Showing papers in "Journal of Cell Science in 2016"

RAS isoforms and mutations in cancer at a glance

Abstract: RAS proteins (KRAS4A, KRAS4B, NRAS and HRAS) function as GDP–GTP-regulated binary on-off switches, which regulate cytoplasmic signaling networks that control diverse normal cellular processes. Gain-of-function missense mutations in RAS genes are found in ∼25% of human cancers, prompting interest in identifying anti-RAS therapeutic strategies for cancer treatment. However, despite more than three decades of intense effort, no anti-RAS therapies have reached clinical application. Contributing to this failure has been an underestimation of the complexities of RAS. First, there is now appreciation that the four human RAS proteins are not functionally identical. Second, with >130 different missense mutations found in cancer, there is an emerging view that there are mutation-specific consequences on RAS structure, biochemistry and biology, and mutation-selective therapeutic strategies are needed. In this Cell Science at a Glance article and accompanying poster, we provide a snapshot of the differences between RAS isoforms and mutations, as well as the current status of anti-RAS drug-discovery efforts.

TFEB at a glance.

Abstract: The transcription factor EB (TFEB) plays a pivotal role in the regulation of basic cellular processes, such as lysosomal biogenesis and autophagy. The subcellular localization and activity of TFEB are regulated by mechanistic target of rapamycin (mTOR)-mediated phosphorylation, which occurs at the lysosomal surface. Phosphorylated TFEB is retained in the cytoplasm, whereas dephosphorylated TFEB translocates to the nucleus to induce the transcription of target genes. Thus, a lysosome-to-nucleus signaling pathway regulates cellular energy metabolism through TFEB. Recently, in vivo studies have revealed that TFEB is also involved in physiological processes, such as lipid catabolism. TFEB has attracted a lot of attention owing to its ability to induce the intracellular clearance of pathogenic factors in a variety of murine models of disease, such as Parkinson's and Alzheimer's, suggesting that novel therapeutic strategies could be based on the modulation of TFEB activity. In this Cell Science at a Glance article and accompanying poster, we present an overview of the latest research on TFEB function and its implication in human diseases.

Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a

Abstract: Angiogenesis plays crucial roles in various physiological processes including wound healing and tissue repair. It requires a tight interaction between endothelial cells and their surrounding environment. Mesenchymal stem cells (MSCs), one of the non-endothelial cell types present in the perivascular environment, have been shown to secret exosomes to modulate intercellular communications between MSCs and their target cells. In this study, we initially isolated exosomes secreted by human adipose-derived MSCs (adMSC-Exo) and examined their roles in angiogenesis. We found that adMSC-Exo could be taken up by endothelial cells and significantly promote angiogenesis in vitro and in vivo Further study showed that miR-125a was enriched in adMSC-Exo, and repressed the expression of the angiogenic inhibitor delta-like 4 (DLL4) by targeting its 3' untranslated region. Additionally, adMSC-Exo and its exosomal transferred miR-125a could repress DLL4 expression and modulate endothelial cell angiogenesis through promoting formation of endothelial tip cells. In conclusion, our study indicates that adMSC-Exo can transfer miR-125a to endothelial cells and promote angiogenesis by repressing DLL4. adMSC-Exo, as a pro-angiogenic factor, might be a promising candidate for therapeutical tissue repair.

Ubiquitin chain diversity at a glance

Abstract: Ubiquitin plays an essential role in modulating protein functions, and deregulation of the ubiquitin system leads to the development of multiple human diseases. Owing to its molecular features, ubiquitin can form various homo- and heterotypic polymers on substrate proteins, thereby provoking distinct cellular responses. The concept of multifaceted ubiquitin chains encoding different functions has been substantiated in recent years. It has been established that all possible ubiquitin linkage types are utilized for chain assembly and propagation of specific signals in vivo. In addition, branched ubiquitin chains and phosphorylated ubiquitin molecules have been put under the spotlight recently. The development of novel technologies has provided detailed insights into the structure and function of previously poorly understood ubiquitin signals. In this Cell Science at a Glance article and accompanying poster, we provide an update on the complexity of ubiquitin chains and their physiological relevance.

