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An unexpected role for the clock protein timeless in developmental apoptosis.

O'Reilly LP, Watkins SC, Smithgall TE - PLoS ONE (2011)

Bottom Line: Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate.Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate.As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: Programmed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells. Recent work from our group demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, we identified Timeless (Tim), the mammalian ortholog of a Drosophila circadian rhythm protein, as a binding partner and substrate for c-Src and probed its role in the differentiation of mES cells.

Methodology/principal findings: To determine whether Tim is involved in ES cell differentiation, Tim protein levels were stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and programmed cell death during cavitation.

Conclusions/significance: Our data demonstrate a novel function for the clock protein Tim during a key stage of early development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that circadian rhythms and early development may be intimately linked.

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Related in: MedlinePlus

Identification of Src SH3-binding proteins in ES cells and embryoid bodies (EBs).The mouse c-Src SH3 domain as well as a binding-defective control were expressed in bacteria as GST fusion proteins and immobilized on glutathione-agarose beads. Lysates from self-renewing ES cells and 6 day EBs were incubated with the immobilized wild-type and mutant Src SH3 domains, and associated proteins were eluted and resolved by SDS-PAGE (see Materials and Methods). A, Image of a Coomassie blue-stained gel of the purified wild-type (WT) and mutant (Mut) Src GST-SH3 proteins (No Lysate). SH3 target proteins captured from ES cell and EB lysates are indicated in the next four lanes. Unique bands were excised and identified via tryptic fingerprinting and MALDI-TOF MS, including the known Src SH3 interacting proteins hnRNP K (RNP) and Dynamin II (Dyn), as well as a fragment of Timeless (Tim; arrows). B, Details of the MS data obtained for Tim. The excised band from ES cells in Part A was halved and subjected to two separate MS runs. Tim was the top hit from both runs, with two peptides of identical sequence identified in each case. The sequence as well as the calculated and observed masses for each peptide are shown. C, The six numbered peptides from Part B map within the N-terminal region of the Tim protein. Three major domains of Tim are illustrated, including the N-terminal Timeless region, the DDT (Domain binding homeobox and Different Transcription factors) domain, and the C-terminal Timeless C region.
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pone-0017157-g001: Identification of Src SH3-binding proteins in ES cells and embryoid bodies (EBs).The mouse c-Src SH3 domain as well as a binding-defective control were expressed in bacteria as GST fusion proteins and immobilized on glutathione-agarose beads. Lysates from self-renewing ES cells and 6 day EBs were incubated with the immobilized wild-type and mutant Src SH3 domains, and associated proteins were eluted and resolved by SDS-PAGE (see Materials and Methods). A, Image of a Coomassie blue-stained gel of the purified wild-type (WT) and mutant (Mut) Src GST-SH3 proteins (No Lysate). SH3 target proteins captured from ES cell and EB lysates are indicated in the next four lanes. Unique bands were excised and identified via tryptic fingerprinting and MALDI-TOF MS, including the known Src SH3 interacting proteins hnRNP K (RNP) and Dynamin II (Dyn), as well as a fragment of Timeless (Tim; arrows). B, Details of the MS data obtained for Tim. The excised band from ES cells in Part A was halved and subjected to two separate MS runs. Tim was the top hit from both runs, with two peptides of identical sequence identified in each case. The sequence as well as the calculated and observed masses for each peptide are shown. C, The six numbered peptides from Part B map within the N-terminal region of the Tim protein. Three major domains of Tim are illustrated, including the N-terminal Timeless region, the DDT (Domain binding homeobox and Different Transcription factors) domain, and the C-terminal Timeless C region.

