Cell Biology Experiments Research Articles

Phase Separation of Zonula Occludens Proteins Drives Formation of Tight Junctions

Tight junctions are cell-adhesion complexes that seal tissues and are involved in cell polarity and signaling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands are dependent on the membrane-associated scaffolding proteins ZO1 and ZO2. To understand how zona occludens (ZO) proteins organize junction assembly, we performed quantitative cell biology and invitro reconstitution experiments. We discovered that ZO proteins self-organize membrane-attached compartments via phase separation. Weidentified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight-junction proteins, including adhesion receptors, cytoskeletal adapters, and transcription factors. Our results suggest that an active-phase transition of ZO proteins into a condensed membrane-bound compartment drives claudin polymerization and coalescence of a continuous tight-junction belt.

<p>TCDD-Inducible Poly-ADP-Ribose Polymerase (TIPARP), A Novel Therapeutic Target Of Breast Cancer</p>

BackgroundTIPARP (TCDD-inducible poly-ADP-ribose polymerase), a mono-ADP-ribosyltransferase and a transcriptional repressor of aryl hydrocarbon receptor (AHR), was one of the potential therapeutic targets for human cancers identified by CRISPR–Cas9 screens recently. Studies about TIPARP on cancers are scarce till now, most of which just focus on expressions, while the functions have not been widely reported yet. Moreover, the TIPARP prognostic significance and therapeutic value of breast cancer is also uncertain.MethodsThe present study was performed to comprehensively analyze the expression pattern, prognostic effect, potential therapeutic function of TIPARP in breast cancer by pooling all currently available databases online including Oncomine, UALCAN, bc-GenExMiner, Kaplan–Meier Plotter, COSMIC, UCSC Xena, STRING, DAVID and Comparative Toxicogenomics Database. Further, we also performed several cell biology experiments including RT-qPCR, Western blot and CCK-8 in cellular and clinical sample levels to confirm the conclusions from bioinformatics analysis.ResultsTIPARP was expressed lower in tumor tissues comparing with normal tissues. Meanwhile, several clinical parameters of breast cancer patients were correlated with TIPARP expression. Further, higher TIPARP expression was related to preferable survival. Moreover, the mutations and DNA methylation of TIPARP might contribute to TIPARP dysregulation in breast cancer. Interactors with TIPARP were significantly enriched in telomere maintenance, telomere organization and mainly participated in pathways in cancer. Finally, several common drugs including metformin were observed to up-regulate the expression of TIPARP.ConclusionTIPARP might act as a preferable prognostic marker of breast cancer through multiple biological processes such as DNA methylation, mutation as well as pathway related to telomere and so on. TIPARP could be considered as a potential therapeutic target for breast cancer. However, large-scale and comprehensive research is needed to clarify our results.

Force-Dependent Regulation of Talin-KANK1 Complex at Focal Adhesions.

KANK proteins mediate cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix. KANKs interact with the integrin/actin-binding protein talin and with several components of microtubule-stabilizing cortical complexes. Because of actomyosin contractility, the talin-KANK complex is likely under mechanical force, and its mechanical stability is expected to be a critical determinant of KANK recruitment to focal adhesions. Here, we quantified the lifetime of the complex of the talin rod domain R7 and the KN domain of KANK1 under shear-force geometry and found that it can withstand forces for seconds to minutes over a physiological force range up to 10 pN. Complex stability measurements combined with cell biological experiments suggest that shear-force stretching promotes KANK1 localization to the periphery of focal adhesions. These results indicate that the talin-KANK1 complex is mechanically strong, enabling it to support the cross-talk between microtubule and actin cytoskeleton at focal adhesions.

FRET-based analysis and molecular modeling of the human GPN-loop GTPases 1 and 3 heterodimer unveils a dominant-negative protein complex.

