scholarly journals Dynein-2 Affects the Regulation of Ciliary Length but Is Not Required for Ciliogenesis in Tetrahymena thermophila

2009 ◽  
Vol 20 (2) ◽  
pp. 708-720 ◽  
Author(s):  
Vidyalakshmi Rajagopalan ◽  
Aswati Subramanian ◽  
David E. Wilkes ◽  
David G. Pennock ◽  
David J. Asai
Keyword(s):  
Electron Microscopy ◽  
Cell Lines ◽  
Heavy Chain ◽  
Tetrahymena Thermophila ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Mild Phenotype ◽  
Motile Cilia ◽  
Cilia And Flagella

Eukaryotic cilia and flagella are assembled and maintained by the bidirectional intraflagellar transport (IFT). Studies in alga, nematode, and mouse have shown that the heavy chain (Dyh2) and the light intermediate chain (D2LIC) of the cytoplasmic dynein-2 complex are essential for retrograde intraflagellar transport. In these organisms, disruption of either dynein-2 component results in short cilia/flagella with bulbous tips in which excess IFT particles have accumulated. In Tetrahymena, the expression of the DYH2 and D2LIC genes increases during reciliation, consistent with their roles in IFT. However, the targeted elimination of either DYH2 or D2LIC gene resulted in only a mild phenotype. Both knockout cell lines assembled motile cilia, but the cilia were of more variable lengths and less numerous than wild-type controls. Electron microscopy revealed normally shaped cilia with no swelling and no obvious accumulations of material in the distal ciliary tip. These results demonstrate that dynein-2 contributes to the regulation of ciliary length but is not required for ciliogenesis in Tetrahymena.

scholarly journals Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly

2004 ◽  
Vol 166 (5) ◽  
pp. 637-643 ◽  
Author(s):  
Agata Jurczyk ◽  
Adam Gromley ◽  
Sambra Redick ◽  
Jovenal San Agustin ◽  
George Witman ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Rna Interference ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Transport Proteins ◽  
Immunogold Electron Microscopy ◽  
Basal Bodies ◽  
Motile Cilia ◽  
Cilia And Flagella ◽  
Centrosome Protein

Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

scholarly journals Emerging mechanisms of dynein transport in the cytoplasm versus the cilium

2018 ◽  
Vol 46 (4) ◽  
pp. 967-982 ◽  
Author(s):  
Anthony J. Roberts
Keyword(s):  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Mechanisms Of Action ◽  
Cell Interior ◽  
Long Distance ◽  
Cargo Transport ◽  
Recent Advances ◽  
Human Disorders ◽  
Cilia And Flagella

Two classes of dynein power long-distance cargo transport in different cellular contexts. Cytoplasmic dynein-1 is responsible for the majority of transport toward microtubule minus ends in the cell interior. Dynein-2, also known as intraflagellar transport dynein, moves cargoes along the axoneme of eukaryotic cilia and flagella. Both dyneins operate as large ATP-driven motor complexes, whose dysfunction is associated with a group of human disorders. But how similar are their mechanisms of action and regulation? To examine this question, this review focuses on recent advances in dynein-1 and -2 research, and probes to what extent the emerging principles of dynein-1 transport could apply to or differ from those of the less well-understood dynein-2 mechanoenzyme.

Proteomic analysis of microtubule inner proteins (MIPs) in Rib72 null Tetrahymena cells reveals functional MIPs

2020 ◽  
Author(s):  
Amy S. Fabritius ◽  
Brian A. Bayless ◽  
Sam Li ◽  
Daniel Stoddard ◽  
Westley Heydeck ◽  
...  
Keyword(s):  
Proteomic Analysis ◽  
Tetrahymena Thermophila ◽  
Electron Tomography ◽  
Functional Characterization ◽  
Ciliary Beating ◽  
Ciliary Function ◽  
Cryo Electron Tomography ◽  
Motile Cilia ◽  
Cilia And Flagella ◽  
Stable Populations

