scholarly journals Role of a Class Dhc1b Dynein in Retrograde Transport of Ift Motors and Ift Raft Particles along Cilia, but Not Dendrites, in Chemosensory Neurons of Living Caenorhabditis elegans

1999 ◽  
Vol 147 (3) ◽  
pp. 519-530 ◽  
Author(s):  
Dawn Signor ◽  
Karen P. Wedaman ◽  
Jose T. Orozco ◽  
Noelle D. Dwyer ◽  
Cornelia I. Bargmann ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Neuronal Cell ◽  
Motor Protein ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Neuronal Cell Body ◽  
Transport Pathway ◽  
Chemosensory Neurons ◽  
Sensory Cilia

The heterotrimeric motor protein, kinesin-II, and its presumptive cargo, can be observed moving anterogradely at 0.7 μm/s by intraflagellar transport (IFT) within sensory cilia of chemosensory neurons of living Caenorhabditis elegans, using a fluorescence microscope–based transport assay (Orozco, J.T., K.P. Wedaman, D. Signor, H. Brown, L. Rose, and J.M. Scholey. 1999. Nature. 398:674). Here, we report that kinesin-II, and two of its presumptive cargo molecules, OSM-1 and OSM-6, all move at ∼1.1 μm/s in the retrograde direction along cilia and dendrites, which is consistent with the hypothesis that these proteins are retrieved from the distal endings of the cilia by a retrograde transport pathway that moves them along cilia and then dendrites, back to the neuronal cell body. To test the hypothesis that the minus end–directed microtubule motor protein, cytoplasmic dynein, drives this retrograde transport pathway, we visualized movement of kinesin-II and its cargo along dendrites and cilia in a che-3 cytoplasmic dynein mutant background, and observed an inhibition of retrograde transport in cilia but not in dendrites. In contrast, anterograde IFT proceeds normally in che-3 mutants. Thus, we propose that the class DHC1b cytoplasmic dynein, CHE-3, is specifically responsible for the retrograde transport of the anterograde motor, kinesin-II, and its cargo within sensory cilia, but not within dendrites.

scholarly journals Chlamydomonas Kinesin-II–dependent Intraflagellar Transport (IFT): IFT Particles Contain Proteins Required for Ciliary Assembly in Caenorhabditis elegans Sensory Neurons

1998 ◽  
Vol 141 (4) ◽  
pp. 993-1008 ◽  
Author(s):  
Douglas G. Cole ◽  
Dennis R. Diener ◽  
Amy L. Himelblau ◽  
Peter L. Beech ◽  
Jason C. Fuster ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Sensory Neurons ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Temperature Sensitive ◽  
Basal Bodies ◽  
Sensitive Mutant ◽  
Sensory Cilia ◽  
A Subunit ◽  
Dependent Transport

We previously described a kinesin-dependent movement of particles in the flagella of Chlamydomonas reinhardtii called intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523). When IFT is inhibited by inactivation of a kinesin, FLA10, in the temperature-sensitive mutant, fla10, existing flagella resorb and new flagella cannot be assembled. We report here that: (a) the IFT-associated FLA10 protein is a subunit of a heterotrimeric kinesin; (b) IFT particles are composed of 15 polypeptides comprising two large complexes; (c) the FLA10 kinesin-II and IFT particle polypeptides, in addition to being found in flagella, are highly concentrated around the flagellar basal bodies; and, (d) mutations affecting homologs of two of the IFT particle polypeptides in Caenorhabditis elegans result in defects in the sensory cilia located on the dendritic processes of sensory neurons. In the accompanying report by Pazour, G.J., C.G. Wilkerson, and G.B. Witman (1998. J. Cell Biol. 141:979–992), a Chlamydomonas mutant (fla14) is described in which only the retrograde transport of IFT particles is disrupted, resulting in assembly-defective flagella filled with an excess of IFT particles. This microtubule- dependent transport process, IFT, defined by mutants in both the anterograde (fla10) and retrograde (fla14) transport of isolable particles, is probably essential for the maintenance and assembly of all eukaryotic motile flagella and nonmotile sensory cilia.

