The ciliary inner dynein arm, I1 dynein, is assembled in the cytoplasm and transported by IFT before axonemal docking - PubMed (original) (raw)

. 2014 Oct;71(10):573-86.

doi: 10.1002/cm.21192. Epub 2014 Oct 30.

Affiliations

The ciliary inner dynein arm, I1 dynein, is assembled in the cytoplasm and transported by IFT before axonemal docking

Rasagnya Viswanadha et al. Cytoskeleton (Hoboken). 2014 Oct.

Abstract

To determine mechanisms of assembly of ciliary dyneins, we focused on the Chlamydomonas inner dynein arm, I1 dynein, also known as dynein f. I1 dynein assembles in the cytoplasm as a 20S complex similar to the 20S I1 dynein complex isolated from the axoneme. The intermediate chain subunit, IC140 (IDA7), and heavy chains (IDA1, IDA2) are required for 20S I1 dynein preassembly in the cytoplasm. Unlike I1 dynein derived from the axoneme, the cytoplasmic 20S I1 complex will not rebind I1-deficient axonemes in vitro. To test the hypothesis that I1 dynein is transported to the distal tip of the cilia for assembly in the axoneme, we performed cytoplasmic complementation in dikaryons formed between wild-type and I1 dynein mutant cells. Rescue of I1 dynein assembly in mutant cilia occurred first at the distal tip and then proceeded toward the proximal axoneme. Notably, in contrast to other combinations, I1 dynein assembly was significantly delayed in dikaryons formed between ida7 and ida3. Furthermore, rescue of I1 dynein assembly required new protein synthesis in the ida7 × ida3 dikaryons. On the basis of the additional observations, we postulate that IDA3 is required for 20S I1 dynein transport. Cytoplasmic complementation in dikaryons using the conditional kinesin-2 mutant, fla10-1 revealed that transport of I1 dynein is dependent on kinesin-2 activity. Thus, I1 dynein complex assembly depends upon IFT for transport to the ciliary distal tip prior to docking in the axoneme.

Keywords: Chlamydomonas dikaryon zygotes; I1 dynein; axonemes; cilia; flagella.

© 2014 Wiley Periodicals, Inc.

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Figures

Figure 1

Figure 1. Schematic model depicting steps leading to I1 dynein assembly in the axoneme

We tested the hypotheses that I1 dynein preassembles in the cytoplasm and that the preassembled I1 complex is transported to the distal tip of the cilium by IFT. Based on the characterization of the ida3 mutant in this paper, we hypothesize that IDA3, a cytoplasmic protein is required for the entry or transport of I1 dynein. IDA3 could be a modifier required for I1 dynein transport or an adapter linking I1 dynein to IFT (both possibilities indicated by a “?”). The postulated barrier between the cytoplasm and the ciliary compartment is shown as black boxes.

Figure 2

Figure 2. I1 dynein assembles in the cytoplasm as a 20S complex

(A) Immunoblots of fractions from velocity sedimentation of axonemal high salt extracts (HSE) and cytoplasmic extracts (CE) were analyzed. The I1 dynein subunits IC140, IC138 and IC97 co-sediment at fraction 5 (arrows) in both axonemal high salt extracts and cytoplasmic extracts from wild-type cells. (B) Cytoplasmic extracts derived from bld1, ida1, ida3 and ida7 were fractionated by velocity sedimentation and analyzed by immunoblots using IC138 or IC140 as a marker of I1 dynein. Cytoplasmic I1 dynein from bld1 and ida3 cosediment with wild-type 20S I1 complex. The 20S I1 complex is missing in the I1 dynein heavy chain mutant ida1 and ida2 (data not shown) and in the IC140-null mutant ida7.

Figure 3

Figure 3. The ida3 mutant is not defective in docking of I1 dynein to the axonemes in vitro

(A) High salt extracts (HSE) containing I1 dynein from oda2 axonemes were reconstituted onto salt-extracted I1-deficient ida3 and ida7 axonemes in the presence of 1mM ATP. Varying ratios of axonemal I1 (HSE) to extracted axonemes (I1: Axonemes) were combined in an ATP-containing buffer, incubated on ice for 30 min and probed with IC140 as a marker for I1 dynein in immunoblots analysis. Each mixture was centrifuged to separate the supernatant (S = unbound I1 dynein) and pellet (P = bound I1 dynein) fractions. Axonemal I1 (HSE) reconstituted onto both ida3 and ida7 axonemes results in I1 dynein binding to the axonemes to saturation (P = pellet/bound I1). (B) I1 dynein-containing cytoplasmic extracts (cyt.) from oda2 were reconstituted onto ida3 and ida7 axonemes in the presence of ATP. Cytoplasmic I1 dynein (cyt.) does not bind to the axoneme as indicated by I1 presence in the supernatant (S) only.

