Role of Negative Regulators of Nuclear Import and the Bidirectional Transporter Importin 13 in Spermatogenesis and Lineage specification
Version 2 2016-12-19, 03:03
Version 1 2016-12-19, 01:46
thesis
posted on 2016-12-19, 03:03 authored by Shadma FatimaRegulated
nucleo-cytoplasmic trafficking of macromolecules is central to processes,
including cell proliferation/differentiation, lineage specification and tissue
formation, wherein altered nuclear transport can lead to disease.
Although much research has focused on mechanisms of nuclear transport and the role of cargoes that play in the nucleus, little is known regarding the role of negative regulators of nuclear import (NRNIs), the mechanisms that may control the localization of Importin (Imp) transported cargoes and alter their functions. NRNIs are proteins that usually act as cytoplasmic retention factors for the cargoes that normally localize in the nucleus thereby serving as an inhibitory mechanism to Imp-mediated nuclear transport.
Here the role of two NRNIs, the BRCA1 binding protein 2 (BRAP2) and the N-terminally truncated inhibitory isoform of Imp13 (tImp13) in testis was examined for the first time. Although both BRAP2 and tImp13 are known to be expressed in testis, little was known about their specific roles before the work described in this thesis, which reports the interactome of BRAP2 and tImp13, identified through high throughput yeast-two-hybrid (Y2H) screening conducted using a human testis cDNA library. Novel BRAP2 binding proteins identified included those with roles in diverse cellular processes, including regulation of the actin cytoskeleton, ubiquitinylation, cell cycle/apoptosis and transcription. Interaction with the PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), A-Kinase anchor protein (AKAP3) and DNA methyl transferase 1 (DNMT1) was validated by coimmunoprecipitation assays from adult mouse testis lysate, while confocal microscopy and co-transfection assays confirmed BRAP2’s ability to inhibit PHLPP1 and DNMT1 nuclear localisation. The physiological relevance of these findings was underlined by the fact that PHLPP1, AKAP3 and DNMT1 show cytoplasmic localisation in pachytene spermatocytes and/or round spermatids where BRAP2 is present at high levels, in contrast to spermatogonia, where BRAP2 levels are lower and PHLPP1 and DNMT1 are nuclear. Interestingly, BRAP2 was also present in murine spermatozoa, in part co-localised with AKAP3. The results indicate for the first time that BRAP2 may play an important NRNI role in germ cells of the testis, with an additional, scaffold or structural role in mature spermatozoa.
tImp13 was hypothesized to act as an NRNI inhibiting Imp13-depedent nucleocytoplasmic transport and is thus thought to play a regulatory role in Imp13-cargoes derived cellular process. From a number of tImp13 interactors identified in Y2H screen, interaction of tImp13 with Elongation translation initiation factor 4γ2 (EIF4G2) and High mobility group containing protein 20A (HMG20A) was validated by coimmunoprecipitation from testis lysates, comparing the interactions with that of the glucocorticoid receptor (GR), a previously characterized testis-expressed Imp13 interactor. Analysis by quantitative confocal microscopy revealed the ability of tImp13 to inhibit nuclear localisation of EIF4G2, and HMG20A, and indicated that full length Imp13 can act as a nuclear exporter for both EIF4G2 and HMG20A. The physiological relevance of these results was implied by the cytoplasmic localisation of EIF4G2, HMG20A and GR in pachytene spermatocytes/round spermatids in mouse testis, where tImp13 is present at high levels, in contrast to the nuclear localisation of HMG20A and GR in spermatogonia, where tImp13 is largely absent. Interestingly, Imp13, EIF4G2, HMG20A and GR were found together in the acrosome of murine epididymal spermatozoa, suggesting a functional role in the mature spermatozoa, in addition to that in the germ cells of the testis.
NRNIs are inhibitors of nuclear import mediated by Imps, the facilitators of nuclear import ferrying cargoes between the nucleus and cytoplasm. Imps can be of α or β subtypes. One of the rare members of the Impβ superfamily is Imp13, which not only can mediate nuclear import but also export. Although implicated as a transporter for many important proteins, the role of Imp13 in directing cell-fate decision in embryonic stem cell (ESC) remains unknown. Here the role of Imp13 in the maintenance of stemness, pluripotency and lineage specification was examined in an embryonic stem cell (ESC) model. Undifferentiated Imp13 knock out (Ipo13−/−, KO) ESCs were viable, retained pluripotency and showed similar morphology to undifferentiated Imp13 wild type (Ipo13+/+, WT) ESCs. However, when induced to form embryonic bodies (EBs), KO ESCs displayed slow proliferation with reduced EB colony size. When compared to WT counterparts, the KOs displayed a significant reduction in the expression of proliferation marker KI67, concomitant with an increase in the number of TUNEL positive apoptotic nuclei. KO EBs also showed enhanced loss of the genes determining the pluripotency and germline differentiation potential of stem cells (OCT3/4, DAZL). At day 5 and 10 of differentiation well-defined clusters of OCT3/4 positive cells were readily observed in WT EBs but could be barely detectable in KOs. In parallel, reduced expression of mesodermal (Brachyury) and ectodermal marker (SOX1) but not endodermal markers (TTR) were observed in D5 KOs. Finally, underlying the functional significance, the KO EBs showed reduced haemoglobinised cells, but with an increase in cardiomyocyte differentiation potential. Our findings provide the first evidence that Imp13 contributes to cell survival and early germ layer specification during early embryonic development.
