<i>Chlamydomonas</i>DIP13 and human NA14: a new class of proteins associated with microtubule structures is involved in cell division

Pfannenschmid, Frank; Wimmer, Verena C.; Rios, Rosa-Maria; Geimer, Stefan; Kröckel, Ulla; Leiherer, Andreas; Haller, Katrin; Němcová, Yvonne‬; Mages, Wolfgang

doi:10.1242/jcs.00337

Cited by 55 publications

(55 citation statements)

References 74 publications

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“…As it was previously reported for wild type DIP13 [Pfannenschmid et al, 2003], the fusion was located in the basal apparatus, when expressed moderately. High expression levels caused the formation of DIP13-GFP aggregates elsewhere in the cytoplasm, largely removed wild-type DIP13 from the basal apparatus, and significantly lowered the amount of wild-type DIP13.…”

Section: Discussionsupporting

confidence: 81%

“…In line with this observation, many cell debris were observed suggesting an increased rate of cell lysis (not shown). DIP13 mRNA accumulates after flagellar amputation suggesting a role of the protein during flagellar assembly [Pfannenschmid et al, 2003]. Flagella regeneration in the strains expressing DIP13-GFP was substantially reduced and slower than in wild type (Fig.…”

Section: Dip13mentioning

confidence: 91%

“…7Ba, Da) [Pfannenschmid et al, 2003]. In contrast, overexpression of the fusion caused the formation of DIP13-GFP aggregates in the cytoplasm (Fig.…”

Section: Dip13mentioning

confidence: 95%

“…In this study, the following antibodies and dilutions were used: anti-GFP 290 (abcam; 1:500 in IF, 1:4,000 in WB); polyclonal anti-SFA (1:200 in IF, 1:3,000 in WB); monoclonal anti-centrin (1:20-50 in IF, 1:50 in WB), affinity-purified anti-DIP13 [Pfannenschmid et al, 2003] (1:200 in IF, 1:500 in WB), anti-acetylated tubulin (clone 6-11B-1, Sigma; 1:300 in IF); polyclonal anti--tubulin (1:800 in IF). Secondary antibodies were obtained from Sigma or Dianova and used according to the manufacturer's recommendations.…”

Section: Identification and Analysis Of Transformantsmentioning

confidence: 99%

“…However, expression was not observed with the exception of DIP13-GFP (deflagellation induced protein of 13 kD), the Chlamydomonas homologue of human NA14 [Pfannenschmid et al, 2003]. This small coiled-coil protein was identified as an autoantigen in patients suffering from Sjogren's syndrome [Ramos-Morales et al, 1998] and is a component of the human centrosome [Andersen et al, 2003].…”

Section: Introductionmentioning

confidence: 99%

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GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas

Schoppmeier

Mages

Lechtreck

2005

Cell Motil. Cytoskeleton

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Green fluorescent protein (GFP) was used to analyse three proteins in the flagellar basal apparatus of C. reinhardtii: (1) Striated fiber assemblin (SFA), the major component of the striated microtubule-associated fibers; (2) Centrin, present in the nucleus basal body connectors (NBBCs) and the distal connecting fiber (dCF) between the two basal bodies; and (3) DIP13, the Chlamydomonas homologue of human autoantigen NA14. The fusions co-localized with the wild-type proteins when expressed moderately. Overexpression of centrin-GFP and DIP13-GFP resulted in the formation of large aggregates and disturbed the distribution of the respective wild-type proteins. The amount of wild-type DIP13 was significantly reduced in cells overexpressing DIP13-GFP. Moreover, the cells frequently failed to assemble full-length flagella and flagellar regeneration was delayed, indicating a role of DIP13 during flagellar assembly. In contrast, overexpression of GFP-SFA, which retained more wild-type properties than SFA-GFP, increased the size of the striated fibers without altering the cross-shaped pattern. Abnormal patterns were observed in centrin-deficient cells, suggesting that centrin is required for proper localization of SFA. Photobleaching of GFP-SFA fibers indicated that GFP-SFA in the fibers is turned over slowly. Conditionally expressed centrin-GFP was incorporated into NBBCs in regions close to the basal bodies, but underrepresented in the dCF, indicative of a different dynamic of these two centrin fibers. Bending of the NBBCs was observed in vivo during flagellar motion, indicating that the filaments are flexible. In conclusion, in Chlamydomonas GFP-tagging is a useful tool for yielding new insights into the function and properties of the analyzed proteins.

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Section: Discussionsupporting

confidence: 81%

Section: Dip13mentioning

confidence: 91%

“…7Ba, Da) [Pfannenschmid et al, 2003]. In contrast, overexpression of the fusion caused the formation of DIP13-GFP aggregates in the cytoplasm (Fig.…”

Section: Dip13mentioning

confidence: 95%

Section: Identification and Analysis Of Transformantsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas

Schoppmeier

Mages

Lechtreck

2005

Cell Motil. Cytoskeleton

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TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren's syndrome‐associated protein NA14

Aki

Funakoshi

Nishida-Kitayama

et al. 2008

Journal Cellular Physiology

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TPRA40/GPR175 is an orphan receptor whose physiological functions have not been found to date. In an attempt to generate transgenic mice that express an shRNA of TPRA40, we observed that the cell division of early mouse embryos that injected the short hairpin RNA expression vector was significantly accelerated compared with the control vector. The regulation of cell division by TPRA40 was also observed in HeLa cells. Since the C-terminal region of TPRA40 has been shown to be essential for the regulation of cell division, we performed yeast two-hybrid screening using the C-terminal region as bait. Nuclear antigen of 14 kDa (NA14), an autoantigen of Sjögren's syndrome, was identified as a binding protein to the C-terminal region of TPRA40. The binding of TPRA40 and NA14 was confirmed by GST pull-down assay and co-immunoprecipitation assay. FLAG-TPRA40 is transported from the cytosol to the plasma membrane in time-dependent manner and the translocation was inhibited by GFP-NA14DeltaN, an N-terminal deletion mutant that cannot bind to microtubules but binds to TPRA40. TPRA40DeltaC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found that the effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14DeltaN. These observations indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14.

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Use of Transcriptomic Data to Support Organelle Proteomic Analysis

Marshall

2008

Organelle Proteomics

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Genome-wide transcriptional profiling provides a rich source of data for the validation and annotation of organelle proteomic data. Organelle biogenesis is in most cases accompanied by upregulation of genes encoding organelle-specific proteins. Consequently, identification of genes whose expression correlates with organelle assembly leads to a candidate list that can be cross-checked with a preliminary organelle proteome. When proteins are found in the proteome and the corresponding genes are found to have organelle assembly-correlated expression, this greatly increases our confidence that those proteins are true components of the organelle and not contamination. Such an approach can be used to narrow down a preliminary proteomic data set and help us to focus on a smaller sub-set of proteins that are supported by transcriptomic cross-validation.

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ChlamydomonasDIP13 and human NA14: a new class of proteins associated with microtubule structures is involved in cell division

Cited by 55 publications

References 74 publications

GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas

GFP as a tool for the analysis of proteins in the flagellar basal apparatus of Chlamydomonas

TPRA40/GPR175 regulates early mouse embryogenesis through functional membrane transport by Sjögren's syndrome‐associated protein NA14

Use of Transcriptomic Data to Support Organelle Proteomic Analysis

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