Access all areas: multisensory science exhibitions tailored toward blind, low-vision and diverse-needs communities.

Monash Sensory Science is a scientific outreach initiative specifically tailored to members of the community who are blind, have low vision and have diverse needs. The purpose of this initiative is to showcase Australian science and encourage greater participation in science from these often-overlooked communities. This article presents our experience in establishing Monash Sensory Science at Monash University and inspiring other institutions to launch similar outreach events.

Overlapping binding sites for importin ß1 and suppressor of fused (SuFu) on glioma-associated oncogene homologue 1 (Gli1) regulate its nuclear localization.

A key factor in oncogenesis is the transport into the nucleus of oncogenic signalling molecules, such as Gli1 (glioma-associated oncogene homologue 1), the central transcriptional activator in the Hedgehog signalling pathway. Little is known, however, how factors such as Gli are transported into the nucleus and how this may be regulated by interaction with other cellular factors, such as the negative regulator suppressor of fused (SuFu). In the present study we show for the first time that nuclear entry of Gli1 is regulated by a unique mechanism through mutually exclusive binding by its nuclear import factor Impß1 (importin ß1) and SuFu. Using quantitative live mammalian cell imaging, we show that nuclear accumulation of GFP-Gli1 fusion proteins, but not of a control protein, is specifically inhibited by co-expression of SuFu. Using a direct binding assay, we show that Impß1 exhibits a high nanomolar affinity to Gli1, with specific knockdown of Impß1 expression being able to inhibit Gli1 nuclear accumulation, thus implicating Impß1 as the nuclear transporter for Gli1 for the first time. SuFu also binds to Gli1 with a high nanomolar affinity, intriguingly being able to compete with Impß1 for binding to Gli1, through the fact that the sites for SuFu and Impß1 binding overlap at the Gli1 N-terminus. The results indicate for the first time that the relative intracellular concentrations of SuFu and Impß1 are likely to determine the localization of Gli1, with implications for its action in cancer, as well as in developmental systems.

The nuclear import factor importin α4 can protect against oxidative stress.

The importin (IMP) superfamily of nuclear transport proteins is essential to key developmental pathways, including in the murine testis where expression of the 6 distinct IMPα proteins is highly dynamic. Present predominantly from the spermatocyte stage onwards, IMPα4 is unique in showing a striking nuclear localization, a property we previously found to be linked to maintenance of pluripotency in embryonic stem cells and to the cellular stress response in cultured cells. Here we examine the role of IMPα4 in vivo for the first time using a novel transgenic mouse model in which we overexpress an IMPα4-EGFP fusion protein from the protamine 1 promoter to recapitulate endogenous testicular germ cell IMPα4 expression in spermatids. IMPα4 overexpression did not affect overall fertility, testis morphology/weight or spermatogenic progression under normal conditions, but conferred significantly (>30%) increased resistance to oxidative stress specifically in the spermatid subpopulation expressing the transgene. Consistent with a cell-specific role for IMPα4 in protecting against oxidative stress, haploid germ cells from IMPα4 null mice were significantly (c. 30%) less resistant to oxidative stress than wild type controls. These results from two unique and complementary mouse models demonstrate a novel protective role for IMPα4 in stress responses specifically within haploid male germline cells, with implications for male fertility and genetic integrity.

Intracellular calcium levels can regulate Importin-dependent nuclear import.

We previously showed that increased intracellular calcium can modulate Importin (Imp)ß1-dependent nuclear import of SRY-related chromatin remodeling proteins. Here we extend this work to show for the first time that high intracellular calcium inhibits Impα/ß1- or Impß1-dependent nuclear protein import generally. The basis of this relates to the mislocalisation of the transport factors Impß1 and Ran, which show significantly higher nuclear localization in contrast to various other factors, and RCC1, which shows altered subnuclear localisation. The results here establish for the first time that intracellular calcium modulates conventional nuclear import through direct effects on the nuclear transport machinery.

Importin alpha2-interacting proteins with nuclear roles during mammalian spermatogenesis.