Mechanisms and functions of lysosome positioning

Abstract: Lysosomes have been classically considered terminal degradative organelles, but in recent years they have been found to participate in many other cellular processes, including killing of intracellular pathogens, antigen presentation, plasma membrane repair, cell adhesion and migration, tumor invasion and metastasis, apoptotic cell death, metabolic signaling and gene regulation In addition, lysosome dysfunction has been shown to underlie not only rare lysosome storage disorders but also more common diseases, such as cancer and neurodegeneration The involvement of lysosomes in most of these processes is now known to depend on the ability of lysosomes to move throughout the cytoplasm Here, we review recent findings on the mechanisms that mediate the motility and positioning of lysosomes, and the importance of lysosome dynamics for cell physiology and pathology

Comparison of cancer cells in 2D vs 3D culture reveals differences in AKT-mTOR-S6K signaling and drug responses.

Abstract: Three-dimensional (3D) cancer models are used as preclinical systems to mimic physiologic drug responses. We provide evidence for strong changes of proliferation and metabolic capacity in three dimensions by systematically analyzing spheroids of colon cancer cell lines. Spheroids showed relative lower activities in the AKT, mammalian target of rapamycin (mTOR) and S6K (also known as RPS6KB1) signaling pathway compared to cells cultured in two dimensions. We identified spatial alterations in signaling, as the level of phosphorylated RPS6 decreased from the spheroid surface towards the center, which closely coordinated with the tumor areas around vessels in vivo These 3D models displayed augmented anti-tumor responses to AKT-mTOR-S6K or mitogen-activated protein kinase (MAPK) pathway inhibition compared to those in 2D models. Inhibition of AKT-mTOR-S6K resulted in elevated ERK phosphorylation in 2D culture, whereas under these conditions, ERK signaling was reduced in spheroids. Inhibition of MEK1 (also known as MAP2K1) led to decreased AKT-mTOR-S6K signaling in 3D but not in 2D culture. These data indicate a distinct rewiring of signaling in 3D culture and during treatment. Detached tumor-cell clusters in vessels, in addition to circulating single tumor cells, play a putative role in metastasis in human cancers. Hence, the understanding of signaling in spheroids and the responses in the 3D models upon drug treatment might be beneficial for anti-cancer therapies.

OPA1 processing in cell death and disease – the long and short of it

Abstract: The regulation of mitochondrial dynamics by the GTPase OPA1, which is located at the inner mitochondrial membrane, is crucial for adapting mitochondrial function and preserving cellular health. OPA1 governs the delicate balance between fusion and fission in the dynamic mitochondrial network. A disturbance of this balance, often observed under stress and pathologic conditions, causes mitochondrial fragmentation and can ultimately result in cell death. As discussed in this Commentary, these morphological changes are regulated by proteolytic processing of OPA1 by the inner-membrane peptidases YME1L (also known as YME1L1) and OMA1. Long, membrane-bound forms of OPA1 are required for mitochondrial fusion, but their processing to short, soluble forms limits fusion and can facilitate mitochondrial fission. Excessive OPA1 processing by the stress-activated protease OMA1 promotes mitochondrial fragmentation and, if persistent, triggers cell death and tissue degeneration in vivo The prevention of OMA1-mediated OPA1 processing and mitochondrial fragmentation might thus offer exciting therapeutic potential for human diseases associated with mitochondrial dysfunction.

Nonsense-mediated mRNA decay in humans at a glance

Abstract: Nonsense-mediated mRNA decay (NMD) is an mRNA quality-control mechanism that typifies all eukaryotes examined to date. NMD surveys newly synthesized mRNAs and degrades those that harbor a premature termination codon (PTC), thereby preventing the production of truncated proteins that could result in disease in humans. This is evident from dominantly inherited diseases that are due to PTC-containing mRNAs that escape NMD. Although many cellular NMD targets derive from mistakes made during, for example, pre-mRNA splicing and, possibly, transcription initiation, NMD also targets ∼10% of normal physiological mRNAs so as to promote an appropriate cellular response to changing environmental milieus, including those that induce apoptosis, maturation or differentiation. Over the past ∼35 years, a central goal in the NMD field has been to understand how cells discriminate mRNAs that are targeted by NMD from those that are not. In this Cell Science at a Glance and the accompanying poster, we review progress made towards this goal, focusing on human studies and the role of the key NMD factor up-frameshift protein 1 (UPF1).

Tenascin-C at a glance.

