Kenneth W. Foster

Reversible bleaching of Chlamydomonas reinhardtii rhodopsin in vivo.

Abstract— The effect of hydroxylamine on the phototactic activity of Chlamydomonas reinhardtii was investigated. The following results were obtained: (1) wild type cells, irradiated for 10 min with green light immediately after addition of 1 mM hydroxylamine, showed a 20 min transient loss of phototactic activity, (2) irradiation of cells, preincubated in the dark with 4 mM. hydroxylamine for 30 min, diminished the phototactic sensitivity permanently by more than 100‐fold without loss of cell motility. (3) The phototactic sensitivity completely recovered within 3(1 min of the removal of hydroxylamin from carotenoid‐containing cells or from carotenoid‐negative cells upon addition of 11‐cis or all‐trans retinal. Our explanation is bleaching of rhodopsin by more than 99% and reconstitution by de novo synthesized or by added retinal.

Light induces accumulation of isocitrate lyase mRNA in a carotenoid-deficient mutant of Chlamydomonas reinhardtii

A cDNA with sequence similarity to isocitrate lyase (ICL) genes was isolated from the unicellular eukaryotic green alga Chlamydomonas reinhardtii as a light-induced mRNA in the carotenoid biosynthetic mutant strain FN68. The 416 amino acid open reading frame shows significant sequence similarity to isocitrate lyases of bacteria (70%), molds (48%), yeasts (45%), and plants (47%).Expression of the Chlamydomonas ICL gene was tested in the mutant strain FN68, which when grown in the dark fails to accumulate carotenoids and is deficient in chlorophyll, and in CC400G, a strain that accumulates wild-type levels of carotenoids and chlorophyll. In vegetative CC400G cells, ICL mRNA accumulated to a high level in the dark and declined to a barely detectable level within 30 min of exposure to light. This response was more sensitive to white (tungsten filament) or red light than green or blue light, excluding cryptochrome and rhodopsin as the photoreceptor. These results are consistent with excitation by chlorophyll and/or a phytochrome-related photoreceptor. In vegetative FN68 cells, ICL mRNA abundance was very low in the dark, but increased dramatically in response to light. At intensities above threshold, excitation by far-red or red light-induced ICL mRNA accumulation to the highest levels. The threshold of the response was lowest for far-red and blue light. These results are consistent with excitation of a photochromic far-red-responsive pigment.

Spectral sensitivity, structure and activation of eukaryotic rhodopsins: Activation spectroscopy of rhodopsin analogs in Chlamydomonas

Retinal normally binds opsin forming the chromophore of the visual pigment, rhodopsin. In this investigation synthetic analogs were bound by the opsin of living cells of the alga Chlamydomonas reinhardtii; the effect was assayed by phototaxis to give an activation spectrum for each rhodopsin analog. The results show the influence of different chromophores and the protein on the absorption of light. The maxima of the phototaxis action spectra shifted systematically with the number of double bonds conjugated with the imine (C = N+H) bond of the chromophore. Chromophores lacking a beta-ionone ring, methyl groups and all C = C double bonds photoactivated the rhodopsin of Chlamydomonas with normal efficiency. On the basis of a simple model involving one-electron transitions between occupied and virtual molecular orbitals, we estimate the charge distribution along the chromophore in the binding site. With this restraint we define a unique structural model for eukaryotic rhodopsins and explain the spectral clustering of pigments, the spectral differences between red and green rhodopsins and the molecular basis of color blindness. Our results are consistent with the triggering of the activation of rhodopsin by the light-mediated change in electric dipole moment rather than the steric cis-trans isomerization of the chromophore.

Photoreceptor for Curling Behavior in Peranema trichophorum and Evolution of Eukaryotic Rhodopsins

ABSTRACT When it is gliding, the unicellular euglenoid Peranema trichophorum uses activation of the photoreceptor rhodopsin to control the probability of its curling behavior. From the curled state, the cell takes off in a new direction. In a similar manner, archaea such as Halobacterium use light activation of bacterio- and sensory rhodopsins to control the probability of reversal of the rotation direction of flagella. Each reversal causes the cell to change its direction. In neither case does the cell track light, as known for the rhodopsin-dependent eukaryotic phototaxis of fungi, green algae, cryptomonads, dinoflagellates, and animal larvae. Rhodopsin was identified in Peranema by its native action spectrum (peak at 2.43 eV or 510 nm) and by the shifted spectrum (peak at 3.73 eV or 332 nm) upon replacement of the native chromophore with the retinal analog n-hexenal. The in vivo physiological activity of n-hexenal incorporated to become a chromophore also demonstrates that charge redistribution of a short asymmetric chromophore is sufficient for receptor activation and that the following isomerization step is probably not required when the rest of the native chromophore is missing. This property seems universal among the Euglenozoa, Plant, and Fungus kingdom rhodopsins. The rhodopsins of animals have yet to be studied in this respect. The photoresponse appears to be mediated by Ca2+ influx.

