The Hippo pathway drives the cellular response to hydrostatic pressure.

Cells need to rapidly and precisely react to multiple mechanical and chemical stimuli in order to ensure precise context-dependent responses. This requires dynamic cellular signalling events that ensure homeostasis and plasticity when needed. A less well-understood process is cellular response to elevated interstitial fluid pressure, where the cell senses and responds to changes in extracellular hydrostatic pressure. Here, using quantitative label-free digital holographic imaging, combined with genome editing, biochemical assays and confocal imaging, we analyse the temporal cellular response to hydrostatic pressure. Upon elevated cyclic hydrostatic pressure, the cell responds by rapid, dramatic and reversible changes in cellular volume. We show that YAP and TAZ, the co-transcriptional regulators of the Hippo signalling pathway, control cell volume and that cells without YAP and TAZ have lower plasma membrane tension. We present direct evidence that YAP/TAZ drive the cellular response to hydrostatic pressure, a process that is at least partly mediated via clathrin-dependent endocytosis. Additionally, upon elevated oscillating hydrostatic pressure, YAP/TAZ are activated and induce TEAD-mediated transcription and expression of cellular components involved in dynamic regulation of cell volume and extracellular matrix. This cellular response confers a feedback loop that allows the cell to robustly respond to changes in interstitial fluid pressure.

The LKB1-TSSK1B axis controls YAP phosphorylation to regulate the Hippo-YAP pathway.

The Hippo pathway's main effector, Yes-associated protein (YAP), plays a crucial role in tumorigenesis as a transcriptional coactivator. YAP's phosphorylation by core upstream components of the Hippo pathway, such as mammalian Ste20 kinase 1/2 (MST1/2), mitogen-activated protein kinase kinase kinase kinases (MAP4Ks), and their substrate, large tumor suppressor 1/2 (LATS1/2), influences YAP's subcellular localization, stability, and transcriptional activity. However, recent research suggests the existence of alternative pathways that phosphorylate YAP, independent of these core upstream Hippo pathway components, raising questions about additional means to inactivate YAP. In this study, we present evidence demonstrating that TSSK1B, a calcium/calmodulin-dependent protein kinase (CAMK) superfamily member, is a negative regulator of YAP, suppressing cellular proliferation and oncogenic transformation. Mechanistically, TSSK1B inhibits YAP through two distinct pathways. Firstly, the LKB1-TSSK1B axis directly phosphorylates YAP at Ser94, inhibiting the YAP-TEAD complex's formation and suppressing its target genes' expression. Secondly, the TSSK1B-LATS1/2 axis inhibits YAP via phosphorylation at Ser127. Our findings reveal the involvement of TSSK1B-mediated molecular mechanisms in the Hippo-YAP pathway, emphasizing the importance of multilevel regulation in critical cellular decision-making processes.

Immune complex-induced apoptosis and concurrent immune complex clearance are anti-inflammatory neutrophil functions.

Persistent neutrophilic inflammation drives host damage in autoimmune diseases that are characterized by abundant immune complexes. Insoluble immune complexes (iICs) potently activate pro-inflammatory neutrophil effector functions. We and others have shown that iICs also promote resolution of inflammation via stimulation of neutrophil apoptosis. We demonstrate here that iICs trigger FcγRIIa-dependent neutrophil macropinocytosis, leading to the rapid uptake, and subsequent degradation of iICs. We provide evidence that concurrent iIC-induced neutrophil apoptosis is distinct from phagocytosis-induced cell death. First, uptake of iICs occurs by FcγRII-stimulated macropinocytosis, rather than phagocytosis. Second, production of reactive oxygen species, but not iIC-internalization is a pre-requisite for iIC-induced neutrophil apoptosis. Our findings identify a previously unknown mechanism by which neutrophils can remove pro-inflammatory iICs from the circulation. Together iIC clearance and iIC-induced neutrophil apoptosis may act to prevent the potential escalation of neutrophilic inflammation in response to iICs.

The transcription factor EGR2 is indispensable for tissue-specific imprinting of alveolar macrophages in health and tissue repair.

Alveolar macrophages are the most abundant macrophages in the healthy lung where they play key roles in homeostasis and immune surveillance against airborne pathogens. Tissue-specific differentiation and survival of alveolar macrophages rely on niche-derived factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor­ß (TGF-ß). However, the nature of the downstream molecular pathways that regulate the identity and function of alveolar macrophages and their response to injury remain poorly understood. Here, we identify that the transcription factor EGR2 is an evolutionarily conserved feature of lung alveolar macrophages and show that cell-intrinsic EGR2 is indispensable for the tissue-specific identity of alveolar macrophages. Mechanistically, we show that EGR2 is driven by TGF-ß and GM-CSF in a PPAR-γ­dependent manner to control alveolar macrophage differentiation. Functionally, EGR2 was dispensable for the regulation of lipids in the airways but crucial for the effective handling of the respiratory pathogen Streptococcus pneumoniae. Last, we show that EGR2 is required for repopulation of the alveolar niche after sterile, bleomycin-induced lung injury and demonstrate that EGR2-dependent, monocyte-derived alveolar macrophages are vital for effective tissue repair after injury. Collectively, we demonstrate that EGR2 is an indispensable component of the transcriptional network controlling the identity and function of alveolar macrophages in health and disease.

