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1.
iScience ; 24(4): 102354, 2021 Apr 23.
Article in English | MEDLINE | ID: mdl-33898946

ABSTRACT

Any proposed mechanism for organelle size control should be able to account not only for average size but also for the variation in size. We analyzed cell-to-cell variation and within-cell variation of length for the two flagella in Chlamydomonas, finding that cell-to-cell variation is dominated by cell size, whereas within-cell variation results from dynamic fluctuations. Fluctuation analysis suggests tubulin assembly is not directly coupled with intraflagellar transport (IFT) and that the observed length fluctuations reflect tubulin assembly and disassembly events involving large numbers of tubulin dimers. Length variation is increased in long-flagella mutants, an effect consistent with theoretical models for flagellar length regulation. Cells with unequal flagellar lengths show impaired swimming but improved gliding, raising the possibility that cells have evolved mechanisms to tune biological noise in flagellar length. Analysis of noise at the level of organelle size provides a way to probe the mechanisms determining cell geometry.

3.
Elife ; 3: e01566, 2014 Jan 01.
Article in English | MEDLINE | ID: mdl-24596149

ABSTRACT

Cilia/flagella are assembled and maintained by the process of intraflagellar transport (IFT), a highly conserved mechanism involving more than 20 IFT proteins. However, the functions of individual IFT proteins are mostly unclear. To help address this issue, we focused on a putative IFT protein TTC26/DYF13. Using live imaging and biochemical approaches we show that TTC26/DYF13 is an IFT complex B protein in mammalian cells and Chlamydomonas reinhardtii. Knockdown of TTC26/DYF13 in zebrafish embryos or mutation of TTC26/DYF13 in C. reinhardtii, produced short cilia with abnormal motility. Surprisingly, IFT particle assembly and speed were normal in dyf13 mutant flagella, unlike in other IFT complex B mutants. Proteomic and biochemical analyses indicated a particular set of proteins involved in motility was specifically depleted in the dyf13 mutant. These results support the concept that different IFT proteins are responsible for different cargo subsets, providing a possible explanation for the complexity of the IFT machinery. DOI: http://dx.doi.org/10.7554/eLife.01566.001.


Subject(s)
Algal Proteins/metabolism , Carrier Proteins/metabolism , Cell Movement , Chlamydomonas reinhardtii/metabolism , Cilia/metabolism , Flagella/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Plant Proteins/metabolism , Zebrafish Proteins/metabolism , Algal Proteins/genetics , Animals , Carrier Proteins/genetics , Cell Line , Chlamydomonas reinhardtii/genetics , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Genotype , Intracellular Signaling Peptides and Proteins/genetics , Mice , Mutation , Phenotype , Plant Proteins/genetics , Protein Transport , Transfection , Zebrafish , Zebrafish Proteins/genetics
4.
Proc Natl Acad Sci U S A ; 110(10): 3925-30, 2013 Mar 05.
Article in English | MEDLINE | ID: mdl-23431147

ABSTRACT

Cilia and flagella are microtubule-based organelles that protrude from the cell body. Ciliary assembly requires intraflagellar transport (IFT), a motile system that delivers cargo from the cell body to the flagellar tip for assembly. The process controlling injections of IFT proteins into the flagellar compartment is, therefore, crucial to ciliogenesis. Extensive biochemical and genetic analyses have determined the molecular machinery of IFT, but these studies do not explain what regulates IFT injection rate. Here, we provide evidence that IFT injections result from avalanche-like releases of accumulated IFT material at the flagellar base and that the key regulated feature of length control is the recruitment of IFT material to the flagellar base. We used total internal reflection fluorescence microscopy of IFT proteins in live cells to quantify the size and frequency of injections over time. The injection dynamics reveal a power-law tailed distribution of injection event sizes and a negative correlation between injection size and frequency, as well as rich behaviors such as quasiperiodicity, bursting, and long-memory effects tied to the size of the localized load of IFT material awaiting injection at the flagellar base, collectively indicating that IFT injection dynamics result from avalanche-like behavior. Computational models based on avalanching recapitulate observed IFT dynamics, and we further show that the flagellar Ras-related nuclear protein (Ran) guanosine 5'-triphosphate (GTP) gradient can in theory act as a flagellar length sensor to regulate this localized accumulation of IFT. These results demonstrate that a self-organizing, physical mechanism can control a biochemically complex intracellular transport pathway.


