ACTIN BINDING PROTEINS pathways to sculpt functionally polymorph - - PowerPoint PPT Presentation

Beáta Bugyi

University of Pécs – Medical School Department of Biophysics

UNIVERSITY OF PÉCS – MEDICAL SCHOOL – DEPARTMENT OF BIOPHYSICS CYTOSKELETAL DYNAMICS LAB http://cytoskeletaldynamics.wix.com/mysite

ACTIN BINDING PROTEINS

pathways to sculpt functionally polymorph actin structures

Muscle Biophysics

PhD Summer School Budapest, August 28–30, 2018

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: Tb4/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

Movement of B16 melanoma cell (EGFP-actin). Klemens Rottner Institute of Genetics, University of Bonn, Germany Beating of neonatal cardiomyocyte (α-actinin– AcGFP in Z discs). Shintani SA. et al. Journal of General Physiology 2014

EUKARYOTIC CYTOSKELETON 1942, ~ 1970

Straub FB. Studies 1942 Ishikawa H. et al. J. Cell Biology1969

FUNCTIONAL POLYMORPHISM OF ACTIN NETWORKS – MANIFESTATION

Movement of B16 melanoma cell (EGFP-actin). Klemens Rottner Institute of Genetics, University of Bonn, Germany Beating of neonatal cardiomyocyte (α-actinin– AcGFP in Z discs). Shintani S. A. The Journal of General Physiology 2014

ENGENEERING ~ 2010s

nanofabricated biocomputers

actomyosin machinery Nicolau D. et al. PNAS 2016

metallized actin

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

3D electrical connections

Galland R. et al. Nature Materials 2013

EUKARYOTIC CYTOSKELETON 1942, ~ 1970

Straub FB. Studies 1942 Ishikawa H. et al. J. Cell Biology1969

TOTAL INTERNAL REFLECTION FLUORESCENCE MICROSCOPY (TIRFM) OBSERVATION OF THE ASSEMBLY OF INDIVIDUAL ACTIN POLYMERS

𝑤𝑓𝑚𝑝𝑜𝑕𝑏𝑢𝑗𝑝𝑜 = 𝑢𝑏𝑜𝛽 = = ∆𝑚𝑓𝑜𝑕𝑢ℎ(𝜈𝑛) ∆𝑢𝑗𝑛𝑓 (𝑡) = ∆𝑚𝑓𝑜𝑕𝑢ℎ ∗ 370(𝑡𝑣) ∆𝑢𝑗𝑛𝑓 (𝑡) 𝑤𝑓𝑚𝑝𝑜𝑕𝑏𝑢𝑗𝑝𝑜 = 𝑙𝑃𝑂 𝐻 − 𝑙𝑃𝐺𝐺 = 𝑙𝑃𝑂 𝐻 − 𝑑𝑑 note 𝑂 = 1 !

KYMOGRAPH

+ end

• end

length (bar = 1 mm) time (bar = 10 s)

a

+ end

• end

SPONTANEOUS ASSEMBLY PATHWAYS OF ACTIN STRUCTURES

𝑙𝑃𝑂 𝑐𝑏𝑠𝑐𝑓𝑒 𝑓𝑜𝑒, 𝐵𝑈𝑄 = 11.6 μ𝑁−1𝑡−1 𝑙𝑃𝑂 𝑞𝑝𝑗𝑜𝑢𝑓𝑒 𝑓𝑜𝑒, 𝐵𝐸𝑄 = 1.3 μ𝑁−1𝑡−1 SPONTANEOUS DE NOVO ASSEMBLY OF INDIVIDUAL SUBUNITS INTO POLYMERS ACCOPANIED BY AN INCREASE IN ACTIN’S ATPaseACTIVITY

Bugyi B. Muscle Contraction - A Hungarian Perspective 2018

SIDEWISE ASSOCIATION cross-linking/bundling ENDWISE ASSOCIATION annealing

Bugyi B. Muscle Contraction a Hungarian Perspective 2018

SPONTANEOUS ASSEMBLY PATHWAYS OF ACTIN STRUCTURES

radial thickening lateral growth SPONTANEOUS ASSOCIATION OF INDIVIDUAL POLYMERS INTO HIGHER ORDER STRUCTURES

