Current knowledge on sea urchin temporary adhesion Romana Santos My - - PowerPoint PPT Presentation

Current knowledge on sea urchin temporary adhesion

Romana Santos

2003 Research period Evolutionary Biomaterials Group Max-Planck, Germany Stanislav Gorb Dagmar Voigt

My own adhesion network

1999-2000 Master thesis Laboratory Marine Biology UMH, Belgium Patrick Flammang 2001-2005 PhD thesis Laboratory Marine Biology UMH, Belgium Patrick Flammang Elise Hennebert Pierre Becker 2006-2009 Pos-doc researcher Mass Spectrometry Lab ITQB, Portugal Ana Coelho 2009-2013 Assistant researcher Biomedical and Oral Sciences Research Unit - UICOB Faculty of Dentistry, Portugal Manuela Lopes Marise Almeida

Sea urchin temporary adhesion

W4 Fabrication

• f bio-inspired

adhesives and their evaluation W1 Chemical characterization and synthesis of adhesives W3 Mechanical testing and theory W2 Structural characterization

• f natural and

synthetic adhesives

Sea urchins have: (1) specialized adhesive organs, the oral tube feet

Sea urchin temporary adhesion Structural characterization

mobility resistence contact adhesion stem disc

Sea urchins have: (2) temporary adhesion => attachment / detachment cycles (1) specialized adhesive organs, the oral tube feet adhesive epidermis

Sea urchin temporary adhesion Structural characterization

Sea urchins have: (2) temporary adhesion => attachment / detachment cycles adhesive epidermis (1) specialized adhesive organs, the oral tube feet duo-gland adhesive system

Sea urchin temporary adhesion Structural characterization

Sea urchins have: (2) temporary adhesion => attachment / detachment cycles duo-gland adhesive system (1) specialized adhesive organs, the oral tube feet

HYDRODYNAMISM subtidal subtidal-intertidal soft hard medium-high low DEPTH SUBSTRATE

adhesive cells adhesive granules

∅ 300-700 nm diversity of ultrastructure released for attachment

Sea urchin temporary adhesion Structural characterization

Sea urchins have: (2) temporary adhesion => attachment / detachment cycles duo-gland adhesive system (1) specialized adhesive organs, the oral tube feet de -adhesive cells

HYDRODYNAMISM subtidal subtidal-intertidal soft hard medium-high low DEPTH SUBSTRATE

de - adhesive granules

150x200 nm constant ultrastructure released for detachment

Sea urchin temporary adhesion Structural characterization

Sea urchins have: (3) adhesive secretion remains on the substrate as a footprint

footprint

Sea urchin temporary adhesion Structural characterization

(2) temporary adhesion => attachment / detachment cycles (1) specialized adhesive organs, the oral tube feet

100-300 nm in thickness sponge-like hydrated structure Sea urchin temporary adhesion Structural characterization

Sea urchins have: (3) adhesive secretion remains on the substrate as a footprint (2) temporary adhesion => attachment / detachment cycles (1) specialized adhesive organs, the oral tube feet

Sea urchins have: (1) Tube foot disc tenacities similar to other marine organisms

Sea urchin temporary adhesion Mechanical testing

sea urchin dynamometer

Disc tenacity force/area of contact

tube foot disc subtrate

Sea urchins have: (1) Tube foot disc tenacities similar to other marine organisms

Sea urchin temporary adhesion Mechanical testing

Tenacity (MPa) 0.20 0.29 0.09 Indo-West Pacific Atlantic and Mediterranean Geography Taxonomy

Sphaerechinus granularis Paracentrotus lividus Arbacia lixula Echinometra mathaei Stomopneustes variolaris Heterocentrotus trigonarius Colobocentrotus atratus

subtidal subtidal-intertidal intertidal low very-high medium-high soft hard Depth Hydrodynamism Substrate 0.22 0.21 0.25 0.54

Sea urchins have: (1) Tube foot disc tenacities similar to other marine organisms

Sea urchin temporary adhesion Mechanical testing

(2) Tube foot discs perform better on high-energy polar substrates

Smooth substrates Tenacity (MPa) Glass 0.31ab Polymethylmethacrylate 0.34b Polystyrene 0.29ab Polypropylene 0.17a Critical surface area (mJ m-2) 70 39 33 32 Polarity 0.17 0.02

Sea urchins have: (1) Tube foot disc tenacities similar to other marine organisms

Sea urchin temporary adhesion Mechanical testing

(2) Tube foot discs perform better on high-energy polar substrates (3) Tube foot discs replicates the substrate roughness, behaving like a visco-elastic material

E1 τ E0

0,5 1 1,5 2 2,5 5 10 15 20 25 30 Time (s) Force (mN) 0,5 1 1,5 2 2,5 20 40 60 80 100 120 Displacement (µm) Force (mN)

Sea urchins have: (1) Tube foot disc tenacities similar to other marine organisms

Sea urchin temporary adhesion Mechanical testing

(2) Tube foot discs perform better on high-energy polar substrates (3) Tube foot discs replicates the substrate roughness, behaving like a visco-elastic material

Disc deforms Spreads adhesive in a thin layer

E1 τ E0

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization adhesive discs non-extruded adhesive secretions soluble proteins complex mixture

