Eukaryotic Flagella Research Articles

Diffusion as a Ruler: Modeling Kinesin Diffusion as a Length Sensor for Intraflagellar Transport

An important question in cell biology is whether cells are able to measure size, either whole cell size or organelle size. Perhaps cells have an internal chemical representation of size that can be used to precisely regulate growth, or perhaps size is just an accident that emerges due to constraint of nutrients. The eukaryotic flagellum is an ideal model for studying size sensing and control because its linear geometry makes it essentially one-dimensional, greatly simplifying mathematical modeling. The assembly of flagella is regulated by intraflagellar transport (IFT), in which kinesin motors carry cargo adaptors for flagellar proteins along the flagellum and then deposit them at the tip, lengthening the flagellum. The rate at which IFT motors are recruited to begin transport into the flagellum is anticorrelated with the flagellar length, implying some kind of communication between the base and the tip and possibly indicating that cells contain some mechanism for measuring flagellar length. Although it is possible to imagine many complex scenarios in which additional signaling molecules sense length and carry feedback signals to the cell body to control IFT, might the already-known components of the IFT system be sufficient to allow length dependence of IFT? Here we investigate a model in which the anterograde kinesin motors unbind after cargo delivery, diffuse back to the base, and are subsequently reused to power entry of new IFT trains into the flagellum. By mathematically modeling and simulating such a system, we are able to show that the diffusion time of the motors can in principle be sufficient to serve as a proxy for length measurement. We found that the diffusion model can not only achieve a stable steady-state length without the addition of any other signaling molecules or pathways, but also is able to produce the anticorrelation between length and IFT recruitment rate that has been observed in quantitative imaging studies.

Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation in Chlamydomonas.

Calcium has been implicated in the motility, assembly, disassembly, and deflagellation of the eukaryotic flagellum or cilium (exchangeable terms). Calmodulin (CaM) is known to be critical for flagellar motility; however, it is unknown whether and how CaM is involved in other flagella-related activities. We have studied CaM in Chlamydomonas, a widely used organism for ciliary studies. CaM is present in the cell body and the flagellum, with enrichment in the basal body region. Loss of CaM causes shortening of the nucleus basal body connector and impairs flagellar motility and assembly but not flagellar disassembly. Moreover, the cam mutant is defective in pH shock-induced deflagellation. The mutant deflagellates, however, upon mechanical shearing and treatment with mastoparan or detergent undergo permeabilization in the presence of calcium, indicating the cam mutant is defective in elevations of cytosolic calcium induced by pH shock, rather than by the deflagellation machinery. Indeed, the cam mutant fails to produce inositol 1,4,5-trisphosphate. Biochemical and genetic analysis showed that CaM does not directly activate PLC. Furthermore, CaM interacts with ADF1, a transient receptor channel that functions in acid-induced calcium entry. Thus, CaM is a critical regulator of flagellar activities especially those involved in modulating calcium homeostasis during acidic stress.-Wu, Q., Gao, K., Zheng, S., Zhu, X., Liang, Y., Pan, J. Calmodulin regulates a TRP channel (ADF1) and phospholipase C (PLC) to mediate elevation of cytosolic calcium during acidic stress that induces deflagellation in Chlamydomonas.

Multispecies exclusion process with fusion and fission of rods: A model inspired by intraflagellar transport.

We introduce a multispecies exclusion model where length-conserving probabilistic fusion and fission of the hard rods are allowed. Although all rods enter the system with the same initial length ℓ=1, their length can keep changing, because of fusion and fission, as they move in a step-by-step manner towards the exit. Two neighboring hard rods of lengths ℓ_{1} and ℓ_{2} can fuse into a single rod of longer length ℓ=ℓ_{1}+ℓ_{2} provided ℓ≤N. Similarly, length-conserving fission of a rod of length ℓ^{'}≤N results in two shorter daughter rods. Based on the extremum current hypothesis, we plot the phase diagram of the model under open boundary conditions utilizing the results derived for the same model under periodic boundary condition using mean-field approximation. The density profile and the flux profile of rods are in excellent agreement with computer simulations. Although the fusion and fission of the rods are motivated by similar phenomena observed in intraflagellar transport (IFT) in eukaryotic flagella, this exclusion model is too simple to account for the quantitative experimental data for any specific organism. Nevertheless, the concepts of "flux profile" and "transition zone" that emerge from the interplay of fusion and fission in this model are likely to have important implications for IFT and for other similar transport phenomena in long cell protrusions.

Intraflagellar transporter protein 140 (IFT140), a component of IFT-A complex, is essential for male fertility and spermiogenesis in mice.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, little is known about its role in sperm flagella formation and male fertility. IFT140 is a component of IFT-A complex. In mouse, it is highly expressed in the testis. Ift140 gene was inactivated specifically in mouse spermatocytes/spermatids. The mutant mice did not show any gross abnormalities, but all were infertile and associated with significantly reduced sperm number and motility. Multiple sperm morphological abnormalities were discovered, including amorphous heads, short/bent flagella and swollen tail tips, as well as vesicles along the flagella due to spermiogenesis defects. The epididymides contained round bodies of cytoplasm derived from the sloughing of the cytoplasmic lobes and residual bodies. Knockout of Ift140 did not significantly affect testicular expression levels of selective IFT components but localization of IFT27 and IFT88, two components of IFT-B complex, was changed. Our findings demonstrate that IFT140 is a key regulator for male fertility and normal spermiogenesis in mice. It not only plays a role in sperm flagella assembling, but is also involved in critical assembly of proteins that interface between the germ cell plasma and the Sertoli cell.

Endogenosymbiosis: from hypothesis to empirical evidence towards a Unified Symbiogenetic Theory (UST).

In 1967 Lynn (Sagan) Margulis proposed that mitochondria, photosynthetic plastids and cilia were acquired prokaryotes and evolved symbiotically to form anaerobic bacteria, photosynthetic bacteria and eventually algae. Although most of this theory is well-accepted now, the hypothesis that endosymbiotic spirochaetes developed into eukaryotic flagella and cilia, and the following proposals of an endosymbiotic origin of other eukaryotic organelles such as peroxisomes, glyoxysomes, etc. have not received much acceptance, since evidence suggests they lack a genome and do not show ultrastructural similarities to bacteria or archaea. Nevertheless, the idea that over millennia mitochondria, plastids, prokaryotic and eukaryotic cells and even flagella and peroxisomes, as either primary or secondary endosymbionts, transferred some or all of their own DNA to the host cell’s nucleus through a process called «endogenosymbiosis» (i.e. a symbiotic gene transfer, such as the internalisation of the endosymbiont’s DNA with lateral transfer) has been recently suggested. This endogenosymbiosis could take place during the evolutionary transition from the symbiotic interacting community, invoked by Margulis, to a fully-integrated (either prokaryotic or eukaryotic) cell. This process could explain the missing evidence of the presence of DNA in flagella and peroxisomes whose ancestor endosymbionts, during the long endogenosymbiotic evolution, could have transferred their whole genome to the host cell that subsequently integrated it in its own genome, directly controlling its expression. Furthermore, the endogenosymbiosis hypothesis could be the explanation of the transition between an RNA to a DNA world and of some cases of true sympatric evolution of species, apparently inexplicable by the canonical speciation processes. Here, after an introduction to the theoretical basis of endogenosymbiosis and a discussion of the empirical confirming evidence, I show a graphical summary of the integration between this and the former endosymbiosis theories. The Serial Endosymbiosis Theory and the Secondary Endosymbiosis are merged with the Endogenosymbiosis Theory in a Unified Symbiogenetic Theory (UST).

