1. chat with Fred and Chris Nov. 19 th, 2010 rhombohedra: SEM observation vs time, 24h-72h Bodega Marine Station payments - order Mytilus californianus spicule matrix proteins: produce in yeast (Liam Holt), then harvest and isolate with TAP (twice ). which proteins do we try? can we go for the 5 most abundant in spicules? do we agree about the “protein-function” assay on calcite polished crystals? It only shows ACC-stabilization function. other, better ideas?

2. Mechanism of transformation of amorphous calcium carbonate into calcite in the sea urchin larval spicule Y Politi, RA Metzler, M Abrecht, B Gilbert, F Wilt, I Sagi, L Addadi, S Weiner, and PUPA Gilbert Procs. Natl. Acad. Sci. USA 105, 17362-17366 (2008) sea urchin spicules revealed 3 phases : 2 Amorphous Calcium Carbonate (ACC), and 1 biogenic calcite: type 1, type 2, type 3. type 1  type 2  type 3? spicule shadow Si wafer X-PEEM (photoelectron emission microscopy) image and spectra

3. Transformation and crystallization energetics of synthetic and biogenic ACC AV Radha, TZ Forbes, CE Killian, PUPA Gilbert, and A Navrotsky Procs. Natl. Acad. Sci. USA, 107, 16438-16443, 2010. transitions type 1  2 and type 2  3 are both energetically downhill isothermal acid-solution calorimetry data red = hydrated ACC green = anhydrous ACC blue = crystalline CaCO 3

4. transitions type 1  2 and type 2  3 are both energetically downhill isothermal acid-solution calorimetry data red = hydrated ACC green = anhydrous ACC blue = crystalline CaCO 3 Transformation and crystallization energetics of synthetic and biogenic ACC AV Radha, TZ Forbes, CE Killian, PUPA Gilbert, and A Navrotsky Procs. Natl. Acad. Sci. USA, 107, 16438-16443, 2010.

5. transitions type 1  2 and type 2  3 are both energetically downhill isothermal acid-solution calorimetry data red = hydrated ACC green = anhydrous ACC blue = crystalline CaCO 3 Transformation and crystallization energetics of synthetic and biogenic ACC AV Radha, TZ Forbes, CE Killian, PUPA Gilbert, and A Navrotsky Procs. Natl. Acad. Sci. USA, 107, 16438-16443, 2010.

7. red = type 1, hydrated ACC green = type 2, anhydrous ACC blue = type 3, biogenic calcite R+G=Yellow R+B=Magenta G+B=Cyan Component Analysis of X-PEEM data (X-ray PhotoElectron Emission spectroMicroscopy) CaCO 3 phase transitions in the sea urchin larval spicule YUT Gong, CE Killian, IC Olson, NP Appathurai, RA Metzler, AL Amasino, FH Wilt, and PUPA Gilbert in preparation type 1  type 2  type 3 sequence experimentally confirmed in spicule cross-section (20-nm pixels)

8. 500 nm type 1  2 and 2  3 are fundamentally different transitions red = type 1, hydrated ACC green = type 2, anhydrous ACC blue = type 3, biogenic calcite R+G+B=White R+G=Yellow G+B=Cyan R+B=Magenta spicule cross-section (20-nm pixels) Component Analysis of X-PEEM data (X-ray PhotoElectron Emission spectroMicroscopy) CaCO 3 phase transitions in the sea urchin larval spicule YUT Gong, CE Killian, IC Olson, NP Appathurai, RA Metzler, AL Amasino, FH Wilt, and PUPA Gilbert in preparation

9. R  G  B enabled red = type 1, hydrated ACC green = type 2, anhydrous ACC blue = type 3, biogenic calcite simulation models R  G inhibited type 1  2 and 2  3 are fundamentally different transitions CaCO 3 phase transitions in the sea urchin larval spicule YUT Gong, CE Killian, IC Olson, NP Appathurai, RA Metzler, AL Amasino, FH Wilt, and PUPA Gilbert in preparation

10. R  G  B enabled 1 µm red = type 1, hydrated ACC green = type 2, anhydrous ACC blue = type 3, biogenic calcite experimental datasimulation models R  G inhibited type 1  2 and 2  3 are fundamentally different transitions type 1  2 transition is biologically controlled type 2  3 transition is spontaneous CaCO 3 phase transitions in the sea urchin larval spicule YUT Gong, CE Killian, IC Olson, NP Appathurai, RA Metzler, AL Amasino, FH Wilt, and PUPA Gilbert in preparation

11. RGB map Green edge Red edge Magenta nanoparticles Hydrated ACC Anhydrous ACC Biogenic calcite red or green edges magenta nanoparticles Yutan: I see just as many cyan nanoparticles as magenta nanoparticles. What did you change here? are the above slides no longer valid?

12. 48h and 72h spicules were extracted and deminineralized with acetic acid, neutralized with Tris buffer at pH 8, then dialyzed against DDH 2 O

13. sanity check: a droplet of DDH 2 O was deposited on a calcite crystal, then air-dried

14. sanity check: a droplet of DDH 2 O was deposited on a calcite crystal, then air-dried re-crystallized calcite is more disordered: lower peaks 2 and 4