Shekar S. et al. Current Biology 2017, Carlier MF. et al. Methods in Enzymology 2012

SPONTANEOUS DISASSEMBLY PATHWAYS OF ACTIN STRUCTURES

𝑙𝑃𝐺𝐺 𝑐𝑏𝑠𝑐𝑓𝑒 𝑓𝑜𝑒, 𝐵𝐸𝑄 = 5.4 𝑡−1 𝑙𝑃𝐺𝐺 𝑞𝑝𝑗𝑜𝑢𝑓𝑒 𝑓𝑜𝑒, 𝐵𝐸𝑄 = 0.25 𝑡−1

SPONTANEOUS DEPOLYMERIZATION DISSOCIATION OF INDIVIDUAL SUBUNITS FROM POLYMERS

+ end

• end

MICROFLUIDICS-ASSISTED TIRFM OBSERVATION OF THE DISASSEMBLY OF INDIVIDUAL ACTIN POLYMERS

Footer MJ. et al. PNAS 2007, Kovar DR. et al. PNAS 2004

SPONTANEOUS MECHANICAL FORCE GENERATION OF ACTIN STRUCTURES

Apparent elongation (nm)

FORCE (pN)

𝐺 = 𝜌2 𝐹𝐽 𝑀2 𝐹𝐽 = 𝑀𝑞 𝑙𝐶𝑈

𝑀𝑞 (𝜈𝑛) 𝐹𝐽 (10−26𝑂𝑛2) 𝐺 (𝑞𝑂)

F-actin 9 3.6 0.25 - 0.56 Ph-F-actin 18 7.2 1.3

𝐺

𝑛𝑏𝑦 = 𝑙𝐶𝑈

∆ ln(𝑑 𝑙𝑃𝑂 𝑙𝑃𝐺𝐺 )

Limulus acrosomal bundle microfabricated wall

𝐺 = −𝑙𝑒

Time (s)

𝑀

∆= 2.7 𝑜𝑛

FUNCTIONAL POLYMORPHISM OF ACTIN NETWORKS – ORIGINS

What are the molecular origins of sculpting functionally polymorph actin structures and providing them with spatio-temporal regulation?

SPATIO-TEMPORAL CONTROL FUNCTIONAL DIVERSIFICATION CELL FREE ENVIRONMENT intrinsic dynamic behavior INTRACELLULAR FUNCTIONING functionally distinct structures controlled dynamics

BACTERIA ARCHEA

actin1 ParM

2ZGY

actin4 MreB

1JCE

actin3 MamK

5LJW

actin2 FtsA

4A2B

actin5 Crenactin

4BQL DNA segregation „divisome” membrane anchor cell division „magnetosome” facilitates magnetotaxis „elongasome” cell morphology cell shape ?

Jiang S. et al. Communicative and Integrative Biology 2016

PROKARYOTE’S CONCEPT OF DIVERSIFICATION – ONE ‚ACTIN’ FOR ONE FUNCTION

twisted 1-stranded antiparallel non-twisted right-handed F-actin 4-stranded,

• pen nanutube

supercoiled antiparallel 4-stranded nanotubule

EUKARYOTE (fungi, metazoa)

functionn function1 ABPn ABP1 function2

EUKARYOTE’S CONCEPT OF DIVERSIFICATION – ONE ‚ACTIN’ FOR ALL FUNCTIONS

ABP2

central element/hub

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

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

• 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

• xidation of Met44, Met77 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)

HOW ABPs RECOGNIZE ‚THEIR OWN ACTIN’? SORTING MECHANISMS OF ABPs

ABPs IN CLASSIC MODELS OF THIN FILAMENT REGULATION

‚TO SEE THEM CONTRACT FOR THE FIRST TIME’ Albert Szent-Györgyi 1963

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 muscle to go over into the contracted state, we have to abolish this inhibition.’

