Becoming a resilient scientist series: An intervention program.

Compared to the general population, science trainees experience challenges and heightened stressors that often lead to adverse mental health outcomes. With COVID-19, the stressors of social distancing, isolation, truncated lab time, and uncertainty about the future have all likely exacerbated these issues. Now, more than ever, practical and effective interventions are vitally needed to address the core causes of stress among science trainees and increase their resilience. This paper introduces a new resilience program targeted to biomedical trainees and scientists - Becoming a Resilient Scientist Series (BRS), a multi-part workshop complemented by facilitated group discussions all aimed at bolstering resilience, particularly in the context of academic and research environments. To assess the program's efficacy, participants completed resilience measures and related assessments before and after completing the series. The results demonstrate that BRS significantly enhances trainee resilience (primary outcome) and reduces perceived stress, anxiety, and work-related presenteeism, as well as increased adaptability, self-awareness, and self-efficacy (secondary outcomes). Furthermore, program participants reported a high level of satisfaction, a strong willingness to recommend the program to others, and perceived positive changes in their resilience skills. To the best of our knowledge, this is the first resilience program designed explicitly for biomedical trainees and scientists, tailored to their unique professional culture and work environment.

Sorting nexin 27 (SNX27) associates with zonula occludens-2 (ZO-2) and modulates the epithelial tight junction.

Proteins of the SNX (sorting nexin) superfamily are characterized by the presence of a PX (Phox homology) domain and associate with PtdIns3P (phosphatidylinositol-3-monophosphate)-rich regions of the endosomal system. SNX27 is the only sorting nexin that contains a PDZ domain. In the present study, we used a proteomic approach to identify a novel interaction between SNX27 and ZO-2 [zonula occludens-2; also known as TJP2 (tight junction protein 2)], a component of the epithelial tight junction. The SNX27-ZO-2 interaction requires the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of ZO-2. When tight junctions were perturbed by chelation of extracellular Ca2+, ZO-2 transiently localized to SNX27-positive early endosomes. Depletion of SNX27 in mpkCCD (mouse primary kidney cortical collecting duct) cell monolayers resulted in a decrease in the rate of ZO-2, but not ZO-1, mobility at cell-cell contact regions after photobleaching and an increase in junctional permeability to large solutes. The findings of the present study identify an important new SNX27-binding partner and suggest a role for endocytic pathways in the intracellular trafficking of ZO-2 and possibly other tight junction proteins. Our results also indicate a role for SNX27-ZO-2 interactions in tight junction maintenance and function.

Dileucine and PDZ-binding motifs mediate synaptic adhesion-like molecule 1 (SALM1) trafficking in hippocampal neurons.

Synaptic adhesion-like molecules (SALMs) are a family of cell adhesion molecules involved in neurite outgrowth and synapse formation. Of the five family members, only SALM1, -2, and -3 contain a cytoplasmic C-terminal PDZ-binding motif. We have found that SALM1 is unique among the SALMs because deletion of its PDZ-binding motif (SALM1ΔPDZ) blocks its surface expression in heterologous cells. When expressed in hippocampal neurons, SALM1ΔPDZ had decreased surface expression in dendrites and the cell soma but not in axons, suggesting that the PDZ-binding domain may influence cellular trafficking of SALMs to specific neuronal locations. Endoglycosidase H digestion assays indicated that SALM1ΔPDZ is retained in the endoplasmic reticulum (ER) in heterologous cells. However, when the entire C-terminal tail of SALM1 was deleted, SALM1 was detected on the cell surface. Using serial deletions, we identified a region of SALM1 that contains a putative dileucine ER retention motif, which is not present in the other SALMs. Mutation of this DXXXLL motif allowed SALM1 to leave the ER and enhanced its surface expression in heterologous cells and neurons. An increase in the number of protrusions at the dendrites and cell body was observed when this SALM1 mutant was expressed in hippocampal neurons. With electron microscopy, these protrusions appeared to be irregular, enlarged spines and filopodia. Thus, enrichment of SALM1 on the cell surface affects dendritic arborization, and intracellular motifs regulate its dendritic versus axonal localization.

Sorting nexin 27 protein regulates trafficking of a p21-activated kinase (PAK) interacting exchange factor (ß-Pix)-G protein-coupled receptor kinase interacting protein (GIT) complex via a PDZ domain interaction.

