Mutant Kinesin-2 Motor Subunits Increase Chromosome Loss

Miller, Mark S.; Esparza, Jessica M.; Lippa, Andrew M.; Lux, Fordyce G.; Cole, Douglas G.; Dutcher, Susan K.

doi:10.1091/mbc.e05-05-0404

Cited by 46 publications

(29 citation statements)

References 53 publications

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“…In fla10 null mutants, flagellar structures are never observed (22). Similar to KRP95, mutations in Chlamydomonas KAP (fla3-1) and KRP85 (fla-8) also cause defective flagellar assembly and maintenance (23)(24)(25). In agreement with the Chlamydomonas data, studies in numerous other models, including sea urchins, Tetrahymena and mice all show that kinesin-II is required for normal cilia formation and function (26)(27)(28)(29)(30).…”

Section: Kinesin 2: the Anterograde Ift Motorsupporting

confidence: 56%

“…Thirdly, in cultured mouse NIH3T3 fibroblast cells, dominant negative expression of a KIF3B fragment (lacks C-terminal 33% of KIF3B) fused to GFP caused chromosomal aneuploidy and abnormal spindle formation (138). Consistent with these data are findings which show that kinesin-II subunits associate with the mitotic machinery (centrioles, mitotic spindles and cytokinesis mid-body) (137,(139)(140)(141), and redistribute to the nuclear compartment during mitosis (22,25,29). However, in contrast to the above data, knockout or knockdown of kinesin-II function in Chlamydomonas and sea-urchins does not appear to specifically affect cell cycle progression (22,25,29).…”

Section: Ift and The Cell Cyclementioning

confidence: 72%

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Intraflagellar transport: from molecular characterisation to mechanism

Blacque

Cevik²,

Kaplan³

2008

Front Biosci

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Research from a wide range of model systems such as Chlamydomonas, C. elegans and mice have shown that intraflagellar transport (IFT) is a bidirectional motility of large protein complexes along cilia and flagella that is essential for building and maintaining these organelles. Since its discovery in 1993, much progress has been made in uncovering the molecular and functional basis of IFT. Presently, many components of the core IFT machinery are known, including the anterograde kinesin 2 motor(s), the IFT-dynein retrograde motor and the collection of at least 17 proteins that makes up the IFT particle. Most significantly, discoveries linking IFT to polycystic kidney disease and other developmental phenotypes have broadened the context of IFT research by demonstrating that primary cilia and IFT are required for processes such as kidney tubule and retinal tissue development, limb bud morphogenesis and organ patterning. Central to the functional basis of IFT is its ability to traffic various ciliary protein cargos, which include structural ciliary subunits, as well as non-structural proteins such as transmembrane channels/receptors and sensory signalling molecules. Indeed, exciting data over the past 3-4 years, linking IFT and primary cilia to developmental and growth factor signalling, as well as the cell cycle, indicates that the current repertoire of IFT cargos is likely to expand. Here we present a comprehensive review of IFT, with particular emphasis on its molecular composition and mechanism of action.

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Section: Kinesin 2: the Anterograde Ift Motorsupporting

confidence: 56%

Section: Ift and The Cell Cyclementioning

confidence: 72%

Intraflagellar transport: from molecular characterisation to mechanism

Blacque

Cevik²,

Kaplan³

2008

Front Biosci

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“…Breakpoints in these reads define the cassette insertion site (orange vertical line) in the genome. restrictive temperature (Vashishtha et al, 1996;Miller et al, 2005). Our identification of 1A4 provides an additional mutant allele of FLA10.…”

