FHIP and FTS proteins are critical for dynein-mediated transport of early endosomes in Aspergillus - PubMed (original) (raw)
FHIP and FTS proteins are critical for dynein-mediated transport of early endosomes in Aspergillus
Xuanli Yao et al. Mol Biol Cell. 2014.
Abstract
The minus end-directed microtubule motor cytoplasmic dynein transports various cellular cargoes, including early endosomes, but how dynein binds to its cargo remains unclear. Recently fungal Hook homologues were found to link dynein to early endosomes for their transport. Here we identified FhipA in Aspergillus nidulans as a key player for HookA (A. nidulans Hook) function via a genome-wide screen for mutants defective in early-endosome distribution. The human homologue of FhipA, FHIP, is a protein in the previously discovered FTS/Hook/FHIP (FHF) complex, which contains, besides FHIP and Hook proteins, Fused Toes (FTS). Although this complex was not previously shown to be involved in dynein-mediated transport, we show here that loss of either FhipA or FtsA (A. nidulans FTS homologue) disrupts HookA-early endosome association and inhibits early endosome movement. Both FhipA and FtsA associate with early endosomes, and interestingly, while FtsA-early endosome association requires FhipA and HookA, FhipA-early endosome association is independent of HookA and FtsA. Thus FhipA is more directly linked to early endosomes than HookA and FtsA. However, in the absence of HookA or FtsA, FhipA protein level is significantly reduced. Our results indicate that all three proteins in the FtsA/HookA/FhipA complex are important for dynein-mediated early endosome movement.
© 2014 Yao et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
Figures
FIGURE 1:
Phenotypic analysis of the _eedB_3/_fhipA_Q367STOP mutant. (A) Colony phenotype of the _eedB_3 mutant in comparison to that of a wild-type control strain, the _eedA_1 mutant (_hookA_L150P, E151K), and the ∆p25 mutant. (B) Microscopic images showing the distributions of mCherry-RabA–labeled early endosomes (mCherry-RabA) and GFP-labeled dynein heavy chain (GFP-dynein HC) in wild type and the _eedB_3 mutant. The same cells are shown for both the mCherry-RabA and GFP-dynein HC images. In the _eedB_3 mutant, dynein-HC proteins form normal comet-like structures, representing their microtubule plus end accumulation. However, abnormal accumulation of the mCherry-RabA signals was observed at ∼71% of the hyphal tips of the _eedB_3 mutant (n = 125), whereas none of the wild-type cells showed the same accumulation (n = 100). (C) Images of nuclei stained by a DNA dye, DAPI, in wild type and the _eedB_3 mutant, indicating that nuclear distribution is normal in the _eedB_3 mutant. (D) Colony phenotypes of the _eedB_3 mutant and the strain in which the wild-type fhipA gene has rescued the mutant. (E) A microscopic image showing the distributions of mCherry-RabA–labeled early endosomes (mCherry-RabA) in the _eedB_3 mutant rescued by the wild-type fhipA gene. In this rescued strain, none of the hyphal tips show the abnormal accumulation of mCherry-RabA signals (n = 100). (F) Colony phenotypes of a wild-type strain and the strain in which the _fhipA_Q367STOP mutation was introduced to the wild-type genome by transformation. (G) A microscopic image showing the distributions of mCherry-RabA–labeled early endosomes (mCherry-RabA) in the _fhipA_Q37STOP mutant. In this strain, ∼73% of the hyphal tips show the abnormal accumulation of mCherry-RabA signals (n = 100). Bar, 5 μm.
FIGURE 2:
Phenotypes of the ∆fhipA and ∆ftsA mutants. (A) Colony phenotypes of the ∆fhipA and ∆ftsA mutants in comparison to that of a wild-type control strain. (B) Distributions of mCherry-RabA–labeled early endosomes in the ∆fhipA and ∆ftsA mutants. About 79% of the ∆fhipA and 57% of the ∆ftsA hyphal tips exhibited abnormal accumulation of mCherry-RabA signals, whereas none of wild-type hyphal tips shows this accumulation. GFP-dynein HC signals in the same cells are shown (bottom) to indicate that dynein localization is normal in these cells. (C) Images of DAPI-stained nuclei in the ∆fhipA and ∆ftsA mutants. The pattern of nuclear distribution in the mutants is normal, as none of the mutant cells shows any cluster of four or more nuclei when grown under the same condition that allow us to see the hyphal-tip mCherry-RabA accumulation. Bar, 5 μm.
FIGURE 3:
The ∆fhipA and ∆ftsA mutants exhibit a defect in HookA–early endosome interaction. (A) HookA-GFP signals in the ∆p25, ∆fhipA, and ∆ftsA mutants (top), along with the mCherry-RabA signals in the same cells (bottom). In contrast to the hyphal-tip accumulation of HookA-GFP signals in the ∆p25 mutant, representing colocalization with early endosomes, HookA-GFP signals in the ∆fhipA and ∆ftsA mutants do not colocalize with the hyphal tip–accumulated early endosomes. Bar, 5 μm. (B) Because the GFP signals are largely diffuse in the cytoplasm of the ∆fhipA and ∆ftsA mutants, a Western blot is shown to demonstrate that the HookA proteins are expressed and stable in both mutants. The negative control protein sample was from a strain without HookA-GFP.