scholarly journals Analysis of the Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein-1 Using a Chemical “Toolkit”

2021 ◽  
Vol 22 (14) ◽  
pp. 7704
Author(s):  
Sayi’Mone Tati ◽  
Laleh Alisaraie
Keyword(s):  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Motor Protein ◽  
Structural Data ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Motor Domain ◽  
Conformational Search ◽  
Structural Mechanism

Dynein is a ~1.2 MDa cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The presented work analyzes the structure–activity relationship (SAR) of dynapyrazole A and B, as well as ciliobrevin A and D, in their various protonated states and their 46 analogues for their binding in the AAA1 subunit, the leading ATP hydrolytic site of the motor domain. This study exploits in silico methods to look at the analogues’ effects on the functionally essential subsites of the motor domain of dynein 1, since no similar experimental structural data are available. Ciliobrevin and its analogues bind to the ATP motifs of the AAA1, namely, the walker-A (W-A) or P-loop, the walker-B (W-B), and the sensor I and II. Ciliobrevin A shows a better binding affinity than its D analogue. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. In addition, protonation of the nitrogen atom in ciliobrevin A and D, as well as dynapyrazole A and B, at the N9 site of ciliobrevin and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the “glutamate switch” mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family.

scholarly journals Somatic CRISPR–Cas9-induced mutations reveal roles of embryonically essential dynein chains in Caenorhabditis elegans cilia

2015 ◽  
Vol 208 (6) ◽  
pp. 683-692 ◽  
Author(s):  
Wenjing Li ◽  
Peishan Yi ◽  
Guangshuo Ou
Keyword(s):  
Caenorhabditis Elegans ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Regulatory Mechanisms ◽  
Light Chains ◽  
Induced Mutations ◽  
Functional Studies ◽  
Cilium Formation ◽  
Dynein Intermediate Chain ◽  
Intermediate Chain

Cilium formation and maintenance require intraflagellar transport (IFT). Although much is known about kinesin-2–driven anterograde IFT, the composition and regulation of retrograde IFT-specific dynein remain elusive. Components of cytoplasmic dynein may participate in IFT; however, their essential roles in cell division preclude functional studies in postmitotic cilia. Here, we report that inducible expression of the clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 system in Caenorhabditis elegans generated conditional mutations in IFT motors and particles, recapitulating ciliary defects in their null mutants. Using this method to bypass the embryonic requirement, we show the following: the dynein intermediate chain, light chain LC8, and lissencephaly-1 regulate retrograde IFT; the dynein light intermediate chain functions in dendrites and indirectly contributes to ciliogenesis; and the Tctex and Roadblock light chains are dispensable for cilium assembly. Furthermore, we demonstrate that these components undergo biphasic IFT with distinct transport frequencies and turnaround behaviors. Together, our results suggest that IFT–dynein and cytoplasmic dynein have unique compositions but also share components and regulatory mechanisms.

scholarly journals Caenorhabditis elegans DYF-2, an Orthologue of Human WDR19, Is a Component of the Intraflagellar Transport Machinery in Sensory Cilia

2006 ◽  
Vol 17 (11) ◽  
pp. 4801-4811 ◽  
Author(s):  
Evgeni Efimenko ◽  
Oliver E. Blacque ◽  
Guangshuo Ou ◽  
Courtney J. Haycraft ◽  
Bradley K. Yoder ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Aspartic Acid ◽  
Critical Role ◽  
Intraflagellar Transport ◽  
Bardet Biedl Syndrome ◽  
Evolutionarily Conserved ◽  
Sensory Cilia ◽  
Mutant Phenotypes ◽  
And Function

