The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

Photosynthetic acclimation to CO₂-limiting stress is associated with control of genetic and physiological responses through a signal transduction pathway, followed by integrated monitoring of the environmental changes. Although several CO₂-responsive genes have been previously isolated, genome-wide analysis has not been applied to the isolation of CO₂-responsive genes that may function as part of a carbon-concentrating mechanism (CCM) in photosynthetic eukaryotes. By comparing expression profiles of cells grown under CO₂-rich conditions with those of cells grown under CO₂-limiting conditions using a cDNA membrane array containing 10,368 expressed sequence tags, 51 low-CO₂ inducible genes and 32 genes repressed by low CO₂ whose mRNA levels were changed more than 2.5-fold in Chlamydomonas reinhardtii Dangeard were detected. The fact that the induction of almost all low-CO₂ inducible genes was impaired in the ccm1 mutant suggests that CCM1 is a master regulator of CCM through putative low-CO₂ signal transduction pathways. Among low-CO₂ inducible genes, two novel genes, LciA and LciB, were identified, which may be involved in inorganic carbon transport. Possible functions of low-CO₂ inducible and/or CCM1-regulated genes are discussed in relation to the CCM.

The carbon-concentrating mechanism (CCM) is essential to support photosynthesis under CO2-limiting conditions in aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii. The CCM is assumed to be comprised of inorganic carbon transport systems that, in conjunction with carbonic anhydrases, maintain high levels of CO2 around ribulose-1, 5-bisphosphate carboxylase/oxygenase in a specific compartment called the pyrenoid. A set of transcripts up-regulated during the induction of the CCM was identified previously and designated as low-CO2 (LC)-inducible genes. Although the functional importance of one of these LC-inducible genes, LciB, has been shown recently, the biochemical properties and detailed subcellular localization of its product LCIB remain to be elucidated. Here, using yeast two-hybrid, immunoprecipitation and mass spectrometry analyses we provide evidence to demonstrate that LCIB interacts with the LCIB homologous protein LCIC in yeast and in vivo. We also show that LCIB and LCIC are co-localized in the vicinity of the pyrenoid under LC conditions in the light, forming a hexamer complex of approximately 350 kDa, as estimated by gel filtration chromatography. LCIB localization around the pyrenoid was dependent on light illumination and LC conditions during active operation of the CCM. In contrast, in the dark or under high-CO2 conditions when the CCM was inactive, LCIB immediately diffused away from the pyrenoid. Based on these observations, we discuss possible functions of the LCIB-LCIC complex in the CCM.

Significance: The entry of inorganic carbon (Ci; CO ₂ and HCO ₃ ^– ) into cells involves many biological processes in both animals and plants, and aquaporins as well as bicarbonate transporters play roles in Ci transport. Although transporting external HCO ₃ ^– into the stroma through the chloroplast envelope is one of the rate-limiting factors for aquatic photosynthetic organisms, specific molecular components in this process have not yet been identified experimentally. Molecular identification of proteins essential for Ci uptake located in the chloroplast envelope and in the plasma membrane documented in this study helps in understanding how aquatic photosynthetic organisms developed machinery to acclimate to CO ₂ -limiting environment and to maintain adequate levels of photosynthesis for survival or growth.

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.