Biology subjects

Wakabayashi, K.-i.

Publications and source records attributed to Wakabayashi, K.-i..

5 recordsLinked to original sources

bioRxiv2025

Chlamydomonas γ-tubulin mutations reveal a critical role of γ-TuRC in maintaining the stability of centriolar microtubules

The centriolar triplet microtubule consists of an A-tubule with 13 protofilaments, and B-and C-tubules, each with 10 protofilaments. Although the formation of the triplets has been shown to require {gamma}-tubulin, its specific role in the formation of each tubule remains elusive. We isolated two novel Chlamydomonas reinhardtii mutants, bld13-1 and bld13-2, each expressing {gamma}-tubulin with a single amino-acid substitution (T292I or E89D). Similar to known centriole-deficient mutants, both mutants exhibited defects in ciliary assembly, nuclear number, as well as the number and orientation of cytoplasmic microtubules. Genetic analyses of the mutants, along with the expression of the mutant {gamma}-tubulins in the wild-type cells, suggested that both mutants exert dominant-negative effects over wild-type {gamma}-tubulin. Interestingly, although the centrioles in these mutants retained the typical nine triplet structure, their triplets frequently lacked several protofilaments in specific regions of the A- and C-tubules. The protofilament loss occurs more frequently in the proximal region of the centriole. These structural defects suggest a critical role for {gamma}-tubulin in maintaining the stability of the A- and C-tubules of centriolar triplets. Summary statementNovel Chlamydomonas {gamma}-tubulin mutations cause a partial loss of protofilaments in centriolar microtubules, indicating a critical role of {gamma}-tubulin in structural stabilization of triplet microtubules.

cell biology↗

bioRxiv2024

Membrane-bound Guanylyl Cyclase COP5/HKR1 changes ciliary beat pattern and biases cell steering during chemotaxis in Chlamydomonas reinhardtii

This study investigates the control of ciliary beat patterns during ammonium chemotaxis in the model ciliate microalgae Chlamydomonas reinhardtii. Screening the chemotaxis response of mutant strains with ciliary defects revealed that a strain lacking CAV2, the alpha subunit of the voltage-gated Ca2+ channel, is deficient in ammonium chemotaxis. CAV2 regulates the switching of the ciliary beat pattern from the asymmetric to the symmetric waveform. Strains lacking COP5/HKR1 (chlamyopsin 5/histidine kinase rhodopsin 1), a membrane-bound guanylyl cyclase, are also deficient in ammonium chemotaxis. Conversely, strains defective in phototaxis perform ammonium chemotaxis normally. Cell motility analysis revealed wild-type cells reduce the incidences of switching the ciliary beat pattern from the asymmetric to symmetric waveform when swimming up the ammonium gradient. In contrast, the COP5/HKR1 disrupted strain does not bias ciliary beat pattern switching in the gradient. This finding reveals that a membrane-bound guanylyl cyclase plays a critical role in Chlamydomonas chemotaxis signaling transduction, similarly to animal chemotaxis. On the other hand, ciliary beat pattern switching induces randomized directional changes, analogous to run-and-tumble chemotaxis of bacteria and archaea. This study reveals that Chlamydomonas signaling transduction is similar to the eukaryotic mechanism, yet the cellular locomotion follows the bacteria and archaea mechanism.

microbiology↗

bioRxiv2023

Multicellularity and increasing Reynolds number impact on the evolutionary shift in photo-induced ciliary response in Volvocales

Volvocales include species with different cell numbers and sizes, reflecting a history of gradual size increase evolution. Unicellular species live in low Reynolds-number (Re) environments where viscous forces dominate, whereas multicellular species live in higher Re environments with nonnegligible inertial forces. Despite significant changes in the physical environment, during the evolution of multicellularity they maintained photobehaviors (i.e., photoshock and phototactic responses), which allows them to survive under changing light conditions. In this study, we classified photo-induced ciliary responses in Volvocales into four patterns: temporal waveform conversion, no obvious response, pause in ciliary beating, temporal changes in ciliary beating directions. We found that which species exhibit which pattern depends on Re associated with the individual size of each species rather than phylogenetic relationships. These results suggest that species with increased cell numbers acquired their responses adapted to higher Re fluid environments. Significance StatementVolvocales green algae include species with various cell numbers and are excellent organisms for studying the evolution of multicellularity. They exhibit photobehaviors by changing the pattern of ciliary beating, which could be categorized into four patterns. We found that the difference in patterns among the organisms is due to the Reynolds number, the ratio of viscous and inertia forces, rather than their phylogenetic relationships. This study indicates that the fluid environment was an important factor in natural selection for behavioral changes in microalgae during evolution. The results link evolution and physics while contributing to the design of micromachines.

evolutionary biology↗

bioRxiv2020

Basis for the phototaxis sign reversal in the green alga Chlamydomonas reinhardtii studied by high-speed observation

For organisms that respond to environmental stimuli using taxes, reversal of the tactic sign should be tightly regulated for survival. The biciliate green alga Chlamydomonas reinhardtii is an excellent model for studying reversal between positive and negative phototaxis. C. reinhardtii cells change swimming direction by modulating the balance of beating forces between their two cilia after photoreception at the eyespot; however, it remains unknown how they reverse phototactic sign. In this study, we observed cells undergoing phototactic turns with a high-speed camera and found that two key factors determine the phototactic sign: which of the two cilia beats more strongly for phototactic turning and when the strong beating starts. The timing of the strong ciliary beating is suggested to be regulated by ROS-regulated switching between the light-on and light-off responses at the eyespot, which leads to the switching between positive and negative phototaxis. This idea is supported by a mathematical model that introduces the timing of the strong ciliary beating after photoreception.

cell biology↗

bioRxiv2018

Inner lumen proteins stabilize doublet microtubules in cilia/flagella

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although it is known that microtubules in cilia are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dynein and intraflagellar transport. To address this question, we identified a new class of microtubule-associated proteins, named FAP45 and FAP52, 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 established that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These inner protrusions are located near the inner junctions of doublet microtubules and lack of FAP45, FAP52, and FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrated that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

cell biology↗