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Profiling of the mammalian mitotic spindle proteome reveals an ER protein, OSTD-1, as being necessary for cell division and ER morphology.

Bonner MK, Han BH, Skop A - PLoS ONE (2013)

Bottom Line: Of the candidate mammalian proteins, we determined that 77% had orthologs in Caenorhabditis elegans and 18% were associated with human disease.Of the C. elegans candidates (n=146), we determined that 34 genes functioned in embryonic development and 56% of these were predicted to be membrane trafficking proteins.We determined that OSTD-1 plays a role in maintaining the dynamic morphology of the ER during the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics and Medical Genetics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Cell division is important for many cellular processes including cell growth, reproduction, wound healing and stem cell renewal. Failures in cell division can often lead to tumors and birth defects. To identify factors necessary for this process, we implemented a comparative profiling strategy of the published mitotic spindle proteome from our laboratory. Of the candidate mammalian proteins, we determined that 77% had orthologs in Caenorhabditis elegans and 18% were associated with human disease. Of the C. elegans candidates (n=146), we determined that 34 genes functioned in embryonic development and 56% of these were predicted to be membrane trafficking proteins. A secondary, visual screen to detect distinct defects in cell division revealed 21 genes that were necessary for cytokinesis. One of these candidates, OSTD-1, an ER resident protein, was further characterized due to the aberrant cleavage furrow placement and failures in division. We determined that OSTD-1 plays a role in maintaining the dynamic morphology of the ER during the cell cycle. In addition, 65% of all ostd-1 RNAi-treated embryos failed to correctly position cleavage furrows, suggesting that proper ER morphology plays a necessary function during animal cell division.

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Visual screen of embryonic lethal genes.Confocal images were taken of embryos from a worm strain expressing GFP-PH PLC1delta1; mCherry-HIS-58 after depleting the 34 embryonic lethal genes for 24 h. The feeding vector (vec) was our negative control, and zen-4 [101] was our positive control. The genes are ordered by GO term, shown by text color (blue for membrane, red for actin, green for microtubule, grey for proteasome, lilac for unknown). Within each GO term group, the genes are ordered by strength of phenotype: ***-Very Strong (75%+ multi-nucleate embryos), **-Strong (50% multi-nucleate embryos), *-Weak (25% multi-nucleate embryos), and Later phenotype (no multi-nucleate embryos). The visual screen identified twenty-one genes that were associated with multi-nucleate embryos, which indicates those genes are involved in cell division. The gene, ostd-1 (outlined in blue), was chosen for further characterization in Figures 3-4. (Scale bar: 10 um).
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pone-0077051-g002: Visual screen of embryonic lethal genes.Confocal images were taken of embryos from a worm strain expressing GFP-PH PLC1delta1; mCherry-HIS-58 after depleting the 34 embryonic lethal genes for 24 h. The feeding vector (vec) was our negative control, and zen-4 [101] was our positive control. The genes are ordered by GO term, shown by text color (blue for membrane, red for actin, green for microtubule, grey for proteasome, lilac for unknown). Within each GO term group, the genes are ordered by strength of phenotype: ***-Very Strong (75%+ multi-nucleate embryos), **-Strong (50% multi-nucleate embryos), *-Weak (25% multi-nucleate embryos), and Later phenotype (no multi-nucleate embryos). The visual screen identified twenty-one genes that were associated with multi-nucleate embryos, which indicates those genes are involved in cell division. The gene, ostd-1 (outlined in blue), was chosen for further characterization in Figures 3-4. (Scale bar: 10 um).

Mentions: To determine which of the 34 embryonic lethal genes played specific roles in cell division, a secondary visual RNAi screen was performed. Here, the presence of multi-nucleate embryos suggests that these genes are important for cell division. To identify candidates that were particularly important for cytokinesis, we used a C. elegans strain that labeled the plasma membrane (GFP-PHPLC1delta1) and the chromatin (mCherry-HIS-58). This allowed us to identify multi-nucleate embryos easily. Twenty-one (21) of the 34 embryonic lethal genes resulted in multi-nucleate phenotypes (Figure 2). Phenotypes varied from one-cell embryos with several nuclei (mlc-5, rpn-11, cct-2) to multi-cellular embryos with one or more cells with at least two nuclei (ubq-1, ostd-1, arl-1). We marked strength of phenotype for each gene in Figure 2. Very strong genes resulted in at least 75% multi-nucleate embryos (Figure 2,***), strong genes yielded 50% multi-nucleate embryos (Figure 2,**), and weak genes resulted in 25% multi-nucleate embryos (Figure 2,*). Genes not resulting in multi-nucleate embryos likely arrested from events later in development (rme-8, unc-112, cap 2, K12H4.4). The largest group of genes (10/34) that was necessary for mitosis belonged to the membrane trafficking group (Figure 2, labeled in blue). Given the aberrant placement of cleavage furrows and cytokinesis failures in OSTD-1 RNAi-treated embryos, we sought to further characterize OSTD-1 in the early C. elegans embryo.


