Identification of dyneins that localize exclusively to the proximal portion of <i>Chlamydomonas</i> flagella

Yagi, Toshiki; Uematsu, Keigo; Liu, Zhongmei; Kamiya, Ritsu

doi:10.1242/jcs.045096

Cited by 121 publications

(132 citation statements)

References 49 publications

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“…In Chlamydomonas, the dynein heavy chain DHC11 (belonging to the inner dynein arm) is restricted to a short proximal section of the flagellum. However, the protein covers the whole length of short flagella undergoing assembly (78), in contrast to what was observed here for FLAM6. DHC5, another dynein heavy chain occupies only the distal segment of the flagellum and it has been proposed that it competes with DHC11 for attachment to microtubules.…”

Section: Discussioncontrasting

confidence: 88%

Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics

Subota

Julkowska

Vincensini

et al. 2014

Molecular & Cellular Proteomics

122

195

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Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that flagella severed below the basal body, and were not contaminated by other cytoskeletal structures such as the flagellar pocket collar or the adhesion zone filament. Mass spectrometry analysis identified a total of 751 proteins with high confidence, including 88% of known flagellar components. Comparison with the cell debris fraction revealed that more than half of the flagellum markers were enriched in flagella and this enrichment criterion was taken into account to identify 212 proteins not previously reported to be associated to flagella. Nine of these were experimentally validated including a 14-3-3 protein not yet reported to be associated to flagella and eight novel proteins termed FLAM (FLAgellar Member). Remarkably, they localized to five different subdomains of the flagellum. For example, FLAM6 is restricted to the proximal half of the axoneme, no matter its length. In contrast, FLAM8 is progressively accumulating at the distal tip of growing flagella and half of it still needs to be added after cell division. A combination of RNA interference and Fluorescence Recovery After Photobleaching approaches demonstrated very different dynamics from one protein to the other, but also according to the stage of construction and the age of the flagellum. Structural proteins are added to the distal tip of the elongating flagellum and exhibit slow turnover whereas membrane proteins such as the arginine kinase show rapid turnover without a detectible polarity. Molecular & Cellular

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Section: Discussioncontrasting

confidence: 88%

Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics

Subota

Julkowska

Vincensini

et al. 2014

Molecular & Cellular Proteomics

122

195

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“…To our knowledge, this is the first study to describe a well defined compartment in the proximal region of respiratory cilia. Previous studies using Chlamydomonas have described proximal axonemal localization of IDA proteins, including dynein HC DHC11, the ortholog to human ciliary dynein HC DNAH7 (35,36) and Fa2p, an ortholog to human NIMA-related kinase (NEK) family proteins (37).…”

Section: E1-e8mentioning

confidence: 99%

“…These studies also proposed a specialized role for the proximal axonemal region in bending and waveform initiation (35,36), as well as mechanosensation and regulation of intraflagellar transport at the site of flagellar assembly/autotomy (37). The Chlamydomonas b-HC gene show ambiguous BLAST homologies to three human genes: DNAH9, DNAH11, and DNAH17.…”

Section: E1-e8mentioning

confidence: 99%

DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes

Dougherty¹,

Loges²,

Klinkenbusch³

et al. 2016

Am J Respir Cell Mol Biol

118

129

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Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-offunction DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed highresolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-leftright dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.Keywords: left-right dynein; primary ciliary dyskinesia; normal ciliary ultrastructure; immunofluorescence microscopy; transmission electron microscopy Clinical RelevanceConventional transmission electron microscopy (TEM) is not diagnostic for approximately 30% of primary ciliary dyskinesia (PCD) cases because they have normal ciliary ultrastructure; DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its molecular characterization in human respiratory cilia is completely lacking. We show that DNAH11 distinctly localizes to the proximal region of respiratory cilia, independently of all previously described factors governing dynein arm assembly. TEM tomography detects a partial reduction of outer dynein arms in only the proximal region of DNAH11-deficient cilia. This helps explain why DNAH11 mutations result in normal ciliary ultrastructure and hyperkinetic ciliary beating and suggests a novel mode of axonemal assembly in respiratory cilia.

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“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 Gene duplication and gene fusion provide a molecular basis through which species-specific elaboration of regulatory networks can occur, and again work with Chlamydomonas provides examples for each mechanism. Lineage-specific duplication of dynein heavy chains gives rise to differential localisation of individual isoforms along the flagellum (Yagi et al 2009). The functional significance of proximal-and distal-specific dynein arms is not yet clear but points to a species-restricted, rather than a more widely distributed, regulatory network.…”

Section: Conservation Of the Protein Inventory In Motile Flagellamentioning

confidence: 99%

Eukaryotic Flagella: Variations in Form, Function, and Composition during Evolution

2014

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Identification of dyneins that localize exclusively to the proximal portion of Chlamydomonas flagella

Cited by 121 publications

References 49 publications

Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics

Proteomic Analysis of Intact Flagella of Procyclic Trypanosoma brucei Cells Identifies Novel Flagellar Proteins with Unique Sub-localization and Dynamics

DNAH11 Localization in the Proximal Region of Respiratory Cilia Defines Distinct Outer Dynein Arm Complexes

Eukaryotic Flagella: Variations in Form, Function, and Composition during Evolution

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