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unc-83 encodes a novel component of the nuclear envelope and is essential for proper nuclear migration

Daniel A. Starr¹, Greg J. Hermann², Christian J. Malone^1,*, William Fixsen^3,, James R. Priess², H. Robert Horvitz³ and Min Han^1,

¹ Howard Hughes Medical Institute and Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA
² Howard Hughes Medical Institute and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
³ Howard Hughes Medical Institute and Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
^* Present address: Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706, USA
Present address: Department of Continuing Education, Harvard University, Cambridge, MA 02138, USA

View larger version (71K): [in a new window]   Fig. 1. unc-83 mutations disrupt the nuclear migrations of three cell types. (A) Left lateral view of P-cell nuclear migration in wild-type and unc-83 embryos at 25°C. P-cell cytoplasm is gray and nuclei are white. White X marks dying nuclei. Ventral is downwards. (B-C) Expression of UNC-47::GFP in GABAergic neurons in the ventral cords of adult hermaphrodites. Ventral is downwards, anterior is leftwards. (B) Wild type, showing the 19 neural cell bodies scored between the arrows. (C) unc-83(e1408), with only 11 GABAergic neurons between the arrows. (D) The dorsal surface of a pre-elongation embryo illustrating intercalation and nuclear migration of hyp7 precursors in wild-type and unc-83 embryos. Cytoplasm of the hyp7 precursors is gray, nuclei that migrate from right to left are black, and nuclei that migrate from left to right are white. Anterior towards left, right is upwards. (E,F) Lateral view of L1 hermaphrodites. Dorsal is upwards. (E) Wild type showing no nuclei in the dorsal cord. (F) An unc-83(e1408) hermaphrodite. The arrows mark misplaced hyp7 nuclei in the dorsal cord. (G) A dorsal view, through the middle of a pre-elongation embryo, of nuclear migration during intestinal polarization during the E16 stage in wild-type and unc-83 embryos raised at 15°C. Cytoplasm of embryonic intestinal cells is gray, nuclei right of the midline are black and nuclei left of the midline are white. Anterior is leftwards, right is upwards. (H) Wild-type embryonic intestinal cells. (I) unc-83(e1408) embryonic intestinal cells. M marks the midline where the intestinal lumen will form, the arrows mark examples of intestinal nuclei at the mid-line in wild-type and separated away from the midline in the unc-83(e1408) embryo. Scale bars: 100 µm for B,C; 10 µm for E,F; 10 µm for H,I.

View larger version (76K): [in a new window]   Fig. 2. unc-83 mutations do not disrupt centrosome attachment to the nucleus. Dorsal views through the intestinal primordium (A,B) or through hyp7 precursors (C) in pre-elongation embryos grown at 15°C. Anterior is leftwards. (A) Wild-type embryo. (B,C) unc-83(e1408) embryos. Embryos were stained with the IFA antibody to identify centrosomes (pseudocolored red) and DAPI to identify chromatin (pseudocolored blue). hyp7 precursors were identified using the jam-1::GFP marker to show hypodermal cell boundaries, pseudocolored green. M marks the midline where the intestinal lumen will form. The arrows mark examples of nuclei with centrosomes properly associated. Note that for the wild-type embryo, nuclei have migrated toward the midline, but nuclei are separated away from the midline in the unc-83(e1408) embryo. Because of the limited focal plane of these images, only a subset of nuclei or centrosomes are shown. Scale bars: in B, 5 µm for A,B; in C, 5 µm for C.

View larger version (97K): [in a new window]   Fig. 3. unc-83 mutations do not disrupt the gross structure of the nuclear lamina. (A) Dorsolateral view of a wild-type embryo. (B) Lateral view of an unc-83(e1408) embryo. Anterior is leftwards. Both embryos are in a JAM-1::GFP background to stain hypodermal adherens junctions, colored green. Embryos were stained with an anti-Lamin antibody to identify the nuclear lamina (pseudocolored red) and DAPI to identify chromatin (pseudocolored blue). Arrows mark the dorsal midline. Arrowheads identify hyp7 nuclei. Scale bar: 10 µm.

