Limits...
Putting things in place for fertilization: discovering roles for importin proteins in cell fate and spermatogenesis.

Loveland KL, Major AT, Butler R, Young JC, Jans DA, Miyamoto Y - Asian J. Androl. (2015 Jul-Aug)

Bottom Line: Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets.Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line.In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology;Department of Anatomy and Developmental Biology, Monash University; Hudson Institute of Medical Research, Monash Medical Centre; School of Clinical Sciences, Monash University, Clayton, VIC, Australia, .

ABSTRACT
Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, including via directing proteins to distinct intracellular compartments and by determining cellular stress responses.

No MeSH data available.


Related in: MedlinePlus

Identification of importin-binding proteins of relevance to spermatogenic differentiation. (a) Table outlining the different approaches used to identify IMPα-specific cargoes. Yeast 2 hybrid (Y2H) screening was used to identify over five specific, validated IMPα2 cargoes. Around 130 protein cargoes were identified using recombinant IMPα proteins tagged with GST in pulldown assays from lysates of whole adult rat testis or from Percoll-purified spermatocytes (SC) and spermatids (ST). An antibody-based immunoprecipitation (IP) approach, followed by mass spectrometry (MS) led to identification of up to 22 proteins specific for the different IMPαs. (b) Schematic of isolation processes leading to identification of cargoes from isolated germ cell subtypes. The most comprehensive studies to date have used pulldown assays with GST-conjugated recombinant IMPα proteins or immunoprecipitation from isolated spermatocyte or round spermatid lysates, followed by cargo identification through mass spectrometry.19,32 Around 130 IMPα binding proteins were identified using this approach. (c) Immunoprecipitation from spermatocytes and spermatids reveals different cargoes bound by each IMPa protein at specific stages of spermatogenesis.19 Examples listed include both nuclear and non-nuclear proteins. IMPα3 and IMPα4 both bind to IMPβ in spermatids and in spermatocytes, as expected, but each binds cargo subsets that are distinct in each germ cell type. This approach also identified common IMPα-specific cargoes: for example ZFP386 was identified as an IMPα3 cargo in both spermatocyte and spermatid lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492042&req=5

Figure 2: Identification of importin-binding proteins of relevance to spermatogenic differentiation. (a) Table outlining the different approaches used to identify IMPα-specific cargoes. Yeast 2 hybrid (Y2H) screening was used to identify over five specific, validated IMPα2 cargoes. Around 130 protein cargoes were identified using recombinant IMPα proteins tagged with GST in pulldown assays from lysates of whole adult rat testis or from Percoll-purified spermatocytes (SC) and spermatids (ST). An antibody-based immunoprecipitation (IP) approach, followed by mass spectrometry (MS) led to identification of up to 22 proteins specific for the different IMPαs. (b) Schematic of isolation processes leading to identification of cargoes from isolated germ cell subtypes. The most comprehensive studies to date have used pulldown assays with GST-conjugated recombinant IMPα proteins or immunoprecipitation from isolated spermatocyte or round spermatid lysates, followed by cargo identification through mass spectrometry.19,32 Around 130 IMPα binding proteins were identified using this approach. (c) Immunoprecipitation from spermatocytes and spermatids reveals different cargoes bound by each IMPa protein at specific stages of spermatogenesis.19 Examples listed include both nuclear and non-nuclear proteins. IMPα3 and IMPα4 both bind to IMPβ in spermatids and in spermatocytes, as expected, but each binds cargo subsets that are distinct in each germ cell type. This approach also identified common IMPα-specific cargoes: for example ZFP386 was identified as an IMPα3 cargo in both spermatocyte and spermatid lysates.

Mentions: To determine how and where importin α proteins influence male germline fate, it has been critical to delineate the “α-importome”. Summarized in Figure 2a, our studies have employed several distinct unbiased screening approaches to interrogate either whole rodent testes or isolated germ cells (spermatocytes and round spermatids). A yeast two-hybrid (Y2H) screen for importin α2 binding partners in the adult mouse testis identified and validated several nuclear proteins.41 These include Homologous protein 2 (Hop2), which is essential for fertility because of its role in promoting pairing between homologous chromosomes during meiosis. Two other binding partners were visualized as distinct nuclear speckles: androgen receptor interacting protein 3 (Arip3) was identified in promyelocytic leukemia (PML) bodies and was associated with SUMO1, while the Cysteine and histidine rich protein (Chrp) variant t4 colocalized with the U1snRNP in the spermatocyte model GC2 cell line, thereby indicating a possible role in RNA processing in male germ cells.41 A Y2H screen of the fetal mouse testis identified an additional importin α2 binding partner that is integral to nuclear paraspeckles.42


Putting things in place for fertilization: discovering roles for importin proteins in cell fate and spermatogenesis.