(Albert Szent-Györgyi 1949)

IN VITRO MOTILITY ASSAY OBSERVATION OF ACTIN POLYMER SLINDING ON MYOSIN FUNCTIONALIZED GLASS

autonomous nature of isolated actomyosins F-actin myosin

 CLASSIC MODELS – SLIDING FILAMENT THEORY STERIC BLOCKING THEORY

STRUCTURAL LANDMARKS – SLIDING FILAMENT THEORY

Z line Z line I band A band I band M line ACTIN MYOSIN

Z: Zwischenscheibe, Krause membrane H: Hensen zone M: Mittelscheibe

H zone

+

• +

Gohkin DS. et al. Nature Reviews Molecular Cell Biology 2013

SLIDING SLIDING

TROPOMYOSIN

TROPOMYOSIN (Tpm)

• identified by Bailey K. (Bailey K. Nature 1946)
• “…the presence of tropomyosin in the Straub-type actin preparations is not accidental, but, that

there is an intimate interaction between actin and tropomyosin”

(Kálmán Laki et al. Arch Biochem Biophys 1962)

• > 40 isoforms in mammals, encoded by 4 genes (alternative splicing of different exons)
• archetypal coiled-coil
• specifically binds F-actin but not G-actin
• dimer (KD ~ mM) / polymer (head-to-tail contacts, KD ~ mM)
• binds Troponin (Ca2+-responsive element)
• central ABP in the steric blocking theory

Tpm2 (geneID:500450) Tpm3 (geneID:117557) Tpm4 (geneID:248512) Tpm1 (geneID:24851) EXON STRUCTURE OF THE Tpm ISOFORMS HMW: 1a, 2a, 2b LMW: 1b X RAY STRUCTURE OF Tpm (PDB1C1G) protein locus expression/pathology Tpm1

15q22.2

cardiac, skeletal, smooth cardiomyopathy, familial hypertrophic 3 (CMH3); cardiomyopathy, dilated 1Y (CMD1Y); left ventricular non- compaction 9 (LVNC9) Tpm2

9p13.3

cardiac, skeletal, smooth nemaline myopathy (NEM4); arthrogryposis multiplex congenital, distal, (DA1A, DA2B); cap myopathy 2 (CAPM2) Tpm3

1q21.3

cardiac, skeletal nemaline myopathy (NEM1); cap myopathy (CAPM1); myopathy, congenital, with fiber-type disproportion (CFTD)

Tpm – INTERACTION WITH ACTIN FILAMENTS

Hitchcock-DeGregori SE. Journal of Structural Biology 2010, Dominquez R. Biophysical Journal 2011, Lehman Comprehensive Physiology 2016

Form-function binding – Gestaltbindung (Holmes K., Lehman W.) N-C overlap (Hitchcock-DeGregori SE. PDB2G9J)

acidid Tpm aas basic actin aas

PDB1C1G Atomic model of Tpm bound to F-actin

(Li XE. et al. Biophysical Journal 2011)

Tpm – STRUCUTRAL ASPECTS OF THE STERIC BLOCKING MODEL

Cryo-EM structure of the F-actin-Tpm complex

(von der Ecken Nature 2015)

Tpm A-state Tpm M-state (PDB4A7H) F-actin myosin (PDB1LKX) Ca2+ ↓ Tpm A-state

• Tpm covers myosin

binding site

• myosin binding

blocked

• NO BINDING

Ca2+ ↑ Tpm C-state

• Ca2+ binding to TnC
• movement of Tpm
• myosin binding sites partially

relieved

• WEAK BINDING

Ca2+ ↑ Tpm M-state

• competitive binding of myosin

and Tpm stimulates further movement of Tpm

• myosin binding site uncovered
• STRONG BINDING

ABPs IN RECENT MODELS OF THIN FILAMENT ASSEMBLY AND MAINTENANCE

DYNAMIC LANDMARKS OF THIN FILAMENT ARRAY FORMATION

MYOFIBRIL ASSEMBLY complex sequence of actin dynamics ‚SMEARED’ burst de novo polymerization

