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Mentorship is vital for early career researchers in training positions, allowing them to navigate the challenges of work and life in research environments. However, the quality of mentorship received by trainees can vary by investigator and by institution. One challenge faced by those hoping to improve trainee mentorship is that the extent to which mentorship is offered to and experienced by research trainees is not well characterized. To address this knowledge gap, we conducted a survey to examine the quality of mentorship received by trainees in research environments, to identify characteristics of positive and negative mentorship, and to highlight best practices to improve trainee mentorship. We received 2,114 responses from researchers at graduate and postdoctoral career stages worldwide. Quantitative analysis showed that at least ∼25-45% of respondents were dissatisfied with some aspects of their mentorship. Qualitative responses revealed that common issues in mentorship include unclear expectations in research and mentoring interactions, lack of guidance, and inadequate support of trainee independence and career goals. Our findings also identified key mentorship elements desired by trainee mentees. Based on trainee suggestions, we describe strategies for individual mentors, departments, and institutions to improve the training experience for graduate and postdoctoral researchers.

Faculty at research institutions play a central role in advancing knowledge and careers, as well as promoting the well-being of students and colleagues in research environments. Faculty members must balance a host of activities - such as performing research, teaching, sourcing funds, administrative and service duties - with their roles as educators and mentors. Mentorship from experienced peers has been touted as critical for enabling these myriad roles to allow faculty development, career progression, and satisfaction. However, there is little information available on who supports faculty and best ways to structure a faculty mentorship program for early- and mid-career academics. Furthermore, the extent to which mentorship and mentoring programs have been offered to faculty members has not been widely characterized. It is also unclear what challenges faculty receiving mentorship may face and which aspects could be further improved. In the interest of advocating for increased and enhanced faculty mentoring and mentoring programs, we surveyed faculty (i.e., group leaders) around the world to gather data on whether and how they receive mentoring from peers, senior researchers, informal mentoring programs, or formal mentoring programs at their institutions. We received responses from 457 early- and mid-career faculty and found that a substantial portion of respondents either reported having no mentor or a lack of a formal mentoring scheme. Qualitative responses on the quality of mentorship revealed that the most common complaints regarding mentorship included lack of mentor availability, unsatisfactory commitment to mentorship, and non-specific or non-actionable advice. Our findings further identified key mentorship elements desired by faculty mentees. Based on these suggestions, we identify a need for training for faculty mentors as well as strategies for individual mentors, departments, and institutions for funding and design of more intentional and supportive mentorship programs for early- and mid-career faculty.

The vascular endothelial growth factor (VEGF) family of cytokines are key drivers of blood vessel growth and remodeling. These ligands act via multiple VEGF receptors (VEGFR) and co-receptors such as Neuropilin (NRP) expressed on endothelial cells. These membrane-associated receptors are not solely expressed on the cell surface, they move between the surface and intracellular locations, where they can function differently. The location of the receptor alters its ability to ’see’ (access and bind to) its ligands, which regulates receptor activation; location also alters receptor exposure to subcellularly localized phosphatases, which regulates its deactivation. Thus, receptors in different subcellular locations initiate different signaling, both in terms of quantity and quality. Similarly, the local levels of co-expression of other receptors alters competition for ligands. Subcellular localization is controlled by intracellular trafficking processes, which thus control VEGFR activity; therefore, to understand VEGFR activity, we must understand receptor trafficking. Here, for the first time, we simultaneously quantify the trafficking of VEGFR1, VEGFR2, and NRP1 on the same cells - specifically human umbilical vein endothelial cells (HUVECs). We build a computational model describing the expression, interaction, and trafficking of these receptors, and use it to simulate cell culture experiments. We use new quantitative experimental data to parameterize the model, which then provides mechanistic insight into the trafficking and localization of this receptor network. We show that VEGFR2 and NRP1 trafficking is not the same on HUVECs as on non-human ECs; and we show that VEGFR1 trafficking is not the same as VEGFR2 trafficking, but rather is faster and weighted to intracellular expression, due to faster internalization and lower recycling, resulting in increased overall degradation. As a consequence, the VEGF receptors are not evenly distributed between the cell surface and intracellular locations, with a very low percentage of VEGFR1 being on the cell surface. Our findings have implications both for the sensing of extracellular ligands and for the composition of signaling complexes at the cell surface versus inside the cell.

