molecular)and)cellular)mechanisms)for)ph)sensing,)ion ...€¦ ·...
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Molecular and cellular mechanisms for pH sensing, ion transport and regula6on of metabolism in corals
Mar$n Tresguerres
The Alfred P. Sloan Foundation
Dr. Ka'e Baro+ Dr. Dimitri Deheyn Ms. Megan Barron Ms. Lauren Linsmayer Ms. Sidney Pérez Ms. Jenny Tu Dr. Alex Venn and Dr. Sylvie Tambu+eé (Monaco Scien'fic Center)
THE IMPORTANCE OF KNOWING BASIC BIOLOGY AND MECHANISMS
1) Proton pump (VHA): NOVEL PHYSIOLOGY AND MULTIPLE
LOCALIZATION WITHIN CORAL CELL TYPES
2) NKA AND PMCA ATPases: SPECIES SPECIFIC DIFFERENCES
3) Poten9al applica9ons for field studies
Implica$ons for how we study coral responses to environmental stress (or why large scale proteomics and transcriptomics is not a good
approach in corals –at least yet-‐)
General roles of ATPases
Use ATP to move ca6ons against the concentra6on gradient -‐ac've transport -‐secondary ac've transport -‐membrane poten'al
NKA
Na+
K+
Na+
H+
Na+ aa/glc
H+
VHA
Ca2+ Ca2+ pump H+
Ca2+ Ca2+ pump H+
H+
VHA
Ca2+
Zoccola et al (2015) Scien$fic Reports
Proposed localiza9on (and func9on) in corals
Immunohistochemistry
h+p://www.proteinatlas.org/
VHA is abundantly expressed in the symbiosome membrane
VHA Nucleus
VHA Nuclei
Baro+ et al 2015 (PNAS)
Bafilomicyn: VHA inhibitor
Lysosensor green: accumulates in acidic compartments
Baro+ et al 2015 (PNAS)
Host VHA acidifies the symbiosome and promotes photosynthesis
200
250
300
350
400
0 10 20 30 40 200
250
300
350
400
0 10 20 30 40
Time (min)
Dis
solv
ed o
xyge
n
(µm
ol L
-1)
A DMSO
Time (min)
Dis
solv
ed o
xyge
n
(µm
ol L
-1)
B Bafilomycin
C D
*
VHA inhibi6on in coral nubbins down regulates oxygen produc6on Baro+ et al 2015 (PNAS)
Host pH 7.1 – 7.4 Symbiosome
pH ~4
Alga pH 7.1 – 7.4
Chloroplast
BT ?
HCO3-
HCO3-
HCO3-
HCO3-
HCO3-
H+
H+ H+
H+
H+ H+
H+ H+
HCO3-
CA CA CO2
CO2
CO2
CO2
CO2
CO2
CO2
Sugars Sugars Sugars
O2
O2
O2
O2
O2
O2
O2
Seawater
Coelenteron
pH 8.0, PCO2 450 μatm Seawater
Coelenteron
pH 7.6 PCO2 1800 μatm
VHA Nuclei
1
2
3
4
1
2
ê Symbiosome VHA protein abundance during “OA” (é CO2)
BUT VHA IS NOT ONLY IN THE SYMBIOSOME…………….CORAL TIP?
Co
Sk
Sw VHA Nucleus
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
H+
Remove H+ from the site of calcifica6on?
“Relief of Energy for symbiosome VHA” ?
“More “proton flux/calcifica9on” VHA”?
PMCA Nuclei
Skeleton
Revisi'ng other parts of the model= Calcium pump may not be in the membrane
Baro+ et al in press (Am J Physiol Reg)
NKA=can consume between 20-‐90% of all the energy in a cell/organism Roles? Implica'ons for analyzing results
Differen'al 'ssue localiza'on of NKA in two coral species
NKA Nuclei
-‐Localiza'on and func'on is (obviously) key for interpre'ng data -‐(Protein and mRNA) Responses in one cell type might mask responses in another -‐Other regula'on (PTMs, e.g. phosphoryla'on) -‐Species specific differences (“a coral is not a coral is not a coral”) -‐Relevance for corals in the ‘real world’?
CONCLUSIONS
Davey Kline
1
2
3
4
Acroporacervicornis
Orbicellaannularis
1
23
4
Simple sampling and processing methods
Fixa9ve
Decalcifica9on Buffer (ship)
70% Ethanol (ship)
-‐Storage at 4 oC (archival) -‐Processing (VHA, NKA, PMCA, sAC, 4 other proteins)
Panama
Scripps (San Diego)
Money from NOAA, NSF, Founda6ons Technician (s) microscopy facili6es Place with low overhead and taxes
CORAL
OA
Ectoderm
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