AbstractMotile cilia and flagella are built from stable populations of doublet microtubules that comprise their axonemes. Their unique stability is brought about, at least in part, by a network of Microtubule Inner Proteins (MIPs) found in the lumen of their doublet microtubules. Rib72A and Rib72B were identified as microtubule inner proteins (MIPs) in the motile cilia of Tetrahymena thermophila. Loss of these proteins leads to ciliary defects and loss of multiple MIPs. We performed mass spectrometry coupled with proteomic analysis and bioinformatics to identify the MIPs lost in RIB72A/B knockout (KO) Tetrahymena cells. From this analysis we identified a number of candidate MIPs and pursued one, Fap115, for functional characterization. We find that loss of Fap115 results in disrupted cell swimming and aberrant ciliary beating. Cryo-electron tomography reveals that Fap115 localizes to MIP6a in the A-tubule of the doublet microtubules. Overall, our results highlight the complex relationship between MIPs, ciliary structure, and ciliary function.

scholarly journals Dynein Intermediate Chain Mediated Dynein–Dynactin Interaction Is Required for Interphase Microtubule Organization and Centrosome Replication and Separation inDictyostelium

1999 ◽  
Vol 147 (6) ◽  
pp. 1261-1274 ◽  
Author(s):  
Shuo Ma ◽  
Leda Triviños-Lagos ◽  
Ralph Gräf ◽  
Rex L. Chisholm
Keyword(s):  
Heavy Chain ◽  
Physiological Role ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Microtubule Organization ◽  
Dynein Intermediate Chain ◽  
Organizing Center ◽  
Intermediate Chain

Cytoplasmic dynein intermediate chain (IC) mediates dynein–dynactin interaction in vitro (Karki, S., and E.L. Holzbaur. 1995. J. Biol. Chem. 270:28806–28811; Vaughan, K.T., and R.B. Vallee. 1995. J. Cell Biol. 131:1507–1516). To investigate the physiological role of IC and dynein–dynactin interaction, we expressed IC truncations in wild-type Dictyostelium cells. ICΔC associated with dynactin but not with dynein heavy chain, whereas ICΔN truncations bound to dynein but bound dynactin poorly. Both mutations resulted in abnormal localization to the Golgi complex, confirming dynein function was disrupted. Striking disorganization of interphase microtubule (MT) networks was observed when mutant expression was induced. In a majority of cells, the MT networks collapsed into large bundles. We also observed cells with multiple cytoplasmic asters and MTs lacking an organizing center. These cells accumulated abnormal DNA content, suggesting a defect in mitosis. Striking defects in centrosome morphology were also observed in IC mutants, mostly larger than normal centrosomes. Ultrastructural analysis of centrosomes in IC mutants showed interphase accumulation of large centrosomes typical of prophase as well as unusually paired centrosomes, suggesting defects in centrosome replication and separation. These results suggest that dynactin-mediated cytoplasmic dynein function is required for the proper organization of interphase MT network as well as centrosome replication and separation in Dictyostelium.

Ultrastructural evidence for an unusual mode of ciliogenesis in mouse multiciliated epithelia

Microscopy ◽  
2020 ◽  
Author(s):  
Keishi Narita ◽  
Sen Takeda
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Primary Cilia ◽  
Single Cells ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Brain Ventricles ◽  
Ultrastructural Evidence ◽  
Transmission Electron ◽  
Motile Cilia

Abstract Multiciliogenesis is a cascading process for generating hundreds of motile cilia in single cells. In vertebrates, this process has been investigated in the ependyma of brain ventricles and the ciliated epithelia of the airway and oviduct. Although the early steps to amplify centrioles have been characterized in molecular detail, subsequent steps to establish multicilia have been relatively overlooked. Here, we focused on unusual cilia-related structures previously observed in wild-type mouse ependyma using transmission electron microscopy and analyzed their ultrastructural features and the frequency of their occurrence. In the ependyma, $\sim$5% of cilia existed as bundles; while the majority of the bundles were paired, bundles of more than three cilia were also found. Furthermore, apical protrusions harboring multiple sets of axonemes were occasionally observed (0–2 per section), suggesting an unusual mode of ciliogenesis. In trachea and oviduct epithelia, ciliary bundles were absent, but protrusions containing multiple axonemes were observed. At the base of such protrusions, certain axonemes were completely enwrapped by membranes, whereas others remained incompletely enwrapped. These data suggested that the late steps of multiciliogenesis might include a unique process underlying the development of cilia, which is distinct from the ciliogenesis of primary cilia.

