Figure 1.
Num1 is required for polar growth and septum formation.
Filament formation of AB31 wild-type and AB31Δnum1 deletion strains was monitored 12–14 hours upon induction of the bE1/bW2 heterodimer. (A) AB31 wild-type cells grow as long, straight filaments; only the tip compartment is filled with cytoplasm, separated from highly vacuolated distal compartments by a septum (arrowhead). (B) AB31Δnum1 hyphae grow shorter and more curved. In AB31Δnum1, bipolar (C) and branched (D) hyphae are observed frequently. (E) Septa of AB31Δnum1 are often formed within the compartment filled with cytoplasm (arrowheads denote delocalized septum; in lower panel, Calcofluor White staining was used to visualize the septum). (F) In approx. 2.5% of hyphae delocalized septa (arrowhead) lead to empty tip compartments. Scale bars: 10 µm.
Table 1.
Quantification of phenotypes in AB31 and AB31Δnum1 12 hours after the induction of the bE/bW-heterodimer.
Figure 2.
Num1 interacts with components of the Prp19/Cdc5L complex.
(A) Schematic representation of the protein-protein interactions (blue arrows) within the human hPrp19/CDC5L-complex [21]. Proteins depicted in blue represent the core complex, additional components (yellow) are associated with individual proteins. Green arrows indicate U. maydis protein interactions identified in this study. (B) The Prp19 N-terminus and the Cef1 C-terminus interact with the full length Num1 protein in a yeast two-hybrid analysis. p53/SV40 T-antigen and Lamin/SV40 T-antigen (Clontech) serve as positive and negative control, respectively. (C) In vivo co-immunoprecipitation of HA-tagged Prp19 and Cef1 and 3eGFP-tagged Num1. α-HA coupled agarose beads were used to precipitate Prp19 and Cef1, respectively. The Num1 protein was detected on a Western blot using an α-GFP antibody. The negative control shows no unspecific binding of the Num1 protein to α-HA agarose. (D) Subcellular localization of the Num1:3eGFP fusion protein. In addition to the nuclear localization of Num1 (enclosed by dashed circle), a cytoplasmic distribution to distinct foci can be observed (arrowhead). Scale bar: 5 µm. (E, F) Num1:3eGFP as well as Cef1:RFP and Prp19:RFP were co-expressed in AB31 under control of their native promoters. Num1 co-localizes with both Cef1 and Prp19 in the nuclei. Additional cytoplasmic Num1:3eGFP foci are indicated by arrowheads. Scale bars: 10 µm.
Figure 3.
Num1 influences the cell cycle and cell division as well as the cellular response to DNA damage.
(A) Induction of the bE1/bW2 heterodimer in wild-type cells (AB31) induces a G2 cell cycle arrest, resulting in hyphae with a single nucleus positioned in the tip compartment. In AB31Δnum1, 18% of the hyphae contain more than one nucleus, usually separated by a septum or a septum-like chitin structure, which is not always visible in the DIC-channel (see also Figure S6). In some cases, delocalized septa give rise to compartments that are devoid of any nuclei (bottom panel). Filaments were stained with DAPI and Congo Red, which effectively stains chitin in fungal cell walls [26], to visualize nuclei and septa within the same hypha. Scale bars: 10 µm. (B) Sensitivity of an FB2Δnum1 deletion strain to DNA-damage in comparison to wild-type cells. Serial 10-fold dilutions were spotted on complete medium containing the indicated DNA-damage inducing supplements, or subsequently irradiated with 300 J/m2 UV light (254 nm), respectively. Pictures were taken 2–3 days after incubation at 28°C.
Figure 4.
Reduced splicing efficiency of the rbf1-gene leads to impaired function of the Rbf1 master regulator.
(A) Plot depicting splicing efficiency of the rbf1-gene. Plotted are the FPKM values (fragments per kilobase of sequence per million fragments mapped) across the genomic region indicated (coordinates in nucleotides) of three independent RNA-Seq experiments for AB31 wild-type (blue lines) and AB31Δnum1 (red lines), respectively. Exons (E) and introns (I) are indicated. All four introns show increased intron retention rates in AB31Δnum1. (B) Western analysis showing abundance of Rbf1:3×HA and α-tubulin (loading control) from AB31 wild-type and Δnum1-deletion strains. In AB31Δnum1, Rbf1 is reduced to 30% of wild-type-level (Quantification: ImageJ [109]). (C, D) Gene expression analyses of b- and rbf1-genes (C) as well as rbf1-target genes (D) using qRT-PCR. RNA samples were isolated from strains AB31 and AB31Δnum1 eight hours after induction of the bE1/bW2-heterodimer. Gene expression is shown relative to the highest expression value, using actin and eIF2b for normalization. Shown are the mean values of three biological and two technical replicates. Error bars represent the SD. (E) Venn diagram depicting the total number of genes repressed in AB31Δnum1 and AB31Δrbf1. * RNA-Seq analysis, this study; ** Microarray analysis conducted five hours after induction of the bE1/bW2-heterodimer [2].
Figure 5.
Num1 physically interacts with the Kin1 motor protein.
(A) In vitro expression and co-immunoprecipitation of Myc-tagged Num1 and HA-tagged Kin1. Proteins were labeled with biotin during in vitro synthesis, detected with streptavidin-conjugated alkaline phosphatase, and visualized with NBT/BCIP. In the negative control (Num1:Myc), no unspecific binding of Myc-tagged Num1 to the HA-antibody was detected. In contrast, after co-incubation with HA-tagged Kin1, the Myc-tagged Num1 protein was co-immunoprecipitated (indicated by asterisk), demonstrating that the Num1 protein physically interacts with Kin1 in vitro. (B) In vivo co-immunoprecipitation of U. maydis strains expressing HA-tagged Kin1 and 3eGFP-tagged Num1 under control of their endogenous promoters. After crosslinking of proteins using DSP, α-HA coupled agarose beads were used to precipitate Kin1. The Num1 protein was detected on Western blots using an α-GFP antibody. As negative control, a strain expressing only Kin1:3×HA was used.
