IC97 Is a Novel Intermediate Chain of I1 Dynein That Interacts with Tubulin and Regulates Interdoublet Sliding

Wirschell, Maureen; Chun, Yang; Yang, Pinfen; Fox, Laura A.; Yanagisawa, Haruaki; Kamiya, Ritsu; Witman, George B.; Porter, Mary E.; Sale, Winfield S.

doi:10.1091/mbc.e09-04-0276

Cited by 50 publications

(55 citation statements)

References 72 publications

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“…Our 2DE analysis did not identify additional I1 components, but polypeptides larger than ∼150 kDa are difficult to resolve by this technique. Consistent with previous studies, we identified only a single isoform of IC97 (20,34) (Fig. 6 and Fig.…”

Section: I1 Intermediate Chain Assembly Into the Axoneme Of Wt And I1supporting

confidence: 92%

“…1). A number of I1 components have been identified biochemically, including three intermediate chains (IC140, IC138, and IC97), several light chains (LC8, LC7a, LC7b, Tctex1, and Tctex2b), and the accessory protein FAP120 (11,13,16,(23)(24)(25)(28)(29)(30)(31)(32)(33)(34). A recent study of an IC138 null mutant (bop5-2) revealed that IC138 is required for assembly of an "IC138 subcomplex" consisting of IC138, IC97, LC7b, and FAP120 (21).…”

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confidence: 99%

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Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein

Heuser

Barber

Lin

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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133

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Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility.dynein f | flagella C ilia and flagella are highly conserved organelles with roles in cellular movement and signal transduction. In humans, defects in cilia can lead to a number of diseases, such as polycystic kidney disease and primary ciliary dyskinesia (1, 2). The axoneme core of most motile cilia and flagella consists of nine doublet microtubules (DMTs) surrounding two single microtubules (MTs) known as the central pair complex (CPC) (Fig. 1). DMTs are highly periodic with a 96-nm-long unit that repeats along the MT length. They are decorated with two rows of dynein motors, the inner dynein arms (IDAs) and the outer dynein arms (ODAs), which drive MT sliding and axoneme bending (3). Generating the diverse ciliary and flagellar waveforms requires the precise coordination of the activity of thousands of dynein motors within a single organelle (4).The primary signaling pathway known to regulate dynein function in axonemes involves signals traveling from the CPC through radial spokes (RSs) to the IDAs and ODAs (reviewed in 5, 6). Many CPC and RS mutants are paralyzed (7). However, MT sliding can be restored to WT levels in isolated CPC/RS mutant axonemes using protein kinase inhibitors (8, 9). These observations, together with biochemical evidence of phosphorylation of dynein subunits, have implicated the dyneins as a major signaling target of the CPC/RS signaling pathway (10, 11). However, the regulatory mechanisms and physical interactions that participate in signal transduction to the dynein targets are not well understood.Isolated axonemes require only ATP for reactivation, suggesting that direct interactions between the dyneins and their regulators are physically built into the axoneme (4). Therefore, studies that visualize axonemal structures and their connections at high resolution can provide ...

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Section: I1 Intermediate Chain Assembly Into the Axoneme Of Wt And I1supporting

confidence: 92%

mentioning

confidence: 99%

Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein

Heuser

Barber

Lin

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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133

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“…As noted above, null mutants and RNAi knockdowns are valuable but provide limited information about molecular mechanisms. Recent elegant studies on inner-arm and outer-arm dynein subunits in Chlamydomonas have made important steps toward addressing this issue (16,32,41,44), but investigating specific functions and molecular mechanisms of flagellum proteins remains a major challenge. The inducible system that we have developed in T. brucei offers an opportunity for systematic mutational analysis of virtually any flagellar protein in a background where the endogenous protein is reduced or absent.…”

Section: Discussionmentioning

confidence: 99%

Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei

2011

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The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.

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“…In contrast, the light intermediate chain (DLIC) of the retrograde IFT motor CD1b (25,27), IC140 in I1 (28), p28 in single-headed IDAs (29), and IC78, LC6, and LC9 of the ODA (30) were not obviously affected. Interestingly, IC97 (also called IC110) in I1 (31) was not detectable, indicating IC97 is the missing 110-kDa IDA component in pf5 (20) and is not essential for the assembly of I1.…”

Section: Pf5 Mutant Displays Multiple Flagellar Defects Because Of Amentioning

confidence: 99%

“…The actual target proteins of LC8 in I1 have not been determined. However, neither IC138, a candidate scaffold molecule with WD repeats, nor IC97, which is most sensitive to the LC8 mutations, is required for I1 assembly (31,35). Instead, both are implicated in phosphoregulation of I1 (31,35,36).…”

Section: Lc8 Promotes the Formation Of The Retrograde Ift Motor For Tmentioning

confidence: 99%

Novel LC8 Mutations Have Disparate Effects on the Assembly and Stability of Flagellar Complexes

Yang

Chun

Wirschell

et al. 2009

Journal of Biological Chemistry

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LC8 functions as a dimer crucial for a variety of molecular motors and non-motor complexes. Emerging models, founded on structural studies, suggest that the LC8 dimer promotes the stability and refolding of dimeric target proteins in molecular complexes, and its interactions with selective target proteins, including dynein subunits, is regulated by LC8 phosphorylation, which is proposed to prevent LC8 dimerization. To test these hypotheses in vivo, we determine the impacts of two new LC8 mutations on the assembly and stability of defined LC8-containing complexes in Chlamydomonas flagella. The three types of dyneins and the radial spoke are disparately affected by dimeric LC8 with a C-terminal extension. The defects include the absence of specific subunits, complex instability, and reduced incorporation into the axonemal super complex. Surprisingly, a phosphomimetic LC8 mutation, which is largely monomeric in vitro, is still dimeric in vivo and does not significantly change flagellar generation and motility. The differential defects in these flagellar complexes support the structural model and indicate that modulation of target proteins by LC8 leads to the proper assembly of complexes and ultimately higher level complexes. Furthermore, the ability of flagellar complexes to incorporate the phosphomimetic LC8 protein and the modest defects observed in the phosphomimetic LC8 mutant suggest that LC8 phosphorylation is not an effective mechanism for regulating molecular complexes.

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IC97 Is a Novel Intermediate Chain of I1 Dynein That Interacts with Tubulin and Regulates Interdoublet Sliding

Cited by 50 publications

References 72 publications

Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein

Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein

Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei

Novel LC8 Mutations Have Disparate Effects on the Assembly and Stability of Flagellar Complexes

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