scholarly journals Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility

2012 ◽  
Vol 23 (18) ◽  
pp. 3554-3565 ◽  
Author(s):  
Panteleimon Rompolas ◽  
Ramila S. Patel-King ◽  
Stephen M. King
Keyword(s):  
Heavy Chain ◽  
Beat Frequency ◽  
Tight Binding ◽  
Cytoplasmic Dynein ◽  
High Load ◽  
Flagellar Motility ◽  
Wild Type ◽  
Regulatory Factor ◽  
Flagellar Beat ◽  
Load Conditions

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a “high-load environment,” we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters.

scholarly journals The FLA3 KAP Subunit Is Required for Localization of Kinesin-2 to the Site of Flagellar Assembly and Processive Anterograde Intraflagellar Transport

2005 ◽  
Vol 16 (3) ◽  
pp. 1341-1354 ◽  
Author(s):  
Joshua Mueller ◽  
Catherine A. Perrone ◽  
Raqual Bower ◽  
Douglas G. Cole ◽  
Mary E. Porter
Keyword(s):  
Amino Acid ◽  
Basal Body ◽  
Intraflagellar Transport ◽  
Body Region ◽  
Wild Type ◽  
Gene Encoding ◽  
Flagellar Assembly ◽  
Cilia And Flagella ◽  
Preferential Incorporation ◽  
Dic Microscopy

Intraflagellar transport (IFT) is a bidirectional process required for assembly and maintenance of cilia and flagella. Kinesin-2 is the anterograde IFT motor, and Dhc1b/Dhc2 drives retrograde IFT. To understand how either motor interacts with the IFT particle or how their activities might be coordinated, we characterized a ts mutation in the Chlamydomonas gene encoding KAP, the nonmotor subunit of Kinesin-2. The fla3-1 mutation is an amino acid substitution in a conserved C-terminal domain. fla3-1 strains assemble flagella at 21°C, but cannot maintain them at 33°C. Although the Kinesin-2 complex is present at both 21 and 33°C, the fla3-1 Kinesin-2 complex is not efficiently targeted to or retained in the basal body region or flagella. Video-enhanced DIC microscopy of fla3-1 cells shows that the frequency of anterograde IFT particles is significantly reduced. Anterograde particles move at near wild-type velocities, but appear larger and pause more frequently in fla3-1. Transformation with an epitope-tagged KAP gene rescues all of the fla3-1 defects and results in preferential incorporation of tagged KAP complexes into flagella. KAP is therefore required for the localization of Kinesin-2 at the site of flagellar assembly and the efficient transport of anterograde IFT particles within flagella.

scholarly journals A Dynein Light Intermediate Chain, D1bLIC, Is Required for Retrograde Intraflagellar Transport

2004 ◽  
Vol 15 (10) ◽  
pp. 4382-4394 ◽  
Author(s):  
Yuqing Hou ◽  
Gregory J. Pazour ◽  
George B. Witman
Keyword(s):  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Wild Type ◽  
Phosphate Binding ◽  
Conserved Domain ◽  
Wild Type Phenotype ◽  
Flagellar Assembly ◽  
Bidirectional Movement ◽  
Mutant Cells ◽  
Intermediate Chain

Intraflagellar transport (IFT), the bidirectional movement of particles along flagella, is essential for flagellar assembly. The motor for retrograde IFT in Chlamydomonas is cytoplasmic dynein 1b, which contains the dynein heavy chain DHC1b and the light intermediate chain (LIC) D1bLIC. To investigate a possible role for the LIC in IFT, we identified a d1blic mutant. DHC1b is reduced in the mutant, indicating that D1bLIC is important for stabilizing dynein 1b. The mutant has variable length flagella that accumulate IFT-particle proteins, indicative of a defect in retrograde IFT. Interestingly, the remaining DHC1b is normally distributed in the mutant flagella, strongly suggesting that the defect is in binding of cargo to the retrograde motor rather than in motor activity per se. Cell growth and Golgi apparatus localization and morphology are normal in the mutant, indicating that D1bLIC is involved mainly in retrograde IFT. Like mammalian LICs, D1bLIC has a phosphate-binding domain (P-loop) at its N-terminus. To investigate the function of this conserved domain, d1blic mutant cells were transformed with constructs designed to express D1bLIC proteins with mutated P-loops. The constructs rescued the mutant cells to a wild-type phenotype, indicating that the function of D1bLIC in IFT is independent of its P-loop.