Figure 6.
Num1 and Kin1 are functionally connected.
(A) The num1 deletion leads to aberrant vacuole morphology. Vacuoles were visualized by CellTracker Blue (7-amino-4-chloromethyl-coumarin, CMAC) staining. Both num1 and kin1 deletion strains contain more, but smaller vacuoles in comparison to wild-type sporidia. Scale bars: 5 µm. Right panel: Quantification of the average number of vacuoles of strains indicated. N represents the number of individual cells analyzed. Mean values of three independent experiments are shown. (B) The num1 deletion leads to an aberrant distribution of early endosomes. For visualization of early endosomes, a Yup1:eGFP fusion protein [9] was expressed under control of the constitutively active Potef-promoter in strains AB33 and AB33Δnum1, which harbor b-genes under control of the nitrate-inducible Pnar1-promoter. Endosomes were analyzed 14 hours after bE1/bW2-induction. Septa (arrowhead) were visualized by Calcofluor White staining. Yup1-labelled early endosomes accumulate at basal and apical parts of the hyphae as well as around delocalized septa in AB33Δnum1 in contrast to wild-type hyphae. Scale bars: 10 µm. Movies detecting Yup1:eGFP-fluorescence of the depicted hyphae are shown in Videos S1 and S2. Shown below are the corresponding kymographs (time and distance as indicated) reflecting the motility of early endosomes. In AB33 hyphae, early endosomes travel continuously towards the hyphal tip (anterograde) and reverse direction to travel back towards the septum (retrograde). Pausing organelles are reflected by a vertical line. In contrast, endosomal motility is drastically reduced in AB33Δnum1 hyphae containing additional septa, where the majority of the Yup1:eGFP-signal is non-motile. An arrowhead indicates the septum. (C) The localization of Dynein is dependent on Num1. In AB33 filaments 3eGFP:Dyn2 fusion proteins form comet-like motile structures that accumulate at the hyphal tip and from there move into retrograde direction [9]. In filaments of AB33Δnum1 strains the apical accumulation is significantly reduced (p = 0.002); instead a stronger signal is observed in the cytoplasm. Dynein localization was monitored 14 hours after induction of the bE1/bW2 heterodimer. Scale bars: 10 µm. Right panel: Quantification of the apical accumulation of 3eGFP:Dyn2 fusion proteins. N represents the number of hyphae analyzed. Mean values of three independent experiments are shown.
Table 2.
Reduced endosomal motility in num1-deletion strains.
Figure 7.
Deletion of the gene for a Num1-homologue in the ascomycete Aspergillus nidulans leads to a phenotype reminiscent to the Δnum1 phenotype in U. maydis.
(A) The morphology of vacuoles is affected by the AN4244-deletion. Spore suspensions of a wild-type (TNO2A3) and the AN4244 deletion mutant were incubated for 18 hours at 37°C and the germinated filaments were CellTracker Blue (CMAC)-stained to visualize the vacuoles. Similar to the num1-deletion in U. maydis, the AN4244-deletion in A. nidulans leads to an increased number of vacuoles. (B) Deletion of AN4244 affects nuclear positioning. Spore suspensions of A. nidulans wild-type (TNO2A3) and the AN4244 deletion mutant were incubated for 12 and 18 hours, respectively, at 37°C, and the germinated filaments were DAPI-stained to visualize nuclei. In wild-type filaments, nuclei are evenly spaced, whereas in the mutant nuclei are often found in clusters. (C) The localization of Dynein (NudA) is dependent on AN4244. Spore suspensions of strains LZ12, expressing a GFP:NudA fusion protein, and LZ12ΔAN4244 were incubated for 18 hours at 37°C. In LZ12 the GFP:NudA signal strongly accumulated in tips of germinating hyphae, whereas in mutant filaments, this apical accumulation was reduced; instead a stronger signal was observed in the cytoplasm. Scale bars: 10 µm.
Figure 8.
Hypothetical model for function of Num1 coordinating pre-mRNA splicing with nuclear pore complex-dependent export of mRNP-particles and microtubule-based mRNA-transport.
Num1 is a component of the spliceosome-associated Prp19/CDC5L-complex (NTC) and has a global effect on splicing efficiency. NTC-components interact with the TREX (“transcription-coupled export”)-complex [82], which mediates the export of mature transcripts into the cytoplasm via the nuclear pore complex (NPC) [83]–[86]. Adapter proteins connect the mRNA to NPC-bound export receptors. The Kinesin-1 motor protein has been shown to be associated to the NPC and promotes nucleo-cytoplasmic transport processes [76]. mRNAs within the hyphae are distributed via the RNA-binding protein Rrm4, which shuttles associated with early endosomes along the polar microtubule cytoskeleton [7], [77]. Motility of the Rrm4 associated early endosomes depends both on Kin1 [7], [14] and Num1. Proteins labeled with (?) depict yet unidentified adapter-proteins that might function as connectors between the nuclear splicing machinery and the cytoplasmic NPC-bound Kin1. As Num1 is a component of the NTC complex and interacts with Kin1, it might fulfill a function during mRNA export processes, possibly by connecting exported mRNP to the cytoplasmic transport machinery via its interaction with Kin1.