Browse
Submit Data
Databases
API
Help

Dataset Information

15 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Protein turnover dynamics suggest a diffusion-to-capture mechanism for peri-basal body recruitment and retention of intraflagellar transport proteins.

ABSTRACT: Intraflagellar transport (IFT) is essential for construction and maintenance of cilia. IFT proteins concentrate at the basal body where they are thought to assemble into trains and bind cargoes for transport. To study the mechanisms of IFT recruitment to this peri-basal body pool, we quantified protein dynamics of eight IFT proteins, as well as five other basal body localizing proteins using fluorescence recovery after photobleaching in vertebrate multiciliated cells. We found that members of the IFT-A and IFT-B protein complexes show distinct turnover kinetics from other basal body components. Additionally, known IFT subcomplexes displayed shared dynamics, suggesting shared basal body recruitment and/or retention mechanisms. Finally, we evaluated the mechanisms of basal body recruitment by depolymerizing cytosolic MTs, which suggested that IFT proteins are recruited to basal bodies through a diffusion-to-capture mechanism. Our survey of IFT protein dynamics provides new insights into IFT recruitment to basal bodies, a crucial step in ciliogenesis and ciliary signaling.

SUBMITTER: Hibbard JVK

PROVIDER: S-EPMC8351562 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

The ciliopathy-associated CPLANE proteins direct basal body recruitment of intraflagellar transport machinery.

Project description:Cilia use microtubule-based intraflagellar transport (IFT) to organize intercellular signaling. Ciliopathies are a spectrum of human diseases resulting from defects in cilia structure or function. The mechanisms regulating the assembly of ciliary multiprotein complexes and the transport of these complexes to the base of cilia remain largely unknown. Combining proteomics, in vivo imaging and genetic analysis of proteins linked to planar cell polarity (Inturned, Fuzzy and Wdpcp), we identified and characterized a new genetic module, which we term CPLANE (ciliogenesis and planar polarity effector), and an extensive associated protein network. CPLANE proteins physically and functionally interact with the poorly understood ciliopathy-associated protein Jbts17 at basal bodies, where they act to recruit a specific subset of IFT-A proteins. In the absence of CPLANE, defective IFT-A particles enter the axoneme and IFT-B trafficking is severely perturbed. Accordingly, mutation of CPLANE genes elicits specific ciliopathy phenotypes in mouse models and is associated with ciliopathies in human patients.

| S-EPMC4978421 | biostudies-literature

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

Project description: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.

| S-EPMC5985012 | biostudies-literature

BBS4 is required for intraflagellar transport coordination and basal body number in mammalian olfactory cilia.

Project description:Bardet-Beidl syndrome (BBS) manifests from genetic mutations encoding for one or more BBS proteins. BBS4 loss impacts olfactory ciliation and odor detection, yet the cellular mechanisms remain unclear. Here, we report that Bbs4-/- mice exhibit shorter and fewer olfactory sensory neuron (OSN) cilia despite retaining odorant receptor localization. Within Bbs4-/- OSN cilia, we observed asynchronous rates of IFT-A/B particle movements, indicating miscoordination in IFT complex trafficking. Within the OSN dendritic knob, the basal bodies are dynamic, with incorporation of ectopically expressed centrin-2 and γ-tubulin occurring after nascent ciliogenesis. Importantly, BBS4 loss results in the reduction of basal body numbers separate from cilia loss. Adenoviral expression of BBS4 restored OSN cilia lengths and was sufficient to re-establish odor detection, but failed to rescue ciliary and basal body numbers. Our results yield a model for the plurality of BBS4 functions in OSNs that includes intraciliary and periciliary roles that can explain the loss of cilia and penetrance of ciliopathy phenotypes in olfactory neurons.

| S-EPMC6432715 | biostudies-literature

Dynein and intraflagellar transport.

Project description: Not available

| S-EPMC4433774 | biostudies-literature

Intraflagellar Transport and Ciliary Dynamics.

Project description:Cilia and flagella are microtubule-based organelles whose assembly requires a motile process, known as intraflagellar transport (IFT), to bring tubulin and other components to the distal tip of the growing structure. The IFT system uses a multiprotein complex with components that appear to be specialized for the transport of different sets of cargo proteins. The mechanisms by which cargo is selected for ciliary import and transport by IFT remain an area of active research. The complex dynamics of cilia and flagella are under constant regulation to ensure proper length control, and this regulation appears to involve regulation at the stage of IFT injection into the flagellum, as well as regulation of flagellar disassembly and, possibly, of cargo binding. Cilia and flagella thus represent a convenient model system to study how multiple motile and signaling pathways cooperate to control the assembly and dynamics of a complex cellular structure.

| S-EPMC5334256 | biostudies-literature

Intraflagellar transport (IFT) cargo: IFT transports flagellar precursors to the tip and turnover products to the cell body.

Project description:Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.

| S-EPMC2172340 | biostudies-literature

Intraflagellar transport protein RABL5/IFT22 recruits the BBSome to the basal body through the GTPase ARL6/BBS3.

Project description:Bardet-Biedl syndrome (BBS) is a ciliopathy caused by defects in the assembly or distribution of the BBSome, a conserved protein complex. The BBSome cycles via intraflagellar transport (IFT) through cilia to transport signaling proteins. How the BBSome is recruited to the basal body for binding to IFT trains for ciliary entry remains unknown. Here, we show that the Rab-like 5 GTPase IFT22 regulates basal body targeting of the BBSome in Chlamydomonas reinhardtii Our functional, biochemical and single particle in vivo imaging assays show that IFT22 is an active GTPase with low intrinsic GTPase activity. IFT22 is part of the IFT-B1 subcomplex but is not required for ciliary assembly. Independent of its association to IFT-B1, IFT22 binds and stabilizes the Arf-like 6 GTPase BBS3, a BBS protein that is not part of the BBSome. IFT22/BBS3 associates with the BBSome through an interaction between BBS3 and the BBSome. When both IFT22 and BBS3 are in their guanosine triphosphate (GTP)-bound states they recruit the BBSome to the basal body for coupling with the IFT-B1 subcomplex. The GTP-bound BBS3 likely remains to be associated with the BBSome upon ciliary entry. In contrast, IFT22 is not required for the transport of BBSomes in cilia, indicating that the BBSome is transferred from IFT22 to the IFT trains at the ciliary base. In summary, our data propose that nucleotide-dependent recruitment of the BBSome to the basal body by IFT22 regulates BBSome entry into cilia.

| S-EPMC7007519 | biostudies-literature

Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in Chlamydomonas.

Project description:Tubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP-β-tubulin by altering the IFT74N-IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP-β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP-β-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP-β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

| S-EPMC7502601 | biostudies-literature

Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46.

Project description:Motile cilia are found on unicellular organisms such as the green alga Chlamydomonas reinhardtii, on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16, as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of C. reinhardtii ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed ?-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of C. reinhardtii, that the C-terminal ?-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.

| S-EPMC5418046 | biostudies-literature