scholarly journals Cytoplasmic Dynein Is Required for the Nuclear Attachment and Migration of Centrosomes during Mitosis in Drosophila

1999 ◽  
Vol 146 (3) ◽  
pp. 597-608 ◽  
Author(s):  
John T. Robinson ◽  
Edward J. Wojcik ◽  
Mark A. Sanders ◽  
Maura McGrail ◽  
Thomas S. Hays
Keyword(s):  
Heavy Chain ◽  
Spatial Organization ◽  
Critical Role ◽  
Genetic System ◽  
Cytoplasmic Dynein ◽  
Chain Gene ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
And Migration

Cytoplasmic dynein is a multisubunit minus-end–directed microtubule motor that serves multiple cellular functions. Genetic studies in Drosophila and mouse have demonstrated that dynein function is essential in metazoan organisms. However, whether the essential function of dynein reflects a mitotic requirement, and what specific mitotic tasks require dynein remains controversial. Drosophila is an excellent genetic system in which to analyze dynein function in mitosis, providing excellent cytology in embryonic and somatic cells. We have used previously characterized recessive lethal mutations in the dynein heavy chain gene, Dhc64C, to reveal the contributions of the dynein motor to mitotic centrosome behavior in the syncytial embryo. Embryos lacking wild-type cytoplasmic dynein heavy chain were analyzed by in vivo analysis of rhodamine-labeled microtubules, as well as by immu-nofluorescence in situ methods. Comparisons between wild-type and Dhc64C mutant embryos reveal that dynein function is required for the attachment and migration of centrosomes along the nuclear envelope during interphase/prophase, and to maintain the attachment of centrosomes to mitotic spindle poles. The disruption of these centrosome attachments in mutant embryos reveals a critical role for dynein function and centrosome positioning in the spatial organization of the syncytial cytoplasm of the developing embryo.

scholarly journals A Novel Dynein Light Intermediate Chain Colocalizes with the Retrograde Motor for Intraflagellar Transport at Sites of Axoneme Assembly in Chlamydomonas and Mammalian Cells

2003 ◽  
Vol 14 (5) ◽  
pp. 2041-2056 ◽  
Author(s):  
Catherine A. Perrone ◽  
Douglas Tritschler ◽  
Patrick Taulman ◽  
Raqual Bower ◽  
Bradley K. Yoder ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cultured Cells ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Body Region ◽  
The Novel ◽  
Gene Encoding ◽  
Efferent Duct ◽  
Cilia And Flagella ◽  
Intermediate Chain

The assembly of cilia and flagella depends on bidirectional intraflagellar transport (IFT). Anterograde IFT is driven by kinesin II, whereas retrograde IFT requires cytoplasmic dynein 1b (cDHC1b). Little is known about how cDHC1b interacts with its cargoes or how it is regulated. Recent work identified a novel dynein light intermediate chain (D2LIC) that colocalized with the mammalian cDHC1b homolog DHC2 in the centrosomal region of cultured cells. To see whether the LIC might play a role in IFT, we characterized the gene encoding the Chlamydomonas homolog of D2LIC and found its expression is up-regulated in response to deflagellation. We show that the LIC subunit copurifies with cDHC1b during flagellar isolation, dynein extraction, sucrose density centrifugation, and immunoprecipitation. Immunocytochemistry reveals that the LIC colocalizes with cDHC1b in the basal body region and along the length of flagella in wild-type cells. Localization of the complex is altered in a collection of retrograde IFT and length control mutants, which suggests that the affected gene products directly or indirectly regulate cDHC1b activity. The mammalian DHC2 and D2LIC also colocalize in the apical cytoplasm and axonemes of ciliated epithelia in the lung, brain, and efferent duct. These studies, together with the identification of an LIC mutation, xbx-1(ok279), which disrupts retrograde IFT in Caenorhabditis elegans, indicate that the novel LIC is a component of the cDHC1b/DHC2 retrograde IFT motor in a variety of organisms.

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