The Hippo Pathway, YAP/TAZ, and the Plasma Membrane.

The plasma membrane allows the cell to sense and adapt to changes in the extracellular environment by relaying external inputs via intracellular signaling networks. One central cellular signaling pathway is the Hippo pathway, which regulates homeostasis and plays chief roles in carcinogenesis and regenerative processes. Recent studies have found that mechanical stimuli and diffusible chemical components can regulate the Hippo pathway primarily through receptors embedded in the plasma membrane. Morphologically defined structures within the plasma membrane, such as cellular junctions, focal adhesions, primary cilia, caveolae, clathrin-coated pits, and plaques play additional key roles. Here, we discuss recent evidence highlighting the importance of these specialized plasma membrane domains in cellular feedback via the Hippo pathway.

Listeria monocytogenes Exploits Host Caveolin for Cell-to-Cell Spreading.

Listeria monocytogenes moves from one cell to another using actin-rich membrane protrusions that propel the bacterium toward neighboring cells. Despite cholesterol being required for this transfer process, the precise host internalization mechanism remains elusive. Here, we show that caveolin endocytosis is key to this event as bacterial cell-to-cell transfer is severely impaired when cells are depleted of caveolin-1. Only a subset of additional caveolar components (cavin-2 and EHD2) are present at sites of bacterial transfer, and although clathrin and the clathrin-associated proteins Eps15 and AP2 are absent from the bacterial invaginations, efficient L. monocytogenes spreading requires the clathrin-interacting protein epsin-1. We also directly demonstrated that isolated L. monocytogenes membrane protrusions can trigger the recruitment of caveolar proteins in a neighboring cell. The engulfment of these bacterial and cytoskeletal structures through a caveolin-based mechanism demonstrates that the classical nanometer-scale theoretical size limit for this internalization pathway is exceeded by these bacterial pathogens.IMPORTANCEListeria monocytogenes moves from one cell to another as it disseminates within tissues. This bacterial transfer process depends on the host actin cytoskeleton as the bacterium forms motile actin-rich membranous protrusions that propel the bacteria into neighboring cells, thus forming corresponding membrane invaginations. Here, we examine these membrane invaginations and demonstrate that caveolin-1-based endocytosis is crucial for efficient bacterial cell-to-cell spreading. We show that only a subset of caveolin-associated proteins (cavin-2 and EHD2) are involved in this process. Despite the absence of clathrin at the invaginations, the classical clathrin-associated protein epsin-1 is also required for efficient bacterial spreading. Using isolated L. monocytogenes protrusions added onto naive host cells, we demonstrate that actin-based propulsion is dispensable for caveolin-1 endocytosis as the presence of the protrusion/invagination interaction alone triggers caveolin-1 recruitment in the recipient cells. Finally, we provide a model of how this caveolin-1-based internalization event can exceed the theoretical size limit for this endocytic pathway.

The Hippo Pathway in Prostate Cancer.

Despite recent efforts, prostate cancer (PCa) remains one of the most common cancers in men. Currently, there is no effective treatment for castration-resistant prostate cancer (CRPC). There is, therefore, an urgent need to identify new therapeutic targets. The Hippo pathway and its downstream effectors-the transcriptional co-activators, Yes-associated protein (YAP) and its paralog, transcriptional co-activator with PDZ-binding motif (TAZ)-are foremost regulators of stem cells and cancer biology. Defective Hippo pathway signaling and YAP/TAZ hyperactivation are common across various cancers. Here, we draw on insights learned from other types of cancers and review the latest advances linking the Hippo pathway and YAP/TAZ to PCa onset and progression. We examine the regulatory interaction between Hippo-YAP/TAZ and the androgen receptor (AR), as main regulators of PCa development, and how uncontrolled expression of YAP/TAZ drives castration resistance by inducing cellular stemness. Finally, we survey the potential therapeutic targeting of the Hippo pathway and YAP/TAZ to overcome PCa.

Immunofluorescence Study of Endogenous YAP in Mammalian Cells.

Immunocytochemistry enables determination of cellular localization and relative abundance of proteins. This protocol describes a rapid and cost-effective approach to study the cellular localization of YAP (and TAZ), the transcriptional co activators of the Hippo pathway, in mammalian cells. Cells are seeded onto coated cover slips, cultivated and treated as required. Subsequently, they are chemically fixed, and cellular proteins are fluorescently labeled by means of specific antibodies. Multiplexing antibodies enables ascertaining the subcellular localization of YAP and TAZ and thereby also the activation state of the Hippo pathway in various cell types.