Abstract: Tenascin-C (TNC) is a hexameric, multimodular extracellular matrix protein with several molecular forms that are created through alternative splicing and protein modifications. It is highly conserved amongst vertebrates, and molecular phylogeny indicates that it evolved before fibronectin. Tenascin-C has many extracellular binding partners, including matrix components, soluble factors and pathogens; it also influences cell phenotype directly through interactions with cell surface receptors. Tenascin-C protein synthesis is tightly regulated, with widespread protein distribution in embryonic tissues, but restricted distribution of tenascin-C in adult tissues. Tenascin-C is also expressed de novo during wound healing or in pathological conditions, including chronic inflammation and cancer. First described as a modulator of cell adhesion, tenascin-C also directs a plethora of cell signaling and gene expression programs by shaping mechanical and biochemical cues within the cellular microenvironment. Exploitment of the pathological expression and function of tenascin-C is emerging as a promising strategy to develop new diagnostic, therapeutic and bioengineering tools. In this Cell Science at a Glance article and the accompanying poster we provide a succinct and comprehensive overview of the structural and functional features of tenascin-C and its potential roles in developing embryos and under pathological conditions.

The mechanical regulation of integrin-cadherin crosstalk organizes cells, signaling and forces.

Abstract: Cadherins and integrins are intrinsically linked through the actin cytoskeleton and share common signaling molecules. Although mechanosensing by the integrin-actin axis has long been appreciated, a growing body of literature now demonstrates that cadherins also transduce and respond to mechanical forces. Mounting evidence shows that mechanically driven crosstalk between integrins and cadherins regulates the spatial distribution of these receptors, their signaling intermediates, the actin cytoskeleton and intracellular forces. This interplay between integrins and cadherins can control fibronectin matrix assembly and signaling, and a fine balance between traction forces at focal adhesions and intercellular tension at adherens junctions is crucial for directional collective cell migration. In this Commentary, we discuss two central ideas: (1) how the dynamic interplay between integrins and cadherins regulates the spatial organization of intracellular signals and the extracellular matrix, and (2) the emerging consensus that intracellular force is a central mechanism that dictates cell behavior, guides tissue development and ultimately drives physiology.

Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission

Abstract: Cytosolic dynamin-related protein 1 (Drp1, also known as DNM1L) is required for both mitochondrial and peroxisomal fission. Drp1-dependent division of these organelles is facilitated by a number of adaptor proteins at mitochondrial and peroxisomal surfaces. To investigate the interplay of these adaptor proteins, we used gene-editing technology to create a suite of cell lines lacking the adaptors MiD49 (also known as MIEF2), MiD51 (also known as MIEF1), Mff and Fis1. Increased mitochondrial connectivity was observed following loss of individual adaptors, and this was further enhanced following the combined loss of MiD51 and Mff. Moreover, loss of adaptors also conferred increased resistance of cells to intrinsic apoptotic stimuli, with MiD49 and MiD51 showing the more prominent role. Using a proximity-based biotin labeling approach, we found close associations between MiD51, Mff and Drp1, but not Fis1. Furthermore, we found that MiD51 can suppress Mff-dependent enhancement of Drp1 GTPase activity. Our data indicates that Mff and MiD51 regulate Drp1 in specific ways to promote mitochondrial fission.

The integrin-collagen connection - a glue for tissue repair?

Abstract: The α1β1, α2β1, α10β1 and α11β1 integrins constitute a subset of the integrin family with affinity for GFOGER-like sequences in collagens. Integrins α1β1 and α2β1 were originally identified on a subset of activated T-cells, and have since been found to be expressed on a number of cell types including platelets (α2β1), vascular cells (α1β1, α2β1), epithelial cells (α1β1, α2β1) and fibroblasts (α1β1, α2β1). Integrin α10β1 shows a distribution that is restricted to mesenchymal stem cells and chondrocytes, whereas integrin α11β1 appears restricted to mesenchymal stem cells and subsets of fibroblasts. The bulk of the current literature suggests that collagen-binding integrins only have a limited role in adult connective tissue homeostasis, partly due to a limited availability of cell-binding sites in the mature fibrillar collagen matrices. However, some recent data suggest that, instead, they are more crucial for dynamic connective tissue remodeling events--such as wound healing--where they might act specifically to remodel and restore the tissue architecture. This Commentary discusses the recent development in the field of collagen-binding integrins, their roles in physiological and pathological settings with special emphasis on wound healing, fibrosis and tumor-stroma interactions, and include a discussion of the most recently identified newcomers to this subfamily--integrins α10β1 and α11β1.