Spermatogenesis, the process of generating haploid sperm capable of fertilizing the female gamete, requires the timely transport into the nucleus of transcription and chromatin-remodeling factors, mediated by members of the importin (IMP) superfamily. Previous IMP expression profiling implies a role for IMPalpha2 in testicular germ cells late in spermatogenesis. To identify interacting proteins of IMPalpha2 that are potential drivers of germ cell development, we performed yeast two-hybrid screening of an adult mouse testis library. IMPalpha2 interactions were verified by coimmunoprecipitation approaches, whereas immunohistochemical staining of testis sections confirmed their coexpression with IMPalpha2 in specific testicular cell types. Key interactors identified were a novel isoform of a cysteine and histidine rich protein (Chrp), a protein inhibitor of activated STAT (PIAS) family member involved in transcriptional regulation and sumoylation, Androgen receptor interacting protein 3 (Arip3), and Homologous protein 2 (Hop2), known to be involved in homologous chromosome pairing and recombination, all of which are highly expressed in the testis and show mRNA expression profiles similar to that of IMPalpha2 throughout testicular development. This is the first study to identify binding partners of IMPalpha2 in the developmental context of germ line development, and we propose that the regulated expression and timely IMPalpha2-mediated nuclear transport of these proteins may coordinate events during spermatogenesis, with IMPalpha2-mediated nuclear localization representing a potentially critical developmental switch in the testis.

Nuclear import of HSV-1 DNA polymerase processivity factor UL42 is mediated by a C-terminally located bipartite nuclear localization signal.

The polymerase accessory protein of the human herpes simplex virus type 1 (HSV-1) DNA polymerase UL42 plays an essential role in viral replication, conferring processivity to the catalytic subunit UL30. We show here that UL42 is imported to the nucleus of living cells in a Ran- and energy-dependent fashion, through a process that requires a C-terminally located bipartite nuclear localization signal (UL42-NLSbip; PTTKRGRSGGEDARADALKKPK(413)). Moreover cytoplasmic mutant derivatives of UL42 lacking UL42-NLSbip are partially relocalized into the cell nucleus upon HSV-1 infection or coexpression with UL30, implying that the HSV-1 DNA polymerase holoenzyme can assemble in the cytoplasm before nuclear translocation occurs, thus explaining why the UL42 C-terminal domain is not strictly required for viral replication in cultured cells. However, mutation of both UL30 and UL42 NLS results in retention of the DNA polymerase holoenzyme in the cytoplasm, suggesting that simultaneous inhibition of both NLSs could represent a viable strategy to hinder HSV-1 replication. Intriguingly, UL42-NLSbip is composed of two stretches of basic amino acids matching the consensus for classical monopartite NLSs (NLSA, PTTKRGR(397); NLSB, KKPK(413)), neither of which are capable of targeting GFP to the nucleus on their own, consistent with the hypothesis that P and G residues in position +3 of monopartite NLSs are not compatible with nuclear transport in the absence of additional basic sequences located in close proximity. Our results showing that substitution of G or P of the NLS with an A residue partially confers NLS function will help to redefine the consensus for monopartite NLSs.

Characterization of gametogenetin 1 (GGN1) and its potential role in male fertility through the interaction with the ion channel regulator, cysteine-rich secretory protein 2 (CRISP2) in the sperm tail.

Cysteine-rich secretory protein 2 (CRISP2) is a testis-enriched protein localized to the sperm acrosome and tail. CRISP2 has been proposed to play a critical role in spermatogenesis and male fertility, although the precise function(s) of CRISP2 remains to be determined. Recent data have shown that the CRISP domain of the mouse CRISP2 has the ability to regulate Ca(2+) flow through ryanodine receptors (RyR) and to bind to MAP kinase kinase kinase 11 (MAP3K11). To further define the biochemical pathways within which CRISP2 is involved, we screened an adult mouse testis cDNA library using a yeast two-hybrid assay to identify CRISP2 interacting partners. One of the most frequently identified CRISP2-binding proteins was gametogenetin 1 (GGN1). Interactions occur between the ion channel regulatory region within the CRISP2 CRISP domain and the carboxyl-most 158 amino acids of GGN1. CRISP2 does not bind to the GGN2 or GGN3 isoforms. Furthermore, we showed that Ggn1 is a testis-enriched mRNA and the protein first appeared in late pachytene spermatocytes and was up-regulated in round spermatids before being incorporated into the principal piece of the sperm tail where it co-localized with CRISP2. These data along with data on RyR and MAP3K11 binding define the CRISP2 CRISP domain as a protein interaction motif and suggest a role for the GGN1-CRISP2 complex in sperm tail development and/or motility.