GPN-loop GTPases 1 and 3 are required for RNA polymerase II (RNAPII) nuclear import. Gpn1 and Gpn3 display some sequence similarity, physically associate, and their protein expression levels are mutually dependent in human cells. We performed here Fluorescence Resonance Energy Transfer (FRET), molecular modeling, and cell biology experiments to understand, and eventually disrupt, human Gpn1-Gpn3 interaction in live HEK293-AD cells. Transiently expressed EYFP-Gpn1 and Gpn3-CFP generated a strong FRET signal, indicative of a very close proximity, in the cytoplasm of HEK293-AD cells. Molecular modeling of the human Gpn1-Gpn3 heterodimer based on the crystallographic structure of Npa3, the Saccharomyces cerevisiae Gpn1 ortholog, revealed that human Gpn1 and Gpn3 associate through a large interaction surface formed by internal α-helix 7, insertion 2, and the GPN-loop from each protein. In site-directed mutagenesis experiments of interface residues, we identified the W132D and M227D EYFP-Gpn1 mutants as defective to produce a FRET signal when coexpressed with Gpn3-CFP. Simultaneous but not individual expression of Gpn1 and Gpn3, with either or both proteins fused to EYFP, retained RNAPII in the cytoplasm and markedly inhibited global transcription in HEK293-AD cells. Interestingly, the W132D and M227D Gpn1 mutants that showed an impaired ability to interact with Gpn3 by FRET were also unable to delocalize RNAPII in this assay, indicating that an intact Gpn1-Gpn3 interaction is required to display the dominant-negative effect on endogenous Gpn1/Gpn3 function we described here. Altogether, our results suggest that a Gpn1-Gpn3 strong interaction is critical for their cellular function.

Gravity-regulated localization of PsPIN1 is important for polar auxin transport in etiolated pea seedlings: Relevance to the International Space Station experiment

To clarify the mechanism of gravity-controlled polar auxin transport, we conducted the International Space Station (ISS) experiment “Auxin Transport” (identified by NASA's operation nomenclature) in 2016 and 2017, focusing on the expression of genes related to auxin efflux carrier protein PsPIN1 and its localization in the hook and epicotyl cells of etiolated Alaska pea seedlings grown for three days in the dark under microgravity (μg) and artificial 1 g conditions on a centrifuge in the Cell Biology Experiment Facility (CBEF) in the ISS, and under 1 g conditions on Earth. Regardless of gravity conditions, the accumulation of PsPIN1 mRNA in the proximal side of epicotyls of the seedlings was not different, but tended to be slightly higher as compared with that in the distal side. 2,3,5-Triiodobenzoic acid (TIBA) also did not affect the accumulation of PsPIN1 mRNA in the proximal and distal sides of epicotyls. However, in the apical hook region, TIBA increased the accumulation of PsPIN1 mRNA under μg conditions as compared with that under artificial 1 g conditions in the ISS. The accumulation of PsPIN1 proteins in epicotyls determined by western blotting was almost parallel to that of mRNA of PsPIN1. Immunohistochemical analysis with a specific polyclonal antibody of PsPIN1 revealed that a majority of PsPIN1 in the apical hook and subapical regions of the seedlings grown under artificial 1 g conditions in the ISS localized in the basal side (rootward) of the plasma membrane of the endodermal tissues. Conversely, in the seedlings grown under μg conditions, localization of PsPIN1 was greatly disarrayed. TIBA substantially altered the cellular localization pattern of PsPIN1, especially under μg conditions. These results strongly suggest that the mechanisms by which gravity controls polar auxin transport are more likely to be due to the membrane localization of PsPIN1. This physiologically valuable report describes a close relationship between gravity-controlled polar auxin transport and the localization of auxin efflux carrier PsPIN1 in etiolated pea seedlings based on the μg experiment conducted in space.

Requirements for Documenting Electrical Cell Stimulation Experiments for Replicability and Numerical Modeling∗.

Thorough documentation of biological experiments is necessary for their replicability. This becomes even more evident when individual steps of in vitro wet-lab experiments are to be incorporated into computer simulation models. In the highly interdisciplinary field of electrical stimulation of biological cells, not only biological but also physical aspects play a crucial role. Simulations may help to identify parameters that influence cells and thereby reveal new insights into mechanisms of the cell biological system. However, missing or misleading documentation of the electrical stimulation step within wet-lab experiments may lead to discrepancies between reported and simulated electrical quantities. In addition, this threatens the replicability of electrical stimulation experiments. Thus, we argue that a minimal set of information is needed to enable a translation of electrical stimulation experiments of biological cells into computer simulation experiments and to support replicability. This set includes detailed information about the electronic devices and components, their set-up as well as the applied stimulus and shall be integrated into an existing guideline for cell biological experiments. Ideally, the documentation should also contain measured properties of the cellular and experimental environment. Furthermore, a realization of our proposed documentation requirements within electronic lab notebooks may provide a crucial step toward a more seamless integration of wet-lab data into simulations. Based on two exemplary studies, we demonstrate the relevance of our claim.

