Identification of extracellular vesicles involved in chicken eggshell biomineralization

Joël GAUTRON,

Lilian STAPANE, Nathalie LE ROY, Jacky EZAGAL Valérie LABAS, Lucie COMBES-SOIA

A. RODRIGUEZ-NAVARRO

M.T. HINCKE

Identification of extracellular vesicles involved in chicken eggshell

biomineralization

Initiation of mineralization

Gautron et al., 2016 ; Rodriguez-Navarro et al., 2015 ; Marie et al., 2015

ACC

transformation into calcite aggregates

Crystalline calcite units

formation

ACC (amorphous calcium carbonate)

calcite

Avian eggshell biomineralization

Mammillary knobs Eggshell

membranes

5h 6h 7h 23h

Cuticle deposition

End phase

ACC deposition

Preferential crystal orientation and

growth

Rapid growth 10h

Calcitic eggshell

Extracellular biomineralization (uterine fluid)

Gautron et al., 2016 ; Marie et al., 2015 ; Rodriguez-Navarro et al., 2015 ; Nys et al., 2010

Eggshell structure and organic matrix

Eggshell ultrastructure and mechanical properties

Key roles of the organic matrix:

Stabilize ACC, determine CaCO

polymorph and crystal morphology

CaCO₃ 95% (calcite) Organic matrix 3,5%

edil3 et mfge8 : localization

Identical arrangement of the 2 chromosomes

EDIL3 MFGE8 HAPLN3 HAPLN1

HAPLN1 HAPLN3 ACAN VCAN EDIL3 MFGE8

Role in mineralization

in silico analysis: EDIL3 and MFGE8 evolution

EDIL3/MFGE8 and the nested genes are involved in bone formation

Stapane et al., 2019, J. Biol. Chemistry

Characterization of Gallus gallus EDIL3 and MFGE8 proteins

Stapane et al., 2019, J. Biol. Chemistry

vesicles

integrins annexins

Balcerzak et al., 2007; Rosenthal et al., 2011; Shapiro et al., 2015

EDIL3 and MFGE8 : biomineralization

Presence in bone/cartilage vesicles (chicken, human, mouse)

chicken uterus

Matrix vesicles identified in CaCO₃ biomineralization organisms: sea urchin, molluscs, …

(Waqas et al., 2016; Dennis et al., 1996;

Fraser et al., 1998)

(Gautron, 2016; Marie et al., 2015)

Characterization of Gallus gallus EDIL3 and MFGE8 proteins

β α annexins

integrins

EDIL3/MFGE8 bind to vesicles ?

Marie et al., 2015

Ca

Stapane et al., 2019, J. Biol. Chemistry

Are vesicles involved in avian eggshell formation?

Vesicle presence?

β α Integrins

Phosphatidylserine

CaCO

inside vesicles? ACC ?

EDIL3 and MFGE8 on vesicles ?

Uterine cells Uterine fluid

Mineralization site 1

2

3

Presence of vesicles in chicken eggshell biomineralization

Strong vesicular activity in oviduct parts where mineralization occured

1

White isthmus

Red

isthmus Uterus Magnum Bone Duodenum Kidney Liver

Stapane et al., Cellular & Molecular Life Sciences, submitted

Uterine fluid accumulates vesicles budding from uterus

Uterine cell

Uterine fluid Uterine fluid Exocytose

Exocytose

Vesicle

Presence of vesicles in chicken eggshell biomineralization

1

Stapane et al., Cellular & Molecular Life Sciences, submitted

Pure fluid

Fluid Ø vesicles

Vesicles

X X

EDIL3 and MFGE8 are present in uterine fluid vesicles ?

Pure fluid

Fluid Ø vesicles

Vesicles

X

1 2 3

1 2 3 1 2 3

Pure fluid Fluid Ø vesicles

Vesicles

2

EDIL3 and MFGE8 are vesicular proteins

Stapane et al., Cellular & Molecular Life Sciences, submitted

Uterine fluid vesicles and calcium carbonate

Uterine fluid vesicles enclose CaCO ₃

Transmission electron microscopy (TEM) on uterine fluid and energy dispersive X- ray Spectroscopy analysis (EDS)

3

Stapane et al., 2019, J. Biol. Chemistry

Uterine fluid vesicles and calcium carbonate

Transmission electron microscopy on uterine fluid And elemental mapping

MET-Electron dispersive X-ray spectroscopy

CaCO

Calcium Calcium

CaCO

Carbone

Oxygène

Carbon Oxygen

Uterine fluid vesicles enclose CaCO ₃ on amorphous form

Amorphous calcium carbonate

SAE-Diffraction

3

Stapane et al., Cellular & Molecular Life Sciences, submitted

EDIL3

Other proteins involved in the vesicular transport

ANXA

1,2,8 CA4

PDCD6IP Syntenin-1

Ezrin

proteomics

ANXA2

ANXA8

PDCD6IP

CA4 CA4 Syntenin-1

Ezrin PDCD6IP

CO

HCO

Ca

CaCO

Uterus

ANXA1

Stapane et al., Cellular & Molecular Life Sciences, submitted

β α

Uterine fluid Uterine cells

Mineralisation site

Calcite

HCO₃^- CO₂ Ca²⁺

Extra/intra-cellular vesicle

ACC

Paracellular ?

Transcellular Vesicular

Carbonic anhydrase 4

Annexins JAM Claudin

Calbindin-1

EDIL3/MFGE8

Blood vessels

SLC4A4-A5-A10

Diffusion ATP2B1-B2

SLC8A1-A3

TRPV2-3 Carbonic anhydrase 2

SLC26A9

Occludin

Transcellular Pathway

Paracellular Pathway ?

Vesicular Pathway

Summary-Conclusion

Infundibulum

Thank you for your attention

Acknowledgements Acknowledgements

Isthmus

• DOVE

• BOA

• PAIB2

• PEAT

• GeT-Genotoul

• Universidad de Granada

• University of Ottawa