We have shown that the C. elegans nucleoporins NPP-1, NPP-3, NPP-4, NPP-11, and NPP-13 interact and are required for the proper orientation of the mitotic spindle in one- and two-cell stage C. elegans embryos. Although several proteins previously have been identified as determinants for spindle positioning, our work is the first to implicate nucleoporins in this process. The yeast two-hybrid interactions and the similar RNAi phenotypes of this subset of nucleoporins suggest that they act as a complex in a common cellular process that is required for proper spindle orientation. Because the interactions between the nucleoporins are conserved, it is possible that their involvement in spindle orientation is also conserved.
The mechanism by which NPP-1,-3,-4,-11, and -13 affect spindle orientation is not clear. The nucleoporins could be affecting spindle orientation either directly by participating in the process of centrosome/nuclear complex rotation or indirectly by affecting the polarity system. Mutation of par-2
results in a spindle orientation phenotype similar to that of npp-1(RNAi)
—failure of the P1 spindle to rotate (Cheng et al., 1995
), but the par-2
effect is indirect. Absence of PAR-2 leads to the abnormal accumulation of anterior complex proteins at the P1 cell cortex (Cuenca et al., 2003
; Etemad-Moghadam et al., 1995
). Because par-3
is epistatic to par-2
for the spindle orientation defect it appears that mislocalized PAR-3 blocks the spindle orientation in P1 in the par-2
mutant (Cheng et al., 1995
, like par-2
mutants, results in abnormal accumulation of cortical PAR-6::GFP in the P1 cell, consistent with a role in localizing the polarity proteins. However, in contrast to the par-2 par-3
double mutant result, the npp-1(RNAi)
spindle phenotype is at least partially epistatic to par-3
. We interpret this partial epsistas to mean that NPP-1 and presumably NPP-3,-4,-11 and -13 are contributing to the mechanics of spindle rotation independently of their effect on distribution of PAR-6.
Determining the exact role for nucleoporins in orienting the mitotic spindle will be a challenge. It is possible that one or more of these proteins interacts directly with factors regulating the mitotic spindle and this interaction is required for proper spindle rotation. Alternatively, the spindle orientation defects could be an indirect effect caused by a partial failure in nuclear function during oogenesis or early embryogenesis.
Homologues of NPP-1, NPP-4, NPP-11, and NPP-13 are clearly involved in nucleo-cytoplasmic transport in vertebrates and yeast. Nsp1p, Nup49p, Nup57p, Nic96p, and Nup192 (homologues of NPP-11, NPP-4, NPP-1, NPP-13, and NPP-3, respectively) all show symmetrical localization to both the cytoplasmic and nucleoplasmic sides of the NPC in yeast (Rout and Aitchison, 2001
) and mutations in Nsp1, Nup49, Nup57, and Nic96 all lead to defects in the nuclear import of reporter proteins (Doye et al., 1994
; Grandi et al., 1995
; Nehrbass et al., 1993
). Nsp1p, Nup49p, and Nup57p form a core complex that is docked to the NPC via an interaction with Nic96p (Grandi et al., 1995
). Similarly, the vertebrate nucleoporins Nup54, Nup58, Nup62 (homologues of NPP-1, NPP-4, and NPP-11, respectively) also form a complex that is involved in nuclear import (Finlay et al., 1991
; Pante et al., 1994
). When the Nup54/Nup58/Nup62 complex is immunodepleted from nuclear reconstruction extracts, reconstructed nuclei form with NPCs but are impaired in their ability to import a reporter protein. Adding the purified rat Nup54/Nup58/Nup62 complex to the extract can restore import activity. The conservation of function between yeast and vertebrate systems, together with our observations, suggests that in C. elegans
, NPP-1, NPP-3, NPP4, NPP-11, and NPP-13 also are involved in nuclear import. Consistent with this, nuclear growth is inhibited when each of these genes are targeted by RNAi (Galy et al., 2003
It is clear from the NPC clustering phenotype, the failure to exclude β-Tubulin::GFP, and the failure to import PIE-1::GFP in npp-1(RNAi) embryos that npp-1(RNAi) causes defects in NPC function. This suggests that the spindle orientation defects could be indirect. However, npp-1 seems not to be required for the localization of the inner nuclear membrane protein GFP::MAN1 nor for the nuclear import of CSN-5, YFP::LMN-1 and PCNA::GFP indicating that NPC function in protein import is not severely compromised. Similarly, terminally differentiated npp-1(RNAi) embryos express a number of cell type-specific GFP markers, indicating that their cells are capable of differentiation. Cells with completely compromised nuclei would likely not be able to differentiate. Therefore, catastrophic defects in NPC function have apparently not occurred in the npp-1(RNAi) embryos. If the spindle defects are indeed indirect effects of compromised NPC function, identifying the affected molecules would be of great interest.