Mentions: Previous studies summarized above point to the c-Src protein-tyrosine kinase as an important regulator of the earliest stages of mES cell differentiation. These findings led to the question of the signaling pathways controlled by c-Src that account for its role in ES cell fate. To address this question, c-Src target protein capture experiments were performed using an immobilized, recombinant c-Src SH3 domain fusion protein and soluble protein extracts from both self-renewing mES cells as well as differentiated EBs. SH3 domains contribute not only to SFK regulation (see Introduction) but also to substrate recruitment by binding to target proteins containing polyproline type II helices [23]. Unique SH3-interacting proteins were captured by the c-Src SH3 domain but not by an inactive mutant control domain, indicative of specific binding (Fig. 1). Three prominent bands were excised from the gel, digested with trypsin, and identified by MALDI-TOF MS and MS/MS sequencing: 1) Dynamin II, a GTPase involved in vesicular trafficking [24]; 2) hnRNPK, which regulates transcription, pre-mRNA processing, mRNA transport and translation [25]; and 3) a 54 kDa N-terminal fragment of Tim. Both Dynamin II and hnRNPK have been identified previously as c-Src SH3-binding proteins [26], [27], validating our experimental approach. In contrast, the SH3-dependent association of Tim with c-Src or other SFKs has not been reported, suggestive of a novel interaction. To confirm that Tim is an SH3-binding partner for c-Src, SH3 capture experiments were repeated using lysates from ES cells and EBs, followed by immunoblotting with an antibody to the Tim protein. Full-length Tim was captured in each case, as well as a prominent cleavage product that corresponds in size to the fragment originally identified by tryptic fingerprinting (Fig. 2A). In contrast, no binding was detected with the inactive mutant of the c-Src SH3 domain or with GST alone, indicative of a specific SH3-mediated binding event.


An unexpected role for the clock protein timeless in developmental apoptosis.

O'Reilly LP, Watkins SC, Smithgall TE - PLoS ONE (2011)

Identification of Src SH3-binding proteins in ES cells and embryoid bodies (EBs).The mouse c-Src SH3 domain as well as a binding-defective control were expressed in bacteria as GST fusion proteins and immobilized on glutathione-agarose beads. Lysates from self-renewing ES cells and 6 day EBs were incubated with the immobilized wild-type and mutant Src SH3 domains, and associated proteins were eluted and resolved by SDS-PAGE (see Materials and Methods). A, Image of a Coomassie blue-stained gel of the purified wild-type (WT) and mutant (Mut) Src GST-SH3 proteins (No Lysate). SH3 target proteins captured from ES cell and EB lysates are indicated in the next four lanes. Unique bands were excised and identified via tryptic fingerprinting and MALDI-TOF MS, including the known Src SH3 interacting proteins hnRNP K (RNP) and Dynamin II (Dyn), as well as a fragment of Timeless (Tim; arrows). B, Details of the MS data obtained for Tim. The excised band from ES cells in Part A was halved and subjected to two separate MS runs. Tim was the top hit from both runs, with two peptides of identical sequence identified in each case. The sequence as well as the calculated and observed masses for each peptide are shown. C, The six numbered peptides from Part B map within the N-terminal region of the Tim protein. Three major domains of Tim are illustrated, including the N-terminal Timeless region, the DDT (Domain binding homeobox and Different Transcription factors) domain, and the C-terminal Timeless C region.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040764&req=5