Flagellar dynamics of chains of active Janus particles fueled by an AC electric field

We study the active dynamics of self-propelled asymmetrical colloidal particles (Janus particles) fueled by an AC electric field. Both the speed and direction of the self-propulsion, and the strength of the attractive interaction between particles can be controlled by tuning the frequency of the applied electric field and the ion concentration of the solution. The strong attractive force at high ion concentration gives rise to chain formation of the Janus particles, which can be explained by the quadrupolar charge distribution on the particles. Chain formation is observed irrespective of the direction of the self-propulsion of the particles. When both the position and the orientation of the heads of the chains are fixed, they exhibit beating behavior reminiscent of eukaryotic flagella. The beating frequency of the chains of Janus particles depends on the applied voltage and thus on the self-propulsive force. The scaling relation between the beating frequency and the self-propulsive force deviates from theoretical predictions made previously on active filaments. However, this discrepancy is resolved by assuming that the attractive interaction between the particles is mediated by the quadrupolar distribution of the induced charges, which gives indirect but convincing evidence on the mechanisms of the Janus particles. This signifies that the dependence between the propulsion mechanism and the interaction mechanism, which had been dismissed previously, can modify the dispersion relations of beating behaviors. In addition, hydrodynamic interaction within the chain, and its effect on propulsion speed, are discussed. These provide new insights into active filaments, such as optimal flagellar design for biological functions.

Flagellar incorporation of proteins follows at least two different routes in trypanosomes.

Eukaryotic cilia and flagella are sophisticated organelles composed of several hundreds of proteins that need to be incorporated at the right time and the right place during assembly. Two methods were used to investigate this process in the model protist Trypanosoma brucei: inducible expression of epitope-tagged labelled proteins and fluorescence recovery after photobleaching of fluorescent fusion proteins. This revealed that skeletal components of the radial spokes (RSP3), the central pair (PF16) and the outer dynein arms (DNAI1) are incorporated at the distal end of the growing flagellum. They display low or even no visible turnover in mature flagella, a finding further confirmed by monitoring a heavy chain of the outer dynein arm. In contrast, the membrane-associated protein arginine kinase 3 (AK3) showed rapid turnover in both growing and mature flagella, without particular polarity and independently of intraflagellar transport. These results demonstrate different modes of incorporation for structural and membrane-associated proteins in flagella. The existence of two distinct modes for incorporation of proteins in growing flagella suggests the existence of different targeting machineries. Moreover, the absence of turnover of structural elements supports the view that the length of the mature flagellum in trypanosomes is not modified after assembly.

150 Runs of Homozygosity Analysis and their Possible Influence on Sperm Motility in Highly Consanguineous Bulls

Inbreeding depression is associated with emergence of deleterious effects and loss of genetic variability. Widespread use of genotyping technologies and new approaches for identification of runs-of-homozygosity (ROH) provide valuable tools to better understand the effects of inbreeding depression. We have previously demonstrated that inbreeding affects sperm motility patterns in cattle (Dorado et al. 2016 Reprod. Fertil. Dev. 29, 712-720; 10.1071/RD15324), with an increase in individuals presenting a hyperactivated-like motility. In this study, we characterised ROH patterns and performed gene ontology analysis of a large, highly consanguineous cattle population. Thirty-three Retinta bulls [average inbreeding percentage FPED = 16.57% (10.25 to 30.62%)] were genotyped using the Axiom® BOS 1 High-Density SNP Array (Thermo Fisher Scientific, Waltham, MA, USA). The ROH were estimated using CGATOH package and classified upon their length into 5 categories: 1-2, 2-4, 4-8, 8-16, and >16 Mb, which are inversely related to inbreeding events occurring 50 to 3 generations before, respectively. The ROH showed an average length of 3.73 Mb (1.48 to 6.71 Mb). Total FROH was partially explained by the increase in ROH fragments longer than 8 Mb, which is consistent with recent inbreeding events that occurred in this population in the last 6 generations. Additionally, the distribution of ROH varied notably between chromosomes. For instance, >16 Mb runs (very recent inbreeding event) were absent in BTA26 and BTA29, whereas certain loci on BTA7, BTA13, and BTA24 showed >16 Mb runs in 9 animals. To identify candidate biological functions affected by inbreeding, we performed functional analysis of the genome areas covered by ROH >8 Mb (our pedigree data covered 5.87 equivalent complete generations) using the Functional Annotation Clustering tool implemented in DAVID. Candidate regions were defined by occurring in ROH >8 in more than 6 animals and by a distance <1 Mb between adjacent single nucleotide polymorphisms. Eight significant gene clusters (enrichment score >1.30; P < 0.05) were identified, with 2 of these clusters related to sperm motility. One of these clusters (score 1.72) contained 23 genes coding microtubule-related proteins, which are associated with cellular movement structures such as flagella. The other cluster (score 1.42), included 4 genes related to dynein and motile cilium assembly ATPase complexes, associated with movement of eukaryotic flagella. We observed an additional cluster representing an ATP-binding feature, which included 77 genes (score 1.43). These results indicate that inbreeding could affect sperm motility by altering microtubule structure and motility. However, the fact that ROH were unevenly distributed across the genome, even in a highly inbred cattle population, also suggests that different metabolic pathways could be affected in individuals with similar inbreeding values. Therefore, the use of inbreeding coefficients as predictors for sperm quality should be approached carefully.