Writing recommendation letters on behalf of students and other early-career researchers is an important mentoring task within academia. An effective recommendation letter describes key candidate qualities such as academic achievements, extracurricular activities, outstanding personality traits, participation in and dedication to a particular discipline, and the mentor’s confidence in the candidate’s abilities. In this Words of Advice, we provide guidance to researchers on composing constructive and supportive recommendation letters, including tips for structuring and providing specific and effective examples, while maintaining a balance in language and avoiding potential biases.

The SARS‐CoV‐2 pandemic has significantly impacted global health. Research on viral mechanisms, highly effective vaccines, and other therapies is in progress. Neuropilins have recently been identified as host cell receptors enabling viral fusion. Here, we provide context to neuropilin's tissue‐specific role in infection and the potential impact of NRP‐based therapeutics. We conclude that the central roles of neuropilins in vascular, neural, and other pathways may render it a less suitable target for treating SARS‐CoV‐2 than agents that target its binding partner, the viral spike protein., The SARS‐CoV‐2 pandemic has significantly impacted global health. Research on viral mechanisms, highly effective vaccines, and other therapies is in progress. Neuropilins have recently been identified as host cell receptors enabling viral fusion. We provide context to the tissue‐specific role of neuropilins in infection and the potential impact of NRP‐based therapeutics. We conclude that the central roles of neuropilins in vascular, neural, and other pathways may render it a less suitable target for treating SARS‐CoV‐2 than agents that target its binding partner, the viral spike protein.

Open and reproducible research practices increase the reusability and impact of scientific research. The reproducibility of research results is influenced by many factors, most of which can be addressed by improved education and training. Here we describe how workshops developed by the Reproducibility for Everyone (R4E) initiative can be customized to provide researchers at all career stages and across most disciplines with education and training in reproducible research practices. The R4E initiative, which is led by volunteers, has reached more than 3000 researchers worldwide to date, and all workshop materials, including accompanying resources, are available under a CC-BY 4.0 license at https://www.repro4everyone.org/.

Mentorship is experience and/or knowledge-based guidance. Mentors support, sponsor and advocate for mentees. Having one or more mentors when you seek advice can significantly influence and improve your research endeavours, well-being and career development. Positive mentee-mentor relationships are vital for maintaining work-life balance and success in careers. Early-career researchers (ECRs), in particular, can benefit from mentorship to navigate challenges in academic and nonacademic life and careers. Yet, strategies for selecting mentors and maintaining interactions with them are often underdiscussed within research environments. In this Words of Advice, we provide recommendations for ECRs to seek and manage mentorship interactions. Our article draws from our experiences as ECRs and published work, to provide suggestions for mentees to proactively promote beneficial mentorship interactions. The recommended practices highlight the importance of identifying mentorship needs, planning and selecting multiple and diverse mentors, setting goals, and maintaining constructive, and mutually beneficial working relationships with mentors.

