2004
FUNCTIONAL POLYMORPHISM OF ACTIN NETWORKS – ORIGINS INTRACELLULAR FUNCTIONING CELL FREE ENVIRONMENT SPATIO-TEMPORAL CONTROL functionally distinct structures intrinsic dynamic behavior FUNCTIONAL DIVERSIFICATION controlled dynamics What are the molecular origins of sculpting functionally polymorph actin structures and providing them with spatio-temporal regulation?
PROKARYOTE’S CONCEPT OF DIVERSIFICATION – ONE ‚ ACTIN ’ FOR ONE FUNCTION BACTERIA ARCHEA actin 1 actin 2 actin 3 actin 4 actin 5 ParM FtsA MamK MreB Crenactin 2ZGY 4A2B 5LJW 1JCE 4BQL DNA „ divisome ” „ magnetosome ” „ elongasome ” cell shape ? segregation membrane anchor facilitates cell morphology cell division magnetotaxis right-handed 4-stranded, supercoiled 4-stranded antiparallel twisted 1-stranded F-actin open nanutube antiparallel nanotubule non-twisted Jiang S. et al. Communicative and Integrative Biology 2016
EUKARYOTE’S CONCEPT OF DIVERSIFICATION – ONE ‚ ACTIN ’ FOR ALL FUNCTIONS EUKARYOTE (fungi, metazoa) central element/hub ABP 1 ABP 2 ABP n function 1 function 2 function n
BASIC/CLASSIC INVENTORY OF ACTIN BINDING PROTEINS adapted from Renault L., Bugyi B., Carlier MF. Trends in Cell Biology 2009, Bugyi B. et al. Annual Reviews in Biophysics 2010
BASIC/CLASSIC INVENTORY OF ACTIN BINDING PROTEINS multifunctional proteins multidomain proteins multiprotein complexes antagonistic/synergic effects protein isoforms links to other cytoskeletal polymers adapted from Renault L., Bugyi B., Carlier MF. Trends in Cell Biology 2009, Bugyi B. et al. Annual Reviews in Biophysics 2010
HOW ABPs RECOGNIZE ‚THEIR OWN ACTIN’? SORTING MECHANISMS OF ABPs INTRINSIC DIFFERENCES IN THE ‚NATURE’ OF ACTIN BIOCHEMICAL DIFFERENCES OF ACTIN ISOFORMS • 6 isoforms encoded by different genes (Perrin B. J. et al. Cytoskeleton 2010) DIFFERENT NUCLEOTIDE STATE OF ACTIN • ADF/cofilin preferential binding to ADP.Pi, ADP actin polymer segments (Suarez C. et al. Current Biology 2011) POSTTRANSLATIONAL MODIFICATIONS oxidation of Met 44 , Met 77 by the redox enzyme Mical impairs polymer stability • (Terman J. R. et al. Current Opininion in Cell Biology 2013) ‚MASTER ABP’ REGULATOR ASSEMBLY/NUCLEATION FACTORS • 15 formin proteins, Arp2/3 complex machinery • structurally different actin polymers ( Bugyi B. et al. Journal of Biological Chemistry 2006 ) TROPOMYOSIN ISOFORMS • > 40 isoforms • functionally distinct actin polymers are associated to different Tpm isoforms (Gunning P. et al. Journal of Cell Science 2015) COMPETITION-MEDIATED SEGREGATION • Competition between ABPs drives their sorting to distinct actin filament networks (Christensen JR. et al. eLIFE 2017) GEOMETRICAL/MECHANICAL CONSTRAINS • nucleation geometry governs actin network architecture • myosin contractility is targeted to branched/ordered antiparallel polymer networks vs. parallel polymers (Blanchoin L. et al. Physiological Reviews 2013, Schramm AC. et al. Biophysical Journal 2017)
ABPs IN CLASSIC MODELS OF THIN FILAMENT REGULATION
‚ TO SEE THEM CONTRACT FOR THE FIRST TIME ’ Albert Szent- Györgyi 1963 F-actin myosin CONTRACTION OF MYOSIN THREADS A: before addition of boiled muscle juice (source of ATP) B: after addition of boiled muscle juice (source of ATP) Szent- Györgyi A. Studies 1942, Szent- Györgyi AG. Journal of General Physiology 2004 ‚ muscle contraction was essentially an interaction of actomyosin and ATP ’ (Albert Szent- Györgyi) ‚… contraction should occur spontaneously wherever the ATP-actomyosinsystem is present in a suitable ionic milieu … In the intact resting muscle, however, we find ATP in an active form, linked to actomyosin, but still the system does not contract-contraction being inhibited by some unknown mechanism. If we want the IN VITRO MOTILITY ASSAY muscle to go over into the contracted state, we OBSERVATION OF ACTIN POLYMER SLINDING ON have to abolish this inhibition. ’ MYOSIN FUNCTIONALIZED GLASS autonomous nature of isolated actomyosins (Albert Szent- Györgyi 1949) CLASSIC MODELS – SLIDING FILAMENT THEORY STERIC BLOCKING THEORY
STRUCTURAL LANDMARKS – SLIDING FILAMENT THEORY MYOSIN ACTIN Z line Z line M line + - - + I band A band I band H zone SLIDING SLIDING Z: Zwischenscheibe, Krause membrane H: Hensen zone M: Mittelscheibe Gohkin DS. et al. Nature Reviews Molecular Cell Biology 2013
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