Section: Validation Of the Insertional Genes As The Causes Of Mutant mentioning

confidence: 73%

MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements

2018

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Insertional mutagenesis, in which a piece of exogenous DNA is randomly integrated into the genomic DNA of the recipient cell, is a useful method to generate new mutants with phenotypes of interest. The unicellular green alga Chlamydomonas reinhardtii is an outstanding model for studying many biological processes. We developed a new computational algorithm, MAPINS (mapping insertions), to efficiently identify insertion sites created by the integration of an APHVIII (aminoglycoside 3'-phosphotransferase VIII) cassette that confers paromomycin resistance. Using whole-genome sequencing (WGS) data, this method eliminates the need for genomic DNA manipulation and retains all the sequencing information provided by paired-end sequencing. We experimentally verified 38 insertion sites out of 41 sites (93%) identified by MAPINS from 20 paromomycin-resistant strains. Using meiotic analysis of 18 of these strains, we identified insertion sites that completely cosegregate with paromomycin resistance. In six of the seven strains with a mutant phenotype, we demonstrated complete cosegregation of the mutant phenotype and the verified insertion site. In addition, we provide direct evidence of complex rearrangements of genomic DNA in five strains, three of which involve the APHVIII insertion site. We suggest that strains obtained by insertional mutagenesis are more complicated than expected from previous analyses in Chlamydomonas. To map the locations of some complex insertions, we designed 49 molecular markers based on differences identified via WGS between wild-type strains CC-124 and CC-125. Overall, MAPINS provides a low-cost, efficient method to characterize insertional mutants in Chlamydomonas.

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“…In fla10-14 , the glutamic acid is changed to lysine but in fla18 (now renamed fla10-16 ) it becomes a glycine. The two alleles have different kinetics of flagellar loss [8]; the E 24 K allele takes over 12 hours to see loss of 50% of the flagella compared to the E 24 G allele that takes only 6 hours to see complete loss (Figure?2A). This glutamic acid is conserved in all kinesin-2 molecules across the ciliated phylogenetic tree (n = 75, data not shown).…”

Section: Discussionmentioning

confidence: 99%

New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas

Lin

Nauman

Albee

et al. 2013

Cilia

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BackgroundThe building of a cilium or flagellum requires molecular motors and associated proteins that allow the relocation of proteins from the cell body to the distal end and the return of proteins to the cell body in a process termed intraflagellar transport (IFT). IFT trains are carried out by kinesin and back to the cell body by dynein.MethodsWe used whole genome sequencing to identify the causative mutations for two temperature-sensitive flagellar assembly mutants in Chlamydomonas and validated the changes using reversion analysis. We examined the effect of these mutations on the localization of IFT81, an IFT complex B protein, the cytoplasmic dynein heavy chain (DHC1b), and the dynein light intermediate chain (D1bLIC).ResultsThe strains, fla18 and fla24, have mutations in kinesin-2 and cytoplasmic dynein, respectively. The fla18 mutation alters the same glutamic acid (E24G) mutated in the fla10-14 allele (E24K). The fla18 strain loses flagella at 32?C more rapidly than the E24K allele but less rapidly than the fla10-1 allele. The fla18 mutant loses its flagella by detachment rather than by shortening. The fla24 mutation falls in cytoplasmic dynein and changes a completely conserved amino acid (L3243P) in an alpha helix in the AAA5 domain. The fla24 mutant loses its flagella by shortening within 6 hours at 32?C. DHC1b protein is reduced by 18-fold and D1bLIC is reduced by 16-fold at 21?C compared to wild-type cells. We identified two pseudorevertants (L3243S and L3243R), which remain flagellated at 32?C. Although fla24 cells assemble full-length flagella at 21?C, IFT81 protein localization is dramatically altered. Instead of localizing at the basal body and along the flagella, IFT81 is concentrated at the proximal end of the flagella. The pseudorevertants show wild-type IFT81 localization at 21?C, but proximal end localization of IFT81 at 32?C.ConclusionsThe change in the AAA5 domain of the cytoplasmic dynein in fla24 may block the recycling of IFT trains after retrograde transport. It is clear that different alleles in the flagellar motors reveal different functions and roles. Multiple alleles will be important for understanding structure-function relationships.

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Mutant Kinesin-2 Motor Subunits Increase Chromosome Loss

Cited by 46 publications

References 53 publications

Intraflagellar transport: from molecular characterisation to mechanism

Intraflagellar transport: from molecular characterisation to mechanism

MAPINS, a Highly Efficient Detection Method That Identifies Insertional Mutations and Complex DNA Rearrangements

New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas

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