Profiling of the mammalian mitotic spindle proteome reveals an ER protein, OSTD-1, as being necessary for cell division and ER morphology.

Bonner MK, Han BH, Skop A - PLoS ONE (2013)

Visual screen of embryonic lethal genes.Confocal images were taken of embryos from a worm strain expressing GFP-PH PLC1delta1; mCherry-HIS-58 after depleting the 34 embryonic lethal genes for 24 h. The feeding vector (vec) was our negative control, and zen-4 [101] was our positive control. The genes are ordered by GO term, shown by text color (blue for membrane, red for actin, green for microtubule, grey for proteasome, lilac for unknown). Within each GO term group, the genes are ordered by strength of phenotype: ***-Very Strong (75%+ multi-nucleate embryos), **-Strong (50% multi-nucleate embryos), *-Weak (25% multi-nucleate embryos), and Later phenotype (no multi-nucleate embryos). The visual screen identified twenty-one genes that were associated with multi-nucleate embryos, which indicates those genes are involved in cell division. The gene, ostd-1 (outlined in blue), was chosen for further characterization in Figures 3-4. (Scale bar: 10 um).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3794981&req=5

pone-0077051-g002: Visual screen of embryonic lethal genes.Confocal images were taken of embryos from a worm strain expressing GFP-PH PLC1delta1; mCherry-HIS-58 after depleting the 34 embryonic lethal genes for 24 h. The feeding vector (vec) was our negative control, and zen-4 [101] was our positive control. The genes are ordered by GO term, shown by text color (blue for membrane, red for actin, green for microtubule, grey for proteasome, lilac for unknown). Within each GO term group, the genes are ordered by strength of phenotype: ***-Very Strong (75%+ multi-nucleate embryos), **-Strong (50% multi-nucleate embryos), *-Weak (25% multi-nucleate embryos), and Later phenotype (no multi-nucleate embryos). The visual screen identified twenty-one genes that were associated with multi-nucleate embryos, which indicates those genes are involved in cell division. The gene, ostd-1 (outlined in blue), was chosen for further characterization in Figures 3-4. (Scale bar: 10 um).
Mentions: To determine which of the 34 embryonic lethal genes played specific roles in cell division, a secondary visual RNAi screen was performed. Here, the presence of multi-nucleate embryos suggests that these genes are important for cell division. To identify candidates that were particularly important for cytokinesis, we used a C. elegans strain that labeled the plasma membrane (GFP-PHPLC1delta1) and the chromatin (mCherry-HIS-58). This allowed us to identify multi-nucleate embryos easily. Twenty-one (21) of the 34 embryonic lethal genes resulted in multi-nucleate phenotypes (Figure 2). Phenotypes varied from one-cell embryos with several nuclei (mlc-5, rpn-11, cct-2) to multi-cellular embryos with one or more cells with at least two nuclei (ubq-1, ostd-1, arl-1). We marked strength of phenotype for each gene in Figure 2. Very strong genes resulted in at least 75% multi-nucleate embryos (Figure 2,***), strong genes yielded 50% multi-nucleate embryos (Figure 2,**), and weak genes resulted in 25% multi-nucleate embryos (Figure 2,*). Genes not resulting in multi-nucleate embryos likely arrested from events later in development (rme-8, unc-112, cap 2, K12H4.4). The largest group of genes (10/34) that was necessary for mitosis belonged to the membrane trafficking group (Figure 2, labeled in blue). Given the aberrant placement of cleavage furrows and cytokinesis failures in OSTD-1 RNAi-treated embryos, we sought to further characterize OSTD-1 in the early C. elegans embryo.

Bottom Line: Of the candidate mammalian proteins, we determined that 77% had orthologs in Caenorhabditis elegans and 18% were associated with human disease.Of the C. elegans candidates (n=146), we determined that 34 genes functioned in embryonic development and 56% of these were predicted to be membrane trafficking proteins.We determined that OSTD-1 plays a role in maintaining the dynamic morphology of the ER during the cell cycle.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Genetics and Medical Genetics, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

ABSTRACT
Cell division is important for many cellular processes including cell growth, reproduction, wound healing and stem cell renewal. Failures in cell division can often lead to tumors and birth defects. To identify factors necessary for this process, we implemented a comparative profiling strategy of the published mitotic spindle proteome from our laboratory. Of the candidate mammalian proteins, we determined that 77% had orthologs in Caenorhabditis elegans and 18% were associated with human disease. Of the C. elegans candidates (n=146), we determined that 34 genes functioned in embryonic development and 56% of these were predicted to be membrane trafficking proteins. A secondary, visual screen to detect distinct defects in cell division revealed 21 genes that were necessary for cytokinesis. One of these candidates, OSTD-1, an ER resident protein, was further characterized due to the aberrant cleavage furrow placement and failures in division. We determined that OSTD-1 plays a role in maintaining the dynamic morphology of the ER during the cell cycle. In addition, 65% of all ostd-1 RNAi-treated embryos failed to correctly position cleavage furrows, suggesting that proper ER morphology plays a necessary function during animal cell division.

Show MeSH
Related in: MedlinePlus