Loveland KL, Major AT, Butler R, Young JC, Jans DA, Miyamoto Y - Asian J. Androl. (2015 Jul-Aug)

Identification of importin-binding proteins of relevance to spermatogenic differentiation. (a) Table outlining the different approaches used to identify IMPα-specific cargoes. Yeast 2 hybrid (Y2H) screening was used to identify over five specific, validated IMPα2 cargoes. Around 130 protein cargoes were identified using recombinant IMPα proteins tagged with GST in pulldown assays from lysates of whole adult rat testis or from Percoll-purified spermatocytes (SC) and spermatids (ST). An antibody-based immunoprecipitation (IP) approach, followed by mass spectrometry (MS) led to identification of up to 22 proteins specific for the different IMPαs. (b) Schematic of isolation processes leading to identification of cargoes from isolated germ cell subtypes. The most comprehensive studies to date have used pulldown assays with GST-conjugated recombinant IMPα proteins or immunoprecipitation from isolated spermatocyte or round spermatid lysates, followed by cargo identification through mass spectrometry.19,32 Around 130 IMPα binding proteins were identified using this approach. (c) Immunoprecipitation from spermatocytes and spermatids reveals different cargoes bound by each IMPa protein at specific stages of spermatogenesis.19 Examples listed include both nuclear and non-nuclear proteins. IMPα3 and IMPα4 both bind to IMPβ in spermatids and in spermatocytes, as expected, but each binds cargo subsets that are distinct in each germ cell type. This approach also identified common IMPα-specific cargoes: for example ZFP386 was identified as an IMPα3 cargo in both spermatocyte and spermatid lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492042&req=5

Figure 2: Identification of importin-binding proteins of relevance to spermatogenic differentiation. (a) Table outlining the different approaches used to identify IMPα-specific cargoes. Yeast 2 hybrid (Y2H) screening was used to identify over five specific, validated IMPα2 cargoes. Around 130 protein cargoes were identified using recombinant IMPα proteins tagged with GST in pulldown assays from lysates of whole adult rat testis or from Percoll-purified spermatocytes (SC) and spermatids (ST). An antibody-based immunoprecipitation (IP) approach, followed by mass spectrometry (MS) led to identification of up to 22 proteins specific for the different IMPαs. (b) Schematic of isolation processes leading to identification of cargoes from isolated germ cell subtypes. The most comprehensive studies to date have used pulldown assays with GST-conjugated recombinant IMPα proteins or immunoprecipitation from isolated spermatocyte or round spermatid lysates, followed by cargo identification through mass spectrometry.19,32 Around 130 IMPα binding proteins were identified using this approach. (c) Immunoprecipitation from spermatocytes and spermatids reveals different cargoes bound by each IMPa protein at specific stages of spermatogenesis.19 Examples listed include both nuclear and non-nuclear proteins. IMPα3 and IMPα4 both bind to IMPβ in spermatids and in spermatocytes, as expected, but each binds cargo subsets that are distinct in each germ cell type. This approach also identified common IMPα-specific cargoes: for example ZFP386 was identified as an IMPα3 cargo in both spermatocyte and spermatid lysates.
Mentions: To determine how and where importin α proteins influence male germline fate, it has been critical to delineate the “α-importome”. Summarized in Figure 2a, our studies have employed several distinct unbiased screening approaches to interrogate either whole rodent testes or isolated germ cells (spermatocytes and round spermatids). A yeast two-hybrid (Y2H) screen for importin α2 binding partners in the adult mouse testis identified and validated several nuclear proteins.41 These include Homologous protein 2 (Hop2), which is essential for fertility because of its role in promoting pairing between homologous chromosomes during meiosis. Two other binding partners were visualized as distinct nuclear speckles: androgen receptor interacting protein 3 (Arip3) was identified in promyelocytic leukemia (PML) bodies and was associated with SUMO1, while the Cysteine and histidine rich protein (Chrp) variant t4 colocalized with the U1snRNP in the spermatocyte model GC2 cell line, thereby indicating a possible role in RNA processing in male germ cells.41 A Y2H screen of the fetal mouse testis identified an additional importin α2 binding partner that is integral to nuclear paraspeckles.42

Bottom Line: Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets.Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line.In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology;Department of Anatomy and Developmental Biology, Monash University; Hudson Institute of Medical Research, Monash Medical Centre; School of Clinical Sciences, Monash University, Clayton, VIC, Australia, .

ABSTRACT
Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems: embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, including via directing proteins to distinct intracellular compartments and by determining cellular stress responses.

No MeSH data available.


Related in: MedlinePlus