Energy alternatives: Metabolic cues regulate algal gene expression

Research Areas:

Plant biology, Gene regulation, Cellular metabolism, Photosystem II

Imaging Needs:

Precise localization, high signal-to-noise ratio

Imaging System:
  • psbA mRNA probes labeled with Alexa Flour 488
  • Alexa Fluor 568 conjugated anti-rabbit secondary antibody to DLA2 protein
  • LeicaDMI6000B microscope
  • 40x/0.75 objective
  • Hamamatsu ORCA-R2 CCD camera
  • Perkin Elmer Volocity acquisition software
  • Colocalization Finder in ImageJ software
Imaging cellular events in real time

Find out how Fang Huang, Jeorg Bewersdorf and colleagues use the sCMOS technology in the ORCA-Flash4.0 camera to achieve video-rate imaging at nanometer scales. Read now.

THE QUESTION

How do cells co-ordinate metabolic activity to suit available energy sources?

The green algae Chlamydomonas reinhardtii has a special talent: it can subsist on sunlight through photosynthesis or live in darkness, given a carbon source such as acetate—or it can use a “mixotrophic” combination of the two. That flexibility makes C. reinhardtii an excellent model organism for studies of energy metabolism and chloroplast gene expression.

When the alga moves from sunlight to dark or vice-versa, how does it adjust its metabolic machinery to emphasize the appropriate energy cycle? Researchers have begun to uncover cellular signals that may lead algae to produce more carbon-consuming enzymes, or to build more light-capturing capacity.

THE BARRIERS

Metabolic influence over gene expression could follow two paths: feedback loops based on metabolite levels, and more direct regulation by a class of metabolic enzymes that also act as RNA binding proteins (RBPs). However, the physiological significance of these RBPs has remained mysterious. Do they play a dual role as metabolic enzymes and regulators of gene expression, and if so, what sets the balance between the two?

THE SOLUTION

Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis
Alexandra-Viola Bohne, Christian Schwarz, Marco Schottkowski, Michael Lidschreiber, Markus Piotrowski, William Zerges, Jorg Nickelsen
PLoS Biology. 2013 Feb; 11(2): e1001482. PMCID: PMC3570535.

Bohne, et al,1 used a combination of genetic, proteomic, and microscopy studies to demonstrate for the first time that one such RBP does act as both enzyme and translation regulator in Chlamydomonas reinhardtii, emphasizing one role or the other depending on available energy sources. The authors showed that the enzyme DLA2, a subunit of a chloroplast complex involved in fatty acid synthesis, binds to the messenger RNA psbA, which encodes the D1 protein of photosystem II. The binding was evident specifically in algae grown with access to both light and acetate. Indeed, DLA2 knock-down cell lines showed stunted growth only under mixotrophic conditions.

To explore the interaction of psbA mRNA with DLA2, the research team localized them within mixotrophic cells using fluorescence in-situ hybridization (FISH) and fluorescence immunostaining, imaged using a Hamamatsu ORCA-R2 camera. Regions with the most intense signal from both psbA and DLA2 most often co-localized to a specific, translation-active zone of the chloroplast membrane, the “T-zone.” The results suggest that DLA2 is required for psbA localization at the T-zone, ensuring that newly synthesized D1 arrives at the photosystem II biogenesis center. The authors propose a model for acetate-driven diversion of DLA2 from its role in lipid synthesis to that of mediating production of the photosynthetic protein D1.

THE POSSIBILITIES

Bohne and colleagues used Hamamatsu’s ORCA-R2 camera to co-localize a protein with its mRNA binding partner within a subcellular organelle. Newer, scientific CMOS technology is enabling ever-finer resolution and localization down to the mid- to low-nanometers. Learn more in Exciting Advances Push the Limits of Visualization.

References

  1. Bohne, et al. Reciprocal Regulation of Protein Synthesis and Carbon Metabolism for Thylakoid Membrane Biogenesis. PLoS Biology. 2013 Feb; 11(2): e1001482. PMCID: PMC3570535.
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