Ratiometric analysis of Acridine Orange staining in the study of acidic organelles and autophagy.

Abstract: Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods.

Cellular functions of the ADF/cofilin family at a glance.

Abstract: The actin depolymerizing factor (ADF)/cofilin family comprises small actin-binding proteins with crucial roles in development, tissue homeostasis and disease. They are best known for their roles in regulating actin dynamics by promoting actin treadmilling and thereby driving membrane protrusion and cell motility. However, recent discoveries have increased our understanding of the functions of these proteins beyond their well-characterized roles. This Cell Science at a Glance article and the accompanying poster serve as an introduction to the diverse roles of the ADF/cofilin family in cells. The first part of the article summarizes their actions in actin treadmilling and the main mechanisms for their intracellular regulation; the second part aims to provide an outline of the emerging cellular roles attributed to the ADF/cofilin family, besides their actions in actin turnover. The latter part discusses an array of diverse processes, which include regulation of intracellular contractility, maintenance of nuclear integrity, transcriptional regulation, nuclear actin monomer transfer, apoptosis and lipid metabolism. Some of these could, of course, be indirect consequences of actin treadmilling functions, and this is discussed.

The integrin adhesome network at a glance

Abstract: The adhesion nexus is the site at which integrin receptors bridge intracellular cytoskeletal and extracellular matrix networks The connection between integrins and the cytoskeleton is mediated by a dynamic integrin adhesion complex (IAC), the components of which transduce chemical and mechanical signals to control a multitude of cellular functions In this Cell Science at a Glance article and the accompanying poster, we integrate the consensus adhesome, a set of 60 proteins that have been most commonly identified in isolated IAC proteomes, with the literature-curated adhesome, a theoretical network that has been assembled through scholarly analysis of proteins that localise to IACs The resulting IAC network, which comprises four broad signalling and actin-bridging axes, provides a platform for future studies of the regulation and function of the adhesion nexus in health and disease

Third harmonic generation microscopy of cells and tissue organization

Abstract: The interaction of cells within their microenvironmental niche is fundamental to cell migration, positioning, growth and differentiation in order to form and maintain complex tissue organization and function. Third harmonic generation (THG) microscopy is a label-free scatter process that is elicited by water-lipid and water-protein interfaces, including intra- and extracellular membranes, and extracellular matrix structures. In applied life sciences, THG delivers a versatile contrast modality to complement multi-parameter fluorescence, second harmonic generation and fluorescence lifetime microscopy, which allows detection of cellular and molecular cell functions in three-dimensional tissue culture and small animals. In this Commentary, we review the physical and technical basis of THG, and provide considerations for optimal excitation, detection and interpretation of THG signals. We further provide an overview on how THG has versatile applications in cell and tissue research, with a particular focus on analyzing tissue morphogenesis and homeostasis, immune cell function and cancer research, as well as the emerging applicability of THG in clinical practice.

Cancer metabolism at a glance.

Abstract: A defining hallmark of cancer is uncontrolled cell proliferation. This is initiated once cells have accumulated alterations in signaling pathways that control metabolism and proliferation, wherein the metabolic alterations provide the energetic and anabolic demands of enhanced cell proliferation. How these metabolic requirements are satisfied depends, in part, on the tumor microenvironment, which determines the availability of nutrients and oxygen. In this Cell Science at a Glance paper and the accompanying poster, we summarize our current understanding of cancer metabolism, emphasizing pathways of nutrient utilization and metabolism that either appear or have been proven essential for cancer cells. We also review how this knowledge has contributed to the development of anticancer therapies that target cancer metabolism.

