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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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Tim is a c-Src SH3 domain binding protein and substrate.A, Lysates were prepared from ES cells and EBs and incubated with immobilized GST, the Src GST-SH3 fusion protein, or the corresponding inactive GST-SH3 mutant. Following washing, bound proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with an anti-peptide antibody to Tim. Full-length Tim and a discrete cleavage product (CP) were found to associate with the GST-SH3 fusion protein, but not with GST alone or with the mutant GST-SH3 domain. B, Tim is a substrate for c-Src. Human 293T cells were transfected wild-type c-Src (Src-WT), a kinase-defective mutant (Src-KD), or with the empty expression plasmid (Con) together with V5 epitope-tagged mouse Tim as indicated. Tim was immunoprecipitated from the transfected cell lysates with a V5 antibody and immunoblotted for Tim protein recovery (Tim), tyrosine phosphorylation (pTyr), and ubiquitin (Ub). Tranfected Src protein expression was confirmed in the cell lysates, with actin as a loading control.
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pone-0017157-g002: Tim is a c-Src SH3 domain binding protein and substrate.A, Lysates were prepared from ES cells and EBs and incubated with immobilized GST, the Src GST-SH3 fusion protein, or the corresponding inactive GST-SH3 mutant. Following washing, bound proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with an anti-peptide antibody to Tim. Full-length Tim and a discrete cleavage product (CP) were found to associate with the GST-SH3 fusion protein, but not with GST alone or with the mutant GST-SH3 domain. B, Tim is a substrate for c-Src. Human 293T cells were transfected wild-type c-Src (Src-WT), a kinase-defective mutant (Src-KD), or with the empty expression plasmid (Con) together with V5 epitope-tagged mouse Tim as indicated. Tim was immunoprecipitated from the transfected cell lysates with a V5 antibody and immunoblotted for Tim protein recovery (Tim), tyrosine phosphorylation (pTyr), and ubiquitin (Ub). Tranfected Src protein expression was confirmed in the cell lysates, with actin as a loading control.

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)

Tim is a c-Src SH3 domain binding protein and substrate.A, Lysates were prepared from ES cells and EBs and incubated with immobilized GST, the Src GST-SH3 fusion protein, or the corresponding inactive GST-SH3 mutant. Following washing, bound proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with an anti-peptide antibody to Tim. Full-length Tim and a discrete cleavage product (CP) were found to associate with the GST-SH3 fusion protein, but not with GST alone or with the mutant GST-SH3 domain. B, Tim is a substrate for c-Src. Human 293T cells were transfected wild-type c-Src (Src-WT), a kinase-defective mutant (Src-KD), or with the empty expression plasmid (Con) together with V5 epitope-tagged mouse Tim as indicated. Tim was immunoprecipitated from the transfected cell lysates with a V5 antibody and immunoblotted for Tim protein recovery (Tim), tyrosine phosphorylation (pTyr), and ubiquitin (Ub). Tranfected Src protein expression was confirmed in the cell lysates, with actin as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040764&req=5

pone-0017157-g002: Tim is a c-Src SH3 domain binding protein and substrate.A, Lysates were prepared from ES cells and EBs and incubated with immobilized GST, the Src GST-SH3 fusion protein, or the corresponding inactive GST-SH3 mutant. Following washing, bound proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with an anti-peptide antibody to Tim. Full-length Tim and a discrete cleavage product (CP) were found to associate with the GST-SH3 fusion protein, but not with GST alone or with the mutant GST-SH3 domain. B, Tim is a substrate for c-Src. Human 293T cells were transfected wild-type c-Src (Src-WT), a kinase-defective mutant (Src-KD), or with the empty expression plasmid (Con) together with V5 epitope-tagged mouse Tim as indicated. Tim was immunoprecipitated from the transfected cell lysates with a V5 antibody and immunoblotted for Tim protein recovery (Tim), tyrosine phosphorylation (pTyr), and ubiquitin (Ub). Tranfected Src protein expression was confirmed in the cell lysates, with actin as a loading control.
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