Scientific conferences are important avenues for researchers to share and discuss research findings, to exchange ideas and insights, and to network for collaboration and career development. Organizing inclusive and useful scientific meetings is a significant responsibility shared by researchers, scientific societies, and other organizations worldwide. Prior to 2020, most conferences were held in-person and provided limited attendance opportunities for many researchers from diverse socioeconomic backgrounds, particularly early career researchers (ECRs), researchers from low- and middle-income countries, and junior principal investigators (PIs)1. Some conferences in select disciplines have implemented valuable changes for the community, becoming more receptive to attendees with families. Due to the COVID-19 pandemic, a major shift has occurred towards virtual conferencing formats, triggering discussions in the scientific community on the current and future formats and organization of conferences. Here we provide recommendations for improving scientific meetings towards more efficient, sustainable, and equitable gatherings. Instituto de Patología Vegetal Fil: Sarabipour, Sarvenaz. Johns Hopkins University. Institute for Computational Medicine. Department of Biomedical Engineering; Estados Unidos Fil: Khan, Aziz. University of Oslo. Nordic EMBL Partnership. Centre for Molecular Medicine Norway (NCMM); Noruega Fil: Khan, Aziz. Stanford University. School of Medicine. Stanford Cancer Institute; Estados Unidos Fil: Yu Fen, Samantha Seah. European Molecular Biology Laboratory. Genome Biology Unit; Alemania Fil: Mwakilili, Aneth D. Swedish University of Agricultural Sciences. Plant Protection Department; Suecia Fil: Mwakilili, Aneth D. University of Dar es Salaam. Department of Molecular Biology and Biotechnology; Tanzania Fil: Mumoki, Fiona N. University of Pretoria. Department of Zoology and Entomology; Sudáfrica Fil: Sáez, Pablo J. Paris Sciences & Lettres Research University. Institut Curie; Francia Fil: Sáez, Pablo J. University Medical Center Hamburg-Eppendorf. Department of Biochemistry and Molecular Cell Biology; Alemania Fil: Schwessinger, Benjamin. Australian National University. Research School of Biology; Australia Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Mestrovic, Tomislav. Dr Zora Profozic Polyclinic. Clinical Microbiology and Parasitology Unit; Croacia Fil: Mestrovic, Tomislav. University Centre Varazdin. University North; Croacia

Reproducibility is a cornerstone of the scientific method and sets apart science from pseudoscience. Unfortunately, a majority of scientists have experienced difficulties in reproducing their own or someone else’s results. This inability to confirm scientific findings negatively impacts individual scientists, funding bodies, academic journals, pharmaceutical drug development and the public’s perception of science. Factors causing irreproducible results can arise from nearly every aspect of the scientific process, and typically reflect a lack of in-depth training in reproducible research practices. Here, we present the Reproducibility for Everyone (R4E) initiative, a collaboration between researchers from diverse scientific disciplines and industry partners who aspire to promote open and reproducible research practices. We have developed a customizable workshop series targeting researchers at all levels and across disciplines. Our workshop series covers the conceptual framework of reproducible research practices followed by an overview of actionable research practices. To date, we have reached more than 2000 researchers through over 25 workshops held at international conferences and local meetings. By incorporating further contributions from the scientific community, we hope to expand this valuable resource for teaching transparent and reproducible research practices. Our initiative demonstrates how a shared set of materials may form the basis for a global initiative to improve reproducibility in science. The workshop materials, including accompanying resources, are available under a CC-BY 4.0 license at www.repro4everyone.org.

Scientists routinely use images to display data. Readers often examine figures first; therefore, it is important that figures are accessible to a broad audience. Many resources discuss fraudulent image manipulation and technical specifications for image acquisition; however, data on the legibility and interpretability of images are scarce. We systematically examined these factors in non-blot images published in the top 15 journals in 3 fields; plant sciences, cell biology, and physiology (n = 580 papers). Common problems included missing scale bars, misplaced or poorly marked insets, images or labels that were not accessible to colorblind readers, and insufficient explanations of colors, labels, annotations, or the species and tissue or object depicted in the image. Papers that met all good practice criteria examined for all image-based figures were uncommon (physiology 16%, cell biology 12%, plant sciences 2%). We present detailed descriptions and visual examples to help scientists avoid common pitfalls when publishing images. Our recommendations address image magnification, scale information, insets, annotation, and color and may encourage discussion about quality standards for bioimage publishing.

Securing research funding is a challenge faced by most scientists in academic institutions worldwide. Funding success rates for all career stages are low, but the burden falls most heavily on early career researchers (ECRs). These are young investigators in training and new principal investigators who have a shorter track record. ECRs are dependent on funding to establish their academic careers. The low number of career development awards and the lack of sustained research funding result in the loss of ECR talent in academia. Several steps in the current funding process, from grant conditions to review, play significant roles in the distribution of funds. Furthermore, there is an imbalance where certain research disciplines and labs of influential researchers receive more funding. As a group of ECRs with global representation, we examined funding practices, barriers, and facilitators to the current funding systems. We also identified alternatives to the most common funding distribution practices, such as diversifying risk or awarding grants on a partly random basis. Here, we detail recommendations for funding agencies and grant reviewers to improve ECR funding prospects worldwide and promote a fairer and more inclusive funding landscape for ECRs. Instituto de Virología Fil: de Winde, Charlotte M. University College London. MRC Laboratory for Molecular Cell Biology; Reino Unido Fil: de Winde, Charlotte M. Amsterdam University Medical Center. Department of Molecular Cell Biology & Immunology; Países Bajos Fil: Sarabipour, Sarvenaz. Johns Hopkins University. Department of Biomedical Engineering. Institute for Computational Medicine; Estados Unidos Fil: Carignano, Hugo Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Virología e Innovaciones Tecnológicas; Argentina Fil: Davla, Sejal. City University of New York. Advanced Science Research Center; Estados Unidos Fil: Eccles, David. Malaghan Institute of Medical Research; Nueva Zelanda Fil: Hainer, Sarah J. University of Pittsburgh. Department of Biological Sciences; Estados Unidos Fil: Haidar, Mansour. Hasselt University; Bélgica Fil: Ilangovan, Vinodh. Aarhus University; Dinamarca Fil: Jadavji, Nafisa M. Midwestern University. Department of Biomedical Sciences; Estados Unidos Fil: Jadavji, Nafisa M. Carleton University. Department of Neuroscience; Canadá Fil: Kritsiligkou, Paraskevi. German Cancer Research Center; Alemania Fil: Lee, Tai-Ying. University of Oxford; Reino Unido Fil: Ólafsdóttir, H. Freyja. Radboud University. Donders Institute for Brain, Cognition and Behaviour; Países Bajos

Scientific conferences provide valuable opportunities for researchers across career stages and disciplines to present their latest work and to network with their peers. The advent of the internet has opened new possibilities for interaction, collaboration and networking, yet the uptake of tools enabling remote participation at scientific meetings has been slow. Academic conferences have become more international with high proliferation in the number of meetings and attendees, but the format and quality of their organization lags behind what is possible. As a result, the experience of attending conferences in many disciplines today is not appreciably different in many respects. A few conferences in some disciplines have recently implemented valuable changes for the community and have become more receptive to attendees with families. Many meetings could still be improved significantly in terms of diversity, inclusivity, promoting early career researcher (ECR) networking and career development, venue accessibility, and more importantly, reducing the meetings’ carbon footprint. These issues are openly visible and to some extent discussed among researchers on social media. It is important to accelerate and mandate these efforts so that researchers in all disciplines, in particular ECRs, consistently benefit from scientific gatherings, for years to come. We examined the current state of over 260 national and international academic meetings in various disciplines for features of inclusivity and sustainability and propose solutions to make conferences more modern, effective, equitable and intellectually productive for the research community and environmentally sustainable for our planet. “They always say time changes things, but you actually have to change them yourself.” — Andy Warhol

The need to protect public health during the current COVID-19 pandemic has necessitated conference cancellations on an unprecedented scale. As the scientific community adapts to new working conditions, it is important to recognize that some of our actions may disproportionately affect early-career researchers and scientists from countries with limited research funding. We encourage all conference organizers, funders and institutions who are able to do so to consider how they can mitigate the unintended consequences of conference and travel cancellations and we provide seven recommendations for how this could be achieved. The proposed solutions may also offer long-term benefits for those who normally cannot attend conferences, and thus lead to a more equitable future for generations of researchers.

All known splice isoforms of vascular endothelial growth factor A (VEGF-A) can bind to the receptor tyrosine kinases VEGFR-1 and VEGFR-2. We focus here on VEGF-A121a and VEGF-A165a, two of the most abundant VEGF-A splice isoforms in human tissue 1 , and their ability to bind the Neuropilin co-receptors NRP1 and NRP2. The Neuropilins are key vascular, immune, and nervous system receptors on endothelial cells, neuronal axons, and regulatory T cells respectively. They serve as co-receptors for the Plexins in Semaphorin binding on neuronal and vascular endothelial cells, and for the VEGFRs in VEGF binding on vascular and lymphatic endothelial cells, and thus regulate the initiation and coordination of cell signaling by Semaphorins and VEGFs. 2 There is conflicting evidence in the literature as to whether only heparin-binding VEGF-A isoforms - that is, isoforms with domains encoded by exons 6 and/or 7 plus 8a - bind to Neuropilins on endothelial cells. While it is clear that VEGF-A165a binds to both NRP1 and NRP2, published studies do not all agree on the ability of VEGF-A121a to bind NRPs. Here, we review and attempt to reconcile evidence for and against VEGF-A121a binding to Neuropilins. This evidence suggests that, in vitro, VEGF-A121a can bind to both NRP1 and NRP2 via domains encoded by exons 5 and 8a; in the case of NRP1, VEGF-A121a binds with lower affinity than VEGF-A165a. In in vitro cell culture experiments, both NRP1 and NRP2 can enhance VEGF-A121a-induced phosphorylation of VEGFR2 and downstream signaling including proliferation. However, unlike VEGFA-165a, experiments have shown that VEGF-A121a does not 'bridge' VEGFR2 and NRP1, i.e. it does not bind both receptors simultaneously at their extracellular domain. Thus, the mechanism by which Neuropilins potentiate VEGF-A121a-mediated VEGFR2 signaling may be different from that for VEGF-A165a. We suggest such an alternate mechanism: interactions between NRP1 and VEGFR2 transmembrane (TM) and intracellular (IC) domains.

Epithelial cadherin (Ecadherin) is responsible for the intercellular cohesion of epithelial tissues. It forms lateral clusters within adherens cell-cell junctions, but its association state outside these clusters is unknown. Here, we use a quantitative Forster resonance energy transfer (FRET) approach to show that Ecadherin forms constitutive dimers and that these dimers exist independently of the actin cytoskeleton or cytoplasmic proteins. The dimers are stabilized by intermolecular contacts that occur along the entire length of Ecadherin, with the intracellular domains having a surprisingly strong favorable contribution. We further show that Ecadherin mutations and calcium depletion induce structural alterations that propagate from the N terminus all the way to the C terminus, without destabilizing the dimeric state. These findings provide context for the interpretation of Ecadherin adhesion experiments. They also suggest that early events of adherens junction assembly involve interactions between from preformed Ecadherin dimers.

Receptor tyrosine kinase (RTK) signal transduction is essential in human skeletal, nervous, and vascular development, in homeostasis, and in disease. RTKs are activated by dimerization in the plasma membrane. The mechanisms of receptor dimerization and activation are multifaceted and complex, and unraveling them remains challenging. Most studies of RTKs have been devoted to crystallographic analysis of their isolated extracellular domain and biochemical analysis of the catalytic domain. However, the past few years have seen direct biophysical studies of (intact) RTK dimerization in native membranes lead to significant progress in our fundamental understanding of the mechanisms of their signal transduction across the plasma membrane. This perspective focuses on recent insights into the mechanisms of fibroblast growth factor receptor and vascular endothelial growth factor receptor transmembrane signaling, derived from studies of wild-type and mutant RTKs in a number of environments, including plasma membrane-derived vesicles. These insights reveal distinct steps in and factors of RTK signaling across the plasma membrane that can guide the drug discovery process for RTK targeting therapeutics.

Many cellular signaling pathways are initiated by cell-surface ligand-sensing complexes that incorporate not just one but multiple receptors. Most studies focus on receptors coexpressed on a single cell (cis interactions), but complexes containing receptors on adjacent cells (trans interactions) are also possible. Recent work by Morin et al published in this journal provides critical evidence for such trans interactions between Neuropilin-1 (NRP1) expressed on human tumor cells and vascular endothelial growth factor receptor 2 (VEGFR2) expressed on adjacent endothelial cells, with the ligand VEGFA binding and bridging the two receptors. They show that the formation of these complexes is correlated with reduced tumor proliferation and increased patient survival. They also observe trans NRP1-VEGFA-VEGFR2 repressing angiogenesis and cis NRP1-VEGFA-VEGFR2 increasing angiogenesis in selected cancers. The distinct molecular signature of each tumor and each patient will determine which type of complexes dominate and will influence prognosis and treatment. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Many postdoctoral researchers apply for faculty positions knowing relatively little about the hiring process or what is needed to secure a job offer. To address this lack of knowledge about the hiring process we conducted a survey of applicants for faculty positions: the survey ran between May 2018 and May 2019, and received 317 responses. We analyzed the responses to explore the interplay between various scholarly metrics and hiring outcomes. We concluded that, above a certain threshold, the benchmarks traditionally used to measure research success – including funding, number of publications or journals published in – were unable to completely differentiate applicants with and without job offers. Respondents also reported that the hiring process was unnecessarily stressful, time-consuming, and lacking in feedback, irrespective of outcome. Our findings suggest that there is considerable scope to improve the transparency of the hiring process.

Applying for a faculty position is a critical phase of many postdoctoral careers, but most postdoctoral researchers in STEM fields enter the academic job market with little knowledge of the process and expectations. A lack of data has made it difficult for applicants to assess their qualifications relative to the general applicant pool and for institutions to develop effective hiring policies. We analyzed responses to a survey of faculty job applicants between May 2018 and May 2019. We establish various background scholarly metrics for a typical faculty applicant and present an analysis of the interplay between those metrics and hiring outcomes. Traditional benchmarks of a positive research track record above a certain threshold of qualifications were unable to completely differentiate applicants with and without offers. Our findings suggest that there is no single clear path to a faculty job offer and that metrics such as career transition awards and publications in high impact factor journals were neither necessary nor sufficient for landing a faculty position. The applicants perceived the process as unnecessarily stressful, time-consuming, and largely lacking in feedback, irrespective of a successful outcome. Our findings emphasize the need to improve the transparency of the faculty job application process. In addition, we hope these and future data will help empower trainees to enter the academic job market with clearer expectations and improved confidence.

The timely and accurate dissemination of scientific discoveries is of utmost importance so that scientific knowledge can be advanced and applied to benefit the public. Scientists communicate amongst themselves at conferences, via journal articles, and, increasingly in the life sciences, in preprint manuscripts which have not been subject to peer review. Journalists translate new research into a language the public can understand, relying on both work presented in scientific forums and interviews with experts. Critically, scientists and journalists both share the ethical principle that publications should be rigorously sourced and fact-checked, with errors subject to publicized corrections. Here we respond to concerns raised about the impact of reporting on results that have not passed through peer review, calling for improved dialogue between scientists and journalists to maintain public trust in research and arguing that imposing limits is against the public interest.

Plasma membrane-derived vesicles are being used in biophysical and biochemical research as a simple, yet native-like model of the cellular membrane. Here we report on the characterization of vesicles produced via two different vesiculation methods from CHO and A431 cell lines. The first method is a recently developed method which utilizes chloride salts to induce osmotic vesiculation. The second is a well established chemical vesiculation method which uses DTT and formaldehyde. We show that both vesiculation methods produce vesicles which contain the lipid species previously reported in the plasma membrane of these cell lines. The two methods lead to small but statistically significant differences in two lipid species only; phosphatidylcholine (PC) and plasmalogen phosphatidylethanolamine (PEp). However, highly significant differences were observed in the degree of incorporation of a membrane receptor and in the degree of retention of soluble cytosolic proteins within the vesicles.

Receptor Tyrosine Kinases (RTKs) conduct biochemical signals upon dimerization in the membrane plane. While RTKs are generally known to be activated in response to ligand binding, many of these receptors are capable of forming unliganded dimers that are likely important intermediates in the signaling process. All 58 RTKs consist of an extracellular domain, a transmembrane (TM) domain, and an intracellular domain which includes a juxtamembrane (JM) sequence and a kinase domain. Here we investigate directly the effect of the JM domain on unliganded dimer stability of FGFR3, a receptor that is critically important for skeletal development. The data suggest that FGFR3 unliganded dimers are stabilized by receptor-receptor contacts that involve the JM domains. The contribution is significant, as it is similar in magnitude to the stabilizing contribution of a pathogenic mutation and the repulsive contribution of the extracellular domain. Furthermore, we show that the effects of the JM domain and a TM pathogenic mutation on unliganded FGFR3 dimer stability are additive. We observe that the JM-mediated dimer stabilization occurs when the JM domain is linked to FGFR3 TM domain and not simply anchored to the plasma membrane. These results point to a coordinated stabilization of the unliganded dimeric state of FGFR3 by its JM and TM domains via a mechanism that is distinctly different from the case of another well studied receptor, EGFR.

Forster resonance energy transfer (FRET) experiments are often used to study interactions between integral membrane proteins in cellular membranes. However, in addition to the FRET of sequence-specific interactions, these experi- ments invariably record a contribution due to proximity FRET, which occurs when a donor and an acceptor approach each other by chance within distances of ~100 Au. This effect does not reflect specific interactions in the membrane and is frequently unappreciated, despite the fact that its magnitude can be significant. Here we develop a computational description of proximity FRET, simulating the cases of proximity FRET when fluorescent proteins are used to tag monomeric, dimeric, trimeric, and tetra- meric membrane proteins, as well as membrane proteins existing in monomer-dimer equilibria. We also perform rigorous exper- imental measurements of this effect, by identifying membrane receptors that do not associate in mammalian membranes. We measure the FRET efficiencies between yellow fluorescent protein and mCherry-tagged versions of these receptors in plasma-membrane-derived vesicles as a function of receptor concentration. Finally, we demonstrate that the experimental mea- surements are well described by our predictions. The work presented here brings additional rigor to FRET-based studies of membrane protein interactions, and should have broad utility in membrane biophysics research.

Isolated receptor tyrosine kinase transmembrane (TM) domains have been shown to form sequence-specific dimers in membranes. Yet, it is not clear whether studies of isolated TM domains yield knowledge that is relevant to full-length receptors or whether the large glycosylated extracellular domains alter the interactions between the TM helices. Here, we address this question by quantifying the effect of the pathogenic A391E TM domain mutation on the stability of the fibroblast growth factor receptor 3 dimer in the presence of the extracellular domain and comparing these results to the case of the isolated TM fibroblast growth factor receptor 3 domains. We perform the measurements in plasma membrane-derived vesicles using a Forster-resonance-energy-transfer-based method. The effect of the mutation on dimer stability in both cases is the same (∼−1.5 kcal/mol), suggesting that the interactions observed in simple TM-peptide model systems are relevant in a biological context.

The activity of receptor tyrosine kinases (RTKs) is controlled through their lateral association in the plasma membrane. RTKs are believed to form both homodimers and heterodimers, and the different dimers are believed to play unique roles in cell signaling. However, RTK heterodimers remain poorly characterized, as compared with homodimers, because of limitations in current experimental methods. Here, we develop a FRET-based methodology to assess the thermodynamics of hetero-interactions in the plasma membrane. To demonstrate the utility of the methodology, we use it to study the hetero-interactions between three fibroblast growth factor receptors-FGFR1, FGFR2, and FGFR3-in the absence of ligand. Our results show that all possible FGFR heterodimers form, suggesting that the biological roles of FGFR heterodimers may be as significant as the homodimer roles. We further investigate the effect of two pathogenic point mutations in FGFR3 (A391E and G380R) on heterodimerization. We show that each of these mutations stabilize most of the heterodimers, with the largest effects observed for FGFR3 wild-type/mutant heterodimers. We thus demonstrate that the methodology presented here can yield new knowledge about RTK interactions and can further our understanding of signal transduction across the plasma membrane.

