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 ◽  
Mary Mirvis ◽  
Hiroaki Ishikawa ◽  
Wallace 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 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 Chlamydomonas IFT70/CrDYF-1 Is a Core Component of IFT Particle Complex B and Is Required for Flagellar Assembly

2010 ◽  
Vol 21 (15) ◽  
pp. 2696-2706 ◽  
Author(s):  
Zhen-Chuan Fan ◽  
Robert H. Behal ◽  
Stefan Geimer ◽  
Zhaohui Wang ◽  
Shana M. Williamson ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Chlamydomonas Reinhardtii ◽  
Posttranslational Modification ◽  
Model Organism ◽  
Intraflagellar Transport ◽  
Apparent Size ◽  
Model Organisms ◽  
Core Component ◽  
B Subunit ◽  
Flagellar Assembly

DYF-1 is a highly conserved protein essential for ciliogenesis in several model organisms. In Caenorhabditis elegans, DYF-1 serves as an essential activator for an anterograde motor OSM-3 of intraflagellar transport (IFT), the ciliogenesis-required motility that mediates the transport of flagellar precursors and removal of turnover products. In zebrafish and Tetrahymena DYF-1 influences the cilia tubulin posttranslational modification and may have more ubiquitous function in ciliogenesis than OSM-3. Here we address how DYF-1 biochemically interacts with the IFT machinery by using the model organism Chlamydomonas reinhardtii, in which the anterograde IFT does not depend on OSM-3. Our results show that this protein is a stoichiometric component of the IFT particle complex B and interacts directly with complex B subunit IFT46. In concurrence with the established IFT protein nomenclature, DYF-1 is also named IFT70 after the apparent size of the protein. IFT70/CrDYF-1 is essential for the function of IFT in building the flagellum because the flagella of IFT70/CrDYF-1–depleted cells were greatly shortened. Together, these results demonstrate that IFT70/CrDYF-1 is a canonical subunit of IFT particle complex B and strongly support the hypothesis that the IFT machinery has species- and tissue-specific variations with functional ramifications.

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 Length regulation of multiple flagella that self-assemble from a shared pool of components

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Thomas G Fai ◽  
Lishibanya Mohapatra ◽  
Prathitha Kar ◽  
Jane Kondev ◽  
Ariel Amir
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Green Algae ◽  
Model Organism ◽  
Size Control ◽  
Intraflagellar Transport ◽  
Canonical Model ◽  
Active Disassembly ◽  
Pool Model ◽  
Length Regulation

The single-celled green algae Chlamydomonas reinhardtii with its two flagella—microtubule-based structures of equal and constant lengths—is the canonical model organism for studying size control of organelles. Experiments have identified motor-driven transport of tubulin to the flagella tips as a key component of their length control. Here we consider a class of models whose key assumption is that proteins responsible for the intraflagellar transport (IFT) of tubulin are present in limiting amounts. We show that the limiting-pool assumption is insufficient to describe the results of severing experiments, in which a flagellum is regenerated after it has been severed. Next, we consider an extension of the limiting-pool model that incorporates proteins that depolymerize microtubules. We show that this ‘active disassembly’ model of flagellar length control explains in quantitative detail the results of severing experiments and use it to make predictions that can be tested in experiments.

scholarly journals Flagellar Length Control System: Testing a Simple Model Based on Intraflagellar Transport and Turnover

2005 ◽  
Vol 16 (1) ◽  
pp. 270-278 ◽  
Author(s):  
Wallace F. Marshall ◽  
Hongmin Qin ◽  
Mónica Rodrigo Brenni ◽  
Joel L. Rosenbaum
Keyword(s):  
Simple Model ◽  
Control Systems ◽  
Size Control ◽  
Intraflagellar Transport ◽  
Balance Point ◽  
Probe Size ◽  
Simple Model System ◽  
Length Regulation ◽  
Flagellar Assembly ◽  
Level Analysis

Flagellar length regulation provides a simple model system for addressing the general problem of organelle size control. Based on a systems-level analysis of flagellar dynamics, we have proposed a mechanism for flagellar length control in which length is set by the balance of continuous flagellar assembly and disassembly. The model proposes that the assembly rate is length dependent due to the inherent length dependence of intraflagellar transport, whereas disassembly is length independent, such that the two rates can only reach a balance point at a single length. In this report, we test this theoretical model by using three different measurements: 1) the quantity of intraflagellar transport machinery as a function of length, 2) the variation of flagellar length as a function of flagellar number, and 3) the rate of flagellar growth as a function of length. We find that the quantity of intraflagellar transport machinery is independent of length, that flagellar length is a decreasing function of flagellar number, and that flagellar growth rate in regenerating flagella depends on length and not on the time since regeneration began. These results are consistent with the balance-point model for length control. The three strategies used here are not limited to flagella and can in principle be adapted to probe size control systems for any organelle.

