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 The DHC1b (DHC2) Isoform of Cytoplasmic Dynein Is Required for Flagellar Assembly

1999 ◽  
Vol 144 (3) ◽  
pp. 473-481 ◽  
Author(s):  
Gregory J. Pazour ◽  
Bethany L. Dickert ◽  
George B. Witman
Keyword(s):  
Molecular Motors ◽  
Cell Movement ◽  
Intraflagellar Transport ◽  
Cytoplasmic Dynein ◽  
Cdna Clones ◽  
Western Blots ◽  
Heavy Chains ◽  
Different Types ◽  
Flagellar Assembly ◽  
Soluble Fractions

Dyneins are microtubule-based molecular motors involved in many different types of cell movement. Most dynein heavy chains (DHCs) clearly group into cytoplasmic or axonemal isoforms. However, DHC1b has been enigmatic. To learn more about this isoform, we isolated Chlamydomonas cDNA clones encoding a portion of DHC1b, and used these clones to identify a Chlamydomonas cell line with a deletion mutation in DHC1b. The mutant grows normally and appears to have a normal Golgi apparatus, but has very short flagella. The deletion also results in a massive redistribution of raft subunits from a peri-basal body pool (Cole, D.G., D.R. Diener, A.L. Himelblau, P.L. Beech, J.C. Fuster, and J.L. Rosenbaum. 1998. J. Cell Biol. 141:993–1008) to the flagella. Rafts are particles that normally move up and down the flagella in a process known as intraflagellar transport (IFT) (Kozminski, K.G., K.A. Johnson, P. Forscher, and J.L. Rosenbaum. 1993. Proc. Natl. Acad. Sci. USA. 90:5519–5523), which is essential for assembly and maintenance of flagella. The redistribution of raft subunits apparently occurs due to a defect in the retrograde component of IFT, suggesting that DHC1b is the motor for retrograde IFT. Consistent with this, Western blots indicate that DHC1b is present in the flagellum, predominantly in the detergent- and ATP-soluble fractions. These results indicate that DHC1b is a cytoplasmic dynein essential for flagellar assembly, probably because it is the motor for retrograde IFT.

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 The UDP-Glc:Glycoprotein Glucosyltransferase Is Essential for Schizosaccharomyces pombe Viability under Conditions of Extreme Endoplasmic Reticulum Stress

1998 ◽  
Vol 143 (3) ◽  
pp. 625-635 ◽  
Author(s):  
Sandra Fanchiotti ◽  
Fabiana Fernández ◽  
Cecilia D'Alessio ◽  
Armando J. Parodi
Keyword(s):  
Cell Viability ◽  
Schizosaccharomyces Pombe ◽  
Double Mutant ◽  
Expression Vector ◽  
The Other ◽  
Wild Type ◽  
Glucosidase Ii ◽  
Wild Type Phenotype ◽  
Polypeptide Chains ◽  
Mutant Cells

Interaction of monoglucosylated oligosaccharides with ER lectins (calnexin and/or calreticulin) facilitates glycoprotein folding but this interaction is not essential for cell viability under normal conditions. We obtained two distinct single Schizosaccharomyces pombe mutants deficient in either one of the two pathways leading to the formation of monoglucosylated oligosaccharides. The alg6 mutant does not glucosy- late lipid-linked oligosaccharides and transfers Man9GlcNAc2 to nascent polypeptide chains and the gpt1 mutant lacks UDP-Glc:glycoprotein glucosyltransferase (GT). Both single mutants grew normally at 28°C. On the other hand, gpt1/alg6 double-mutant cells grew very slowly and with a rounded morphology at 28°C and did not grow at 37°C. The wild-type phenotype was restored by transfection of the double mutant with a GT-encoding expression vector or by addition of 1 M sorbitol to the medium, indicating that the double mutant is affected in cell wall formation. It is suggested that facilitation of glycoprotein folding mediated by the interaction of monoglucosylated oligosaccharides with calnexin is essential for cell viability under conditions of extreme ER stress such as underglycosylation of proteins caused by the alg6 mutation and high temperature. In contrast, gls2/alg6 double-mutant cells that transfer Man9GlcNAc2 and that are unable to remove the glucose units added by GT as they lack glucosidase II (GII), grew at 37°C and had, when grown at 28°C, a phenotype of growth and morphology almost identical to that of wild-type cells. These results indicate that facilitation of glycoprotein folding mediated by the interaction of calnexin and monoglucosylated oligosaccharides does not necessarily require cycles of reglucosylation–deglucosylation catalyzed by GT and GII.