An integration design of gas exchange, bubble separation, and flow control in a space cell culture system on board the SJ-10 satellite.

Pathophysiological changes of astronauts under space microgravity involve complex factors and require an integrative perspective to fully understand the mechanisms. The readouts from space cell biology experiments strongly depend on the hardware and especially the cell bioreactor that is used in distinct spacecraft. Herein, a specialized cell culture bioreactor is designed for culturing mammalian cells on board the SJ-10 satellite. This hardware focuses mainly on satisfying the requirements of gas exchange, bubble separation, and flow control, as well as their functional and structural integration on cell culture within the technical and environmental constraints of the spacecraft platform under microgravity. A passive bubble separator is constructed and is connected in series to an individual cell culture chamber to remove the bubbles that were produced in orbit during cell growth. A moderate flow rate is preset to provide sufficient mass transfer and low shear stress in a well-designed flow circuit. Together with other modules of temperature control, in situ microscopic imaging, and online imaging acquisition, this novel space cell culture system is successfully used to culture human endothelial cells and rat bone marrow-derived mesenchymal stem cells in the SJ-10 mission. The advantages and shortcomings of the integration design are discussed for this type of the hardware.

Systematically characterize the clinical and biological significances of 1p19q genes in 1p/19q non-codeletion glioma.

1p/19q codeletion, which leads to the abnormal expression of 1p19q genes in oligodendroglioma, is associated with chemosensitivity and favorable prognosis. Here, we aimed to explore the clinical implications of 1p19q gene expression in 1p/19q non-codel gliomas. We analyzed expression of 1p19q genes in 668 1p/19q non-codel gliomas obtained from The Cancer Genome Atlas (n = 447) and the Chinese Glioma Genome Atlas (n = 221) for training and validation, respectively. The expression of 1p19q genes was significantly correlated with the clinicopathological features and overall survival of 1p/19q non-codel gliomas. Then, we derived a risk signature of 25 selected 1p19q genes that not only had prognosis value in total 1p/19q non-codel gliomas but also had prognosis value in stratified gliomas. The prognosis value of the risk signature was superior than known clinicopathological features in 1p/19q non-codel gliomas and was also highly associated with the following features: loss of CDKN2A/B copy number in mutant-IDH-astrocytoma; telomerase reverse transcriptase (TERT) promoter mutation, combined chromosome 7 gain/chromosome 10 loss and epidermal growth factor receptor amplification in wild-type-IDH-astrocytoma; classical and mesenchymal subtypes in glioblastoma. Furthermore, genes enriched in the biological processes of cell division, extracellular matrix, angiogenesis significantly correlated to the signature risk score, and this is also supported by the immunohistochemistry and cell biology experiments. In conclusion, the expression profile of 1p19q genes is highly associated with the malignancy and prognosis of 1p/19q non-codel gliomas. A 25-1p19q-gene signature has powerfully predictive value for both malignant molecular pathological features and prognosis across distinct subgroups of 1p/19q non-codel gliomas.

Drosophila ADCK1 is critical for maintaining mitochondrial structures and functions in the muscle.

The function of AarF domain-containing kinase 1 (ADCK1) has not been thoroughly revealed. Here we identified that ADCK1 utilizes YME1-like 1 ATPase (YME1L1) to control optic atrophy 1 (OPA1) and inner membrane mitochondrial protein (IMMT) in regulating mitochondrial dynamics and cristae structure. We firstly observed that a serious developmental impairment occurred in Drosophila ADCK1 (dADCK1) deletion mutant, resulting in premature death before adulthood. By using temperature sensitive ubiquitously expression driver tub-Gal80ts/tub-Gal4 or muscle-specific expression driver mhc-Gal4, we observed severely defective locomotive activities and structural abnormality in the muscle along with increased mitochondrial fusion in the dADCK1 knockdown flies. Moreover, decreased mitochondrial membrane potential, ATP production and survival rate along with increased ROS and apoptosis in the flies further demonstrated that the structural abnormalities of mitochondria induced by dADCK1 knockdown led to their functional abnormalities. Consistent with the ADCK1 loss-of-function data in Drosophila, ADCK1 over-expression induced mitochondrial fission and clustering in addition to destruction of the cristae structure in Drosophila and mammalian cells. Interestingly, knockdown of YME1L1 rescued the phenotypes of ADCK1 over-expression. Furthermore, genetic epistasis from fly genetics and mammalian cell biology experiments led us to discover the interactions among IMMT, OPA1 and ADCK1. Collectively, these results established a mitochondrial signaling pathway composed of ADCK1, YME1L1, OPA1 and IMMT, which has essential roles in maintaining mitochondrial morphologies and functions in the muscle.

FOXM1 promotes hepatocellular carcinoma progression by regulating KIF4A expression

BackgroundForkhead box M1 (FOXM1) is a proliferation-associated transcription factor of the forkhead box proteins superfamily, which includes four isoforms FOXM1a, b, c, and d. FOXM1 has been implicated in hepatocellular carcinoma (HCC) progression, but the underlying molecular mechanism remains elusive. In this study, we aim to clarify the molecular basis for FOXM1-mediated HCC progression.MethodsBioinformatic analysis was used to explore the differentially expressed genes predicting HCC proliferation. The expression of FOXM1 and kinesin family member (KIF)4A was confirmed by western blotting and immunohistochemistry in HCC tissues. Kaplan-Meier survival analysis was conducted to analyze the clinical impact of FOXM1 and KIF4A on HCC. The effect of FOXM1 on the regulation of KIF4A expression was studied in cell biology experiments. The interaction between KIF4A and FOXM1 was analyzed by chromatin immunoprecipitation and luciferase experiments. A series of experiments was performed to explore the functions of FOXM1/KIF4A in HCC progression, such as cell proliferation, cell growth, cell viability, and cell cycle. A xenograft mouse model was used to explore the regulatory effect of FOXM1-KIF4A axis on HCC tumor growth.ResultsFOXM1 and KIF4A were overexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and are significant risk factors for HCC recurrence and shorter survival. We found that KIF4A was dominantly regulated by FOXM1c among the four isoforms, and further identified KIF4A as a direct downstream target of FOXM1c. Inhibiting FOXM1 decreased KIF4A expression in HCC cells, whereas its overexpression had the opposite effect. FOXM1-induced HCC cell proliferation was dependent on elevated KIF4A expression as KIF4A knockdown abolished FOXM1-induced proliferation of HCC cells both in vitro and in vivo.ConclusionThe FOXM1–KIF4A axis mediates human HCC progression and is a potential therapeutic target for HCC treatment.

Evolution as a guide for experimental cell biology.

Evolution as a guide for experimental cell biology.

Nutrient exchange in arbuscular mycorrhizal symbiosis from a thermodynamic point of view.

Summary To obtain insights into the dynamics of nutrient exchange in arbuscular mycorrhizal (AM) symbiosis, we modelled mathematically the two‐membrane system at the plant–fungus interface and simulated its dynamics.In computational cell biology experiments, the full range of nutrient transport pathways was tested for their ability to exchange phosphorus (P)/carbon (C)/nitrogen (N) sources.As a result, we obtained a thermodynamically justified, independent and comprehensive model of the dynamics of the nutrient exchange at the plant–fungus contact zone. The predicted optimal transporter network coincides with the transporter set independently confirmed in wet‐laboratory experiments previously, indicating that all essential transporter types have been discovered.The thermodynamic analyses suggest that phosphate is released from the fungus via proton‐coupled phosphate transporters rather than anion channels. Optimal transport pathways, such as cation channels or proton‐coupled symporters, shuttle nutrients together with a positive charge across the membranes. Only in exceptional cases does electroneutral transport via diffusion facilitators appear to be plausible. The thermodynamic models presented here can be generalized and adapted to other forms of mycorrhiza and open the door for future studies combining wet‐laboratory experiments with computational simulations to obtain a deeper understanding of the investigated phenomena.

Effect and mechanism of long non-coding RNA ZEB2-AS1 in the occurrence and development of colon cancer.

