α-Importome in germline: towards identification of novel roles and binding candidates for importin α family members in mammalian germ cell maturation
2017-02-28T03:46:10Z (GMT) by
Importin α family proteins are the cargo receptor component of the classical nuclear import machinery in eukaryotic cells. In the cytoplasm importin αs bind to cargo proteins through amino acid motifs known as nuclear localization signals (NLSs) and thereby mediate association with the nuclear import transporter, importin β1. This trimeric complex then enters the nucleus through interactions of importin β1 with components of the nuclear pore complex, and nuclear cargo is delivered into the nucleus to perform its function. During evolution, importin α genes have increased in number with increasing organismal complexity. Thus, 7 and 6 importin α family members have been identified in human and mouse, respectively, while lower eukaryotes contain 3 importin α family members and yeast has only 1. Despite sharing a high level of conservation in sequence, structure and function, these family members have acquired non-redundant roles in developmental systems. The functional specialization imparted to importin α proteins is, in part, effected by regulated spatial and temporal expression of individual family members as well as through their acquired cargo binding specificity. Adult spermatogenesis is a unique maturation process in which spermatogonial stem cells produce spermatozoa through highly regulated cell division and differentiation events. We hypothesized that importin α family members serve distinct functions in spermatogenesis, predicting initially that their roles in nucleocytoplasmic transport would require them to recognize distinct cargo subsets at key germline differentiation steps. We also proposed that based on known differences in their transcription patterns individual importin α proteins would exist in different proportions and levels at distinct steps of spermatogenesis. The purpose of the research conducted as part of this thesis was to first delineate the protein levels and deduce the relative stoichiometry of three importin α family members, (α2, α3 and α4) in addition to importin β1 in testicular germ cells of two sequential developmental stages, pachytene spermatocytes and round spermatids, through quantitative Western blot analyses. The second aim was to define the germline "α-importome" through identification of interacting partners for importin α family members in pachytene spermatocytes and round spermatids by means of recombinant protein pull-down and mass spectrometric analysis. The third aim of this thesis was to validate the interaction of importin α with a newly identified candidate binding partner, Mtmr4, a protein with known cytoplasmic localization. The results of the protein detection analyses supported our hypothesis that importin αs serve distinct and dynamic roles in spermatogenesis as reflected by regulated protein synthesis. Amongst the importin α family members examined, importin α2 had the highest variation in total concentration between spermatocytes and spermatids, followed by importin α3, with both α2 and α3 present at higher levels in spermatocytes than in spermatids. In contrast, the concentration of importin α4 and importin β1 did not differ between these two cell types. Comparison of the protein levels in these germ cell lysates to that in a lysate of total adult rat testis demonstrated that importin α family members have distinctly higher levels in the germ cell isolates than in the total testis, while the difference in importin β1 protein levels between isolated germ cells and total adult testis was marginal. Amongst the examined importin α family members, the most abundant was importin α2 followed by importin α3 and α4. Intriguingly however, these analyses revealed that importin αs are collectively more abundant than importin β1. This is the first study in which the differential production of importin proteins in a developmental context has been examined quantitatively, and the outcomes demonstrate that nucleocytoplasmic transport and other importin-mediated functions are potentially governed by processes that specify and modulate intracellular levels of specific importin proteins during development. The recombinant protein pull-down and mass spectrometry analyses led to identification of numerous binding candidates for importin α2 and α4 from spermatocyte and spermatids. Noticeably, the majority of candidates identified by the different importin αs or from each cell type were distinct. Several bioinformatics analyses of the candidate binding partners supported the hypothesis that importin αs serve distinct functions in each stage of spermatogenesis. Computational analysis aimed at discerning potential nuclear localization motifs verified that, within the list of all identified candidates, there is a clear enrichment in the proportion of proteins containing a putative NLS. However the level of this enrichment was not uniform between candidate cohorts identified by different importins from spermatocytes and spermatids. Specifically, the protein lists generated by importin α4 from spermatocytes and importin α2 from spermatids contained the highest proportion of candidates with a putative nuclear localization signal (52% each), followed by cohorts of candidates identified by importin α2 in spermatocytes and α4 in spermatids (39% and 30%, respectively). Bioinformatics examination of the molecular pathways represented by different importin α binding candidates showed that importin α2 binding candidate in spermatocytes are predicted to be involved in processes concerning RNA stability and differentiation, while importin α4 binding candidates in spermatocytes enriched many terms related to protein trafficking. The candidate binding partners for both importin α2 and α4 in spermatids, enriched many terms related to chromosomal and nuclear dynamics. These results align with the few recently published studies of a similar nature, which also give insight into the potential diversity in importin α family members function. The final body of work presented in this thesis is concerned with characterization of a putative interaction between importin α and Mtmr4, a known cytoplasmic protein. Mtmr4 was identified as a binding candidate of importin α4 from spermatocytes lysate in a previous study. One of the Mtmr4 family members (Mtmr2) has essential roles in spermatogenesis. Thus we aimed to accumulate evidence on the authenticity of Mtmr4 interaction with importin α in the context of spermatogenesis. Northern blot analysis showed that Mtmr4 transcript is present in a wide range of tissues; in situ hybridization on testis sections showed that this transcript is most readily detectable in pachytene spermatocytes in which importin α4 transcript and protein are present. Subsequently a putative nuclear localization signal was identified within the amino acid sequence of Mtmr4 which was shown to interact with both recombinant importin α2 and α4 in vitro. Pull-down and immunoprecipitation experiments provided further support for authenticity of the interaction between importin α and Mtmr4. Interestingly Mtmr4 was more readily detected in the pull-down product of an importin α2 truncate with higher NLS binding capacity and was barely detectable in the pull-down product of an importin α2 mutant, defective in NLS binding activity; this observation suggests that interaction of Mtmr4 and importin α occurs in NLS binding region of importin α. Further experimental approaches devised in this thesis could verify the functional nature of this intriguing interaction between importin α family members and Mtmr4, once appropriate reagents become available. Overall the results of this study have shed light on the importance of regulated synthesis and levels of individual importin α family members in developmental processes such as spermatogenesis. Many novel binding candidates for importin α family members have been identified in testicular germ cells which indicate that importin α proteins serve a broad spectrum of roles relating to cellular metabolism, potentially linked to a molecular scaffolding capacity. Advances made in characterizing the previously indicated interaction between importin α and Mtmr4 provide a clear demonstration that importin α proteins have significant interactions with proteins that are predominantly cytoplasmic. The potential for importins to mediate new germline-specific and general cellular metabolic roles is supported by the findings of this thesis.