The Gene Ontology of eukaryotic cilia and flagella

BackgroundRecent research into ciliary structure and function provides important insights into inherited diseases termed ciliopathies and other cilia-related disorders. This wealth of knowledge needs to be translated into a computational representation to be fully exploitable by the research community. To this end, members of the Gene Ontology (GO) and SYSCILIA Consortia have worked together to improve representation of ciliary substructures and processes in GO.MethodsMembers of the SYSCILIA and Gene Ontology Consortia suggested additions and changes to GO, to reflect new knowledge in the field. The project initially aimed to improve coverage of ciliary parts, and was then broadened to cilia-related biological processes. Discussions were documented in a public tracker. We engaged the broader cilia community via direct consultation and by referring to the literature. Ontology updates were implemented via ontology editing tools.ResultsSo far, we have created or modified 127 GO terms representing parts and processes related to eukaryotic cilia/flagella or prokaryotic flagella. A growing number of biological pathways are known to involve cilia, and we continue to incorporate this knowledge in GO. The resulting expansion in GO allows more precise representation of experimentally derived knowledge, and SYSCILIA and GO biocurators have created 199 annotations to 50 human ciliary proteins. The revised ontology was also used to curate mouse proteins in a collaborative project. The revised GO and annotations, used in comparative ‘before and after’ analyses of representative ciliary datasets, improve enrichment results significantly.ConclusionsOur work has resulted in a broader and deeper coverage of ciliary composition and function. These improvements in ontology and protein annotation will benefit all users of GO enrichment analysis tools, as well as the ciliary research community, in areas ranging from microscopy image annotation to interpretation of high-throughput studies. We welcome feedback to further enhance the representation of cilia biology in GO.

Synchronization and Collective Dynamics of Flagella and Cilia as Hydrodynamically Coupled Oscillators

Cooperative motion of flagella and cilia faciliates swimming of microorganisms and material transport in the body of multicellular organisms. Using minimal models, we address the roles of hydrodynamic interaction in synchronization and collective dynamics of flagella and cilia. Collective synchronization of bacterial flagella is studied with a model of bacterial carpets. Cilia and eukaryotic flagella are characterized by periodic modulation of their driving forces, which produces various patterns of two-body synchronization and metachronal waves. Long-range nature of the interaction introduces novel features in the dynamics of these model systems. The flagella of a swimmer synchronize also by a viscous drag force mediated through the swimmer’s body. Recent advance in experimental studies of the collective dynamics of flagella, cilia and related artificial systems are summarized.

Intraflagellar transporter protein (IFT27), an IFT25 binding partner, is essential for male fertility and spermiogenesis in mice