View larger version (31K): [in a new window]   Fig. 4. Cloning unc-83. (A) The genomic region on chromosome V between exp-2 and dpy-11 is shown. The names of cosmids in the region that rescue exp-2 or dpy-11 or that contain part of unc-83 are indicated. (B) Cosmid W01A11 and its derivatives are shown on the left. The deleted parts of W01A11 in pD19 and pD21 are indicated by broken lines. The numbers above the lines are the sites of breakpoints of the deletions of W01A11 in pD19 and pD21 and of the region of W01A11 subcloned into pBS to create pD22 (see Materials and Methods). On the right are data concerning the rescue of the unc-83(e1408) nuclear migration defect of hyp7 cells and P cells. (C) The genomic regions of three predicted transcripts of unc-83 are shown. The white boxes represent exons, which are connected by introns. The predicted translational start site (ATG) in each transcript and any detected SL1 splice leader sequences of each transcript are indicated. The numbers at the bottom label exons 1 to 16 of the longest unc-83 transcript. (D) The molecular lesions of 16 unc-83 alleles are shown. The arrows indicate the approximate sites of the lesions. For nonsense mutations, the affected amino acid is numbered. Black boxes indicate alleles that did not disrupt hyp7 nuclear migration, while a white background indicates alleles that disrupted both P cell and hyp7 nuclear migrations. Sequence data for the unc-83 transcripts are available from GenBank/EMBL/DDBJ under Accession Number AF338767.

View larger version (93K): [in a new window]   Fig. 5. UNC-83 is localized to the nuclear envelope. In all panels, anterior is leftwards, and dorsal is upwards. UNC-83 antibody localization is pseudocolored red, and DAPI staining is in blue. In some panels, JAM-1::GFP is used to show adherens junctions in green. (A-C) Pre-elongation embryos, examples of hyp7 nuclei are marked with arrowheads. (A) unc-83(e1408), a putative null. (B) Wild-type. (C) unc-83(ku18), an allele in which hyp7 nuclear migration is not disrupted. (D-F) Lateral views of sections through a single wild-type embryo at the onset of elongation (bean stage). (D) A section near the top with P-cell nuclei marked by arrowheads. (E) A section 1 µm lower showing hyp7 nuclei marked with arrowheads. (F) A section 4 µm lower showing UNC-83 staining in intestinal cells marked with arrowheads. (G-I) Wild-type worms. (G) A lateral view of a hermaphrodite late in the first larval stage showing UNC-83 in marked P cells. The arrow marks an intestinal cell in the same plane. (H) The tail of an adult worm. UNC-83 stained many hypodermal cells, examples are marked with arrowheads, a large intestinal cell (arrow) and other unidentified cells. (I) The head of an adult worm showing UNC-83 staining in hypodermal (examples marked with arrowheads) and other unidentified cells. Scale bars: in F, 10 µm for A-F; in G, 9 µm for G; in I, 25 µm for H,I.

View larger version (58K): [in a new window]   Fig. 6. UNC-83 co-localization with Lamin and UNC-84. (A) Co-localization of UNC-83 (left panel) and Lamin (center panel) shown merged together (right panel) with UNC-83 pseudocolored red and Lamin in green. (B) Co-localization of UNC-83 (left panel) and UNC-84::GFP (center panel) shown merged together (right panel) with UNC-83 pseudocolored red and UNC-84::GFP green. Scale bar: 2 µm.

View larger version (22K): [in a new window]   Fig. 7. UNC-84 is required to target UNC-83 to the nuclear envelope. (A-D) Localization of UNC-83 antibody in pre-elongation embryos in the four classes of unc-84 embryos. Anterior is leftwards. (A) unc-84(n369), a class 1 or null allele. (B) unc-84(n371), a class 2 allele. (C) unc-84(n399), a class 3 allele. (D) unc-84(e1411), a class 4 allele. Scale bar: 10 µm. (E) The molecular lesions in UNC-84 of the seven alleles tested for UNC-83 localization. Only one allele from each class is shown in A-D, although other alleles were tested (see text). (F) UNC-83 and the SUN domain of UNC-84 interact in vitro. The 35S-methionine-labeled UNC-83 that was pulled down by the bait-coated beads is shown. Labels designate the GST fusion protein attached to beads used as bait, or the presence of cold UNC-83/MBP in the middle lane.