• individual myofibrils ↑↑ to the fibers
• irregular Z disk
• homogeneous distribution of newly

generated actin filaments

‚PATCHY’ lateral assembly at filament (pointed) ends

• repeating sarcomeric units
• more regular Z disk

‚FRAME’ lateral assembly at pointed ends, radial assembly at the circumference, barbed/pointed end turnover arrays

• characteristic striated pattern
• more regular Z disk

MYOFIBRIL MAINTENANCE

Shwartz A. eLIFE 2016

PRE MYOFIBRILS NASCENT MYOFIBRILS MATURE MYOFIBRILS

𝐻 ~25 𝜈𝑁 𝐻 ~1 𝜈𝑁

Tb4 – THE FOUNDING MEMBER OF THE WH2-DOMAIN PROTEIN FAMILY

Tb4/WH2

PDB2A41

LLxxI hydrophobic triplet LKKT/V canonical motif intrinsically disordered region IDR

Tb4 – THE FOUNDING MEMBER OF THE WH2-DOMAIN PROTEIN FAMILY

𝒕𝒎𝒑𝒒𝒇 = 𝑻𝑩 𝑻𝟏 = 𝒅𝒅 𝒅𝒅 + 𝑳𝑬 𝒅− 𝑭𝑶𝑬~ 𝟏. 𝟕 𝝂𝑵 𝒅+ 𝑭𝑶𝑬~ 𝟏. 𝟐 𝝂𝑵 𝑻𝑩 = 𝑻𝟏 𝒅𝒅 𝒅𝒅 + 𝑳𝑬

• binds G-actin (KD ~ 0.1 mM)
• incompatible with both nucleation and elongation

SUPPRESSION OF ANARCHIC FILAMENT ASSEMBLY

Tóth M. Bugyi B. et al Journal of Biological Chemistry 2016

pyrenyl actin kinetics steady-state critical concentration plot (J(c) plot) steady-state measurement of F-actin

FUNCTIONAL VARIEGATION OF THE WH2-DOMAIN PROTEIN FAMILY

Dominquez R. Trends in Biochemical Sciences 2016 vertebrate Sarcomere Lenght Short (SALS) diptera polarity in early embryogenesis neuromorphogenesis Vibrio parahaemolyticus pathogen infection Spotted fever group rickettsiae infection muscle sarcomere organization, thin filament lengthening cell motility ER Golgi transport Rikettsiaepathogen motility, infection muscle thin filament dynamics muscle sarcomere organization, membrane organization

PROFILIN

PROFILIN:ACTIN

1HLU ACTIN PROFILIN ACTIN

• binds G-actin (KD ~ 0.1 mM)
• suppresses both nucleation and elongation

SUPPRESSION OF ANARCHIC FILAMENT ASSEMBLY

• directs actin assembly towards barbed ends, inhibits pointed end assembly

COOPERATIVE POINTED END ASSEMBLY REGULATION WITH TROPOMODULIN

barbed pointed barbed pointed STERIC CLASH

𝑙𝑃𝑂 𝑏𝑑𝑢𝑗𝑜 = 11.6 μ𝑁−1𝑡−1 𝑙𝑃𝑂 𝑞𝑠𝑝𝑔𝑗𝑚𝑗𝑜:𝑏𝑑𝑢𝑗𝑜 = 7.3 μ𝑁−1𝑡−1

Vig A. Bugyi B. et al Journal of Biological Chemistry 2017, Pintér R., Bugyi B. unpublished

PROFILIN

CLOSED-CLEFT CONFORMATION

• PROFILIN

3U4L OPEN-CLEFT CONFORMATION + PROFILIN 3UB5

• catalyzes nucleotide exchange

REPLENISHMENT OF THE G-ACTIN POOL

• binds to poly-L-prolin stretches

FH1:PROFILIN AS A MOLECULAR SWITCH IN FORMIN-ASSISTED ACTIN ASSEMBLY MAINTAINING THIN FILAMENT ELONGATION IN THE PRESENCE OF CAPPING PROTEIN

POLY-L-PROLIN (FH1):PROFILIN:ACTIN 2V8F FH2 domain

PROFILIN

ATP ATP Ca2+ Ca2+ nucleotide SD4 SD3 SD1 SD2

subfamily proteins locus expression/pathology (* human ** mouse) localization Diaphanous- related formin

Dia1

5q31.3

heart no obvious sarcomeric localization

Dia2

Xq21.33

heart maturation of premyofibrils Z band, M band

Dia3

13q21.2

skeletal Disheveled- associated activator of morphogenesis

DAAM1

14q23.1

heart, skeletal DAAM1-/-: ventricular noncompaction** hypomorphic mutation: congenital heart defects* Z band

DAAM2

6p21.2

Formin-like protein

FMNL1

17q21.31

heart Fmnl1-/-: longer sarcomeres, fewer thin filaments, myofibril repair** I band region enveloping Z band

FMNL2

2q23.3

Fmnl2-/-: saromere structure failed to form, initial

• rganization/formation of F-actin**

throughout sarcomere \ {Z disk}

FMNL3

12q13.12

heart no obvious sarcomeric localization Inverted formin

INF1

14q32.33

INF2

4q31.3

heart Z band FH1/FH2 domain- containing protein

FHOD1

16q22.1

heart intercalated disks, costameres

FHOD3

18q12.2

heart, skeletal Fhod3-/-: embryonic lethal, failed myocardial development** V1151I: increased incidence of hyperthrophic cardiomyopathy* Z disk, along thin filaments, near pointed ends Delphilin

Delphilin

7p22.1

heart Formin

FMN1

15q13.3

heart Z band

FMN2

1q43

heart

FORMINS

Rosado M. Molecular Biology of the Cell 2014

FORMINS

G: GTPase binding DID: Diaphanous inhibitory domain DD: dimerization domain CC: coiled-coil FH1: formin homology 1 domain FH2: formin homology 2 domain DAD: Diaphanous autoregulatory domain C: C terminal

INACTIVE ACTIVE

Rho family of GTPase BINDING

90o

PROFILIN ACTIN

barbed end

FH2 FH2 ACTIN ACTIN

AUTOINHIBITORY CONSTRUCTION

Vig A., Bugyi B. et al Journal of Biological Chemistry 2017, Barkó Sz., Bugyi B. et al. Journal of Biological Chemistry 2010

FORMINS – FH1:PROFILIN AS A MOLECULAR SWITCH IN THE ACTIN ACTIVITIES OF FORMINS

FH1-FH2

FH1 FH2

𝑙𝑃𝑂 𝑏𝑑𝑢𝑗𝑜 < 11.6 μ𝑁−1𝑡−1 𝑙𝑃𝑂 𝑞𝑠𝑝𝑔𝑗𝑚𝑗𝑜:𝑏𝑑𝑢𝑗𝑜 = 11.6 − 100 μ𝑁−1𝑡−1

Barkó Sz., Bugyi B. et al. Journal of Biological Chemistry 2010, Paul A. Pollard TD. Journal of Biological Chemistry 2009

FORMINS – AS PROCESSIVE BARBED END TRACKING ACTIN POLYMERASES

100 s 10 mm

mDia1

Goode B. et al. Annual Review of Biochemistry 2007

FORMINS – CLASSIC MODEL OF FORMIN-ASSISTED ACTIN DYNAMICS

FH2 FH2 actin binding site actin actin actin F-actin F-actin

• pen (down) / closed (up)

CAPPING PROTEINS – CapZ, CapG, Gelsolin

𝑳𝑬~ 𝟏. 𝟐 − 𝟐 𝒐𝑵 𝑙𝑃𝑂 ~ 3 ∙ 10−6 𝑁−1 ∙ 𝑡−1 𝑙𝑃𝐺𝐺 ~ 3 ∙ 10−4 𝑡−1

Kuhn JR. et al. JBC 2007 Carlier MF. CMLS 2015, Narita A. et. al EMBO Journal 2006, Tóth M. Bugyi B. et al Journal of Biological Chemistry 2016, Pintér R., Bugyi B. unpublished