The Hippo Pathway Regulates Caveolae Expression and Mediates Flow Response via Caveolae.

The Hippo pathway plays major roles in development, regeneration, and cancer. Its activity is tightly regulated by both diffusible chemical ligands and mechanical stimuli. The pathway consists of a series of kinases that can control the sub-cellular localization and stability of YAP or TAZ, homologous transcriptional co-factors. Caveolae, small (60-100 nm) bulb-like invaginations of the plasma membrane, are comprised predominantly of caveolin and cavin proteins and can respond to mechanical stimuli. Here, we show that YAP/TAZ, the major transcriptional mediators of the Hippo pathway, are critical for expression of caveolae components and therefore caveolae formation in both mammalian cells and zebrafish. In essence, without YAP/TAZ, the cell loses an entire organelle. CAVEOLIN1 and CAVIN1, the two essential caveolar genes, are direct target genes of YAP/TAZ, regulated via TEA domain (TEAD) transcription factors. Notably, YAP/TAZ become nuclear enriched and facilitate target gene transcription in cells with diminished levels of caveolae. Furthermore, caveolar-mediated shear stress response activates YAP/TAZ. These data link caveolae to Hippo signaling in the context of cellular responses to mechanical stimuli and suggest activity-based feedback regulation between components of caveolae and the outputs of the Hippo pathway.

Binding of serotonin to the human serotonin transporter. Molecular modeling and experimental validation.

Molecular modeling and structure-activity relationship studies were performed to propose a model for binding of the neurotransmitter serotonin (5-HT) to the human serotonin transporter (hSERT). Homology models were constructed using the crystal structure of a bacterial homologue, the leucine transporter from Aquifex aeolicus, as the template and three slightly different sequence alignments. Induced fit docking of 5-HT into hSERT homology models resulted in two different binding modes. Both show a salt bridge between Asp98 and the charged primary amine of 5-HT, and both have the 5-HT C6 position of the indole ring pointing toward Ala173. The difference between the two orientations of 5-HT is an enantiofacial discrimination of the indole ring, resulting in the 5-hydroxyl group of 5-HT being vicinal to either Ser438/Thr439 or Ala169/Ile172/Ala173. To assess the binding experimentally, binding affinities for 5-HT and 17 analogues toward wild type and 13 single point mutants of hSERT were measured using an approach termed paired mutant-ligand analogue complementation (PaMLAC). The proposed ligand-protein interaction was systematically examined by disrupting it through site-directed mutagenesis and re-establishing another interaction via a ligand analogue matching the mutated residue, thereby minimizing the risk of identifying indirect effects. The interactions between Asp98 and the primary amine of 5-HT and the interaction between the C6-position of 5-HT and hSERT position 173 was confirmed using PaMLAC. The measured binding affinities of various mutants and 5-HT analogues allowed for a distinction between the two proposed binding modes of 5-HT and biochemically support the model for 5-HT binding in hSERT where the 5-hydroxyl group is in close proximity to Thr439.

EHD Proteins Cooperate to Generate Caveolar Clusters and to Maintain Caveolae during Repeated Mechanical Stress.

Caveolae introduce flask-shaped convolutions into the plasma membrane and help to protect the plasma membrane from damage under stretch forces. The protein components that form the bulb of caveolae are increasingly well characterized, but less is known about the contribution of proteins that localize to the constricted neck. Here we make extensive use of multiple CRISPR/Cas9-generated gene knockout and knockin cell lines to investigate the role of Eps15 Homology Domain (EHD) proteins at the neck of caveolae. We show that EHD1, EHD2, and EHD4 are recruited to caveolae. Recruitment of the other EHDs increases markedly when EHD2, which has been previously detected at caveolae, is absent. Construction of knockout cell lines lacking EHDs 1, 2, and 4 confirms this apparent functional redundancy. Two striking sets of phenotypes are observed in EHD1,2,4 knockout cells: (1) the characteristic clustering of caveolae into higher-order assemblies is absent; and (2) when the EHD1,2,4 knockout cells are subjected to prolonged cycles of stretch forces, caveolae are destabilized and the plasma membrane is prone to rupture. Our data identify the first molecular components that act to cluster caveolae into a membrane ultrastructure with the potential to extend stretch-buffering capacity and support a revised model for the function of EHDs at the caveolar neck.

MAP4K family kinases act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway.