In addition to nuclear import and export, nucleoporins have been implicated in a number of cellular processes including chromatin organization (Galy et al., 2000
), chromosome segregation (Kerscher et al., 2001
), and interaction with the spindle checkpoint machinery (Iouk et al., 2002
). In addition to their nuclear envelope enrichment, some nucleoporins also localize to regions outside of the NPC. Vertebrate Nup98 is found concentrated in foci within the nucleus (Griffis et al., 2002
). Yeast Ndc1 is an essential component of the spindle pole body and is required for its duplication (Chial et al., 1998
; Winey et al., 1993
). We have localized two C. elegans
nucleoporins to the kinetochore during mitosis (V. Galy, P.A., I.W.M. unpublished). Vertebrate Nup62 interacts with a general transcription factor Sp1 and a putative transcription factor rtSox23 suggesting a requirement of Nup62 for proper transcription in certain situations (Han et al., 1998
; Yamashita et al., 1998
). Nup62 has also been suggested to be involved NF-kB signaling (Gamper et al., 2000
). The diversity of functions ascribed to nucleoporins, especially homologs of NPP-11, raises the possibility that C. elegans
nucleoporins could have roles away from the NPC and thus could play a direct role in orienting the mitotic spindle.
Evidence in both yeast and vertebrate models suggests the possibility of a more direct role for nucleoporins in spindle orientation. Nup57p, the yeast homologue of npp-1
, interacts with Jnm1p, a member of the dynactin complex (Ito et al., 2001
null mutants display defects in nuclear migration and defects in spindle orientation that are analogous to spindle orientation defects seen in npp-1(RNAi)
embryos (McMillan and Tatchell, 1994
). Jnm1p is the functional homologue of the vertebrate dynactin component p50/Dynamitin (Kahana et al., 1998
). A recent study demonstrated that five nucleoporins, including nucleoporin Nup62 (NPP-11), co-immunoprecipitated with the dynactin p150(Glued) from bovine oocytes (Payne et al., 2003
). Dynactin p150(Glued) also co-localizes to with nucleoporin Nup62 on the nuclear envelope in vertebrates (Payne et al., 2003
). In C. elegans
, depletion of the dynactin components DNC-1/p150(Glued) or DNC-2/dynamitin via RNAi led to a failed rotation of the mitotic spindle in P0 and P1 similar to the spindle orientation defects observed in npp-1(RNAi)
embryos (Skop and White, 1998
). The interaction between Jnm1p/dynamitin and Nup57p in S. cerevisiae
predicts that NPP-1 may interact with DNC-2 in C. elegans
. The interaction between vertebrate nucleoporin Nup62 and dynactin p150(Glued) predicts that NPP-11 and DNC-1 may also interact. If these interactions are required for the function of the dynactin complex, then depleting either NPP-1 or NPP-11 could lead to the spindle orientation defects observed in this study. Similarly, if the physical interactions between NPP-1, NPP-3, NPP-4, NPP-11, and NPP-13 are important for this function, then depletion of any of these components could lead to the observed spindle orientation defects. Although we were unable to detect an interaction with any of the nucleoporins and DNC-1or DNC-2 using the yeast two-hybrid system, such an interaction remains possible.
We can only speculate as to how these NPC components might contribute to spindle orientation. One simple model is a direct interaction with the dynactin complex to anchor the centrosomes to the nuclear envelope during rotation. Alternatively NPPs could influence microtubule dynamics either at the minus ends or at the plus ends by modifying proteins that migrate from the centrosomes to the cortex.
Analysis of the relationship between the NPPs and OOC-3 and OOC-5 could provide some insight. The spindle orientation defects, the failure to maintain cell polarity, and the smaller embryo size observed in npp-1(RNAi)
embryos resembles phenotypes observed in ooc-3
embryos (Basham and Rose, 1999
; Rose and Kemphues, 1998
). OOC-3 and the Torsin-related protein OOC-5 both localize to the endoplasmic reticulum and the nuclear envelope (Basham and Rose, 2001
). A recent study demonstrated that the ATP-bound form of TorsinA localizes to the nuclear envelope in COS-7 cells while the ATP-free form is distributed throughout the endoplasmic reticulum (Naismith et al., 2004
). This suggests that TorsinA binding partners are found at the nuclear envelope and may include nucleoporins or other nuclear envelope proteins. Thus OOC-5 could interact with nuclear envelope proteins including nucleoporins. OOC-5 activity at the nuclear envelope could then facilitate the nucleoporin activity required for proper rotation of the mitotic spindle.
In conclusion, we have shown that the C. elegans nucleoporins NPP-1, NPP-3, NPP-4, NPP-11, and NPP-13 physically interact and are required for spindle orientation in early embryos.