The zebra fish cassiopeia mutant reveals that SIL is required for mitotic spindle organization

Abstract

A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly.

FIG. 1.

Characterization of the cassiopeia zebrafish mutant, which has mitotic defects and increased apoptosis. (A) At 36 hpf, csp mutant embryos are ventrally curved, have increased cell death that gives the head an opaque gray appearance, and begin to develop cardiac edema. (B) Phospho-H3 immunostaining on 28-hpf embryos shows an increased number of mitotic cells in csp mutants. (C) Acridine orange staining detects high levels of cell death in the head and dorsal neurons of csp mutant embryos. Note that the bright green of the yolk is autofluorescence. (D) Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) indicates that the dorsal cell death in csp mutants is apoptotic. (E) Representative DNA content analysis of wild-type (wt) and csp mutant embryos showing increased populations of 4N and sub-G₁ populations. Scale bars = 250 μm. Max, maximum.

FIG. 2.

Loss of SIL gene function causes the csp mutant phenotype. (A, top) Schematic representation of the genetic mapping interval containing the csp mutation on zebrafish linkage group 22. Recombinants for each microsatellite or single-strand conformation polymorphism marker across the region are shown. (A, middle) Physical map of the fully sequenced BACs coinciding with the csp interval on linkage group 22. (A, bottom) Genes identified by BLAST searches based on the BAC sequences. Note that microsatellite marker zkp48 falls within the sil locus and has 0 recombinants. (B) Sequencing of the csp^cz65 and csp^cz299 mutants revealed they possessed independent nonsense mutations. csp^cz65 mutants have a G-to-A mutation at nt 820 of the coding sequence, which results in a UGA stop codon corresponding to amino acid 273 replacing the wild-type (wt) UGG (Trp) sequence. A C-to-T mutation at nt 1834 of the csp^cz299 locus altered the wild-type CAG (Gln) sequence corresponding to amino acid 612 to a UAG stop codon. (C) Morpholino oligonucleotides targeted against sil cause increased phospho-H3 staining. moA and moB are independent splice site morpholino oligonucleotides that synergize to induce a more robust phenocopy. Scale bars = 250 μm. cntrl, control. (D) Whole-mount in situ hybridization with a sil riboprobe on 14-somite (top) and 24-hpf (bottom) embryos shows that csp mutants (right) lack positive staining, which is the earliest observable phenotype of the csp mutants. Scale bars = 100 μm.

FIG. 3.

The mitotic spindles of csp mutants are numerous and highly disorganized. (A) Microtubule staining detected by immunostaining against α-tubulin (red) and phospho-H3 (green) shows that all of the phospho-H3-positive cells in the csp mutants have an abnormal spindle (bottom) compare to wild-type bipolar spindles (top). The phospho-H3 staining demonstrates that the DNA is not properly aligned along a metaphase plate in the mitotic cells of the csp mutant. Scale bar = 5 μm. (B) Centrosome immunostaining with γ-tubulin (green) reveals that the disorganized mitotic cells of csp mutants identified with α-tubulin (in red) often have only one centrosome (bottom), which often stains with weaker intensity than wild-type centrosomes (top). (C) Immunoblotting for γ-tubulin shows that csp mutants have wild-type (WT) levels of γ-tubulin protein. (D) csp mutants are positive for BrdU incorporation. BrdU-labeled (red) embryos were costained for phospho-H3 (green) to distinguish the wild type from csp mutants. Scale bar = 200 μm.

FIG. 4.

csp mutant zebrafish do not exhibit left-right symmetry defects. In situ hybridization for cardiac myosin light chain 2 (cmlc2) (A) and lefty2 (B) showed robust staining in the heart field of 24-hpf wild-type (wt) and csp mutant embryos. Top, dorsal views of embryos with the anterior surface to the top. Bottom, left-side lateral views of the same embryos. Scale bars = 100 μm.

FIG. 5.

SIL localizes to the mitotic spindle in HeLa cells. (A) Affinity-purified anti-SIL detects a single band of roughly 150 kDa in transfected HEK293T cells. (B) Immunostaining for α-tubulin (red) and endogenous SIL (green) in mitotic HeLa cells shows accumulation of SIL at the mitotic spindle poles during metaphase. The hazy cytoplasmic staining of anti-SIL is not seen in interphase cells and is likely detecting cytosolic SIL protein expression during mitosis. (C) Costaining for centrosomes with γ-tubulin (red) indicates that SIL localizes to the pericentriolar region but does not completely colocalize with the centrosome. (D) Immunostaining for dynactin (red) and SIL (green) reveals that the spindle pole localization of SIL has some overlap with that of dynactin. Scale bar = 10 μm. DAPI, 4′,6′-diamidino-2-phenylindole.

FIG. 6.

SIL is expressed in the cytoplasm when overexpressed, and SIL does not localize to the spindle poles during anaphase. (A) HeLa cells were cotransfected with pCMV-HA-SIL and pCMV-SIL and immunostained with anti-HA (red) and anti-SIL (green) antibodies. SIL was present in the cytoplasm and stains positively with both antibodies. As in the cell shown, high levels of overexpression resulted in the formation of positively stained foci, which may be accumulation of proteins at the ribosomes or protein aggregates. CMV, cytomegalovirus. (B) A cell in anaphase stained with the anti-SIL antibody reveals that SIL does not localize to the spindle poles when cells are in anaphase. Scale bars = 10 μm.

FIG. 7.

shRNA knockdown of SIL in HeLa cells causes disorganized spindles. (A) Two hairpins, SH1 and SH3, knock down cytomegalovirus (CMV) SIL overexpression in HEK293T cells. The loading control a the bottom is a 70-kDa band detected with the 12CA5 anti-HA antibody. (B) Quantification of mitotic cells stained for α-tubulin reveals that SH1 and SH3 have a statistically significant increase in cells with disorganized mitotic spindles. (C) The majority of SIL knockdown cells with disorganized spindles lack SIL staining. SH4, a nonfunctional shRNA, was used as a secondary negative control in panels B and C. **, P < 0.001. (D) Examples of SH1-transfected cells with abnormal spindles and loss of SIL staining. (Top) A cell in which the chromatin fails to form a metaphase plate, the spindle lacks proper organization, and spindle poles are not defined. (Bottom) A cell in which a metaphase plate is formed and the bipolar mitotic spindle is not focused at the spindle pole. Scale bar = 10 μm. DAPI, 4′,6′-diamidino-2-phenylindole. (E) An SH1-transfected cell stained for α-tubulin and γ-tubulin shows an example of a knockdown cell in which the centrosomes are associated with the spindle but fail to localize to spindle poles.

FIG. 8.

Dynactin localizes to the spindle poles and remains associated with the mitotic spindle in SIL knockdown cells. (A) Costaining with α-tubulin (red) and dynactin (green) shows that dynactin localizes to the spindle pole during metaphase and has a broader region of expression than SIL. Note that dynactin is highly expressed on the astral side of the spindle. SIL expression is not seen on the astral side of the spindle. (B) A representative SH3-transfected cell with disorganized spindles shows that SIL knockdown does not result in dynactin dissociation from the spindle. Greater than 100 knockdown cells were examined, and none of them lost dynactin expression on the mitotic spindle. Scale bars = 10 μm. DAPI, 4′,6′-diamidino-2-phenylindole.