Accumulation of cytoplasmic dynein and dynactin at microtubule plus ends in Aspergillus nidulans is kinesin dependent

Abstract

The mechanism(s) by which microtubule plus-end tracking proteins are targeted is unknown. In the filamentous fungus Aspergillus nidulans, both cytoplasmic dynein and NUDF, the homolog of the LIS1 protein, localize to microtubule plus ends as comet-like structures. Herein, we show that NUDM, the p150 subunit of dynactin, also forms dynamic comet-like structures at microtubule plus ends. By examining proteins tagged with green fluorescent protein in different loss-of-function mutants, we demonstrate that dynactin and cytoplasmic dynein require each other for microtubule plus-end accumulation, and the presence of cytoplasmic dynein is also important for NUDF's plus-end accumulation. Interestingly, deletion of NUDF increases the overall accumulation of dynein and dynactin at plus ends, suggesting that NUDF may facilitate minus-end-directed dynein movement. Finally, we demonstrate that a conventional kinesin, KINA, is required for the microtubule plus-end accumulation of cytoplasmic dynein and dynactin, but not of NUDF.

Figure 1

(A) Dynamics of GFP-NUDM dynactin comets in vivo. Bar, ∼5 μm. (B) Immunostaining of GFP-NUDM and microtubules. This image resulted from a merge of a pseudocolored microtubule image (red) and a GFP-NUDM image (green).

Figure 2

Images of GFP-NUDM dynactin in wild-type (GFP-NUDM) and the ΔnudA mutant (GFP-NUDM/ΔnudA) cells, GFP-TUBA (α tubulin) in the ΔnudA mutant (GFP-TUBA/ΔnudA), and GFP-NUDA dynein heavy chain in wild-type (GFP-NUDA) and the nudM116 mutant (GFP-NUDA/nudM116) cells.

Figure 3

(A) Images of GFP-NUDF (LIS1-like protein) in wild-type and the ΔnudA mutant cells. (B) Western blot analysis on the level of GFP-NUDF in protein extract from the wild-type and the ΔnudA mutant cells. A monoclonal anti-GFP antibody (BD Biosciences Clontech) was used at 1/500. The level of protein loading was shown by Ponceau S staining of the membrane.

Figure 4

Images of GFP-NUDA (dynein HC), GFP-NUDI (dynein IC), and GFP-NUDM (p150 dynactin) in wild-type and the ΔnudF mutant cells.

Figure 5

Graphic presentation of the fluorescence intensities of GFP comets near the hyphal tip. Values of mean and SD were generated based on 12 individual comets of each strain.

Figure 6

Images of GFP-NUDA (dynein HC), GFP-NUDI (dynein IC), GFP-NUDM (p150 dynactin), and GFP-NUDF (LIS1-like protein) in wild-type and the ΔkinA mutant cells.

Figure 7

NudA/kinA double mutant analyses. (A) Growth of the wild-type, ΔkinA, GFP-nudA, and ΔkinA/GFP-nudA (JZ4) strains on YUU that represses the expression of the alcA-driven GFP-nudA fusion gene, the only intact version of nudA in the genome. The plate was incubated at 32°C for 2 d. (B) 4,6-Diamidino-2-phenylindole staining of the wild-type, ΔkinA, GFP-nudA, and ΔkinA/GFP-nudA (JZ4) cells that were grown in YUU at 32°C for 7.5 h.

Figure 8

Model providing an explanation for the observation that accumulation of dynein/dynactin at the microtubule plus ends is increased in the nudF deletion mutant. NUDF/LIS1 may facilitate dynein-mediated cargo departure from the plus end toward the minus end of the microtubule. Failure of such departure is expected to cause a more intense plus-end accumulation of dynein/dynactin, which is what has been found in absence of NUDF.