Sorting nexin 27 (SNX27) is a 62-kDa protein localized to early endosomes and known to regulate the intracellular trafficking of ion channels and receptors. In addition to a PX domain, SNX27 is the only sorting family member that contains a PDZ domain. To identify novel SNX27-PDZ binding partners, we performed a proteomic screen in mouse principal kidney cortical collecting duct cells using a GST-SNX27 fusion construct as bait. We found that ß-Pix (p21-activated kinase-interactive exchange factor), a guanine nucleotide exchange factor for the Rho family of small GTPases known to regulate cell motility directly interacted with SNX27. The association of ß-Pix and SNX27 is specific for ß-Pix isoforms terminating in the type-1 PDZ binding motif (ETNL). In the same screen we also identified Git1/2 as a potential SNX27 interacting protein. The interaction between SNX27 and Git1/2 is indirect and mediated by ß-Pix. Furthermore, we show recruitment of the ß-Pix·Git complex to endosomal sites in a SNX27-dependent manner. Finally, migration assays revealed that depletion of SNX27 from HeLa and mouse principal kidney cortical collecting duct cells significantly decreases cell motility. We propose a model by which SNX27 regulates trafficking of ß-Pix to focal adhesions and thereby influences cell motility.

Yes-associated protein 65 (YAP) expands neural progenitors and regulates Pax3 expression in the neural plate border zone.

Yes-associated protein 65 (YAP) contains multiple protein-protein interaction domains and functions as both a transcriptional co-activator and as a scaffolding protein. Mouse embryos lacking YAP did not survive past embryonic day 8.5 and showed signs of defective yolk sac vasculogenesis, chorioallantoic fusion, and anterior-posterior (A-P) axis elongation. Given that the YAP knockout mouse defects might be due in part to nutritional deficiencies, we sought to better characterize a role for YAP during early development using embryos that develop externally. YAP morpholino (MO)-mediated loss-of-function in both frog and fish resulted in incomplete epiboly at gastrulation and impaired axis formation, similar to the mouse phenotype. In frog, germ layer specific genes were expressed, but they were temporally delayed. YAP MO-mediated partial knockdown in frog allowed a shortened axis to form. YAP gain-of-function in Xenopus expanded the progenitor populations in the neural plate (sox2(+)) and neural plate border zone (pax3(+)), while inhibiting the expression of later markers of tissues derived from the neural plate border zone (neural crest, pre-placodal ectoderm, hatching gland), as well as epidermis and somitic muscle. YAP directly regulates pax3 expression via association with TEAD1 (N-TEF) at a highly conserved, previously undescribed, TEAD-binding site within the 5' regulatory region of pax3. Structure/function analyses revealed that the PDZ-binding motif of YAP contributes to the inhibition of epidermal and somitic muscle differentiation, but a complete, intact YAP protein is required for expansion of the neural plate and neural plate border zone progenitor pools. These results provide a thorough analysis of YAP mediated gene expression changes in loss- and gain-of-function experiments. Furthermore, this is the first report to use YAP structure-function analyzes to determine which portion of YAP is involved in specific gene expression changes and the first to show direct in vivo evidence of YAP's role in regulating pax3 neural crest expression.

Activation of the epithelial sodium channel by the metalloprotease meprin ß subunit.

The Epithelial Na(+) Channel (ENaC) is an apical heteromeric channel that mediates Na(+) entry into epithelial cells from the luminal cell surface. ENaC is activated by proteases that interact with the channel during biosynthesis or at the extracellular surface. Meprins are cell surface and secreted metalloproteinases of the kidney and intestine. We discovered by affinity chromatography that meprins bind γ-ENaC, a subunit of the ENaC hetero-oligomer. The physical interaction involves NH(2)-terminal cytoplasmic residues 37-54 of γ-ENaC, containing a critical gating domain immediately before the first transmembrane domain, and the cytoplasmic COOH-terminal tail of meprin ß (residues 679-704). This potential association was confirmed by co-expression and co-immunoprecipitation studies. Functional assays revealed that meprins stimulate ENaC expressed exogenously in Xenopus oocytes and endogenously in epithelial cells. Co-expression of ENaC subunits and meprin ß or α/ß in Xenopus oocytes increased amiloride-sensitive Na(+) currents approximately two-fold. This increase was blocked by preincubation with an inhibitor of meprin activity, actinonin. The meprin-mediated increase in ENaC currents in oocytes and epithelial cell monolayers required meprin ß, but not the α subunit. Meprin ß promoted cleavage of α and γ-ENaC subunits at sites close to the second transmembrane domain in the extracellular domain of each channel subunit. Thus, meprin ß regulates the activity of ENaC in a metalloprotease-dependent fashion.

Cystic fibrosis transmembrane conductance regulator interacts with multiple immunoglobulin domains of filamin A.

Mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) that impair its apical localization and function cause cystic fibrosis. A previous report has shown that filamin A (FLNa), an actin-cross-linking and -scaffolding protein, interacts directly with the cytoplasmic N terminus of CFTR and that this interaction is necessary for stability and confinement of the channel to apical membranes. Here, we report that the CFTR N terminus has sequence similarity to known FLNa-binding partner-binding sites. FLNa has 24 Ig (IgFLNa) repeats, and a CFTR peptide pulled down repeats 9, 12, 17, 19, 21, and 23, which share sequence similarity yet differ from the other FLNa Ig domains. Using known structures of IgFLNa.partner complexes as templates, we generated in silico models of IgFLNa.CFTR peptide complexes. Point and deletion mutants of IgFLNa and CFTR informed by the models, including disease-causing mutations L15P and W19C, disrupted the binding interaction. The model predicted that a P5L CFTR mutation should not affect binding, but a synthetic P5L mutant peptide had reduced solubility, suggesting a different disease-causing mechanism. Taken together with the fact that FLNa dimers are elongated ( approximately 160 nm) strands, whereas CFTR is compact (6 approximately 8 nm), we propose that a single FLNa molecule can scaffold multiple CFTR partners. Unlike previously defined dimeric FLNa.partner complexes, the FLNa-monomeric CFTR interaction is relatively weak, presumptively facilitating dynamic clustering of CFTR at cell membranes. Finally, we show that deletion of all CFTR interacting domains from FLNa suppresses the surface expression of CFTR on baby hamster kidney cells.

Isoform divergence of the filamin family of proteins.

The vertebrate filamin family (A, B, and C) is part of the spectrin family of actin cross-linking proteins. Family members share high sequence similarity (>64%) and have both common and isoform-distinct functionalities. To identify the basis for isoform-specific functionality, we perform an evolutionary trace of chordate filamin at the granularity of single residues. Our trace methodology is constrained to focus on neofunctionality by requiring that one isoform remain the ancestral type, whereas at least one isoform has an accepted mutation. We call divergence meeting these characteristics "class-distinctive." To obtain a temporal and spatial context for class-distinctive residues, we derive an all-atom model of full-length filamin A by homology modeling and joining individual domains. We map onto our model both conserved and class-distinctive residues along with the period (Teleostei, Amphibian, and Mammalian) in which they diverged. Our phylogenetic analysis suggests that filamins diverged from a common ancestral gene between urochordate and vertebrate lineages. Filamins also diverged the most just after gene duplication, in the Teleostei period, with filamin C remaining closest to ancestral filamin. At the residue level, domains with well-characterized interfaces, IgFLN 17 and IgFLN 21 (immunoglobulin, Ig), have diverged in potentially critical residues in their adhesion protein-binding interfaces, signifying that isoforms may bind or regulate ligand binding differentially. Similarly, isoform divergence in a region associated with F actin-binding regulation suggests that isoforms differentially regulate F-actin binding. In addition, we observe some class-distinctive residues in the vicinity of missense mutations that cause filamin A and B-associated skeletal disorders. Our analysis, utilizing both spatial and temporal granularity, has identified potentially important residues responsible for vertebrate filamin isoform-specific divergence-significantly in regions where few binding partners have been discovered to date- and suggests yet to be discovered filamin-binding partners and isoform-specific differential regulation with these binding partners.

Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR.

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation.

Transient anchorage of cross-linked glycosyl-phosphatidylinositol-anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides.

How outer leaflet plasma membrane components, including glycosyl-phosphatidylinositol-anchored proteins (GPIAPs), transmit signals to the cell interior is an open question in membrane biology. By deliberately cross-linking several GPIAPs under antibody-conjugated 40-nm gold particles, transient anchorage of the gold particle-induced clusters of both Thy-1 and CD73, a 5' exonucleotidase, occurred for periods ranging from 300 ms to 10 s in fibroblasts. Transient anchorage was abolished by cholesterol depletion, addition of the Src family kinase (SFK) inhibitor PP2, or in Src-Yes-Fyn knockout cells. Caveolin-1 knockout cells exhibited a reduced transient anchorage time, suggesting the partial participation of caveolin-1. In contrast, a transmembrane protein, the cystic fibrosis transmembrane conductance regulator, exhibited transient anchorage that occurred without deliberately enhanced cross-linking; moreover, it was only slightly inhibited by cholesterol depletion or SFK inhibition and depended completely on the interaction of its PDZ-binding domain with the cytoskeletal adaptor EBP50. We propose that cross-linked GPIAPs become transiently anchored via a cholesterol-dependent SFK-regulatable linkage between a transmembrane cluster sensor and the cytoskeleton.