Various methods can provide a readout of the physical interaction between two biomolecules. A recently described tripartite split-GFP system has the potential to report by direct visualization via a fluorescence signal the intimate association of minimally tagged proteins expressed at their endogenous level in their native cellular milieu and can capture transient or weak interactions. Here we document the utility of this tripartite split-GFP system to assess in living cells protein-protein interactions in a dynamic cytoskeletal structure-the septin collar at the yeast bud neck. We show, first, that for septin-septin interactions, this method yields a robust signal whose strength reflects the known spacing between the subunits in septin filaments and thus serves as a "molecular ruler." Second, the method yields little or no spurious signal even with highly abundant cytosolic proteins readily accessible to the bud neck (including molecular chaperone Hsp82 and glycolytic enzyme Pgk1). Third, using two proteins (Bni5 and Hsl1) that have been shown by other means to bind directly to septins at the bud neck in vivo, we validate that the tripartite split-GFP method yields the same conclusions and further insights about specificity. Finally, we demonstrate the capacity of this approach to uncover additional new information by examining whether three other proteins reported to localize to the bud neck (Nis1, Bud4, and Hof1) are able to interact physically with any of the subunits in the septin collar and, if so, with which ones.

Unlabelled

Septins are a conserved family of GTP-binding proteins that form heterooctameric complexes that assemble into higher-order structures. In yeast, septin superstructure at the bud neck serves as a barrier to separate a daughter cell from its mother and as a scaffold to recruit the proteins that execute cytokinesis. However, how septins recruit specific factors has not been well characterized. In the accompanying article in this issue, (Finnigan et al. 2015), we demonstrated that the C-terminal extensions (CTEs) of the alternative terminal subunits of septin heterooctamers, Cdc11 and Shs1, share a role required for optimal septin function in vivo. Here we describe our use of unbiased genetic approaches (both selection of dosage suppressors and analysis of synthetic interactions) that pinpointed Bni5 as a protein that interacts with the CTEs of Cdc11 and Shs1. Furthermore, we used three independent methods-construction of chimeric proteins, noncovalent tethering mediated by a GFP-targeted nanobody, and imaging by fluorescence microscopy-to confirm that a physiologically important function of the CTEs of Cdc11 and Shs1 is optimizing recruitment of Bni5 and thereby ensuring efficient localization at the bud neck of Myo1, the type II myosin of the actomyosin contractile ring.Related article in

Genetics

Finnigan, G. C. et al., 2015 Comprehensive Genetic Analysis of Paralogous Terminal Septin Subunits Shs1 and Cdc11 in Saccharomyces cerevisiae. Genetics 200: 841-861.

Objective

Identify the age at diagnosis and intervention of immigrant and/or non-English-speaking children with hearing loss (HL) and risk factors associated with delays. Identify barriers for non-English-speaking caregivers of deaf/hard-of-hearing children.

Study design

Sequential mixed methods.

Setting

Tertiary care center in an urban city.

Methods

The analysis includes descriptive statistics, and 1-way and 2-way analysis of variance of the retrospective chart review. The quantitative study demonstrated foreign-born experienced disparities, so we conducted semistructured interviews on a subset of non-English-speaking families in the cohort that was then thematically analyzed using a human-centered design strategy.

Results

We divided 532 children into 3 groups: US-born with English as the preferred language (N = 294), US-born and non-English language preferred (N = 173), and foreign-born (N = 67). The laterality of HL and pure-tone averages were similar among the groups (p = .972 and .071, respectively). Age at diagnosis and time to the intervention were significantly different (39.7, 31.5, 75.8 months, p < .001 and 24.6, 29.2, 48.9 months, p = .001, respectively). Ages at diagnosis and intervention were associated with birthplace (p = .005, p = .0005, respectively) but not preferred language (p = .667, p = .343, respectively). Included in the qualitative interviews were Mandarin- (n = 5), Arabic- (n = 4), and Spanish-speaking families (n = 3). Insights revealed participants' quest for anticipatory guidance and social support, the consequences of cultural stigma, and the complexity of caring for a child with HL in an immigrant family.

Conclusion

Foreign-born children with HL have significant delays in diagnosis and intervention compared to US-born children. For non-English-speaking parents, the diagnosis of HL presents challenges beyond that of the immigrant experience.

Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences.