TI - Results And Discussion . AB - Alp7 localizes to the nucleus only during mitosis ( Fig 1A ) . There are two main possible ways to achieve mitosis-specific nuclear accumulation of Alp7 : (i) the nuclear entry of Alp7 is inhibited during interphase but potentiated during mitosis , and ( ii ) the nuclear entry of Alp7 is not restricted to mitosis , but it is only during mitosis that Alp7 is retained in the nucleus . To discriminate between these two possibilities , we used leptomycin B ( LMB ) , an inhibitor of the nuclear export factor Exportin/Crm1 ( Kudo et al , 1998 ) . Cells bearing Alp7-YFP ( yellow fluorescent protein ) with the SPB marker Cut12-CFP ( cyan fluorescent protein ) and the microtubule marker mRFP-Atb2 ( monomeric red fluorescent protein ( RFP ) -tagged alpha2-tubulin ) were prepared and LMB was added . Within 1 h , nuclear accumulation of Alp7-YFP was observed , although cells were still in interphase , as judged from their microtubule structures (Fig 1B) . This suggests that Alp7 undergoes nucleocytoplasmic shuttling--Alp7 enters the nucleus during interphase but is immediately exported to the cytoplasm by Exportin/Crm1 . Alp7 was also identified independently in a genome -wide screen for proteins that accumulate in the nucleus in an LMB -dependent manner ( Matsuyama et al , 2006 ) . In general , TACC is required for the localization of TOG to the mitotic centrosome . This is also true for fission yeast , as Alp7/TACC is essential for the localization of Alp14G to mitotic SPBs ( Sato et al , 2004 ) . As an NLS is present in Alp7 ( Sato & Toda , 2007 ) , the role of the Alp7/TACC subunit might be to import the Alp14G subunit into the nucleus . To explore this possibility , Alp14 was visualized together with CFP-tubulin in the alp7-RARA ( R122A/R124A ) mutant , in which the intrinsic NLS has been made inactive ( Sato & Toda , 2007 ) . Alp14-rRFP co-localized with Alp7-RARA-GFP ( green fluorescent protein ) to the cytoplasmic microtubules even 1.5 h after LMB addition , at which time wild-type Alp7-GFP and Alp14-rRFP ( refoldable RFP ) had accumulated in the nucleus ( Fig 1C ) . These results verify that the nuclear entry of Alp14G is dependent on a functional NLS in Alp7/TACC . Many of the cargo proteins that are exported by Exportin/Crm1 contain a nuclear export signal ( NES ) , which consists of a characteristic cluster of leucine residues . To identify the NES in Alp7 , we performed a domain analysis by deleting the amino terminal or carboxy terminal part of Alp7 . Deletion of the C-terminal 45 amino-acid residues caused constitutive accumulation of Alp7 in the nucleus , irrespective of the cell -cycle stage , which suggests that NES activity resides in this region ( Alp7-DeltaC45-GFP ; Fig 2A , B ) . This region contains two putative NES-like sequences with clustered leucine and hydrophobic residues ( Leu 430-Leu 440 and Leu 454-Leu 470 ; supplementary Fig S1A online ) . To delineate the NES sequence , Leu 433 and Leu 435 , Leu 461 or other hydrophobic residues were mutated to alanine . The Alp7-L433A/L435A-GFP protein behaved in a manner similar to wild-type Alp7-GFP ( supplementary Fig S1B , C online ) . Furthermore , the Alp7-L454A - , Alp7-M457A - and Alp7-V462-GFP proteins localized normally to the cytoplasmic dots with Alp14-rRFP ( supplementary Fig S2 online ) . By sharp contrast , the Alp7-L461A-GFP construct led to Alp7 accumulation mostly in the nucleus during interphase , although localization to the cytoplasmic microtubules was still observed ( Fig 2C ; supplementary Figs S1D and S2 online ) , indicating that the intrinsic NES activity of Alp7 was significantly impaired . As this domain is located in the TACC domain , the L461A mutation might also abrogate the function of Alp7 . In fact , the alp7-L461A mutant showed few microtubule bundles in the cytoplasm ( supplementary Fig S1D online ) , which is reminiscent of the alp7Delta mutant ( Sato et al , 2004 ; Zheng et al , 2006 ) . Consistent with this , Alp7-L461A failed to complement either the hypersensitivity of the alp7Delta mutant to thiabendazole ( TBZ ) ; ( a microtubule depolymerizing drug ) or growth at high temperature ( supplementary Fig 1E online ) . Thus , the alp7-L461A mutant lost its intrinsic NES activity and its ability to organize cytoplasmic microtubules . Next , we sought to determine why Alp7-L461A was not functional . Given that our previous study had shown that the C-terminal TACC domain containing Leu 461 is responsible for Alp14 binding ( Sato et al , 2004 ) , we looked for an interaction between Alp7-L461A and Alp14 . Intriguingly , Alp14 co-localized neither with Alp7-DeltaC45 nor with Alp7-L461A , which is particularly evident during mitosis ( Fig 2B , C ) . Even during interphase , Alp14 localization to interphase microtubules was greatly compromised ( Fig 2B , C ) . These observations raised the possibility that the interaction between Alp7-L461A and Alp14 was impaired . A yeast two hybrid assay showed that this was indeed the case ( Fig 2D ) . The short C-terminal fragment of Alp14 (Alp14 Delta696) and the Alp7 TACC fragment , which lacks the N terminus (Alp7 DeltaN) , showed elevated affinities for full-length Alp7 and Alp14 , respectively ( Sato et al , 2004 ) . Alp7-L461A did not , however , interact even with Alp14 Delta696 (Fig 2D) . Similarly , introduction of the L461A mutation into Alp7 DeltaN (Alp7 DeltaN-L461A) abolished the interaction with both full-length Alp14 and Alp14 Delta696 (Fig 2E) . Collectively , these results show that L461 has a dual function in Alp7 function : it is a part of the NES , and it is involved in the interaction with its binding partner Alp14G . Alternatively , Alp7 might need to interact with Alp14 to be exported to the cytoplasm . Alp7 did not , however , accumulate in the nucleus in the alp14Delta mutant , which excludes the possibility that Alp14 functions as a nuclear export factor for Alp7 ( supplementary Fig S3 online ) . Alp7 shuttles between the cytoplasm and the nucleus but is retained to the cytoplasmic microtubules during interphase and to the nuclear spindle during mitosis . It is , therefore , possible that the microtubule structure per se might function as an anchoring platform . The absence of cytoplasmic microtubules did not , however , cause nuclear accumulation of Alp7 ( supplementary Fig S4 online ) . Thus , interphase microtubule structures do not function as tethering devices . We then questioned whether altering the localization of Alp7 would , in turn , affect microtubule structure . Therefore , we forced Alp7 to accumulate in the nucleus by adding a robust canonical NLS sequence to the N terminus of this protein ( PKKKRKV ; NLS-Alp7 ) . On fusing an inactive NLS peptide (PAAARKV) to Alp7 (NLSmut-Alp7) as a control , the protein co-localized with Alp14-rRFP to the cytoplasmic microtubules ( Fig 3A ) , as was the case for wild-type Alp7 . By contrast , NLS-Alp7-GFP accumulated in the nucleus and recruited Alp14 to the nucleus ( Fig 3B ) . It should be noted that in these cells cytoplasmic microtubules were short and fragmented ( Fig 3B ) . This is probably because the Alp7-Alp14 complex was sequestered in the nucleus , which caused a shortage of this complex in the cytoplasm that led to the subsequent disorganization of the microtubules ; this is reminiscent of the alp14 deletion mutant ( Garcia et al , 2001 ) . The shorter cytoplasmic microtubules seen in NLS-Alp7-GFP-containing cells were distinct from those observed in the NES-defective alp7-DeltaC45 ( or alp7-L461A ) mutant ( Fig 2B , C ) , in which the nuclear accumulation of Alp7 was also observed but Alp14 was still localized to the cytoplasmic microtubules . This difference suggests that Alp14 on the cytoplasmic microtubules exerts its ability to stabilize microtubules independently of the Alp7/TACC subunit . Alternatively , some population of Alp7 might still associate with Alp14 in the Alp7-DeltaC45 ( or alp7-L461A ) mutant on the microtubules , thereby providing the activity to stabilize microtubules . Whichever is the case , these results show that nuclear export of the Alp7-Alp14 complex is essential for maintaining the cytoplasmic microtubule structure . Therefore , nucleocytoplasmic shuttling of the Alp7-Alp14 complex is required for cell cycle -dependent microtubule assembly , interphase cytoplasmic microtubule arrays and nuclear mitotic spindles . Then what determines the cytoplasmic and nuclear retention of Alp7 during interphase and mitosis , respectively? Two possible , albeit not mutually exclusive , situations might occur on entry into mitosis : (i) the acceleration of nuclear import , and ( ii ) the inhibition of nuclear export . To distinguish between these two possibilities , the signal intensity of GFP fluorescence from wild-type or truncated Alp7-GFP fusion proteins , the latter of which had a defective NES , was quantified during both interphase and mitosis . A line was drawn along the long axis of a cell , along which the signal intensity of Alp7-GFP was measured . Nuclear accumulation of the full-length Alp7-GFP ( Alp7FL-GFP ) signal clearly occurred during mitosis but not during interphase , which validates these measurements ( Fig 4A ) . Importantly , the nuclear intensity along the section of the NES-lacking Alp7-DeltaC45-GFP , which was detectable during interphase , did not increase on mitotic entry ( Fig 4B ) , although total nuclear volume might have slightly increased . Hence , instead of the NLS , NES activity might be responsible for the mitosis-specific nuclear accumulation of Alp7 . To verify the downregulation of NES during mitosis , LMB was added to both interphase and mitotic cells and Alp7-YFP signals were quantified as described above ( Fig 4C ) . As LMB blocks nuclear export , this experiment should evaluate whether nuclear export of Alp7 is active during mitosis . Although the Alp7-YFP signal was increased in the interphase nucleus in response to the addition of LMB ( Fig 4C , left ) , it was not augmented further in the mitotic nucleus ( Fig 4C , right ) . This strongly suggests that nuclear export of Alp7-YFP does not occur during mitosis . We conclude that the inhibition of NES activity leads to Alp7 accumulation in the nucleus in a mitosis-specific manner , although we cannot completely exclude the possibility that activation of the NLS , as well as inhibition of the NES , occurs . It should be noted that the NES coincides with the Alp14 -binding site ( Leu 461 ) . It is , therefore , tempting to speculate that Alp14 and Crm1 might compete with one another for Alp7 binding . If so , an increased affinity between Alp14 and Alp7 in the nucleus might be able to inhibit nuclear export of the Alp7-Alp14 complex by blocking the accessibility of Crm1 . Further studies are required to test the competition model between Alp14 and Crm1 . Which factor inactivates the NES of Alp7 during mitosis? Possible candidates are mitotic kinases . Cdc2/Cdc13 (CDK/cyclin B) is localized at mitotic SPBs and spindles ( Alfa et al , 1990 ; Decottignies et al , 2001 ) . Given this localization , we speculated that CDK might well be such a regulator . To assess this possibility , we observed Alp7-GFP in temperature-sensitive cdc2 or cdc13 mutants , but the ambiguous localization of Alp7 in these conditional mutants hampered our ability to draw a solid conclusion ( data not shown ) . As an alternative approach , the analogue -sensitive cdc2-as mutant ( Dischinger et al , 2008 ) was used . Addition of the analogue 1NM-PP1 to inhibit CDK caused a significant loss of nuclear Alp7 during mitosis ( Fig 5 ; supplementary Fig S5 online ) . Alp7 in particular was lost from the nucleoplasm and spindle microtubules , although a certain amount of Alp7 remained at the SPBs . These results indicate that the nuclear accumulation of Alp7 requires CDK activity . Experiments using the cdc13 shut-off mutant ( cdc13so ) during meiotic prophase also supports the requirement of CDK activities for nuclear accumulation of Alp7 ( supplementary Fig S6 online ) . Is there any reason for Alp7 to enter the nucleus during interphase , apparently without a specific purpose? We envisage that in fission yeast the system to accelerate or decelerate the velocity of nuclear import depending on the cell -cycle stage might not be well developed or advantageous . Instead , the fission yeast might have evolved to devise the NES , the activities of which are specifically downregulated during mitosis , which is under the control of CDK . Kinesin-8 Klp5 and Klp6 shuttle between the nucleus and the cytoplasm ; as is the case for Alp7 and Alp14 , they also localize to cytoplasmic microtubules during interphase and to the nuclear spindle during mitosis ( Unsworth et al , 2008 ) . Furthermore , the protein phosphatase Clp1/Flp1 is retained in the cytoplasm during late mitosis by the Sid2 kinase and the 14-3-3 proteins ( Chen et al , 2008 ) . It would be intriguing to address how these proteins establish nuclear and cytoplasmic retention in a cell cycle -dependent manner . Ran maintains the nucleocytoplasmic balance of microtubule formation through the transport of Alp7 ( and Alp14 ) . CDK shifts this nucleocytoplasmic shuttling towards nuclear retention on entering mitosis . Our findings here , therefore , have revealed that Ran , Exportin and CDK coordinate the spatiotemporal regulation of microtubule formation in which Alp7/TACC , together with Alp14G , is a crucial component . It is still unknown how CDK affects NES activity , as no consensus sites for PHOSphorylation by CDK were found around the NES of Alp7 . Further studies are awaited to clarify the molecular mechanism by which CDK regulates the mitotic nuclear retention of Alp7-Alp14 .