Movement of cargos along microtubules plays key roles in diverse cellular processes, from signalling to mitosis. In cilia, rapid movement of ciliary components along the microtubules to and from the ...assembly site is essential for the assembly and disassembly of the structure itself
. This bidirectional transport, known as intraflagellar transport (IFT)
, is driven by the anterograde motor kinesin-2
and the retrograde motor dynein-1b (dynein-2 in mammals)
. However, to drive retrograde transport, dynein-1b must first be delivered to the ciliary tip by anterograde IFT
. Although, the presence of opposing motors in bidirectional transport processes often leads to periodic stalling and slowing of cargos
, IFT is highly processive
. Using cryo-electron tomography, we show that a tug-of-war between kinesin-2 and dynein-1b is prevented by loading dynein-1b onto anterograde IFT trains in an autoinhibited form and by positioning it away from the microtubule track to prevent binding. Once at the ciliary tip, dynein-1b must transition into an active form and engage microtubules to power retrograde trains. These findings provide a striking example of how coordinated structural changes mediate the behaviour of complex cellular machinery.
The Cilium Secretes Bioactive Ectosomes Wood, Christopher R.; Huang, Kaiyao; Diener, Dennis R. ...
CB/Current biology,
05/2013, Volume:
23, Issue:
10
Journal Article
Peer reviewed
Open access
The release of membrane vesicles from the surface of cells into their surrounding environment is now recognized as an important pathway for the delivery of proteins to extracellular sites of ...biological function. Membrane vesicles of this kind, termed exosomes and ectosomes, are the result of active processes and have been shown to carry a wide array of biological effector molecules that can play roles in cell-to-cell communication and remodeling of the extracellular space 1–7. Degradation of the extracellular matrix (ECM) through the regulated release of proteolytic enzymes is a key process for development, morphogenesis, and cell migration in animal and plant cells. Here we show that the unicellular alga Chlamydomonas achieves the timely degradation of its mother cell wall, a type of ECM, through the budding of ectosomes directly from the membranes of its flagella. Using a combination of immunoelectron microscopy, immunofluorescence microscopy, and functional analysis, we demonstrate that these vesicles, which we term ciliary ectosomes, act as carriers of the proteolytic enzyme necessary for the liberation of daughter cells following mitosis 8, 9. Chlamydomonas has proven to be the key unicellular model for the highly conserved mechanisms of mammalian cilia, and our results suggest that cilia may be an underappreciated source of bioactive, extracellular membrane vesicles.
•Ectosomes can bud directly from the membranes of cilia•Ciliary ectosomes carry the protease necessary for hatching in Chlamydomonas•Cilia may function to release signals, in addition to their role as sensory antennae
The transition zone (TZ) is a specialized region of the cilium characterized by Y-shaped connectors between the microtubules of the ciliary axoneme and the ciliary membrane 1. Located near the base ...of the cilium, the TZ is in the prime location to act as a gate for proteins into and out of the ciliary compartment, a role supported by experimental evidence 2–6. The importance of the TZ has been underscored by studies showing that mutations affecting proteins located in the TZ result in cilia-related diseases, or ciliopathies, presenting symptoms including renal cysts, retinal degeneration, and situs inversus 7–9. Some TZ proteins have been identified and shown to interact with each other through coprecipitation studies in vertebrate cells 4, 10, 11 and genetics studies in C. elegans 3. As a distinct approach to identify TZ proteins, we have taken advantage of the biology of Chlamydomonas to isolate TZs. Proteomic analysis identified 115 proteins, ten of which were known TZ proteins related to ciliopathies, indicating that the preparation was highly enriched for TZs. Interestingly, six proteins of the endosomal sorting complexes required for transport (ESCRT) were also associated with the TZs. Identification of these and other proteins in the TZ will provide new insights into functions of the TZ, as well as candidate ciliopathy genes.
•Ciliary transition zones were isolated from Chlamydomonas•Proteomic analysis of the transition zones identified 115 proteins•Six ESCRT proteins were identified associated with the transition zone
Defects in the transition zone, a specialized region found at the base of cilia, result in cilia-related diseases. Diener et al. report a proteomic analysis of isolated transition zones that revealed the presence of ESCRT proteins, suggesting that these proteins play a previously unrecognized role in ciliary membrane dynamics.
