Biology subjects

Lau, C. S.

Publications and source records attributed to Lau, C. S..

5 recordsLinked to original sources

bioRxiv2023

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2- concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules. One-sentence summaryIn Chlamydomonas, the pyrenoid-localized bestrophin-like protein BST4 is a putative ion channel involved in pH regulation of the thylakoid lumen, possibly by mediating bicarbonate transport.

plant biology↗

bioRxiv2022

The phase separated CO2-fixing pyrenoid proteome determined by TurboID

Phase separation underpins many biologically important processes such as RNA metabolism, signaling and CO2 fixation. However, determining the composition of a phase separated organelle is often challenging due to their sensitivity to environmental conditions which limits the application of traditional proteomics techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID based proximity labeling technique in Chlamydomonas chloroplasts, where proximal proteins are labeled by biotin radicals generated from the TurboID-tagged protein. Through the expression of two core pyrenoid components fused with the TurboID tag, we have generated a high confidence pyrenoid proxiome that contains the majority of known pyrenoid proteins plus a number of novel pyrenoid candidates. Fluorescence protein tagging of 8 previously uncharacterized TurboID-identified proteins showed 7 were localized to a range of sub-pyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox sensitive iron-sulfur cluster metabolism. This developed pipeline opens the possibility of investigating a broad range of biological processes in Chlamydomonas especially at a temporally resolved sub-organellar resolution.

plant biology↗

bioRxiv2020

Assembly of the algal CO2-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif

Approximately one-third of the Earths photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid, and how the organelles sub-compartments - membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath - are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we discovered that pyrenoid proteins share a sequence motif. We show that the motif is sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoids three sub-compartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multi-layered phase-separated organelle. One Sentence SummaryA ubiquitous Rubisco-binding motif targets proteins to the pyrenoid and holds together the pyrenoids three sub-compartments.

cell biology↗

bioRxiv2020

A recombineering pipeline to clone large and complex genes in Chlamydomonas

The ability to clone genes has driven fundamental advances in cell and molecular biology, enabling researchers to introduce precise mutations, generate fluorescent protein fusions for localization and to confirm genetic causation by mutant complementation. Most gene cloning is PCR or DNA synthesis dependent, which can become costly and technically challenging as genes increase in size and particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, with a high percentage of genes containing complex sequence structures, an average genomic GC content of 64% and gene expression requiring regular introns for stable transcription. Here we overcome these challenges via the development of a recombineering pipeline that enables the rapid parallel cloning of genes from a Chlamydomonas BAC collection. We show the method can successfully retrieve large and complex genes that PCR-based methods have previously failed to clone, including genes as large as 23 kilobases, thus making previously technically challenging genes to study now amenable to cloning. We initially applied the pipeline to 12 targets with a 92% cloning success rate. We then developed a high-throughput approach and targeted 191 genes relating to the Chlamydomonas CO2 concentrating mechanism (CCM) with an overall cloning success rate of 77% that is independent of gene size. Localization of a subset of CCM targets has confirmed previous mass spectrometry data and identified new pyrenoid components. To expand the functionality of our system, we developed a series of localization vectors that enable complementation of Chlamydomonas Library Project mutants and enable protein tagging with a range of fluorophores. Vectors and detailed protocols are available to facilitate the easy adoption of this method by the Chlamydomonas research community. We envision that this technology will open up new possibilities in algal and plant research and be complementary to the Chlamydomonas mutant library.

plant biology↗

bioRxiv2020

Identification of 38 novel loci for systemic lupus erythematosus and genetic heterogeneity that may underly population disparities in this disease

Systemic lupus erythematosus (SLE), a worldwide autoimmune disease with high heritability, shows differences in prevalence, severity and age of onset among different ancestral groups. Previous genetic studies have focused more on European populations, which appear to be the least affected. Consequently, the genetic variations that underly the commonalities, differences and treatment options in SLE among ancestral groups have not been well elucidated. To address this, we undertook a genome-wide association study, increasing the sample size of Chinese populations to the level of existing European studies. Thirty-eight novel SLE-associated loci and incomplete sharing of genetic architecture were identified. Nine disease loci showed clear ancestral group heterogeneity and implicated antibody production as a potential mechanism for differences in disease manifestation. Polygenic risk scores performed significantly better when trained on matched ancestral data sets. These analyses help to reveal the genetic bases for disparities in SLE among ancestral groups.

genetics↗