FIGURE 4:
FhipA-GFP and FtsA-GFP colocalize with early endosomes. (A) FhipA-GFP colocalizes with early endosomes. Top, images of FhipA-GFP and mCherry-RabA in the same wild-type cell. Arrows indicate the FhipA-GFP signals that appear to colocalize with the mCherry-RabA signals. Bottom, images of FhipA-GFP and mCherry-RabA in the same ∆p25 mutant cell. Note that in the ∆p25 mutant, FhipA-GFP signals are concentrated at the hyphal tip, where early endosomes accumulate. (B) FtsA-GFP colocalizes with early endosomes. Top, images of FtsA-GFP and mCherry-RabA in the same wild-type cell. Arrows indicate the FtsA-GFP signals that appear to colocalize with the mCherry-RabA signals. Bottom, images of FtsA-GFP and mCherry-RabA in the same ∆p25 mutant cell. Note that in the ∆p25 mutant, FtsA-GFP signals are concentrated at the hyphal tip, where early endosomes accumulate. Bar, 5 μm.
FIGURE 5:
FtsA-GFP does not colocalize with early endosomes in the ∆hookA and ∆fhipA mutants. (A) FtsA-GFP does not colocalize with mCherry-RabA–labeled early endosomes in the ∆hookA mutant. (B) FtsA-GFP does not colocalize with mCherry-RabA–labeled early endosomes in the ∆fhipA mutant. Bar, 5 μm. (C) Because the FtsA-GFP signals are largely diffuse in the cytoplasm, a Western blot is shown to demonstrate that the FtsA proteins are expressed and stable in the ∆hookA and ∆fhipA mutants. The negative control protein sample was from a strain without FtsA-GFP. Bands above the indicated FtsA-GFP band represent proteins that cross-reacted with the anti-GFP antibody.
FIGURE 6:
FhipA localization/stability in the ∆hookA and ∆ftsA mutants and FhipA–HookA interaction in the ∆ftsA mutant. (A) FhipA-GFP colocalizes with mCherry-RabA–labeled early endosomes in the ∆hookA mutant. (B) FhipA-GFP colocalizes with mCherry-RabA–labeled early endosomes in the ∆ftsA mutant. Bar, 5 μm. (C) A Western blot showing the FhipA-GFP protein level in the ∆hookA and ∆ftsA mutants. (D) Quantification of the Western results showing that the FhipA-GFP protein level is decreased in the ∆hookA and ∆ftsA mutants (n = 3, p < 0.001 for both mutants). This analysis was done by measuring protein signal intensity on the western blots in relation to protein loading as indicated by Ponceau S staining. The ratio of the FhipA-GFP band intensity to the Ponceau S–stained loading control was calculated. Values presented are relative to the wild-type value, which is set at 1. The mean ± SD values for the Δ_hookA_ and ∆ftsA mutants are shown. (E) A Western blot showing that HookA-GFP is able to pull down FhipA-S from wild-type extract, as well as from ∆ftsA mutant extract. The amount of FhipA-S pulled down is significantly lower from the ∆ftsA extract, which is most likely due to a decrease in the protein level of FhipA-S in the ∆ftsA total extract. A Western blot of HookA-GFP in the ∆ftsA and wild-type extracts is presented as loading control. (F) Quantification of the Western results, showing that the amount of FhipA-S pulled down with HookA-GFP is significantly decreased in the ∆ftsA mutant (n = 3, p < 0.005). Values presented are relative to the wild type value, which is set at 1. The mean ± SD values for the ∆ftsA mutant are shown.
FIGURE 7:
Model showing that the FtsA/HookA/FhipA complex is required for linking HookA to early endosomes. HookA (blue, depicted as a dimer) interacts with dynein–dynactin complexes, as revealed by a previous study (Zhang et al., 2014). The C-terminus of HookA most likely interacts with FtsA (brown) directly, based on the yeast two-hybrid data on human FTS interaction with the C-termini of Hook1 and Hook3 (Xu et al., 2008). FhipA (red) is able to interact with early endosome in the absence of either HookA or FtsA (brown). However, FhipA protein integrity/stability depends on both HookA and FtsA. FhipA-HookA interaction is most likely direct, based on the pull-down data showing this interaction in the absence of FtsA (Figure 6E). The FhipA–HookA interaction may involve the C-terminus of HookA, which is the domain mediating HookA-early endosome interaction (Zhang et al., 2014). In addition, based on data from U. maydis, a region of Hook upstream of the C-terminal early endosome–binding domain may enhance Hook-FTS-FHIP interactions (Bielska et al., 2014).
References
- Abenza JF, Pantazopoulou A, Rodriguez JM, Galindo A, Peñalva MA. Long-distance movement of Aspergillus nidulans early endosomes on microtubule tracks. Traffic. 2009;10:57–75. - PubMed
- Balderhaar HJ, Ungermann C. CORVET and HOPS tethering complexes—coordinators of endosome and lysosome fusion. J Cell Sci. 2013;126:1307–1316. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources