Temperature-dependent augmentation of ciliary motility by the TRP2 channel in Chlamydomonas reinhardtii.

Temperature is a critical factor for living organisms. Many microorganisms migrate toward preferable temperatures, and this behavior is called thermotaxis. In this study, the molecular and physiological bases for thermotaxis are examined in Chlamydomonas reinhardtii.

Mechanoresponses mediated by the TRP11 channel in cilia of .

Cilia are organelles involved in motility and sensory transduction, but how these two functions coexist has not been elucidated in depth. Here, the involvement of the ciliary transient receptor potential (TRP) channel TRP11 in mechanoresponses is studied in using a TRP11-knockout mutant. The mutant has defects in the conversion of the bending mode of the cilium from forward to reverse when tapped with a glass rod, the detachment of cilia when shear is applied, the increase in ciliary beat frequency upon application of mechanical agitation by vortex mixing, and the initiation of gliding while both cilia are attached in opposite directions to a glass surface.

The four-celled Volvocales green alga Tetrabaena socialis exhibits weak photobehavior and high-photoprotection ability.

Photo-induced behavioral responses (photobehaviors) are crucial to the survival of motile phototrophic organisms in changing light conditions. Volvocine green algae are excellent model organisms for studying the regulatory mechanisms of photobehavior. We recently reported that unicellular Chlamydomonas reinhardtii and multicellular Volvox rousseletii exhibit similar photobehaviors, such as phototactic and photoshock responses, via different ciliary regulations.

MCAs in Arabidopsis are Ca-permeable mechanosensitive channels inherently sensitive to membrane tension.

Mechanosensitive (MS) ion channels respond to mechanical stress and convert it into intracellular electric and ionic signals. Five MS channel families have been identified in plants, including the Mid1-Complementing Activity (MCA) channel; however, its activation mechanisms have not been elucidated in detail. We herein demonstrate that the MCA2 channel is a Ca-permeable MS channel that is directly activated by membrane tension.

Transgenic Expressing Human Transient Receptor Potential Ankyrin 1 (TRPA1) Channels to Assess the Effect of Agonists and Antagonists.

Transient receptor potential ankyrin 1 (TRPA1) channel is an ion channel whose gating is controlled by agonists, such as allyl isothiocyanate (AITC), and temperature. Since TRPA1 is associated with various disease symptoms and chemotherapeutic side effects, it is a frequent target of drug development. To facilitate the screening of TRPA1 agonists and antagonists, this study aimed to develop a simple bioassay for TRPA1 activity.

Responses to transient receptor potential (TRP) channel agonists in .

Pungent substances, such as capsaicin and gingerol, activate the transient receptor potential (TRP)-V1 channel and affect the feeding behaviors of animals. To gain insight into how living organisms have acquired a sense for pungent substances, we explored the response to TRP agonists in a protist, When capsaicin or gingerol was applied to wild-type cells, they became immotile, with flagella detaching from the cell body. The degree of deflagellation was nearly halved in a mutant defective in the TRP channel ADF1.

Elucidation of the Interaction between Flavan-3-ols and Bovine Serum Albumin and Its Effect on Their In-Vitro Cytotoxicity.

Flavan-3-ols (FLs), specifically catechin and its oligomer B-type procyanidins, are suggested to potently bind to bovine serum albumin (BSA). We examined the interaction between BSA and FLs by fluorescence quenching and found the following order of binding activities to BSA: cinnamtannin A2 (A2; tetramer) > procyanidin C1 (C1; trimer) ≈ procyanidin B2 (B2, dimer) > (-)epicatechin (EC, monomer). Docking simulations between BSA and each compound at the binding site showed that the calculated binding energies were consistent with the results of our experimental assay.

Temperature-sensitive mutants of MscL mechanosensitive channel.

MscL is a mechanosensitive channel that undergoes a global conformational change upon application of membrane stretching. To elucidate how the structural stability and flexibility occur, we isolated temperature-sensitive (Ts) mutants of Escherichia coli MscL that allowed cell growth at 32°C but not at 42°C. Two Ts mutants, L86P and D127V, were identified.

Thermotaxis in Chlamydomonas is brought about by membrane excitation and controlled by redox conditions.

Temperature is physiologically critical for all living organisms, which cope with temperature stress using metabolic and behavioral responses. In unicellular and some multicellular organisms, thermotaxis is a behavioral response to avoid stressful thermal environments and promote accumulation in an optimal thermal environment. In this study, we examined whether Chlamydomonas reinhardtii, a unicellular green alga, demonstrated thermotaxis.

Voltage-Dependent Inactivation of MscS Occurs Independently of the Positively Charged Residues in the Transmembrane Domain.

MscS (mechanosensitive channel of small conductance) is ubiquitously found among bacteria and plays a major role in avoiding cell lysis upon rapid osmotic downshock. The gating of MscS is modulated by voltage, but little is known about how MscS senses membrane potential. Three arginine residues (Arg-46, Arg-54, and Arg-74) in the transmembrane (TM) domain are possible to respond to voltage judging from the MscS structure.

cAMP controls the balance of the propulsive forces generated by the two flagella of Chlamydomonas.

The motility of cilia and flagella of eukaryotic cells is controlled by second messengers such as Ca(2+), cAMP, and cGMP. In this study, the cAMP-dependent control of flagellar bending of Chlamydomonas is investigated by applying cAMP through photolysis of 4,5-dimethoxy-2-nitrobenzyl adenosine 3',5'-cyclicmonophosphate (caged cAMP). When cAMP is applied to demembranated and reactivated cells, cells begin to swim with a larger helical path.

A cytoplasmic helix is required for pentamer formation of the Escherichia coli MscL mechanosensitive channel.

Many membrane proteins such as ion channels are oligomers, but the determinants of the degree of oligomerization are not fully understood. Mechanosensitive channel with large conductance (MscL), which is ubiquitous in bacteria, is a homopentamer with two transmembrane helices and a cytoplasmic helix in each subunit. The carboxyl-terminal cytoplasmic helices assemble into a pentameric bundle that resembles cartilage oligomeric matrix protein.

The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding.

Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension.

Electrophysiological characterization of the mechanosensitive channel MscCG in Corynebacterium glutamicum.

Corynebacterium glutamicum MscCG, also referred to as NCgl1221, exports glutamate when biotin is limited in the culture medium. MscCG is a homolog of Escherichia coli MscS, which serves as an osmotic safety valve in E. coli cells.

Organellar mechanosensitive channels in fission yeast regulate the hypo-osmotic shock response.

A key molecule of sensing machineries essential for survival upon hypo-osmotic shock is the mechanosensitive channel. The bacterial mechanosensitive channel MscS functions directly for this purpose by releasing cytoplasmic solutes out of the cell, whereas plant MscS homologues are found to function in chloroplast organization. Here we show that the fission yeast MscS homologues, designated Msy1 and Msy2, participate in the hypo-osmotic shock response by a mechanism different from that operated by the bacterial MscS.

A gain-of-function mutation in gating of Corynebacterium glutamicum NCgl1221 causes constitutive glutamate secretion.

The A-to-V mutation at position 111 (A111V) in the mechanosensitive channel NCgl1221 (MscCG) causes constitutive glutamate secretion in Corynebacterium glutamicum. Patch clamp experiments revealed that NCgl1221 (A111V) had a significantly smaller gating threshold than the wild-type counterpart and displayed strong hysteresis, suggesting that the gain-of-function mutation in the gating of NCgl1221 leads to the oversecretion of glutamate.

Mechanoreception in motile flagella of Chlamydomonas.

Ciliates and flagellates temporarily swim backwards on collision by generating a mechanoreceptor potential. Although this potential has been shown to be associated with cilia in Paramecium, the molecular entity of the mechanoreceptor has remained unknown. Here we show that Chlamydomonas cells express TRP11, a member of the TRP (transient receptor potential) subfamily V, in the proximal region of the flagella, and that suppression of TRP11 expression results in loss of the avoiding reaction.