Intraflagellar transport (IFT) is an evolutionarily conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. In mice, mutations in IFT proteins have been shown to cause several ciliopathies including retinal degeneration, polycystic kidney disease, and hearing loss. However, little is known about its role in the formation of the sperm tail, which has the longest flagella of mammalian cells. IFT27 is a component of IFT-B complex and binds to IFT25 directly. In mice, IFT27 is highly expressed in the testis. To investigate the role of IFT27 in male germ cells, the floxed Ift27 mice were bred with Stra8−iCre mice so that the Ift27 gene was disrupted in spermatocytes/spermatids. The Ift27: Stra8−iCre mutant mice did not show any gross abnormalities, and all of the mutant mice survived to adulthood. There was no difference between testis weight/body weight between controls and mutant mice. All adult homozygous mutant males examined were completely infertile. Histological examination of the testes revealed abnormally developed germ cells during the spermiogenesis phase. The epididymides contained round bodies of cytoplasm. Sperm number was significantly reduced compared to the controls and only about 2% of them remained significantly reduced motility. Examination of epididymal sperm by light microscopy and SEM revealed multiple morphological abnormalities including round heads, short and bent tails, abnormal thickness of sperm tails in some areas, and swollen tail tips in some sperm. TEM examination of epididymal sperm showed that most sperm lost the “9+2″ axoneme structure, and the mitochondria sheath, fibrous sheath, and outer dense fibers were also disorganized. Some sperm flagella also lost cell membrane. Levels of IFT25 and IFT81 were significantly reduced in the testis of the conditional Ift27 knockout mice, and levels of IFT20, IFT74, and IFT140 were not changed. Sperm lipid rafts, which were disrupted in the conditional Ift25 knockout mice, appeared to be normal in the conditional Ift27 knockout mice. Our findings suggest that like IFT25, IFT27, even though not required for ciliogenesis in somatic cells, is essential for sperm flagella formation, sperm function, and male fertility in mice. IFT25 and IFT27 control sperm formation/function through many common mechanisms, but IFT25 has additional roles beyond IFT27.

Dynamics of the IFT machinery at the ciliary tip.

Intraflagellar transport (IFT) is essential for the elongation and maintenance of eukaryotic cilia and flagella. Due to the traffic jam of multiple trains at the ciliary tip, how IFT trains are remodeled in these turnaround zones cannot be determined by conventional imaging. Using PhotoGate, we visualized the full range of movement of single IFT trains and motors in Chlamydomonas flagella. Anterograde trains split apart and IFT complexes mix with each other at the tip to assemble retrograde trains. Dynein-1b is carried to the tip by kinesin-II as inactive cargo on anterograde trains. Unlike dynein-1b, kinesin-II detaches from IFT trains at the tip and diffuses in flagella. As the flagellum grows longer, diffusion delays return of kinesin-II to the basal body, depleting kinesin-II available for anterograde transport. Our results suggest that dissociation of kinesin-II from IFT trains serves as a negative feedback mechanism that facilitates flagellar length control in Chlamydomonas.

Mitochondria are not captive bacteria

Lynn Sagan's conjecture (1967) that three of the fundamental organelles observed in eukaryote cells, specifically mitochondria, plastids and flagella were once free-living primitive (prokaryotic) cells was accepted after considerable opposition. Even though the idea was swiftly refuted for the specific case of origins of flagella in eukaryotes, the symbiosis model in general was accepted for decades as a realistic hypothesis to describe the endosymbiotic origins of eukaryotes. However, a systematic analysis of the origins of the mitochondrial proteome based on empirical genome evolution models now indicates that 97% of modern mitochondrial protein domains as well their homologues in bacteria and archaea were present in the universal common ancestor (UCA) of the modern tree of life (ToL). These protein domains are universal modular building blocks of modern genes and genomes, each of which is identified by a unique tertiary structure and a specific biochemical function as well as a characteristic sequence profile. Further, phylogeny reconstructed from genome-scale evolution models reveals that Eukaryotes and Akaryotes (archaea and bacteria) descend independently from UCA. That is to say, Eukaryotes and Akaryotes are both primordial lineages that evolved in parallel. Finally, there is no indication of massive inter-lineage exchange of coding sequences during the descent of the two lineages. Accordingly, we suggest that the evolution of the mitochondrial proteome was autogenic (endogenic) and not endosymbiotic (exogenic).

Electrostatic interaction between polyglutamylated tubulin and the nexin-dynein regulatory complex regulates flagellar motility.