PROFILIN:ACTIN + ~ 1nM Capping Protein Note: Inhibition of elongation is not direct proof! Check: [IC50], J(c) plot 𝒅− 𝑭𝑶𝑬~ 𝟏.𝟕 𝝂𝑵 𝒅+ 𝑭𝑶𝑬~ 𝟏.𝟐 𝝂𝑵

• inhibits subunit association/dissociation at barbed ends

ALIGNMENT OF THIN FILAMENT BARBED ENDS AT Z DISK LIMITING THIN FILAMENT ELONGATION NEAR Z DISKS

FORMIN – CAPPING PROTEIN ANTAGONISM: decision complex at filament barbed ends

Bombardier JP. et al. Nature Communications 2015, Shekar S. et al. Nature Communications 2015 POSITION RECORDS FOR BARBED ENDS BF BFC BF colocalization of FORMIN and CP at barbed ends CPb – post of FH21 CPb – knob of FH21 CPb – knob of FH21

TROPOMODULIN – Tmod

Fowler SE. et al. Biophysical Journal 2017 proteins locus expression/pathology (* human ** mouse) localization

Tmod1

9q22.33

cardiac, skeletal, smooth, mouse embryonic lethal, mouse skeletal muscle weakness** thin filament pointed ends

Tmod3

15q21.2

skeletal mouse embryonic lethal** thin filament pointed ends

Tmod4

1q21.3

skeletal mouse skeletal muscle normal compensated by Tmod1 thin filament pointed ends

LEIOMODIN – Lmod: WH2 domain protein family, Tmod homologue protein family

proteins locus expression/pathology (* human ** mouse) localization

Lmod1

1q32.1

smooth, cardiac postnatal/juvenile lethality du to megacyst microcolon intestinal hypoperistaltis syndrome (MMIHS)* diffuse localization in F-actin rich regions between dense bodies

Lmod2

7q31.32

heart, skeletal progressive development of dilated cardiomyopathy** near pointed ends and along the thin filaments, A band

Lmod3

3p14.1

skeletal, cardiac nemaline myopathy (NEM2)* ** near pointed ends and along the thin filaments, A band BARBED END BINDING lack of D loop binding site Fowler SE. et al. Biophysical Journal 2017

BIOCHEMICAL ACTIVITIES OF Tmod AND Lmod

• promotes filament formation

ASSEMBLING SSARCOMERIC THIN FILAMENTS (nucleation, circumferential growth)

• inhibits subunit association/dissociation at pointed ends in a tropomyosin-dependent fashion

LIMITING THIN FILAMENT ELONGATION NEAR M LINE

Fowler SE. et al. Biophysical Journal 2017

CONCERTED ACTION OF Tmod AND Lmod AT POINTED ENDS

Fowler SE. et al. Biophysical Journal 2017

Tmod1-/-

• failure of myofibril assembly

Lmod2-/-

• no failure of myofibril assembly
• shorter thin filaments

PROPOSED MODEL OF ACTIN DYNAMICS REGULATION BY Lmod AND Tmod

Fowler SE. et al. Biophysical Journal 2017

ABPs – ADF/cofilin

Suarez C. et al. Current Biology 2011, McCullough BR. et al. Biophysical Journal 2011 Alexa-568-actin Alexa-488-cofilin

local asymmetry in filament topology and mechanics at boundaries of bare and cofilin-decorated segments → localizes mechanical stress → fragmentation