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.

scholarly journals Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas

2003 ◽  
Vol 163 (3) ◽  
pp. 597-607 ◽  
Author(s):  
Lai-Wa Tam ◽  
William L. Dentler ◽  
Paul A. Lefebvre
Keyword(s):  
Immunofluorescence Microscopy ◽  
Intraflagellar Transport ◽  
Wild Type ◽  
Western Blots ◽  
Functional Complex ◽  
Length Regulation ◽  
Flagellar Assembly ◽  
Unequal Length ◽  
Null Mutants ◽  
Novel Protein

Four long-flagella (LF) genes are important for flagellar length control in Chlamydomonas reinhardtii. Here, we characterize two new null lf3 mutants whose phenotypes are different from previously identified lf3 mutants. These null mutants have unequal-length flagella that assemble more slowly than wild-type flagella, though their flagella can also reach abnormally long lengths. Prominent bulges are found at the distal ends of short, long, and regenerating flagella of these mutants. Analysis of the flagella by electron and immunofluorescence microscopy and by Western blots revealed that the bulges contain intraflagellar transport complexes, a defect reported previously (for review see Cole, D.G., 2003. Traffic. 4:435–442) in a subset of mutants defective in intraflagellar transport. We have cloned the wild-type LF3 gene and characterized a hypomorphic mutant allele of LF3. LF3p is a novel protein located predominantly in the cell body. It cosediments with the product of the LF1 gene in sucrose density gradients, indicating that these proteins may form a functional complex to regulate flagellar length and assembly.

scholarly journals Intraflagellar transport (IFT) cargo

2004 ◽  
Vol 164 (2) ◽  
pp. 255-266 ◽  
Author(s):  
Hongmin Qin ◽  
Dennis R. Diener ◽  
Stefan Geimer ◽  
Douglas G. Cole ◽  
Joel L. Rosenbaum
Keyword(s):  
Cell Body ◽  
Soluble Fraction ◽  
Intraflagellar Transport ◽  
Radial Spokes ◽  
Flagellar Assembly ◽  
Protein Particles ◽  
Bidirectional Movement

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.

scholarly journals The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth.

2021 ◽  
Author(s):  
Karina Perlaza ◽  
Mariya Mirvis ◽  
Hiroaki Ishikawa ◽  
Wallace F Marshall
Keyword(s):  
Model Organism ◽  
Size Control ◽  
Intraflagellar Transport ◽  
Loss Of Function ◽  
Wild Type ◽  
Cytoplasmic Microtubule ◽  
Flagellar Assembly ◽  
Flagellar Regeneration ◽  
Domain Protein ◽  
Late Stages

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined. We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

scholarly journals The role of the dynein light intermediate chain in retrograde IFT and flagellar function in Chlamydomonas

2016 ◽  
Vol 27 (15) ◽  
pp. 2404-2422 ◽  
Author(s):  
Jaimee Reck ◽  
Alexandria M. Schauer ◽  
Kristyn VanderWaal Mills ◽  
Raqual Bower ◽  
Douglas Tritschler ◽  
...  
Keyword(s):  
Intraflagellar Transport ◽  
Small Subset ◽  
Dependent Manner ◽  
Microtubule Motor ◽  
Flagellar Assembly ◽  
Cilia And Flagella ◽  
The Stability ◽  
Mutant Phenotypes ◽  
Intermediate Chain

The assembly of cilia and flagella depends on the activity of two microtubule motor complexes, kinesin-2 and dynein-2/1b, but the specific functions of the different subunits are poorly defined. Here we analyze Chlamydomonas strains expressing different amounts of the dynein 1b light intermediate chain (D1bLIC). Disruption of D1bLIC alters the stability of the dynein 1b complex and reduces both the frequency and velocity of retrograde intraflagellar transport (IFT), but it does not eliminate retrograde IFT. Flagellar assembly, motility, gliding, and mating are altered in a dose-dependent manner. iTRAQ-based proteomics identifies a small subset of proteins that are significantly reduced or elevated in d1blic flagella. Transformation with D1bLIC-GFP rescues the mutant phenotypes, and D1bLIC-GFP assembles into the dynein 1b complex at wild-type levels. D1bLIC-GFP is transported with anterograde IFT particles to the flagellar tip, dissociates into smaller particles, and begins processive retrograde IFT in <2 s. These studies demonstrate the role of D1bLIC in facilitating the recycling of IFT subunits and other proteins, identify new components potentially involved in the regulation of IFT, flagellar assembly, and flagellar signaling, and provide insight into the role of D1bLIC and retrograde IFT in other organisms.

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 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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