Subject(s)
Chlamydomonas reinhardtii/physiology , Cilia/physiology , Biological Transport, Active , Chlamydomonas reinhardtii/genetics , Flagella/physiology , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Kinesins/genetics , Kinesins/metabolism , Microscopy, Fluorescence , Microscopy, Video , Models, Biological , Plant Proteins/genetics , Plant Proteins/metabolism
5.
J Cell Biol ; 199(1): 151-67, 2012 Oct 01.
Article in English | MEDLINE | ID: mdl-23027906

ABSTRACT

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.


Subject(s)
Flagella/metabolism , Biological Transport , Cells, Cultured , Chlamydomonas reinhardtii/cytology , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Cloning, Molecular , Dyneins/genetics , Dyneins/isolation & purification , Dyneins/metabolism , Flagella/genetics , Kinetics , Mutation , Temperature
6.
Cytoskeleton (Hoboken) ; 67(8): 504-18, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20540087

ABSTRACT

The functional role of centrioles or basal bodies in mitotic spindle assembly and function is currently unclear. Although supernumerary centrioles have been associated with multipolar spindles in cancer cells, suggesting centriole number might dictate spindle polarity, bipolar spindles are able to assemble in the complete absence of centrioles, suggesting a level of centriole-independence in the spindle assembly pathway. In this report we perturb centriole number using mutations in Chlamydomonas reinhardtii, and measure the response of the mitotic spindle to these perturbations in centriole number. Although altered centriole number increased the frequency of monopolar and multipolar spindles, the majority of spindles remained bipolar regardless of the centriole number. But even when spindles were bipolar, abnormal centriole numbers led to asymmetries in tubulin distribution, half-spindle length and spindle pole focus. Half spindle length correlated directly with number of centrioles at a pole, such that an imbalance in centriole number between the two poles of a bipolar spindle correlated with increased asymmetry between half spindle lengths. These results are consistent with centrioles playing an active role in regulating mitotic spindle length. Mutants with centriole number alteration also show increased cytokinesis defects, but these do not correlate with centriole number in the dividing cell and may therefore reflect downstream consequences of defects in preceding cell divisions.


Subject(s)
Centrioles/ultrastructure , Chlamydomonas reinhardtii/ultrastructure , Spindle Apparatus/ultrastructure , Centrioles/physiology , Spindle Apparatus/physiology
7.
Int Rev Cytol ; 260: 175-212, 2007.
Article in English | MEDLINE | ID: mdl-17482906

ABSTRACT

A fundamental unsolved question in cell biology is how the cell controls the size of its organelles. Cilia and flagella are an ideal test case to study the mechanism of organelle size control, because their size is easily measured and can be represented by a single number-length. Moreover, the involvement of cilia in many developmental and physiological processes suggests an understanding of their size control system is critical for understanding ciliary diseases, many of which (e.g., autosomal recessive polycystic kidney disease) are known to involve abnormally short cilia. The flagella of the model organism Chlamydomonas reinhardtii provide the best genetic and cell-biological system to study length control of cilia. Studies in this organism using genetics, biochemistry, imaging, and mathematical modeling have revealed many genes involved in length control of cilia and flagella, and have suggested several testable models for length regulation.


Subject(s)
Chlamydomonas reinhardtii/cytology , Flagella , Animals , Biological Transport/physiology , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/physiology , Cilia/physiology , Cilia/ultrastructure , Disease Models, Animal , Feedback, Physiological , Flagella/physiology , Flagella/ultrastructure , Genotype , Humans , Nanotechnology , Phenotype , Regeneration , Signal Transduction/physiology
8.
Curr Biol ; 14(23): R992-3, 2004 Dec 14.
Article in English | MEDLINE | ID: mdl-15589146

ABSTRACT

Eukaryotic flagella produce a swimming force by coordinating thousands of dynein motor proteins. Recent work provides new clues into how this coordination is achieved.


Subject(s)
Dyneins/physiology , Flagella/physiology , Molecular Motor Proteins/physiology , Signal Transduction/physiology , Flagella/chemistry , Microtubules/physiology , Models, Biological
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