The Hippo pathway plays a central role in tissue homoeostasis, and its dysregulation contributes to tumorigenesis. Core components of the Hippo pathway include a kinase cascade of MST1/2 and LATS1/2 and the transcription co-activators YAP/TAZ. In response to stimulation, LATS1/2 phosphorylate and inhibit YAP/TAZ, the main effectors of the Hippo pathway. Accumulating evidence suggests that MST1/2 are not required for the regulation of YAP/TAZ. Here we show that deletion of LATS1/2 but not MST1/2 abolishes YAP/TAZ phosphorylation. We have identified MAP4K family members--Drosophila Happyhour homologues MAP4K1/2/3 and Misshapen homologues MAP4K4/6/7-as direct LATS1/2-activating kinases. Combined deletion of MAP4Ks and MST1/2, but neither alone, suppresses phosphorylation of LATS1/2 and YAP/TAZ in response to a wide range of signals. Our results demonstrate that MAP4Ks act in parallel to and are partially redundant with MST1/2 in the regulation of LATS1/2 and YAP/TAZ, and establish MAP4Ks as components of the expanded Hippo pathway.

Cavin-3 knockout mice show that cavin-3 is not essential for caveolae formation, for maintenance of body composition, or for glucose tolerance.

The cavins are a family of proteins associated with caveolae, cavin-1, -2 and -3 being widely expressed while cavin-4 is restricted to striated muscle. Deletion of cavin-1 results in phenotypes including metabolic changes consistent with adipocyte dysfunction, and caveolae are completely absent. Deletion of cavin-2 causes tissue-specific loss of caveolae. The consequences of cavin-3 deletion are less clear, as there are divergent data on the abundance of caveolae in cavin-3 null mice. Here we examine the consequences of cavin-3 deficiency in vivo by making cavin-3 knockout mice. We find that loss of cavin-3 has minimal or no effects on the levels of other caveolar proteins, does not appear to play a major role in formation of protein complexes important for caveolar morphogenesis, and has no significant effect on caveolae abundance. Cavin-3 null mice have the same body weight and fat mass as wild type animals at ages 8 through 30 weeks on both normal chow and high fat diets. Likewise, the two mouse strains exhibit identical glucose tolerance tests on both diets. Microarray analysis from adipose tissue shows that the changes in mRNA expression between cavin-3 null and wild type mouse are minimal. We conclude that cavin-3 is not absolutely required for making caveolae, and suggest that the mechanistic link between cavin-3 and metabolic regulation remains uncertain.

Exploring the caves: cavins, caveolins and caveolae.

Caveolae are ampullate (flask-shaped) invaginations that are abundant in the plasma membrane of many mammalian cell types. Although caveolae are implicated in a wide range of processes including endothelial transcytosis, lipid homeostasis and cellular signalling, a detailed molecular picture of many aspects of their function has been elusive. Until recently, the only extensively characterised protein components of caveolae were the caveolins. Recently, data from several laboratories have demonstrated that a family of four related proteins, termed cavins 1-4, plays key roles in caveolar biogenesis and function. Salient properties of the cavin family include their propensity to form complexes with each other and their different but overlapping tissue distribution. This review summarises recent data on the cavins, and sets them in the context of open questions on the construction and function of caveolae. The discovery of cavins implies that caveolae might have unexpectedly diverse structural properties, in accord with the wide range of functions attributed to these 'little caves'.

Molecular mechanisms of clathrin-independent endocytosis.

There is good evidence that, in addition to the canonical clathrin-associated endocytic machinery, mammalian cells possess multiple sets of proteins that are capable of mediating the formation of endocytic vesicles. The identity, mechanistic properties and function of these clathrin-independent endocytic pathways are currently under investigation. This Commentary briefly recounts how the field of clathrin-independent endocytosis has developed to date. It then highlights recent progress in identifying key proteins that might define alternative types of endocytosis. These proteins include CtBP (also known as BARS), flotillins (also known as reggies) and GRAF1. We argue that a combination of information about pathway-specific proteins and the ultrastructure of endocytic invaginations provides a means of beginning to classify endocytic pathways.

SDPR induces membrane curvature and functions in the formation of caveolae.

Caveolae are plasma membrane invaginations with a characteristic flask-shaped morphology. They function in diverse cellular processes, including endocytosis. The mechanism by which caveolae are generated is not fully understood, but both caveolin proteins and PTRF (polymerase I and transcript release factor, also known as cavin) are important. Here we show that loss of SDPR (serum deprivation protein response) causes loss of caveolae. SDPR binds directly to PTRF and recruits PTRF to caveolar membranes. Overexpression of SDPR, unlike PTRF, induces deformation of caveolae and extensive tubulation of the plasma membrane. The B-subunit of Shiga toxin (STB) also induces membrane tubulation and these membrane tubes also originate from caveolae. STB colocalizes extensively with both SDPR and caveolin 1. Loss of caveolae reduces the propensity of STB to induce membrane tubulation. We conclude that SDPR is a membrane-curvature-inducing component of caveolae, and that STB-induced membrane tubulation is facilitated by caveolae.