Roles of cyclic AMP in regulation of phototaxis in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii swims toward or away from light (phototaxis) in a graded way depending on various conditions. Activation of rhodopsin provides signals to control the steering of this unicellular organism relative to a light source and to up-regulate rhodopsin biosynthesis. Intracellular cAMP and cGMP concentrations were measured in positive (1117, swims toward light) and negative (806, swims away from light) phototactic strains with and without light stimulation or 3-isobutyl-1-methylxanthine (IBMX). In the dark, the levels of cAMP and cGMP were significantly higher in the strain with positive phototaxis than in the strain with negative phototaxis. To test whether either cyclic nucleotide influenced the direction, their pre-stimulus levels were pharmacologically manipulated. Higher pre-stimulus levels of cAMP biased the cells to swim toward green light and lower levels biased the cells to swim away. In addition, green-light activation of rhodopsin or addition of IBMX causes a sustained increase in cAMP in both strains. As a consequence of this increase in cAMP, carotenogenesis is induced, as shown by recovery of phototaxis in a carotenoid mutant. Thus, two functions for cAMP were identified: high pre-stimulus level biases swimming toward a light source and sustained elevation following rhodopsin activation increases rhodopsin biosynthesis.

The DPY-30 domain and its flanking sequence mediate the assembly and modulation of flagellar radial spoke complexes.

ABSTRACT RIIa is known as the dimerization and docking (D/D) domain of the cyclic AMP (cAMP)-dependent protein kinase. However, numerous molecules, including radial spoke protein 2 (RSP2) in Chlamydomonas flagella, also contain an RIIa or a similar DPY-30 domain. To elucidate new roles of D/D domain-containing proteins, we investigated a panel of RSP2 mutants. An RSP2 mutant had paralyzed flagella defective in RSP2 and multiple subunits near the spokehead. New transgenic strains lacking only the DPY-30 domain in RSP2 were also paralyzed. In contrast, motility was restored in strains that lacked only RSP2s calmodulin-binding C-terminal region. These cells swam normally in dim light but could not maintain typical swimming trajectories under bright illumination. In both deletion transgenic strains, the subunits near the spokehead were restored, but their firm attachment to the spokestalk required the DPY-30 domain. We postulate that the DPY-30–helix dimer is a conserved two-prong linker, required for normal motility, organizing duplicated subunits in the radial spoke stalk and formation of a symmetrical spokehead. Further, the dispensable calmodulin-binding region appears to fine-tune the spokehead for regulation of “steering” motility in the green algae. Thus, in general, D/D domains may function to localize molecular modules for both the assembly and modulation of macromolecular complexes.

Analysis of the ciliary/flagellar beating of Chlamydomonas.

Eukaryotic flagella and cilia are alternative names, for the slender cylindrical protrusions of a cell (240nm diameter, approximately 12,800nm-long in Chlamydomonas reinhardtii) that propel a cell or move fluid. Cilia are extraordinarily successful complex organelles abundantly found in animals performing many tasks. They play a direct or developmental role in the sensors of fluid flow, light, sound, gravity, smells, touch, temperature, and taste in mammals. The failure of cilia can lead to hydrocephalus, infertility, and blindness. However, in spite of their large role in human function and pathology, there is as yet no consensus on how cilia beat and perform their many functions, such as moving fluids in brain ventricles and lungs and propelling and steering sperm, larvae, and many microorganisms. One needs to understand and analyze ciliary beating and its hydrodynamic interactions. This chapter provides a guide for measuring, analyzing, and interpreting ciliary behavior in various contexts studied in the model system of Chlamydomonas. It describes: (1) how cilia work as self-organized beating structures (SOBSs), (2) the overlaid control in the cilia that optimizes the SOBS to achieve cell dispersal, phototaxis steering, and avoidance of obstacles, (3) the assay of a model intracellular signal processing system that responds to multiple external and internal inputs, choosing mode of behavior and then controlling the cilia, (4) how cilia sense their environment, and (5) potentially an assay of ciliary performance for toxicology or medical assessment.

Eye Evolution: Two Eyes Can Be Better Than One

The development of our eyes is owed in part to ancestral structures which functioned in phototaxis. With the origin of bilateral annelid larva, two eyes co-evolved with neurons to improve phototaxis performance.

Dynamics of a sensory signaling network in a unicellular eukaryote

The processing components and the dynamic signaling network that an individual cell uses to do signal integration and make decisions based on multiple sensory inputs are being identified in a well studied free-swimming unicellular green algal model organism, Chlamydomonas. It has many sensory photoreceptors and measurable behavior associated with its orienting and swimming with respect to light sources in its environment. Study of the dynamics of the beating of its two steering cilia reveals their complex specialization

Making a robust biomolecular time scale for phylogenetic studies.

The further evolution of informational molecular sequences should depend on the number of viable alternatives possible for the sequences as set by selection, the unrepaired mutation rate, and time. Most biomolecular clocks are based on Kimuras nearly neutral mutation random-drift hypothesis. This clock assumes that informational sequences are in equilibrium, i.e., the nucleotides mutate at a uniform rate and the number of nucleotides unconstrained by selection remains constant. Correcting for deviations from these assumptions should produce a more accurate clock. Informational molecules probably formed from polynucleotides having some other function such as nitrogen or nucleotide storage, thus being initially functionally unselected. At any time the rate of development of functionality in a protein may be expected to be proportional to the number of viable alternatives of sequence in its potentially interacting regions. Assuming the rate of unrepaired mutations is constant, these clocks should exponentially slow as they evolve, each with a different rate toward individual equilibria. Also if the degree of selection changes, its clock rate should change. For a more precise clock two approaches are suggested to estimate these time dependent changes in evolutionary rate. An improved clock could improve estimation of phylogeny and put a time scale on that phylogeny.