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 Protein arginine methyltransferases interact with intraflagellar transport particles and change location during flagellar growth and resorption

2017 ◽  
Vol 28 (9) ◽  
pp. 1208-1222 ◽  
Author(s):  
Katsutoshi Mizuno ◽  
Roger D. Sloboda
Keyword(s):  
Posttranslational Modifications ◽  
Intraflagellar Transport ◽  
Temperature Sensitive ◽  
Protein Arginine Methyltransferases ◽  
Cellular Processes ◽  
Arginine Residues ◽  
Flagellar Assembly ◽  
Flagellar Regeneration ◽  
Flagellar Proteins ◽  
Punctate Pattern

Changes in protein by posttranslational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMT 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature-sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.

scholarly journals Promoters of the dhs-21 gene encoding dicarbonyl/l-xylulose reductase in Caenorhabditis elegans

2018 ◽  
Vol 15 (2) ◽  
pp. 359-365
Author(s):  
Lê Thọ Sơn ◽  
Joohong Ahnn ◽  
Jeong Hoon Cho ◽  
Nguyễn Huy Hoàng
Keyword(s):  
Caenorhabditis Elegans ◽  
Model Organism ◽  
Biological Research ◽  
Loss Of Function ◽  
Wild Type ◽  
C Elegans ◽  
Larval Stages ◽  
Vital Functions

Dicarbonyl/L-xylulose (DCXR) was identified as a dehydrogenase. This type of enzyme was presented in various forms of lives including bacteria, fungi, plants and animals. Generally, it converts L-xylulose to xylitol in the presence of either cofactor NADH or NADPH in vitro. Previous studies reported the biochemistry properties and crystal structure but largely uncovered biological roles of DCXRs. It was impossible to dissect the functions in mice or human cells that had many DCXR homologs in their genomes. Interestingly, the wild-type Caenorhabditis elegans, a well-known model organism in biological research, has only nuclear genomic dhs-21 that encodes a unique homologous DCXR. Thus Ce.dhs-21 and the host C. elegans were relevant for investigation of the physiologically-vital functions of the DCXR. This research aimed to the expression of dhs-21 in vivo. We defined three promoters , manipulated three relative reporter-constructs that conjugated the dhs-21 gene and Green Flouresent Protein (known as GFP) one. The construct vectors were transferred into wild-type C. elegans N2 and as well as the hermaphroditic loss of function dhs-21(jh129) by microinjection. In the results, we found that the expression pattern of dhs-21 under the only p2-promoter construct was stable and similar to immunogold Electric Microscopy (EM) images. The dhs-21 gene was expressed in both sexes of at all larval stages till the deaths of worms. DHS-21 was expressed in the cytosol of the intestinal, gonad sheath and uterous seam cell (utse).

scholarly journals Intraflagellar transport particle size scales inversely with flagellar length: revisiting the balance-point length control model

2009 ◽  
Vol 187 (1) ◽  
pp. 81-89 ◽  
Author(s):  
Benjamin D. Engel ◽  
William B. Ludington ◽  
Wallace F. Marshall
Keyword(s):  
Intraflagellar Transport ◽  
Control Model ◽  
Differential Interference Contrast Microscopy ◽  
Protein Traffic ◽  
Balance Point ◽  
Interference Contrast Microscopy ◽  
Flagellar Assembly ◽  
Transport Particle ◽  
Flagellar Regeneration ◽  
Kinetics Of

The assembly and maintenance of eukaryotic flagella are regulated by intraflagellar transport (IFT), the bidirectional traffic of IFT particles (recently renamed IFT trains) within the flagellum. We previously proposed the balance-point length control model, which predicted that the frequency of train transport should decrease as a function of flagellar length, thus modulating the length-dependent flagellar assembly rate. However, this model was challenged by the differential interference contrast microscopy observation that IFT frequency is length independent. Using total internal reflection fluorescence microscopy to quantify protein traffic during the regeneration of Chlamydomonas reinhardtii flagella, we determined that anterograde IFT trains in short flagella are composed of more kinesin-associated protein and IFT27 proteins than trains in long flagella. This length-dependent remodeling of train size is consistent with the kinetics of flagellar regeneration and supports a revised balance-point model of flagellar length control in which the size of anterograde IFT trains tunes the rate of flagellar assembly.

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