stiffer 𝐺

𝑡ℎ𝑓𝑏𝑠 ↑

less stiff 𝐺

𝑡ℎ𝑓𝑏𝑠 ↓

~ 30o ~ 60o

HOW TO RECONCILE ABPs ACTIVITIES WITH THIN FILAMENT STRUCTURE/DYNAMICS

Arp2 Arp3

OUTLOOK – AUTOCATALYTIC BRANCHING BY THE ARP2/3 COMPLEX MACHINERY

movie courtesy Brad Nolen Lamellipodial actin morphology in fish keratinocyte. Robinson RC. et al. Science 2001 (PDB1K8K), Vinzenz M. et al. Journal of Cell Science 2012, Bugyi B. et al. EMBO Journal 2010, Lai F. et al. EMBO Journal 2008, Reymann AC. et al. Molecular Biology of the Cell 2012

actin Arp2/3 Cofilin

Gunning P. Journal of Cell Biology 2017

OUTLOOK – MASTER REGULATOR CONCEPT IN ACTIN’S FUNCTIONAL DIVERSITY

actin1 actin2 Tpm2 Tpm1 Formin2 Formin1

• 1. Actin isoform specific conformation of

polymers

↓

Recruitment of tropomyosin

• 2. Formin specific conformation of nucleated

actin polymers

↓

Recruitment of tropomyosin

↓

Tropomyosin specific interactions with ABPs

• different binding dynamics
• different position on actin filaments

Laurent Blanchoin: Cytomorpholab Grenoble, Azioune et al. Lab on a Chip 2009, Reymann AC. et al. Nature Materials 2010

CELL/PROTEIN REPULSIVE LAYER CELL/PROTEIN ADHESIVE PATTERN fibronectin streptavidin/ biotin

OUTLOOK – MICROPATTERNING PRINCIPLES

OUTLOOK – NUCLEATION GEOMETRY GOVERNS ORDERED ACTIN NETWORK STRUCTURES

0o

PARALLEL ANTIPARALLEL BRANCHED

180o DETERMINISTIC PROCESS + SPATIAL BOUNDARY CONDITIONS  NETWORK ORGANIZAITON

• Assembly of geometrically controlled and polarized actin polymer networks.
• Spatial boundary conditions drives autonomous self-organization of actin networks

Reyman AC. et al. Science 2012

OUTLOOK – ACTIN NETWORK ARCHITECTURE DIRECT THE ACTIVITIES OF ABPs

Gressin L. et al. Current Biology 2015

ADF/cofilin-driven actin network disassembly depends on network architecture

OUTLOOK – ACTIN NETWORK ARCHITECTURE DIRECT THE ACTIVITIES OF ABPs

Reyman AC. et al. Science 2012, Ennomani Current Biology 2016

Myosin-driven actin network contractility depends on network architecture

lamellipodium sarcomere cytokinetic ring

ACTIN BINDING PROTEINS pathways to sculpt functionally polymorph actin structures

• Prokaryotes and eukaryotes have evolved different strategies for cytoskeleton

diversification.

• The dynamic behavior is intrinsic to actin.
• ABPs are at the heart of eukaryotic actin’s diversification, which harness the

intrinsic properties of actin to assemble functionally distinct actin networks.

• Different models of ABPs sorting are likely not to be mutually exclusive, indeed

facets of each of them must be incorporated into a working model.

• As continuous technical developments and muscle/non-muscle actin research

are interwoven, modern genetic approaches and super-resolution imaging techniques are likely to advance our mechanistic understanding of the origins of the functional polymorphism of actin structures.

Post-docs Tamás Huber Andrea Vig PhD Students Réka Pintér Mónika Tóth Veronika Kollár Undergraduate Students Péter Gaszler

THANK YOU FOR YOUR ATTENTION!

COLLABORATIONS

József Mihály Biological Research Centre Szeged, Hungary Alf Månsson Linnæus University Department of Chemistry and Biomedical Sciences Kalmar, Sweden Robert C. Robinson AStar, Institute of Molecular and Cell Biology Singapore Anikó Pintér-Keller University of Szeged, Faculty of Medicine, Department

• f Biochemistry

Szeged, Hungary

http://cytoskeletaldynamics.wix.com/mysite http://biofizika.aok.pte.hu