The results shed new light on the role of NRNIs in the process of spermatogenesis in the testis, the importance of the counterbalance between the activity of full length Imp13 and NRNIs such as tImp13 and BRAP2, can unearth a key role for Imp13 in lineage specification.
Although much research has focused on mechanisms of nuclear transport and the role of cargoes that play in the nucleus, little is known regarding the role of negative regulators of nuclear import (NRNIs), the mechanisms that may control the localization of Importin (Imp) transported cargoes and alter their functions. NRNIs are proteins that usually act as cytoplasmic retention factors for the cargoes that normally localize in the nucleus thereby serving as an inhibitory mechanism to Imp-mediated nuclear transport.
Here the role of two NRNIs, the BRCA1 binding protein 2 (BRAP2) and the N-terminally truncated inhibitory isoform of Imp13 (tImp13) in testis was examined for the first time. Although both BRAP2 and tImp13 are known to be expressed in testis, little was known about their specific roles before the work described in this thesis, which reports the interactome of BRAP2 and tImp13, identified through high throughput yeast-two-hybrid (Y2H) screening conducted using a human testis cDNA library. Novel BRAP2 binding proteins identified included those with roles in diverse cellular processes, including regulation of the actin cytoskeleton, ubiquitinylation, cell cycle/apoptosis and transcription. Interaction with the PH domain and leucine rich repeat protein phosphatase 1 (PHLPP1), A-Kinase anchor protein (AKAP3) and DNA methyl transferase 1 (DNMT1) was validated by coimmunoprecipitation assays from adult mouse testis lysate, while confocal microscopy and co-transfection assays confirmed BRAP2’s ability to inhibit PHLPP1 and DNMT1 nuclear localisation. The physiological relevance of these findings was underlined by the fact that PHLPP1, AKAP3 and DNMT1 show cytoplasmic localisation in pachytene spermatocytes and/or round spermatids where BRAP2 is present at high levels, in contrast to spermatogonia, where BRAP2 levels are lower and PHLPP1 and DNMT1 are nuclear. Interestingly, BRAP2 was also present in murine spermatozoa, in part co-localised with AKAP3. The results indicate for the first time that BRAP2 may play an important NRNI role in germ cells of the testis, with an additional, scaffold or structural role in mature spermatozoa.
tImp13 was hypothesized to act as an NRNI inhibiting Imp13-depedent nucleocytoplasmic transport and is thus thought to play a regulatory role in Imp13-cargoes derived cellular process. From a number of tImp13 interactors identified in Y2H screen, interaction of tImp13 with Elongation translation initiation factor 4γ2 (EIF4G2) and High mobility group containing protein 20A (HMG20A) was validated by coimmunoprecipitation from testis lysates, comparing the interactions with that of the glucocorticoid receptor (GR), a previously characterized testis-expressed Imp13 interactor. Analysis by quantitative confocal microscopy revealed the ability of tImp13 to inhibit nuclear localisation of EIF4G2, and HMG20A, and indicated that full length Imp13 can act as a nuclear exporter for both EIF4G2 and HMG20A. The physiological relevance of these results was implied by the cytoplasmic localisation of EIF4G2, HMG20A and GR in pachytene spermatocytes/round spermatids in mouse testis, where tImp13 is present at high levels, in contrast to the nuclear localisation of HMG20A and GR in spermatogonia, where tImp13 is largely absent. Interestingly, Imp13, EIF4G2, HMG20A and GR were found together in the acrosome of murine epididymal spermatozoa, suggesting a functional role in the mature spermatozoa, in addition to that in the germ cells of the testis.
NRNIs are inhibitors of nuclear import mediated by Imps, the facilitators of nuclear import ferrying cargoes between the nucleus and cytoplasm. Imps can be of α or β subtypes. One of the rare members of the Impβ superfamily is Imp13, which not only can mediate nuclear import but also export. Although implicated as a transporter for many important proteins, the role of Imp13 in directing cell-fate decision in embryonic stem cell (ESC) remains unknown. Here the role of Imp13 in the maintenance of stemness, pluripotency and lineage specification was examined in an embryonic stem cell (ESC) model. Undifferentiated Imp13 knock out (Ipo13−/−, KO) ESCs were viable, retained pluripotency and showed similar morphology to undifferentiated Imp13 wild type (Ipo13+/+, WT) ESCs. However, when induced to form embryonic bodies (EBs), KO ESCs displayed slow proliferation with reduced EB colony size. When compared to WT counterparts, the KOs displayed a significant reduction in the expression of proliferation marker KI67, concomitant with an increase in the number of TUNEL positive apoptotic nuclei. KO EBs also showed enhanced loss of the genes determining the pluripotency and germline differentiation potential of stem cells (OCT3/4, DAZL). At day 5 and 10 of differentiation well-defined clusters of OCT3/4 positive cells were readily observed in WT EBs but could be barely detectable in KOs. In parallel, reduced expression of mesodermal (Brachyury) and ectodermal marker (SOX1) but not endodermal markers (TTR) were observed in D5 KOs. Finally, underlying the functional significance, the KO EBs showed reduced haemoglobinised cells, but with an increase in cardiomyocyte differentiation potential. Our findings provide the first evidence that Imp13 contributes to cell survival and early germ layer specification during early embryonic development.
The results shed new light on the role of NRNIs in the process of spermatogenesis in the testis, the importance of the counterbalance between the activity of full length Imp13 and NRNIs such as tImp13 and BRAP2, can unearth a key role for Imp13 in lineage specification.