The intraflagellar transport (IFT) machinery required to build functional cilia consists of a multisubunit complex whose molecular composition, organization, and function are poorly understood. Here, we describe a novel tryptophan-aspartic acid (WD) repeat (WDR) containing IFT protein from Caenorhabditis elegans, DYF-2, that plays a critical role in maintaining the structural and functional integrity of the IFT machinery. We determined the identity of the dyf-2 gene by transgenic rescue of mutant phenotypes and by sequencing of mutant alleles. Loss of DYF-2 function selectively affects the assembly and motility of different IFT components and leads to defects in cilia structure and chemosensation in the nematode. Based on these observations, and the analysis of DYF-2 movement in a Bardet–Biedl syndrome mutant with partially disrupted IFT particles, we conclude that DYF-2 can associate with IFT particle complex B. At the same time, mutations in dyf-2 can interfere with the function of complex A components, suggesting an important role of this protein in the assembly of the IFT particle as a whole. Importantly, the mouse orthologue of DYF-2, WDR19, also localizes to cilia, pointing to an important evolutionarily conserved role for this WDR protein in cilia development and function.

scholarly journals Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

2006 ◽  
Vol 172 (5) ◽  
pp. 663-669 ◽  
Author(s):  
James E. Evans ◽  
Joshua J. Snow ◽  
Amy L. Gunnarson ◽  
Guangshuo Ou ◽  
Henning Stahlberg ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Intraflagellar Transport ◽  
Full Length ◽  
Sensory Stimuli ◽  
Sensory Cilia ◽  
Section Electron ◽  
Functional Modulation ◽  
Serial Section Electron Microscopy ◽  
Section Electron Microscopy ◽  
Ciliated Neurons

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure–frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a “canonical” IFT motor, whereas OSM-3 is an “accessory” IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.

scholarly journals The WD Repeat-containing Protein IFTA-1 Is Required for Retrograde Intraflagellar Transport

2006 ◽  
Vol 17 (12) ◽  
pp. 5053-5062 ◽  
Author(s):  
Oliver E. Blacque ◽  
Chunmei Li ◽  
Peter N. Inglis ◽  
Muneer A. Esmail ◽  
Guangshuo Ou ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Bardet Biedl Syndrome ◽  
C Elegans ◽  
Gene Mutant ◽  
Strong Homology ◽  
Sensory Cilia ◽  
Wd Repeat ◽  
Mammalian Protein

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet–Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.

Intraflagellar transport motors in Caenorhabditis elegans neurons

2004 ◽  
Vol 32 (5) ◽  
pp. 682-684 ◽  
Author(s):  
J.M. Scholey ◽  
G. Ou ◽  
J. Snow ◽  
A. Gunnarson
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Time Lapse ◽  
Protein Fusion ◽  
C Elegans ◽  
Sensory Cilia ◽  
Macromolecular Protein ◽  
Green Fluorescent

IFT (intraflagellar transport) assembles and maintains sensory cilia on the dendritic endings of chemosensory neurons within the nematode Caenorhabditis elegans. During IFT, macromolecular protein complexes called IFT particles (which carry ciliary precursors) are moved from the base of the sensory cilium to its distal tip by anterograde IFT motors (kinesin-II and Osm-3 kinesin) and back to the base by retrograde IFT-dynein [Rosenbaum and Witman (2002) Nat. Rev. Mol. Cell Biol. 3, 813–825; Scholey (2003) Annu. Rev. Cell Dev. Biol. 19, 423–443; and Snell, Pan and Wang (2004) Cell 117, 693–697]. In the present study, we describe the protein machinery of IFT in C. elegans, which we have analysed using time-lapse fluorescence microscopy of green fluorescent protein-fusion proteins in concert with ciliary mutants.

scholarly journals Ectosome uptake by glia sculpts Caenorhabditis elegans sensory cilia

2021 ◽  
Author(s):  
Adrià Razzauti ◽  
Patrick Laurent
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Surface ◽  
Glial Cells ◽  
Intraflagellar Transport ◽  
Membrane Receptors ◽  
Sensory Function ◽  
C Elegans ◽  
Sensory Cilia