Figure 4

Figure 4. The 20S I1 dynein assembled in the ida3 mutant cytoplasm is defective in entry to the ciliary compartment

(A) Immunoblots comparing I1 intermediate chains, IC138 and IC140, in wild-type and ida3 cytoplasmic extracts. NAB1 was used a marker and loading control (Mussgnug et al. 2005) (B) Immunoblots of ciliary (Cil), axonemal (Axo) and membrane + matrix (M+M) fractions were analyzed using antibodies to the IC138 and IC140 subunits of I1 dynein. For detection of axonemal subunits (IC69, IC140, IC138), the M+M was loaded at five times the relative amount of cilia and axonemes. Analysis showed a significant reduction in ciliary and axonemal I1 dynein in ida3 compared to wild-type. In addition, M+M fractions show that I1 dynein is absent in ida3 compared to wild-type indicating inefficient entry into the ciliary compartment. The M+M samples from WT and ida3 were also examined by immunoblots at twice the protein load (data not shown), and I1 dynein subunits were never present in ida3. The outer arm component, IC69, is present in equivalent amounts in WT and ida3 M+M and serves as a control for IFT cargo. The IC69 immunoblot also validates that the defect in ida3 is specific to I1 dynein. IFT46 serves as a positive control for M+M fractionation. CBB = Coomassie Brilliant Blue loading control.

Figure 5

Figure 5. Dikaryon rescue of I1 dynein assembly occurs from the distal tip

(A) Immunofluorescence of IC140 in ida7 x wild-type (WT) dikaryons. Cytoplasmic complementation in WT and I1-deficient dikaryons results in the rescue of I1 dynein assembly (arrows) from the distal tip to base in the ida7 mutant cilia. Scale bar = 2μm. (B) Quantification of I1 dynein staining along the cilia of I1 mutants at various time points post mating. Normalized fluorescence intensity is plotted against the distance of rescue from the tip for one rescuing mutant cilium of the WT x ida7 dikaryon shown in part A. The length of I1 assembly at the ciliary tip (vertical dotted line) increased progressively over time: 2.5 μm at 30m, 5.25 μm at 60m, and 8.75 μm at 90m. (C) Comparison of dikaryon rescue rates of I1 assembly in various mutant combinations. The length of IC140 staining at the distal end of the cilium is plotted versus time. WT x ida3 (blue circles, n = 83) and WT x ida7 (black circles, n = 29) dikaryons demonstrate progressive rescue of axonemal I1 dynein assembly with complete recovery at 90m. Dikaryons between ida3 and ida1 show this same pattern of rescue along all four cilia (black triangles, n = 59). Rescue of axonemal I1 dynein assembly is delayed in ida3 x ida7 dikaryons (red squares, n = 30), but occurs from the distal tip like other dikaryon combinations.

Figure 6

Figure 6. Dikaryon rescue of axonemal I1 assembly requires protein synthesis of IC140 and/or IDA3

(A) Immunofluorescence of IC140 in ida3 x ida7 dikaryons 60 minutes after mixing gametes. Rescue of axonemal I1 dynein assembly is seen at the distal end as in Figure 5A (arrows, top panel). In contrast, in the presence of the protein synthesis inhibitor, cyclohexamide (CHX), the recovery of I1 dynein is not seen in the cilia (dotted circle, bottom panel). Scale bar = 2μm. (B) Quantification of IC140 staining from the distal tip of I1-deficient cilia as in Figure 5B in the presence of CHX. Axonemal I1 dynein assembly occurs progressively from the distal tip to base in WT x ida3 (blue circles, n = 25), wild-type x ida7 (black circles, n = 25) and ida3 x ida1 (black triangles, n = 22) dikaryons. In contrast I1 dynein is not assembled on the axoneme in ida3 x ida7 dikaryons (red squares, n = 29).

Figure 7

Figure 7. Transport of I1 to the tip of the cilium requires kinesin-2

(A) Diagram of dikaryon rescue experimental design using the temperature sensitive mutant, fla10-1. (B) Immunofluorescence of IC140 in fla10-1 x ida3; fla10-1 dikaryons 60 minutes after mixing gametes. At the permissive temperature (21°C), recovery of axonemal I1 dynein assembly occurs from the distal end of ida3 cilia (arrows). At the restrictive temperature (32°C), I1 dynein assembly on the axoneme is not seen in ida3; fla10-1 cilia (dotted circles). Scale bar = 2μm. (C) Quantification of IC140 staining at the distal end of I1-deficient cilia at permissive (21°C) and restrictive (32°C) temperatures. Axonemal I1 dynein assembly occurs progressively from the distal tip to base in wild-type x ida3 (n = 19) and fla10-1 x ida3 (black diamonds and black circles, n = 39) dikaryons at both permissive and restrictive temperatures. While I1 assembly at the distal tip is seen at the permissive temperature in fla10-1 x ida3; fla10-1 dikaryons (blue circles, n = 52), I1 assembly at the tip does not occur at restrictive temperature (red circles, n = 19). As a control, ida3; fla10-1 x ida3 dikaryons were tested for a lack of I1 axonemal assembly at both temperatures (black dotted triangles and squares, n = 46).

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