Tubulins undergo various posttranslational modifications. Among them, polyglutamylation is involved in the motility of eukaryotic flagella and the stability of the axonemal microtubules. However, it remains unclear where polyglutamylated tubulin localizes precisely within the axoneme and how tubulin polyglutamylation affects flagellar motility. In this study, we identified the three-dimensional localization of the polyglutamylated tubulin in Chlamydomonas flagella using antibody labeling and cryo-electron tomography. Polyglutamylated tubulins specifically located in close proximity to a microtubule-cross-bridging structure called the nexin-dynein regulatory complex (N-DRC). Because N-DRC is positively charged, we hypothesized that there is an electrostatic interaction between the polyglutamylated tubulin and the N-DRC, and therefore we mutated the amino acid sequences of DRC4 to modify the charge of the N-DRC. We found that both augmentation and reduction of the positive charge on DRC4 resulted in reduced flagellar motility. Moreover, reduced motility in a mutant with a structurally defective N-DRC was partially restored by increasing the positive charge on DRC4. These results clearly indicate that beating motion of flagella is maintained by the electrostatic cross-bridge formed between the negatively charged polyglutamylated tubulins and the positively charged N-DRC.

Evidence for a self-organized compliant mechanism for the spontaneous steady beating of cilia.

Cilia or eukaryotic flagella are slender 200-nm-diameter organelles that move the immersing fluid relative to a cell and sense the environment. Their core structure is nine doublet microtubules (DMTs) arranged around a central-pair. When motile, thousands of tiny motors slide the DMTs relative to each other to facilitate traveling waves of bending along the cilium's length. These motors provide the energy to change the shape of the cilium and overcome the viscous forces of moving in the surrounding fluid. In planar beating, motors walk toward where the cilium is attached to the cell body. Traveling waves are initiated by motors bending the elastic cilium back and forth, a self-organized mechanical oscillator. We found remarkably that the energy in a wave is nearly constant over a wide range of (ATP) and medium viscosities and inter-doublet springs operate only in the central and not in the basal region. Since the energy in a wave does not depend on its rate of formation, the control mechanism is likely purely mechanical. Further the torque per length generated by the motors acting on the doublets is proportional to and nearly in phase with the microtubule sliding velocity with magnitude dependent on the medium. We determined the frequency-dependent elastic moduli and strain energies of beating cilia. Incorporation of these in an energy-based model explains the beating frequency, wavelength, limiting of the wave amplitude and the overall energy of the traveling wave. Our model describes the intricacies of the basal-wave initiation as well as the traveling wave.

Lynn Margulis and the endosymbiont hypothesis: 50 years later.

The 1967 article “On the Origin of Mitosing Cells” in the Journal of Theoretical Biology by Lynn Margulis (then Lynn Sagan) is widely regarded as stimulating renewed interest in the long-dormant endosymbiont hypothesis of organelle origins. In her article, not only did Margulis champion an endosymbiotic origin of mitochondria and plastids from bacterial ancestors, but she also posited that the eukaryotic flagellum (undulipodium in her usage) and mitotic apparatus originated from an endosymbiotic, spirochete-like organism. In essence, she presented a comprehensive symbiotic view of eukaryotic cell evolution (eukaryogenesis). Not all of the ideas in her article have been accepted, for want of compelling evidence, but her vigorous promotion of the role of symbiosis in cell evolution unquestionably had a major influence on how subsequent investigators have viewed the origin and evolution of mitochondria and plastids and the eukaryotic cell per se.

IFT25, an intraflagellar transporter protein dispensable for ciliogenesis in somatic cells, is essential for sperm flagella formation.

Intraflagellar transport (IFT) is a conserved mechanism essential for the assembly and maintenance of most eukaryotic cilia and flagella. However, IFT25, a component of the IFT complex, is not required for the formation of cilia in somatic tissues. In mice, the gene is highly expressed in the testis, and its expression is upregulated during the final phase when sperm flagella are formed. To investigate the role of IFT25 in sperm flagella formation, the gene was specifically disrupted in male germ cells. All homozygous knockout mice survived to adulthood and did not show any gross abnormalities. However, all homozygous knockout males were completely infertile. Sperm numbers were reduced and these sperm were completely immotile. Multiple morphological abnormalities were observed in sperm, including round heads, short and bent tails, with some tails showing branched flagella and others with frequent abnormal thicknesses, as well as swollen tips of the tail. Transmission electron microscopy revealed that flagellar accessory structures, including the fibrous sheath and outer dense fibers, were disorganized, and most sperm had also lost the "9+2" microtubule structure. In the testis, IFT25 forms a complex with other IFT proteins. In Ift25 knockout testes, IFT27, an IFT25 binding partner, was missing, and IFT20 and IFT81 levels were also reduced. Our findings suggest that IFT25, although not necessary for the formation of cilia in somatic cells, is indispensable for sperm flagellum formation and male fertility in mice.