pone-0017157-g001: Identification of Src SH3-binding proteins in ES cells and embryoid bodies (EBs).The mouse c-Src SH3 domain as well as a binding-defective control were expressed in bacteria as GST fusion proteins and immobilized on glutathione-agarose beads. Lysates from self-renewing ES cells and 6 day EBs were incubated with the immobilized wild-type and mutant Src SH3 domains, and associated proteins were eluted and resolved by SDS-PAGE (see Materials and Methods). A, Image of a Coomassie blue-stained gel of the purified wild-type (WT) and mutant (Mut) Src GST-SH3 proteins (No Lysate). SH3 target proteins captured from ES cell and EB lysates are indicated in the next four lanes. Unique bands were excised and identified via tryptic fingerprinting and MALDI-TOF MS, including the known Src SH3 interacting proteins hnRNP K (RNP) and Dynamin II (Dyn), as well as a fragment of Timeless (Tim; arrows). B, Details of the MS data obtained for Tim. The excised band from ES cells in Part A was halved and subjected to two separate MS runs. Tim was the top hit from both runs, with two peptides of identical sequence identified in each case. The sequence as well as the calculated and observed masses for each peptide are shown. C, The six numbered peptides from Part B map within the N-terminal region of the Tim protein. Three major domains of Tim are illustrated, including the N-terminal Timeless region, the DDT (Domain binding homeobox and Different Transcription factors) domain, and the C-terminal Timeless C region.
Mentions: Previous studies summarized above point to the c-Src protein-tyrosine kinase as an important regulator of the earliest stages of mES cell differentiation. These findings led to the question of the signaling pathways controlled by c-Src that account for its role in ES cell fate. To address this question, c-Src target protein capture experiments were performed using an immobilized, recombinant c-Src SH3 domain fusion protein and soluble protein extracts from both self-renewing mES cells as well as differentiated EBs. SH3 domains contribute not only to SFK regulation (see Introduction) but also to substrate recruitment by binding to target proteins containing polyproline type II helices [23]. Unique SH3-interacting proteins were captured by the c-Src SH3 domain but not by an inactive mutant control domain, indicative of specific binding (Fig. 1). Three prominent bands were excised from the gel, digested with trypsin, and identified by MALDI-TOF MS and MS/MS sequencing: 1) Dynamin II, a GTPase involved in vesicular trafficking [24]; 2) hnRNPK, which regulates transcription, pre-mRNA processing, mRNA transport and translation [25]; and 3) a 54 kDa N-terminal fragment of Tim. Both Dynamin II and hnRNPK have been identified previously as c-Src SH3-binding proteins [26], [27], validating our experimental approach. In contrast, the SH3-dependent association of Tim with c-Src or other SFKs has not been reported, suggestive of a novel interaction. To confirm that Tim is an SH3-binding partner for c-Src, SH3 capture experiments were repeated using lysates from ES cells and EBs, followed by immunoblotting with an antibody to the Tim protein. Full-length Tim was captured in each case, as well as a prominent cleavage product that corresponds in size to the fragment originally identified by tryptic fingerprinting (Fig. 2A). In contrast, no binding was detected with the inactive mutant of the c-Src SH3 domain or with GST alone, indicative of a specific SH3-mediated binding event.

Bottom Line: Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate.Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate.As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America.

ABSTRACT

Background: Programmed cell death is critical not only in adult tissue homeostasis but for embryogenesis as well. One of the earliest steps in development, formation of the proamniotic cavity, involves coordinated apoptosis of embryonic cells. Recent work from our group demonstrated that c-Src protein-tyrosine kinase activity triggers differentiation of mouse embryonic stem (mES) cells to primitive ectoderm-like cells. In this report, we identified Timeless (Tim), the mammalian ortholog of a Drosophila circadian rhythm protein, as a binding partner and substrate for c-Src and probed its role in the differentiation of mES cells.

Methodology/principal findings: To determine whether Tim is involved in ES cell differentiation, Tim protein levels were stably suppressed using shRNA. Tim-defective ES cell lines were then tested for embryoid body (EB) formation, which models early mammalian development. Remarkably, confocal microscopy revealed that EBs formed from the Tim-knockdown ES cells failed to cavitate. Cells retained within the centers of the failed cavities strongly expressed the pluripotency marker Oct4, suggesting that further development is arrested without Tim. Immunoblots revealed reduced basal Caspase activity in the Tim-defective EBs compared to wild-type controls. Furthermore, EBs formed from Tim-knockdown cells demonstrated resistance to staurosporine-induced apoptosis, consistent with a link between Tim and programmed cell death during cavitation.

Conclusions/significance: Our data demonstrate a novel function for the clock protein Tim during a key stage of early development. Specifically, EBs formed from ES cells lacking Tim showed reduced caspase activity and failed to cavitate. As a consequence, further development was halted, and the cells present in the failed cavity remained pluripotent. These findings reveal a new function for Tim in the coordination of ES cell differentiation, and raise the intriguing possibility that circadian rhythms and early development may be intimately linked.

Show MeSH
Related in: MedlinePlus