Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments.

Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated whether two Caenorhabditis elegans α- and β-tubulin isotypes, identified through mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules. One isotype, TBB-4, assembles into microtubules at the tips of the axoneme core and distal segments, where the microtubule tip tracker EB1 is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, fluorescence recovery after photobleaching analysis and modelling indicate that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady-state length

Revealing the hexokinase step of glycolysis in lactic acid producer fungus Rhizopus oryzae

Rhizopus oryzae is a filamentous fungus which can grow on some renewable carbon sources and this makes it a good candidate for production of lactic acid and ethanol. However, in R. oryzae the production yields of these products are lower compared to alternative organisms. The utilization of the carbon source through glycolysis might end up in lactic acid or ethanol production. This makes the glycolytic pathway a focus of attention for our search for a way to increase the speed and efficiency of this pathway thus increasing the yield of desired end product. Hexokinase catalyse the first and irreversible step in glycolysis and implicated as one of the major control points in this pathway. In this study we are trying to investigate the regulation of glycolytic pathway of R. oryzae, in the light of studies done for Saccharomyces cerevisiae. The probable hexokinase genes were found from the annotated genome database of R. oryzae by doing a blast search using the known protein sequences of closely related S. cerevisiae hexokinases and glucokinase. Five of our ten probable hexokinase genes were obtained by using PCR. Sequencing results of three of them proved that these genes have no introns meaning they were expressed in R. oryzae. In the complementation study done in hexokinaless mutant of S. cerevisiae for one of these genes the growth of the transformant was observed on glucose or fructose containing media. Same experiments will be carried out for the rest of these genes

Interplay between Ciliary Ultrastructure and IFT-Train Dynamics Revealed by Single-Molecule Super-resolution Imaging

Summary: Cilia are built and maintained by intraflagellar transport (IFT), driving IFT trains back and forth along the ciliary axoneme. How IFT brings about the intricate ciliary structure and how this structure affects IFT are not well understood. We identify, using single-molecule super-resolution imaging of IFT components in living C. elegans, ciliary subdomains, enabling correlation of IFT-train dynamics to ciliary ultra-structure. In the transition zone, IFT dynamics are impaired, resulting in frequent pauses. At the ciliary base and tip, IFT trains show intriguing turnaround dynamics. Surprisingly, deletion of IFT motor kinesin-II not only affects IFT-train dynamics but also alters ciliary structure. Super-resolution imaging in these mutant animals suggests that the arrangement of IFT trains with respect to the axonemal microtubules is different than in wild-type animals. Our results reveal a complex, mutual interplay between ciliary ultrastructure and IFT-train dynamics, highlighting the importance of physical cues in the control of IFT dynamics. : Oswald et al. use trajectories of single intraflagellar transport proteins in the chemosensory cilia of C. elegans to generate super-resolution fluorescence maps. Local motility properties can be correlated to ciliary ultrastructure. They find that in the absence of kinesin-II, the ciliary ultrastructure is substantially altered. Keywords: single-molecule fluorescence microscopy, super-resolution microscopy, intraflagellar transport, intracellular transport, cilia, motor cooperation, molecular motor protein

Single-Molecule Turnarounds of Intraflagellar Transport at the C. elegans Ciliary Tip

Summary: Cilia are microtubule-based sensing hubs that rely on intraflagellar transport (IFT) for their development, maintenance, and function. Kinesin-2 motors transport IFT trains, consisting of IFT proteins and cargo, from ciliary base to tip. There, trains turn around and are transported back by IFT dynein. The mechanism of tip turnaround has remained elusive. Here, we employ single-molecule fluorescence microscopy of IFT components in the tips of phasmid cilia of living C. elegans. Analysis of the trajectories reveals that while motor proteins and IFT-A particle component CHE-11 mostly turn around immediately, the IFT-B particle component OSM-6 pauses for several seconds. Our data indicate that IFT trains disassemble into at least IFT-A, IFT-B, IFT-dynein, and OSM-3 complexes at the tip, where OSM-6 is temporarily retained or undergoes modification, prior to train reassembly and retrograde transport. The single-molecule approach used here is a valuable tool to study how directional switches occur in microtubule-based transport processes. : Using single-molecule fluorescence microscopy, Mijalkovic et al. visualize the dynamics of IFT components at the tips of C. elegans chemosensory cilia. They find that the motors and the IFT-A particle component CHE-11 reverse almost immediately, while the IFT-B component OSM-6 is temporarily retained before reassembly and reversal. Keywords: intracellular transport, IFT, kinesin, dynein, single-molecule biophysics, ciliary tip turn

The crowding dynamics of the motor protein kinesin-II

Intraflagellar transport (IFT) in C. elegans chemosensory cilia is an example of functional coordination and cooperation of two motor proteins with distinct motility properties operating together in large groups to transport cargoes: a fast and processive homodimeric kinesin-2, OSM-3, and a slow and less processive heterotrimeric kinesin-2, kinesin-II. To study the mechanism of the collective dynamics of kinesin-II of C. elegans cilia in an in vitro system, we used Total Internal Reflection Fluorescence microscopy to image the motility of truncated, heterodimeric kinesin-II constructs at high motor densities. Using an analysis technique based on correlation of the fluorescence intensities, we extracted quantitative motor parameters, such as motor density, velocity and average run length, from the image. Our experiments and analyses show that kinesin-II motility parameters are far less affected by (self) crowding than OSM-3. Our observations are supported by numerical calculations based on the TASEP-LK model (Totally Asymmetric Simple Exclusion Process-Langmuir Kinetics). From a comparison of data and modelling of OSM-3 and kinesin-II, a general picture emerges of the collective dynamics of the kinesin motors driving IFT in C. elegans chemosensory cilia and the way the motors deal with crowding

Intraflagellar transport delivers tubulin isotypes to sensory cilium middle and distal segments.

Sensory cilia are assembled and maintained by kinesin-2-dependent intraflagellar transport (IFT). We investigated whether two Caenorhabditis elegans α- and β-tubulin isotypes, identified through mutants that lack their cilium distal segments, are delivered to their assembly sites by IFT. Mutations in conserved residues in both tubulins destabilize distal singlet microtubules. One isotype, TBB-4, assembles into microtubules at the tips of the axoneme core and distal segments, where the microtubule tip tracker EB1 is found, and localizes all along the cilium, whereas the other, TBA-5, concentrates in distal singlets. IFT assays, fluorescence recovery after photobleaching analysis and modelling indicate that the continual transport of sub-stoichiometric numbers of these tubulin subunits by the IFT machinery can maintain sensory cilia at their steady-state length