In this paper, flat plate film cooling with two rows of compound angle cylindrical film cooling holes was investigated. A data processing method was evaluated which could determine the film cooling ...effectiveness and heat transfer coefficient simultaneously from the transient wall temperature data. The method was based on solving an inverse problem of the one-dimensional transient heat conduction equation. To evaluate the performance of the method, wall temperature data were obtained using the known film cooling effectiveness and heat transfer coefficient data as the convection boundary condition. Then, the method was applied to calculate the film cooling effectiveness and heat transfer coefficient based on the wall temperature data. Different blowing ratios, heat transfer coefficients, mainstream temperatures, and material thermal conductivities were investigated. In general, the data and calculation were in good agreement. It was found that the error decreased when the heat transfer coefficient increased and the material thermal conductivity decreased. The percentage error of the span-wise averaged film cooling effectiveness was mainly between 0% and 10%, and the percentage error of the span-wise averaged heat transfer coefficient was mainly between 0% and 4%.
Liquid–liquid phase separation (LLPS) facilitates the formation of condensed biological assemblies with well-delineated physical boundaries, but without lipid membrane barriers. LLPS is increasingly ...recognized as a common mechanism for cells to organize and maintain different cellular compartments in addition to classical membrane-delimited organelles. Membraneless condensates have many distinct features that are not present in membrane-delimited organelles and that are likely indispensable for the viability and function of living cells. Malformation of membraneless condensates is increasingly linked to human diseases. In this review, we summarize commonly used methods to investigate various forms of LLPS occurring both in 3D aqueous solution and on 2D membrane bilayers, such as LLPS condensates arising from intrinsically disordered proteins or structured modular protein domains. We then discuss, in the context of comparisons with membrane-delimited organelles, the potential functional implications of membraneless condensate formation in cells. We close by highlighting some challenges in the field devoted to studying LLPS-mediated membraneless condensate formation.
PDZ domains are highly abundant protein-protein interaction modules and are often found in multidomain scaffold proteins. PDZ-domain-containing scaffold proteins regulate multiple biological ...processes, including trafficking and clustering receptors and ion channels at defined membrane regions, organizing and targeting signalling complexes at specific cellular compartments, interfacing cytoskeletal structures with membranes, and maintaining various cellular structures. PDZ domains, each with ~90-amino-acid residues folding into a highly similar structure, are best known to bind to short C-terminal tail peptides of their target proteins. A series of recent studies have revealed that, in addition to the canonical target-binding mode, many PDZ-target interactions involve amino acid residues beyond the regular PDZ domain fold, which we refer to as extensions. Such extension sequences often form an integral structural and functional unit with the attached PDZ domain, which is defined as a PDZ supramodule. Correspondingly, PDZ-domain-binding sequences from target proteins are frequently found to require extension sequences beyond canonical short C-terminal tail peptides. Formation of PDZ supramodules not only affords necessary binding specificities and affinities demanded by physiological functions of PDZ domain targets, but also provides regulatory switches to be built in the PDZ-target interactions. At the 20th anniversary of the discovery of PDZ domain proteins, we try to summarize structural features and target-binding properties of such PDZ supramodules emerging from studies in recent years.
Synapses are semi-membraneless, protein-dense, sub-micron chemical reaction compartments responsible for signal processing in each and every neuron. Proper formation and dynamic responses to ...stimulations of synapses, both during development and in adult, are fundamental to functions of mammalian brains, although the molecular basis governing formation and modulation of compartmentalized synaptic assemblies is unclear. Here, we used a biochemical reconstitution approach to show that, both in solution and on supported membrane bilayers, multivalent interaction networks formed by major excitatory postsynaptic density (PSD) scaffold proteins led to formation of PSD-like assemblies via phase separation. The reconstituted PSD-like assemblies can cluster receptors, selectively concentrate enzymes, promote actin bundle formation, and expel inhibitory postsynaptic proteins. Additionally, the condensed phase PSD assemblies have features that are distinct from those in homogeneous solutions and fit for synaptic functions. Thus, we have built a molecular platform for understanding how neuronal synapses are formed and dynamically regulated.
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•Biochemical reconstitution reveals PSD assembly formation via phase separation•The ePSD condensates cluster NMDA receptor and promote actin bundle formation•The ePSD condensates selectively enrich SynGAP and actively exclude gephyrin•The ePSD condensates can be modulated by activity-dependent protein modifications
Phase transition-mediated formation of excitatory postsynaptic density condensates revealed by biochemical reconstitutions.
Asymmetric local concentration of protein complexes on distinct membrane regions is a fundamental property in numerous biological processes and is a hallmark of cell polarity. Evolutionarily ...conserved core polarity proteins form specific and dynamic networks to regulate the establishment and maintenance of cell polarity, as well as distinct polarity-driven cellular events. This review focuses on the molecular and structural basis governing regulated formation of several sets of core cell polarity regulatory complexes, as well as their functions in epithelial cell polarization and asymmetric cell division.