Mechanosensitivity of ion channels based on protein-lipid interactions.

Ion channels form a group of membrane proteins that pass ions through a pore beyond the energy barrier of the lipid bilayer. The structure of the transmembrane segment of membrane proteins is influenced by the charges and the hydrophobicity of the surrounding lipids and the pressure on its surface. A mechanosensitive channel is specifically designed to change its conformation in response to changes in the membrane pressure (tension).

Chlamydomonas CAV2 encodes a voltage- dependent calcium channel required for the flagellar waveform conversion.

Cilia and flagella can alter their beating patterns through changes in membrane excitation mediated by Ca(2+) influx. The ion channel that generates this Ca(2+) influx and its cellular distribution have not been identified. In this study, we analyzed the Chlamydomonas ppr2 mutant, which is deficient in the production of a flagellar Ca(2+) current and consequently has a defective photophobic response and mechanoshock response.

Gating-associated conformational changes in the mechanosensitive channel MscL.

Bacterial cells avoid lysis in response to hypoosmotic shock through the opening of the mechanosensitive channel MscL. Upon channel opening, MscL is thought to expand in the plane of the membrane and form a large pore with an estimated diameter of 3-4 nm. Here, we set out to analyze the closed and open structure of cell-free MscL.

Interaction between the cytoplasmic and transmembrane domains of the mechanosensitive channel MscS.

The bacterial mechanosensitive channel MscS protects the bacteria from rupture on hypoosmotic shock. MscS is composed of a transmembrane domain with an ion permeation pore and a large cytoplasmic vestibule that undergoes significant conformational changes on gating. In this study, we investigated whether specific residues in the transmembrane and cytoplasmic domains of MscS influence each other during gating.

Molecular and electrophysiological characterization of a mechanosensitive channel expressed in the chloroplasts of Chlamydomonas.

MscS is a mechanosensitive channel that is ubiquitous among bacteria. Recent progress in the genome projects has revealed that homologs of MscS are also present in eukaryotes, but whether they operate as ion channels is unknown. In this study we cloned MSC1, a homolog of MscS in Chlamydomonas, and examined its function when expressed in Escherichia coli.

Lipid-protein interaction of the MscS mechanosensitive channel examined by scanning mutagenesis.

The mechanosensitive channel of small conductance (MscS) is a bacterial mechanosensitive channel that opens in response to rapid hypoosmotic stress. Since MscS can be opened solely by membrane stretch without help from any accessory protein, the lipid-protein interface must play a crucial role in sensing membrane tension. In this study, the hydrophobic residues in the lipid-protein interface were substituted one by one with a hydrophilic amino acid, asparagine, to modify the interaction between the protein and the lipid.

Phototactic activity in Chlamydomonas 'non-phototactic' mutants deficient in Ca2+-dependent control of flagellar dominance or in inner-arm dynein.

In the mechanism underlying the phototactic behavior of Chlamydomonas, Ca(2+) has been thought to control the dominance between the two flagella so as to steer the cell to correct directions. A newly isolated mutant, lsp1, that displays weak phototaxis was found to be defective in this Ca(2+)-dependent shift in flagellar dominance; in demembranated and reactivated cell models, the trans flagellum (the flagellum farthest from the eyespot) beat more strongly than the other (the cis flagellum) in about half of the cells regardless of the Ca(2+) concentration between <10(-9) M and 10(-6) M, a range over which wild-type cell models display switching of flagellar dominance. This is unexpected because ptx1, another mutant that is also deficient in flagellar dominance control, has been reported to lack phototactic ability.

Loss-of-function mutations at the rim of the funnel of mechanosensitive channel MscL.

MscL is a bacterial mechanosensitive channel that is activated directly by membrane stretch. Although the gene has been cloned and the crystal structure of the closed channel has been defined, how membrane tension causes conformational changes in MscL remains largely unknown. To identify the site where MscL senses membrane tension, we examined the function of the mutants generated by random and scanning mutagenesis.