#	Pagina
attuale pagina	/open-h2020/projects/198707/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - SEACELLS (Marine phytoplankton as biogeochemical drivers: Scaling from membranes and single cells to populations)

Teaser

Summary

The oceans and the life therein play a key role in regulating the Earthâ€™s climate as well as providing a vast resource for human society. The single-celled photosynthetic eukaryotic phytoplankton, along with the cyanobacteria are responsible for around half of the total global primary productivity and removal of carbon dioxide from the atmosphere. Similar to land plants they fix inorganic carbon into organic molecules that form the building blocks of life. They form the basis of the marine food web on which all marine life depends. The phytoplankton also drive other major biogeochemical processes in the ocean such as calcification by coccolithophores and silicification by diatoms. These processes all require transport of ions and molecules across the membrane of the phytoplankton cells We are beginning to gain exciting new insights into the mechanisms and evolution of membrane transport, cell signalling and metabolic regulation in phytoplankton species, such as diatoms and coccolithophores. The SeaCells project addresses fundamental questions in phytoplankton biology from cellular to population scales and builds on a number of recent findings. These include the discovery of cell membrane properties that were thought to be typical of animal cells but now must be considered to be of much more ancient origin. SeaCells brings together single cell biophysics, imaging and state of the art molecular biology with studies of natural oceanic phytoplankton populations. A significant aim is to gain critical mechanistic understanding at the molecular and single cell level along with information on the microenvironment that surrounds cells. In order to understand how the physiological properties of single cells in the laboratory translate to behaviour of natural populations we will transfer single cell technologies developed in the laboratory to ship-board platforms. The project also aims to understand how the variability in the responses of individual phytoplankton cells to environmental perturbations is likely to determine the overall effects of changing ocean conditions on natural phytoplankton populations.

Work performed

1. Cell surface carbonate chemistry.
We have completed a detailed experimental microelectrode monitoring and modelling analysis of total carbonate chemistry at the cell surface of a photosynthetic diatom. This work has revealed dramatic fluctuations in pH and carbonate at the surface of a large diatom as a direct response to photosynthetic carbon uptake. In contrast, small diatoms that do not suffer from diffusion limitation at their cell surface do not show such pronounced flucturations. We have shown that for large cells the changes in chemistry are far more rapid and pronounced at the cell surface than in the bulk medium, forcing a reconsideration of phytoplankton productivity models that rely on bulk seawater parameters. Moreover, we have shown a critical role for the enzyme carbonic anhydrase at the external cell surface in facilitating the uptake of carbon dioxide and overcoming the diffusion barrier limitation to carbon acquisition in large diatoms. This work is currently under review with a high impact journal.

2. Molecular tools development.
We have generated a range of genetically encoded fluorescent reporters expressed in the diatom Phaeodactylum tricornutum. These include reporters for cytosolic calcium, cellular pH, cellular phosphate, membrane potential and reactive oxygen generation.
We have also established gene knock out approaches for ion channels in diatoms, primarily using the model diatom P. tricornutum, but extending this approach to other tractable diatom species. We have used this approach to knock out an increasing number of ion channel genes, allowing for the first time detailed functional analysis of their functions.
3. Calcium signalling.
Using the genetically fluorescent calcium â€“probe R-Geko, we have carried out extensive analysis of calcium signalling in response to a range of external cues in the diatom P. tricornutum. This work is providing new insights into the regulation of cytosolic calcium and its role in signalling in diatoms.
4. A new class of eukaryote ion channels.
A major finding is the discovery of a novel class of eukaryotic voltage-dependent cation channels (VDCCs) in diatoms and coccolithophores. These have close similarity to primitive bacterial voltage-dependent sodium channels (NavBac). They also resemble single units of the more complex 4-domain VDCC calcium and sodium channels that impart electrical excitability and underlie calcium signalling in metazoans. By using electrophysiological characterization in a heterologous expression system (human HEK cells) we have shown unexpectedly that the NavBac-like (NavBac-L) channels cloned from the diatom P. tricornutum behaves as a calcium channel whereas the coccolithophore NavBac-L channel behaves as a sodium channel. These results are allowing new insights into the evolution of channel selectivity in eukaryotes.
5. Functional role of NavBac-L channels in diatoms.
By using Crispr-Cas gene knockdown we have generated a number of strains of P. tricornutum with deletions in the NavBac-L channel gene. By using these knockouts in cells expressing the calcium reporter R-Geko, we have been able to show that deletion of this channel results in defective calcium signal generation, reduced growth and impaired cell motility. This provides a powerful system for studying the physiological roles of these novel channels.

Final results

1. New approaches in microscopy. In collaboration with Dr. Brad Amon (LMB Cambridge) and Professor Gail McConnel (University of Strathclyde) we have investigated the feasibility of adapting a novel microscope to monitor in vivo physiological parameters of every single cell in a large population of cells. The Mesoscope presents for the first time a high resolution imaging system with a very wide field of view. We have shown that it is possible to gain sub-cellular fluorescence resolution of every cell in a population of more than 10,000 cells. This system offers a number of advantages over current fluorescence and confocal systems for monitoring both laboratory cultures and natural populations. The system has no moving parts and produces fast ultra high resolution images. The system will be delivered in Summer 2017 for trial in laboratory and on board ship.

We have had much interest in this system from biological oceanographers who are keen to explore the application of this system for monitoring individual cell behaviour in situ in large populations of phytoplankton. We expect that this approach will provide much novel information on the responses of marine plankton to changes in climate and ocean chemistry.

2. Generation of new genetically encoded reporter strains. We have generated a range of fluorescent genetically-encoded reporter lines for cell physiology, including reporters for cellular calcium, pH membrane potential, cellular phosphate, reactive oxygen. We expect that these will be of wide interest to the diatom research field.

3. A new class of primitive eukaryotic ion channels. Our discovery of single-domain cation channels in diatoms and coccolithophores sheds new light on the evolution of ion channels. It also provides new models for the elucidation of voltage dependent cation channel function and regulation. These novel channels most likely bear similarities to primitive ancestral ion channels from which the 4-domain voltage dependent cation channels evolved in animals.

4. Development of gene editing approaches in diatoms. We have applied the Crispr -Cas gene editing protocol to knock out novel calcium channels in diatoms, revealing their physiological and regulatory role in calcium signalling and cell motility.