Nuclear-localized Asunder regulates cytoplasmic dynein localization via its role in the integrator complex

Abstract

We previously reported that Asunder (ASUN) is essential for recruitment of dynein motors to the nuclear envelope (NE) and nucleus-centrosome coupling at the onset of cell division in cultured human cells and Drosophila spermatocytes, although the mechanisms underlying this regulation remain unknown. We also identified ASUN as a functional component of Integrator (INT), a multisubunit complex required for 3'-end processing of small nuclear RNAs. We now provide evidence that ASUN acts in the nucleus in concert with other INT components to mediate recruitment of dynein to the NE. Knockdown of other individual INT subunits in HeLa cells recapitulates the loss of perinuclear dynein in ASUN-small interfering RNA cells. Forced localization of ASUN to the cytoplasm via mutation of its nuclear localization sequence blocks its capacity to restore perinuclear dynein in both cultured human cells lacking ASUN and Drosophila asun spermatocytes. In addition, the levels of several INT subunits are reduced at G2/M when dynein is recruited to the NE, suggesting that INT does not directly mediate this step. Taken together, our data support a model in which a nuclear INT complex promotes recruitment of cytoplasmic dynein to the NE, possibly via a mechanism involving RNA processing.

FIGURE 1:

INT subunits are individually required for dynein recruitment to the NE. HeLa cells were transfected with siRNA as indicated. (A–N) After siRNA treatment, cells were incubated in nocodazole, fixed, and stained for DIC and DNA. Loss of perinuclear dynein was observed upon individual knockdown of the majority of INT subunits. Scale bar, 10 μm. (O) Quantification of perinuclear dynein in cells after knockdown of individual INT subunits. *p < 0.0001 (compared with NT control). (P) hASUN immunoblot analysis of cell lysates after knockdown of individual INT subunits. Tubulin was used as loading control.

FIGURE 2:

INT subunits localize to the nucleus, cytoplasm, or both. HeLa cells were transfected with the indicated expression constructs encoding tagged versions of INT subunits. (A–L) After fixation, cells were stained with phalloidin and DAPI; cells expressing Myc-hASUN were also immunostained for Myc. (A) Myc-hASUN was either exclusively nuclear (A1), predominantly cytoplasmic (A2), or distributed throughout the cell (A3). (B) GFP (control) was diffusely present throughout the cell. (C–L) GFP-tagged versions of other INT subunits localized to the nucleus (E, G, I, J, L), cytoplasm (C, H), or both (D, F). Scale bar, 10 μm. (M) Immunoblot analysis of lysates of transfected cells using antibodies against Myc or GFP tags revealed fusion proteins of the predicted sizes. Tubulin was used as loading control.

FIGURE 3:

Levels of a subset of INT subunits are reduced at G2/M. HeLa cells were transfected with the indicated GFP- or Myc-fusion constructs and either left untreated (asynchronous population; –) or treated with a CDK1 inhibitor (G2/M-arrested population; +) for 16 h. Immunoblot analysis of lysates of transfected cells using antibodies against GFP or Myc tags revealed decreased levels of several INT subunits at G2/M (A), whereas no changes were observed for other subunits (B). hASUN antibodies were used to assess endogenous hASUN levels. (C) No changes were observed in the levels of GFP (control), GFP-BICD2, or endogenous CENP-F at G2/M. Tubulin was used as loading control.

FIGURE 4:

Identification of a functional NLS in human ASUN. (A) Schematic of Myc-tagged hASUN with predicted NLS (yellow box) in the C-terminal region of the protein. Letter A (red) indicates each charged residue of the endogenous NLS mutated to alanine in Myc-hASUN^mutNLS and Myc-NLS-hASUN^mutNLS. An exogenous NLS (blue box) was added between the Myc tag and either hASUN or hASUN^mutNLS to generate Myc-NLS-hASUN and Myc-NLS-hASUN^mutNLS, respectively. (B) Myc immunoblot analysis of lysates of transfected HeLa cells revealed Myc-hASUN fusion proteins of the predicted sizes. Tubulin was used as loading control. (C) Representative images showing predominant localizations of Myc-hASUN fusions in transfected HeLa cells. Scale bars, 10 μm. (D) Quantification of localization patterns of Myc-hASUN fusion proteins in transfected HeLa cells.

FIGURE 5:

Functional NLS is conserved in Drosophila ASUN homologue. (A) Schematic of CHY-tagged dASUN with predicted NLS (yellow box) in the C-terminal region of the protein. Letter A indicates each charged residue of the endogenous NLS mutated to alanine in CHY-dASUN^mutNLS. An exogenous NLS (blue box) was added to the N-terminal end of CHY-tagged dASUN to generate NLS-CHY-dASUN. (B) CHY immunoblot analysis of testes lysates from asun males with or without germline expression of CHY-dASUN fusions revealed proteins of the predicted sizes. An intervening lane just left of the last lane was removed. Wild-type (WT) males and asun males lacking a transgene were used as negative controls. Tubulin was used as loading control. (C) Representative images showing localizations of CHY-dASUN fusions in transgenic G2 spermatocytes. Scale bar, 10 μm. (D) Quantification of localization patterns of CHY-dASUN fusion proteins in transgenic G2 spermatocytes.

FIGURE 6:

Nuclear pool of hASUN is required for dynein recruitment to the NE in HeLa cells. HeLa cells were transfected with NT or hASUN siRNA plus Myc (vector control) or Myc-hASUN expression constructs as indicated. (A) After nocodazole treatment, cells were fixed and stained for DIC and DNA. Representative images of cells. Perinuclear dynein was restored only by expression of hASUN fusion proteins with nuclear localization. Scale bar, 10 μm. (B) Quantification of cells with perinuclear dynein after the indicated siRNA and DNA transfections. *p < 0.0001 (pairwise comparisons indicated). N.S., nonsignificant statistical differences. (C) hASUN immunoblot of lysates of transfected HeLa cells confirmed depletion of hASUN in hASUN-siRNA cells, and Myc immunoblot confirmed expression of Myc-hASUN fusion proteins of the predicted sizes. Tubulin was used as loading control.

FIGURE 7:

Nuclear pool of dASUN is required for dynein recruitment to the NE in Drosophila spermatocytes. (A, B) Testes dissected from wild-type (WT) or asun males with or without germline expression of CHY-tagged dASUN fusion proteins were stained for dynein heavy chain and DNA. (A) Representative images of G2 spermatocytes. Perinuclear dynein was restored to asun G2 spermatocytes only by expression of dASUN fusion proteins with nuclear localization. Scale bar, 10 μm. (B) Quantification of G2 spermatocytes with perinuclear dynein. (C) Fertility assay shows the average number of progeny per fertile male. *p < 0.0001 (compared with wild-type control).