Objective: Clarify the expression changes, biological functions and related mechanisms of long non-coding RNA (lncRNA) ZEB2-AS1 in colon cancer tissues. Methods: The expression levels of ZEB2-AS1 in colon cancer tissues and adjacent tissues were detected by qRT-PCR and in situ hybridization methods. Cell biology experiments were performed to detect the proliferation, migration and apoptosis of colon cancer cells when the level of ZEB2-AS1 was overexpression or silencing. Then, Western blot was performed to analyze the effect of ZEB2-AS1 on the expression levels of β-catenin protein and related genes in the signal pathway. Results: We found that the expression level of ZEB2-AS1 in colon cancer tissues was significantly up-regulated compared with that in adjacent normal tissues. In colon cancer cell line of HCT8, overexpression of ZEB2-AS1 could promote cell proliferation and migration, while silencing ZEB2-AS1 would enhance cell apoptosis and inhibit proliferation. Study on the mechanism of ZEB2-AS1 showed that it could promote the expression of β-catenin, activate downstream genes to be transcribed and promote the occurrence and development of tumors. Conclusion: ZEB2-AS1 could promote colon cancer cell proliferation and inhibit apoptosis to promote the progression of colon cancer by upregulating the expression of β-catenin protein. ZEB2-AS1 may be a useful new target for treating colon cancer patients.

Verticillium dahliae transcription factors Som1 and Vta3 control microsclerotia formation and sequential steps of plant root penetration and colonisation to induce disease.

Verticillium dahliae nuclear transcription factors Som1 and Vta3 can rescue adhesion in aFLO8-deficient Saccharomyces cerevisiae strain. Som1 and Vta3 induce the expression of the yeast FLO1 and FLO11 genes encoding adhesins. Som1 and Vta3 are sequentially required for root penetration and colonisation of the plant host by V.dahliae. The SOM1 and VTA3 genes were deleted and their functions in fungus-induced plant pathogenesis were studied using genetic, cell biology, proteomic and plant pathogenicity experiments. Som1 supports fungal adhesion and root penetration and is required earlier than Vta3 in the colonisation of plant root surfaces and tomato plant infection. Som1 controls septa positioning and the size of vacuoles, and subsequently hyphal development including aerial hyphae formation and normal hyphal branching. Som1 and Vta3 control conidiation, microsclerotia formation, and antagonise in oxidative stress responses. Themolecular function of Som1 is conserved between the plant pathogen V.dahliae and the opportunistic human pathogen Aspergillus fumigatus. Som1 controls genes for initial steps of plant root penetration, adhesion, oxidative stress response and VTA3 expression to allow subsequent root colonisation. Both Som1 and Vta3 regulate developmental genetic networks required for conidiation, microsclerotia formation and pathogenicity of V.dahliae.

Comparative biological effects of spherical noble metal nanoparticles (Rh, Pd, Ag, Pt, Au) with 4-8 nm diameter.

For a comparative cytotoxicity study, nanoparticles of the noble metals Rh, Pd, Ag, Pt, and Au (spherical, average diameter 4 to 8 nm) were prepared by reduction in water and colloidally stabilized with poly(N-vinyl pyrrolidone) (PVP). Thus, their shape, size, and surface functionalization were all the same. Size and morphology of the nanoparticles were determined by dynamic light scattering (DLS), analytical disc centrifugation (differential centrifugal sedimentation, DCS), and high-resolution transmission electron microscopy (HRTEM). Cell-biological experiments were performed to determine the effect of particle exposure on the viability of human mesenchymal stem cells (hMSCs). Except for silver, no adverse effect of any of the metal nanoparticles was observed for concentrations up to 50 ppm (50 mg L−1) incubated for 24 h, indicating that noble metal nanoparticles (rhodium, palladium, platinum, gold) that do not release ions are not cytotoxic under these conditions.

Optimal localization patterns in bacterial protein synthesis

In $\textit{Escherichia coli}$ bacterium, the molecular compounds involved in protein synthesis, messenger RNAs (mRNAs) and ribosomes, show marked intracellular localization patterns. Yet a quantitative understanding of the physical principles which would allow one to control protein synthesis by designing, bioengineering, and optimizing these localization patterns is still lacking. In this study, we consider a scenario where a synthetic modification of mRNA reaction-diffusion properties allows for controlling the localization and stoichiometry of mRNAs and polysomes$\mathrm{-}$complexes of multiple ribosomes bound to mRNAs. Our analysis demonstrates that protein synthesis can be controlled, e.g., optimally enhanced or inhibited, by leveraging mRNA spatial localization and stoichiometry only, without resorting to alterations of mRNA expression levels. We identify the physical mechanisms that control the protein-synthesis rate, highlighting the importance of colocalization between mRNAs and freely diffusing ribosomes, and the interplay between polysome stoichiometry and excluded-volume effects due to the DNA nucleoid. The genome-wide, quantitative predictions of our work may allow for a direct verification and implementation in cell-biology experiments, where localization patterns and protein-synthesis rates may be monitored by fluorescence microscopy in single cells and populations.