Paracentrotus lividus

footprints extruded adhesive secretions insoluble proteins simpler mixture

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins

High concentration of denaturing (2% SDS) and reducing agents (0.5M DTT) => noncovalent (hydrophobic and electrostatic interactions) + disulfide bonds 6 proteins identified by MS – actin, tubulin and histone 7 unidentified proteins - 5 de novo generated sequences with no homology with known proteins

45% 6% 2% 3% 44% inorganic residues proteins neutral sugars lipids unknown

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins

7 unidentified proteins - 5 de novo generated sequences with no homology with known proteins

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins (3) Tube feet discs is more soluble but more complex and contains at least 204 unique proteins

Response to stimulus; 5,2% Regulation; 38,2% Metabolic process; 11,5% Localization; 20,1% Interaction with cells/organisms; 8,4% Immune system process; 8,4% Growth ; 3,1% Developmental process; 10,5% Cellular process; 72,3% Other; 41,2%

sliced 40 bands 32 fractions/band

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins (3) Tube feet discs is more soluble but more complex and contains at least 204 unique proteins

Response to stimulus; 5,2% Regulation; 38,2% Metabolic process; 11,5% Localization; 20,1% Interaction with cells/organisms; 8,4% Immune system process; 8,4% Growth ; 3,1% Developmental process; 10,5% Cellular process; 72,3% Other; 41,2%

More abundant proteins: 15% actins => cellular cytoskeleton 11% histones => nucleosome assembly + antibacterial activity 9% ras-related proteins => signal Transduction + vesicle-mediated protein transport + regulation of exocytosis 8% tubulins => microtubule based- movement + protein polymerization

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins (3) Tube feet discs is more soluble but more complex and contains at least 204 unique proteins

Other interesting protein groups: Protein biosynthesis (ribossomal protein, eukaryotic

initiation factor, elongation factor 1-alpha and ubiquitin)

Protein folding and glycosylation

(peptidyl- prolyl cis-trans isomerase; protein disulfide-isomerase, dolichyl-diphosphooligosaccharide protein glycosyltransferase )

adhesive protein secretion

Response to stimulus; 5,2% Regulation; 38,2% Metabolic process; 11,5% Localization; 20,1% Interaction with cells/organisms; 8,4% Immune system process; 8,4% Growth ; 3,1% Developmental process; 10,5% Cellular process; 72,3% Other; 41,2%

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins (3) Tube feet discs is more soluble but more complex and contains at least 204 unique proteins

Other interesting protein groups: Spatial learning (Hippocalcin-like protein 1 ) Visual perception + response to stimulus

(Hemicentin-1 percursor)

Signal transduction (guanine nucleotide-binding

protein ; GTPase Kras; Rab GDP dissociation inhibitor, Ras- related protein, isoform Long of Flotillin-1)

Substrate recognition Triggering of attachment/detachment

Response to stimulus; 5,2% Regulation; 38,2% Metabolic process; 11,5% Localization; 20,1% Interaction with cells/organisms; 8,4% Immune system process; 8,4% Growth ; 3,1% Developmental process; 10,5% Cellular process; 72,3% Other; 41,2%

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins (3) Tube feet discs is more soluble but more complex and contains at least 204 unique proteins

Other interesting protein groups: Exocytosis (annexin, ras-related proteins ) Release of adhesive/de-adhesive secretions

Response to stimulus; 5,2% Regulation; 38,2% Metabolic process; 11,5% Localization; 20,1% Interaction with cells/organisms; 8,4% Immune system process; 8,4% Growth ; 3,1% Developmental process; 10,5% Cellular process; 72,3% Other; 41,2%

Sea urchins have: (1) Two sources of adhesive material – extruded and non-extruded

Sea urchin temporary adhesion Biochemical characterization

(2) Footprint material is highly insoluble, being composed of at least 13 proteins (3) Tube feet discs is more soluble but more complex and contains at least 204 unique proteins

Other interesting protein groups: Cell/substrate adhesion

(nahoda protein, echinonectin, galectin)

Constituents of the adhesive secretions + Unidentified proteins with de novo sequences similar to the ones found in footprints

Response to stimulus; 5,2% Regulation; 38,2% Metabolic process; 11,5% Localization; 20,1% Interaction with cells/organisms; 8,4% Immune system process; 8,4% Growth ; 3,1% Developmental process; 10,5% Cellular process; 72,3% Other; 41,2%

Sea urchin temporary adhesion Conclusions

Sea urchin adhesive secretions have: (1) Tenacities between 0.09-0.54 MPa (2) Efficient in substrates with different chemistries => perform better on high-energy polar substrates (3) Tube feet discs have visco-elastic properties to cope with substrate roughness => replicate substrate profile and spread adhesive in a thin layer (4) Footprint material is highly insoluble, being composed of at least 13 proteins => some new or highly modified (5) Tube feet discs is composed of at least 204 unique proteins => involved in protein biosynthesis, folding and glycosylation + sensory perception and response to stimulus + protein exocytosis + cell/substrate adhesion + new

• r highly modified

Sea urchin temporary adhesion Acknowledgments