AbstractCilia are sensory organelles protruding from cell surface. A tight regulation of membrane receptors traffic in and out of cilia is achieved by the action of Intraflagellar Transport (IFT). Here, we show that ectosomes bud from a subset of C. elegans sensory cilia. Packing and disposal of ciliary receptors in ectosomes complement their retrieval by IFT. Mutations in ciliary retrieval genes increase export of the salt sensor GCY-22 from ASER neurons by ectosomes, preventing its accumulation in ASER cilia. Ectosomes are produced from two ciliary locations: cilia tip and/or cilia base. Ectosomes budding from cilia tip are released in the environment. Ectosomes produced from the cilia base are concomitantly phagocytosed by the associated glial cells. Although ectocytosis does not require glia to occur, ectosome phagocytosis by the contacting glia contributes to maintain cilia shape and sensory function. We suggest this coordinated neuron-glia interaction is required for proper cilia function.

scholarly journals Elucidation of Structural Mechanism of ATP Inhibition at the AAA1 Subunit of Cytoplasmic Dynein 1 Using a Chemical "Toolkit"

2021 ◽  
Author(s):  
Sayi'Mone Martinet Tati ◽  
Laleh Alisaraie
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Atp Hydrolysis ◽  
Critical Role ◽  
Motor Protein ◽  
Structural Data ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Motor Domain ◽  
Conformational Search

Dynein is a cytoskeletal motor protein that carries organelles via retrograde transport in eukaryotic cells. The motor protein belongs to the ATPase family of proteins associated with diverse cellular activities and plays a critical role in transporting cargoes to the minus end of the microtubules. The motor domain of dynein possesses a hexameric head, where ATP hydrolysis occurs. The AAA1 binding site is the leading ATP hydrolytic site, followed by the AAA3 subsite. Small-molecule ATP competitive inhibitors of dynein are thought to target the AAA1 site. The presented work elucidates the structure-activity relationship of dynapyrazole A and B, ciliobrevin A and D in their various protonated states and their 46 analogs for their binding properties in the nucleotide-binding site of the AAA1 subunit and their effects on the functionally essential subsites of the motor domain of cytoplasmic dynein 1, as there is currently no similar experimental structural data available. Ciliobrevin and its analogs bind to the ATP motifs of the AAA1, namely the Walker-A or P-loop, the Walker-B, and the sensor I and II. Ciliobrevin A shows a better binding affinity to the AAA1 binding site of dynein 1 than its D analog. Although the double bond in ciliobrevin A and D was expected to decrease the ligand potency, they show a better affinity to the AAA1 binding site than dynapyrazole A and B, lacking the bond. Protonation of the nitrogen in ciliobrevin A, D, dynapyrazole A, and B at the N9 site of ciliobrevin, and the N7 of the latter increased their binding affinity. Exploring ciliobrevin A geometrical configuration suggests the E isomer has a superior binding profile over the Z due to binding at the critical ATP motifs. Utilizing the refined structure of the motor domain obtained through protein conformational search in this study exhibits that Arg1852 of the yeast cytoplasmic dynein could involve in the "glutamate switch" mechanism in cytoplasmic dynein 1 in lieu of the conserved Asn in AAA+ protein family, as the guanidine moiety of the Arg engages in an H-bond with the carboxylate moiety of Glu1849.

scholarly journals Protein Particles in Chlamydomonas Flagella Undergo a Transport Cycle Consisting of Four Phases

2001 ◽  
Vol 153 (1) ◽  
pp. 13-24 ◽  
Author(s):  
Carlo Iomini ◽  
Veronica Babaev-Khaimov ◽  
Massimo Sassaroli ◽  
Gianni Piperno
Keyword(s):  
Phase I ◽  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Phase Iii ◽  
Cycle Phase ◽  
Flagellar Assembly ◽  
Protein Particles ◽  
Velocity Changes ◽  
Phase I And Ii

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.

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