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•Epithelial apical–basal polarity is regulated by a set of core polarity regulatory complexes.•The assembly mechanisms and regulations of the polarity complexes are reviewed.•Epithelial polarity is tightly coupled with asymmetric cell division.•Connections of the polarity complexes with the mitotic spindle orientation machineries are discussed.
As the major components of the postsynaptic density of excitatory neuronal synapses, PDZ-domain-containing scaffold proteins regulate the clustering of surface glutamate receptors, organize synaptic ...signalling complexes, participate in the dynamic trafficking of receptors and ion channels, and coordinate cytoskeletal dynamics. These scaffold proteins often contain multiple PDZ domains, with or without other protein-binding modules, and they usually lack intrinsic enzymatic activities. Recent biochemical and structural studies have shown that tandemly arranged PDZ domains often serve as structural and functional supramodules that could regulate the organization and dynamics of synaptic protein complexes, thus contributing to the broad range of neuronal activity.
Apical–basal polarity is the basic organizing principle of epithelial cells, and endows epithelial cells to function as defensive barriers and as mediators of vectorial transport of nutrients in and ...out of organisms. Apical–basal polarity is controlled by a number of conserved polarity factors that regulate cytoskeletal organizations, asymmetric distributions of cellular components, and directional transports across cells. Polarity factors often occupy specific membrane regions in response to the adhesion forces generated by cell–cell and cell–extracellular matrix interactions. Both internal polarity factors and the external extracellular matrices play fundamental roles in epithelial cell polarity establishment and maintenance. This review focuses on recent developments of the Par3/Par6/aPKC complex and its interacting proteins in epithelial cell polarity.
•The Par3/Par6/aPKC complex is a major cell polarity regulatory engine in all eukaryotes.•The Par complex functions as a hub interacting with many other cell polarity factors.•Epithelia is an ideal model system for studying the Par complex function.
Both the timing and kinetics of neurotransmitter release depend on the positioning of clustered Ca2+ channels in active zones to docked synaptic vesicles on presynaptic plasma membranes. However, how ...active zones form is not known. Here, we show that RIM and RIM-BP, via specific multivalent bindings, form dynamic and condensed assemblies through liquid-liquid phase separation. Voltage-gated Ca2+ channels (VGCCs), via C-terminal-tail-mediated direct binding to both RIM and RIM-BP, can be enriched to the RIM and RIM-BP condensates. We further show that RIM and RIM-BP, together with VGCCs, form dense clusters on the supported lipid membrane bilayers via phase separation. Therefore, RIMs and RIM-BPs are plausible organizers of active zones, and the formation of RIM and RIM-BP condensates may cluster VGCCs into nano- or microdomains and position the clustered Ca2+ channels with Ca2+ sensors on docked vesicles for efficient and precise synaptic transmissions.
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•RIM and RIM-BP mixture forms liquid-liquid phase-separation-mediated condensates•Specific multivalent interaction between RIM and RIM-BP is essential for the LLPS•RIM and RIM-BP condensates cluster Ca2+ channels in solution and on membrane surface•RIM and RIM-BP are plausible organizers of presynaptic active zones
Clustering of Ca2+ channels at presynaptic active zones is critical for precise control of neurotransmitter release. Wu et al. show that the presynaptic active zone scaffold proteins RIM and RIM-BP form self-assembled condensates via liquid-liquid phase separations capable of clustering voltage-gated Ca2+ channels on lipid membrane bilayers.
Inhibitory synapses are also known as symmetric synapses due to their lack of prominent postsynaptic densities (PSDs) under a conventional electron microscope (EM). Recent cryo-EM tomography studies ...indicated that inhibitory synapses also contain PSDs, albeit with a rather thin sheet-like structure. It is not known how such inhibitory PSD (iPSD) sheet might form. Here, we demonstrate that the key inhibitory synapse scaffold protein gephyrin, when in complex with either glycine or GABA
receptors, spontaneously forms highly condensed molecular assemblies via phase separation both in solution and on supported membrane bilayers. Multivalent and specific interactions between the dimeric E-domain of gephyrin and the glycine/GABA
receptor multimer are essential for the iPSD condensate formation. Gephyrin alone does not form condensates. The linker between the G- and E-domains of gephyrin inhibits the iPSD condensate formation via autoinhibition. Phosphorylation of specific residues in the linker or binding of target proteins such as dynein light chain to the linker domain regulates gephyrin-mediated glycine/GABA
receptor clustering. Thus, analogous to excitatory PSDs, iPSDs are also formed by phase separation-mediated condensation of scaffold protein/neurotransmitter receptor complexes.
Extensive studies in the past few years have shown that nonmembrane bound organelles are likely assembled via liquid–liquid phase separation (LLPS), a process that is driven by multivalent ...protein–protein and/or protein–nucleic acid interactions. Both stoichiometric molecular interactions and intrinsically disordered region (IDR)-driven interactions can promote the assembly of membraneless organelles, and the field is currently dominated by IDR-driven biological condensate formation. Here we discuss recent studies that demonstrate the importance of specific biomolecular interactions for functions of diverse physiological condensates. We suggest that phase separation based on combinations of specific interactions and promiscuous IDR-driven interactions is likely a general feature of biological condensation under physiological conditions.
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