Tumoral Acidic pH-Responsive cis-Diaminodichloroplatinum-Incorporated Cy5.5-PEG- g-A-HA Nanoparticles for Targeting Delivery of CDDP against Cervical Cancer.

Cisplatin (CDDP) has been considered as one of the most effective anticancer drugs against cervical cancer, but the lack of selectivity of CDDP to tumor tissues often leads to serious toxic side effects. In this study, CDDP-incorporated Cy5.5-PEG- g-A-HA nanoparticles were prepared to endue CDDP the ability to selectively target tumors and fluorescence imaging in vivo. The nanoparticles exhibited a spherical shape with particle sizes between 216.4 and 281.5 nm and had a pH and Cl- concentration dependence on controlled and sustained CDDP release, which was favorable for nanoparticles to release more drugs at acidic tumor microenvironment. Cell biology experiments demonstrated that the nanoparticles had good biocompatibility and tumor targeting; the nanoparticles could selectively bind and internalize into HeLa cells and induce apoptosis, but lead to less cytotoxicity on NIH3T3 cells. What is more, the nanoparticles could be clearly fluorescent-imaged in vivo and showed an effective accumulation at the tumor site. Antitumor test in vivo displayed that the nanoparticles had good antitumor efficiency and low systemic toxicity which improved the life quality of mice. Hence, the CDDP-incorporated Cy5.5-PEG- g-A-HA nanoparticles were a potential delivery system for targeting delivery of CDDP against cervical cancer.

Stochastic models of cell invasion with fluorescent cell cycle indicators

Fluorescent cell cycle labelling in cell biology experiments provides real time information about the location of individual cells, as well as the phase of the cell cycle of individual cells. We develop a stochastic, lattice-based random walk model of a two-dimensional scratch assay where the total population is composed of three distinct subpopulations which we visualise as red, yellow and green subpopulations. Our model mimics FUCCI technology in which cells in the G1 phase of the cell cycle fluoresce red, cells in the early S phase fluoresce yellow, and cells in the S/G2/M phase fluoresce green. The model is an exclusion process so that any potential motility or proliferation event that would place an agent on an occupied lattice site is aborted. Using experimental images and previous experimental measurements we explain how to apply the stochastic model to simulate a scratch assay initialised with a low to moderate density monolayer of human melanoma cell line. We obtain additional mathematical insight by deriving an approximate partial differential equation (PDE) description of the stochastic model, leading to a novel system of three coupled nonlinear reaction diffusion equations. Comparing averaged simulation data with the solution of the continuum limit model confirms that the PDE description is accurate for biologically-relevant parameter combinations.

Robust imaging and gene delivery to study human lymphoblastoid cell lines

Lymphoblastoid cell lines (LCLs) have been by far the most prevalent cell type used to study the genetics underlying normal and disease-relevant human phenotypic variation, across personal to epidemiological scales. In contrast, only few studies have explored the use of LCLs in functional genomics and mechanistic studies. Two major reasons are technical, as (1) interrogating the sub-cellular spatial information of LCLs is challenged by their non-adherent nature, and (2) LCLs are refractory to gene transfection. Methodological details relating to techniques that overcome these limitations are scarce, largely inadequate (without additional knowledge and expertise), and optimisation has never been described. Here we compare, optimise, and convey such methods in-depth. We provide a robust method to adhere LCLs to coverslips, which maintained cellular integrity, morphology, and permitted visualisation of sub-cellular structures and protein localisation. Next, we developed the use of lentiviral-based gene delivery to LCLs. Through empirical and combinatorial testing of multiple transduction conditions, we improved transduction efficiency from 3% up to 48%. Furthermore, we established strategies to purify transduced cells, to achieve sustainable cultures containing >85% transduced cells. Collectively, our methodologies provide a vital resource that enables the use of LCLs in functional cell and molecular biology experiments. Potential applications include the characterisation of genetic variants of unknown significance, the interrogation of cellular disease pathways and mechanisms, and high-throughput discovery of genetic modifiers of disease states among others.

Specific Eph receptor-cytoplasmic effector signaling mediated by SAM-SAM domain interactions.

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here, we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain many Eph SAM mutations identified in patients suffering from cancers and other diseases.