Sepsis remains a significant health burden and a major clinical need exists for therapeutics to dampen the excessive and uncontrolled immune activation. Nuclear protein high mobility group box ...protein 1 (HMGB1) is released following cell death and is a late mediator in sepsis pathogenesis. While approaches targeting HMGB1 have demonstrated reduced mortality in pre-clinical models of sepsis, the impact of HMGB1 blockade on the complex septic inflammatory milieu and the development of subsequent immunosuppression remain enigmatic. Analysis of plasma samples obtained from septic shock patients established an association between increased HMGB1 and non-survival, higher APACHE II scores, and increased pro-inflammatory cytokine responses. Pre-clinically, administration of neutralising ovine anti-HMGB1 polyclonal antibodies improved survival in murine endotoxaemia and caecal ligation and puncture-induced sepsis models, and altered early cytokine profiles to one which corresponded to patterns observed in the surviving patient cohort. Additionally, anti-HMGB1 treated murine sepsis survivors were significantly more resistant to secondary bacterial infection and exhibited altered innate immune cell phenotypes and cytokine responses. These findings demonstrate that anti-HMGB1 antibodies alter inflammation in murine sepsis models and reduce sepsis mortality without potentiating immunosuppression.
Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of ...the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule—membrane links there. Ultrastructural analysis revealed defects in these microtubule—membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule—membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.
Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The "Sementis Copenhagen Vector" (SCV) system is a recently developed ...vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR
mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR
mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR
mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced "shot burden" for these often co-circulating arboviruses.
Context. Optical long baseline interferometry is a unique way to study astronomical objects at milli-arcsecond resolutions not attainable with current single-dish telescopes. Yet, the significance of ...its scientfic return strongly depends on a dense coverage of the uv-plane and a highly stable transfer function of the interferometric instrument. In the last few years, integrated optics (IO) beam combiners have facilitated the emergence of 4-telescope interferometers such as PIONIER or GRAVITY, boosting the imaging capabilities of the VLTI. However, the spectral range beyond 2.2 μm is not ideally covered by the conventional silica based IO. Here, we consider new laser-written IO prototypes made of gallium lanthanum sulfide (GLS) glass, a material that permits access to the mid-infrared spectral regime. Aims. Our goal is to conduct a full characterization of our mid-IR IO two-telescope coupler in order to measure the performance levels directly relevant for long-baseline interferometry. We focus in particular on the exploitation of the L and M astronomical bands. Methods. We use a dedicated Michelson-interferometer setup to perform Fourier transform spectroscopy on the coupler and measure its broadband interferometric performance. We also analyze the polarization properties of the coupler, the differential dispersion and phase degradation, as well as the modal behavior and the total throughput. Results. We measure broadband interferometric contrasts of 94.9% and 92.1% for unpolarized light in the L and M bands. Spectrally integrated splitting ratios are close to 50%, but show chromatic dependence over the considered bandwidths. Additionally, the phase variation due to the combiner is measured and does not exceed 0.04 rad and 0.07 rad across the L and M band, respectively. The total throughput of the coupler including Fresnel and injection losses from free-space is 25.4%. Furthermore, differential birefringence is low (<0.2 rad), in line with the high contrasts reported for unpolarized light. Conclusions. The laser-written IO GLS prototype combiners prove to be a reliable technological solution with promising performance for mid-infrared long-baseline interferometry. In the next steps, we will consider more advanced optical functions, as well as a fiber-fed input, and we will revise the optical design parameters in order to further enhance the total throughput and achromatic behavior.
Eukaryotic cilia and flagella are microtubule-based organelles whose relatively simple shape makes them ideal for investigating the fundamental question of organelle size regulation. Most of the ...flagellar materials are transported from the cell body via an active transport process called intraflagellar transport (IFT). The rate of IFT entry into flagella, known as IFT injection, has been shown to negatively correlate with flagellar length. However, it remains unknown how the cell measures the length of its flagella and controls IFT injection. One of the most-discussed theoretical models for length sensing to control IFT is the ion-current model, which posits that there is a uniform distribution of Ca
channels along the flagellum and that the Ca
current from the flagellum into the cell body increases linearly with flagellar length. In this model, the cell uses the Ca
current to negatively regulate IFT injection. The recent discovery that IFT entry into flagella is regulated by the phosphorylation of kinesin through a calcium-dependent protein kinase has provided further impetus for the ion-current model. To test this model, we measured and manipulated the levels of Ca
inside of
flagella and quantified IFT injection. Although the concentration of Ca
inside of flagella was weakly correlated with the length of flagella, we found that IFT injection was reduced in calcium-deficient flagella, rather than increased as the model predicted, and that variation in IFT injection was uncorrelated with the occurrence of flagellar Ca
spikes. Thus, Ca
does not appear to function as a negative regulator of IFT injection, hence it cannot form the basis of a stable length control system.
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