Protein transport in growing and steady-state cilia.

Cilia and eukaryotic flagella are threadlike cell extensions with motile and sensory functions. Their assembly requires intraflagellar transport (IFT), a bidirectional motor-driven transport of protein carriers along the axonemal microtubules. IFT moves ample amounts of structural proteins including tubulin into growing cilia likely explaining its critical role for assembly. IFT continues in non-growing cilia contributing to a variety of processes ranging from axonemal maintenance and the export of non-ciliary proteins to cell locomotion and ciliary signaling. Here, we discuss recent data on cues regulating the type, amount and timing of cargo transported by IFT. A regulation of IFT-cargo interactions is critical to establish, maintain and adjust ciliary length, protein composition and function.

Flagellar Length Control can be Achieved through a Simple Diffusion-Based Mechanism

An important question in cell biology is how cells know how big to make their organelles. The eukaryotic flagellum is an ideal model for studying size control because its linear geometry makes it essentially one-dimensional, greatly simplifying mathematical modeling. The assembly of flagella is regulated by intraflagellar transport (IFT), in which trains of kinesin motors walk to the tip of the flagellum and deposit the cargo necessary for the flagellum to grow. The competing length control factor is a length-independent decay of the flagellum. In Chlamydomonas reinhardtii flagella, this process results in initial rapid growth followed by convergence to a steady-state length. Curiously, the rate at which motors are recruited to begin transport is indirectly proportional to the length, implying some kind of communication between the base and the tip. We propose a model in which motors unbind after cargo delivery and diffuse back to the base, and are reused in IFT. In this model, the diffusion time of the motors serves as a proxy for length measurement. To explore the viability of this diffusion-based length control, we computationally built this model in three different ways. First, we built an agent-based model in which we used object-oriented programming to explicitly model flagella and motors, including time dynamics. Second, we modeled the number density along the flagellum as a vector, and built a stochastic matrix to simulate time dynamics and determine a steady-state. Third, we used differential equations to directly solve for the steady-state length. In all three, we found that the diffusion model can achieve steady-state length and an inverse relationship between length and recruitment rate. This is remarkable because this is perhaps the simplest explanation of length control, giving it credence in light of evolution.

The Flagella Beat of Chlamydomonas Has Distinctly Regulated Static and Dynamic Components, which Accord with Curvature Controlled Dynein Activity

The axoneme, the mechanical core of eukaryotic cilia and flagella, is a huge macromolecular assembly of microtubules, cross-links and dynein motors. The coordinated activity of the dyneins produces a diversity of flagellar waveforms that drive fluid flows across tissue surfaces or propel micro-swimmers. While it is believed that the flagellar beat is a result of feedback - based on the ability of motors to produce and sense forces - the mechanism by which motors are controlled remains unknown.To elucidate how molecular motors are involved in the generation of flagellar waveforms, we studied the beat of axonemes isolated from Chlamydomonas reinhardtii, a single cell green alga. Chlamydomonas flagella can exhibit both of the most common waveforms found in nature: the asymmetrical breaststroke-like beat as well as the symmetrical sperm-like beat.Using high speed microscopy and image analysis we precisely determine axonemal shapes in space and time. A characterization of the waveform properties at different ATP concentrations shows that the static asymmetry of the breaststroke - which has an approximately circular shape - can be separated from the dynamic beat component. This analysis reveals that the breaststroke can essentially be viewed as a sperm-like beat traveling around a circular shape and allows us to examine both waveform components independently.By comparing the dynamic component of experimentally measured waveforms to a mechanical model of the axoneme, we found that the shapes were consistent with a model in which dynein motors respond to changes in axonemal curvature. We furthermore show that the static asymmetric shape underlying the beat could also result from curvature controlled dynein activity.Together our findings present novel insights into how molecular motors shape the asymmetric waveform of Chlamydomonas flagella.