Missense mutations that introduce or remove cysteine residues in receptor tyrosine kinases are believed to cause pathologies by stabilizing the active receptor tyrosine kinase dimers. However, the magnitude of this stabilizing effect has not been measured for full-length receptors. Here, we characterize the dimer stabilities of three full-length fibroblast growth factor receptor (FGFR) mutants harboring pathogenic cysteine substitutions: the C178S FGFR1 mutant, the C342R FGFR2 mutant, and the C228R FGFR3 mutant. We find that the three mutations stabilize the FGFR dimers. We further see that the mutations alter the configuration of the FGFR transmembrane dimers. Thus, both aberrant dimerization and perturbed dimer structure likely contribute to the pathological phenotypes arising due to these mutations.

The G380R mutation in the transmembrane domain of FGFR3 is a germline mutation responsible for most cases of Achondroplasia, a common form of human dwarfism. Here we use quantitative Fӧster Resonance Energy Transfer (FRET) and osmotically derived plasma membrane vesicles to study the effect of the achondroplasia mutation on the early stages of FGFR3 signaling in response to the ligands fgf1 and fgf2. Using a methodology that allows us to capture structural changes on the cytoplasmic side of the membrane in response to ligand binding to the extracellular domain of FGFR3, we observe no measurable effects of the G380R mutation on FGFR3 ligand-bound dimer configurations. Instead, the most notable effect of the achondroplasia mutation is increased propensity for FGFR3 dimerization in the absence of ligand. This work reveals new information about the molecular events that underlie the achondroplasia phenotype, and highlights differences in FGFR3 activation due to different single amino-acid pathogenic mutations.

Receptor tyrosine kinase (RTK) heterodimers are necessary for proper cell function and have properties unique from those of the better-studied homodimer. However, the heterodimer species’ propensity for formation and physicochemical properties remain largely unknown, because traditional methods struggle to distinguish between hetero- and homo-dimers. In this project, we overcome that challenge by fluorescently labeling each receptor in the heterodimer and employing Forster Resonance Energy Transfer (FRET); this technique reports on the formation of heterodimers but not homodimers. Experiments are performed in cell-derived vesicles, which closely mimic the structure and composition of the native membrane, and over a wide range of receptor concentrations. In this system, homodimers also form, though they do not contribute to FRET, and are accounted for in thermodynamic equilibrium equations. Here, we examine heterodimers in the Fibroblast Growth Factor Receptor (FGFR) family of RTKs, which regulates development of the skeletal system. In addition to the wild-type FGFR1, FGFR2, and FGFR3 heterodimer species, we consider the heterodimers formed in the presence of two different mutations in the transmembrane domain of FGFR3. The first mutation, FGFR3 G380R, is responsible for achondroplasia, the most common form of human dwarfism, and disrupts proper development of the long bones. The second mutation, FGFR3 A391E, is responsible for Crouzon syndrome with acanthosis nigricans, a disorder characterized by prematurely ossified skull bones accompanied by a skin disorder. We show that heterodimers form between both wild-type and mutant receptors. This work enhances our understanding of heterodimerization in the FGFR family.

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Here we introduce a fast, cost-effective, and highly efficient method for production of soluble fluorescent proteins from bacteria. The method does not require optimization and does not use isopropyl β-d-1-thiogalactopyranoside (IPTG) induction. The method relies on uninduced expression in the BL21-Gold (DE3) strain of Escherichia coli and yields large amounts (up to 0.4 μmol) of fluorescent protein from a 250-ml culture. This method is much simpler than published methods and can be used to produce any fluorescent protein that is needed in biomedical research.