The kindlin family: functions, signaling properties and implications for human disease

Abstract: The kindlin (or fermitin) family of proteins comprises three members (kindlin-1,-2 and -3) of evolutionarily conserved focal adhesion (FA) proteins, whose best-known task is to increase integrin affinity for a ligand (also referred as integrin activation) through binding of β-integrin tails. The consequence of kindlin-mediated integrin activation and integrin-ligand binding is cell adhesion, spreading and migration, assembly of the extracellular matrix (ECM), cell survival, proliferation and differentiation. Another hallmark of kindlins is their involvement in disease. Mutations in the KINDLIN-1 (also known as FERMT1) gene cause Kindler syndrome (KS)--in which mainly skin and intestine are affected, whereas mutations in the KINDLIN-3 (also known as FERMT3) gene cause leukocyte adhesion deficiency type III (LAD III), which is characterized by impaired extravasation of blood effector cells and severe, spontaneous bleedings. Also, aberrant expression of kindlins in various forms of cancer and in tissue fibrosis has been reported. Although the malfunctioning of integrins represent a major cause leading to kindlin-associated diseases, increasing evidence also point to integrin-independent functions of kindlins that play an important role in the pathogenesis of certain disease aspects. Furthermore, isoform-specific kindlin functions have been discovered, explaining, for example, why loss of kindlins differentially affects tissue stem cell homeostasis or tumor development. This Commentary focuses on new and isoform-specific kindlin functions in different tissues and discusses their potential role in disease development and progression.

The inner workings of stress fibers - from contractile machinery to focal adhesions and back.

Abstract: Ventral stress fibers and focal adhesions are physically coupled structures that play key roles in cellular mechanics and force sensing. The tight functional interdependence between the two is manifested not only by their apparent proximity but also by the fact that ventral stress fibers and focal adhesions are simultaneously diminished upon actomyosin relaxation, and grow when subjected to external stretching. However, whereas the apparent co-regulation of the two structures is well-documented, the underlying mechanisms remains poorly understood. In this Commentary, we discuss some of the fundamental, yet still open questions regarding ventral stress fiber structure, its force-dependent assembly, as well as its capacity to generate force. We also challenge the common approach - i.e. ventral stress fibers are variants of the well-studied striated or smooth muscle machinery - by presenting and critically discussing alternative venues. By highlighting some of the less-explored aspects of the interplay between stress fibers and focal adhesions, we hope that this Commentary will encourage further investigation in this field.

Uses and abuses of macropinocytosis.

Abstract: Macropinocytosis is a means by which eukaryotic cells ingest extracellular liquid and dissolved molecules. It is widely conserved amongst cells that can take on amoeboid form and, therefore, appears to be an ancient feature that can be traced back to an early stage of evolution. Recent advances have highlighted how this endocytic process can be subverted during pathology - certain cancer cells use macropinocytosis to feed on extracellular protein, and many viruses and bacteria use it to enter host cells. Prion and prion-like proteins can also spread and propagate from cell to cell through macropinocytosis. Progress is being made towards using macropinocytosis therapeutically, either to deliver drugs to or cause cell death by inducing catastrophically rapid fluid uptake. Mechanistically, the Ras signalling pathway plays a prominent and conserved activating role in amoebae and in mammals; mutant amoebae with abnormally high Ras activity resemble tumour cells in their increased capacity for growth using nutrients ingested through macropinocytosis. This Commentary takes a functional and evolutionary perspective to highlight progress in understanding and use of macropinocytosis, which is an ancient feeding process used by single-celled phagotrophs that has now been put to varied uses by metazoan cells and is abused in disease states, including infection and cancer.

Transcriptional regulation of mammalian autophagy at a glance.

Abstract: Macroautophagy, hereafter referred to as autophagy, is a catabolic process that results in the lysosomal degradation of cytoplasmic contents ranging from abnormal proteins to damaged cell organelles. It is activated under diverse conditions, including nutrient deprivation and hypoxia. During autophagy, members of the core autophagy-related (ATG) family of proteins mediate membrane rearrangements, which lead to the engulfment and degradation of cytoplasmic cargo. Recently, the nuclear regulation of autophagy, especially by transcription factors and histone modifiers, has gained increased attention. These factors are not only involved in rapid responses to autophagic stimuli, but also regulate the long-term outcome of autophagy. Now there are more than 20 transcription factors that have been shown to be linked to the autophagic process. However, their interplay and timing appear enigmatic as several have been individually shown to act as major regulators of autophagy. This Cell Science at a Glance article and the accompanying poster highlights the main cellular regulators of transcription involved in mammalian autophagy and their target genes.

Collective migration and cell jamming in asthma, cancer and development

Abstract: Collective cellular migration within the epithelial layer impacts upon development, wound healing and cancer invasion, but remains poorly understood. Prevailing conceptual frameworks tend to focus on the isolated role of each particular underlying factor - taken one at a time or at most a few at a time - and thus might not be tailored to describe a cellular collective that embodies a wide palette of physical and molecular interactions that are both strong and complex. To bridge this gap, we shift the spotlight to the emerging concept of cell jamming, which points to only a small set of parameters that govern when a cellular collective might jam and rigidify like a solid, or instead unjam and flow like a fluid. As gateways to cellular migration, the unjamming transition (UJT) and the epithelial-to-mesenchymal transition (EMT) share certain superficial similarities, but their congruence - or lack thereof - remains unclear. In this Commentary, we discuss aspects of cell jamming, its established role in human epithelial cell layers derived from the airways of non-asthmatic and asthmatic donors, and its speculative but emerging roles in development and cancer cell invasion.

Computational 'microscopy' of cellular membranes.

Abstract: Computational 'microscopy' refers to the use of computational resources to simulate the dynamics of a molecular system. Tuned to cell membranes, this computational 'microscopy' technique is able to capture the interplay between lipids and proteins at a spatio-temporal resolution that is unmatched by other methods. Recent advances allow us to zoom out from individual atoms and molecules to supramolecular complexes and subcellular compartments that contain tens of millions of particles, and to capture the complexity of the crowded environment of real cell membranes. This Commentary gives an overview of the main concepts of computational 'microscopy' and describes the state-of-the-art methods used to model cell membrane processes. We illustrate the power of computational modelling approaches by providing a few in-depth examples of large-scale simulations that move up from molecular descriptions into the subcellular arena. We end with an outlook towards modelling a complete cell in silico.

Ca2+ is a key factor in α-synuclein-induced neurotoxicity.

Abstract: Aggregation of α-synuclein leads to the formation of oligomeric intermediates that can interact with membranes to form pores. However, it is unknown how this leads to cell toxicity in Parkinson's disease. We investigated the species-specific effects of α-synuclein on Ca(2+) signalling in primary neurons and astrocytes using live neuronal imaging and electrophysiology on artificial membranes. We demonstrate that α-synuclein induces an increase in basal intracellular Ca(2+) in its unfolded monomeric state as well as in its oligomeric state. Electrophysiology of artificial membranes demonstrated that α-synuclein monomers induce irregular ionic currents, whereas α-synuclein oligomers induce rare discrete channel formation events. Despite the ability of monomeric α-synuclein to affect Ca(2+) signalling, it is only the oligomeric form of α-synuclein that induces cell death. Oligomer-induced cell death was abolished by the exclusion of extracellular Ca(2+), which prevented the α-synuclein-induced Ca(2+) dysregulation. The findings of this study confirm that α-synuclein interacts with membranes to affect Ca(2+) signalling in a structure-specific manner and the oligomeric β-sheet-rich α-synuclein species ultimately leads to Ca(2+) dysregulation and Ca(2+)-dependent cell death.

Atg9A trafficking through the recycling endosomes is required for autophagosome formation

Abstract: Autophagy is an intracellular degradation pathway conserved in eukaryotes. Among core autophagy-related (Atg) proteins, mammalian Atg9A is the sole multi-spanning transmembrane protein, and both of its N- and C-terminal domains are exposed to the cytoplasm. It is known that Atg9A travels through the trans-Golgi network (TGN) and the endosomal system under nutrient-rich conditions, and transiently localizes to the autophagosome upon autophagy induction. However, the significance of Atg9A trafficking for autophagosome formation remains elusive. Here, we identified sorting motifs in the N-terminal cytosolic stretch of Atg9A that interact with the adaptor protein AP-2. Atg9A with mutations in the sorting motifs could not execute autophagy and was abnormally accumulated at the recycling endosomes. The combination of defects in autophagy and Atg9A accumulation in the recycling endosomes was also found upon the knockdown of TRAPPC8, a specific subunit of the TRAPPIII complex. These results show directly that the trafficking of Atg9A through the recycling endosomes is an essential step for autophagosome formation.

HEAT repeats – versatile arrays of amphiphilic helices working in crowded environments?

Abstract: Cellular proteins do not work in isolation. Instead, they often function as part of large macromolecular complexes, which are transported and concentrated into specific cellular compartments and function in a highly crowded environment. A central theme of modern cell biology is to understand how such macromolecular complexes are assembled efficiently and find their destinations faithfully. In this Opinion article, we will focus on HEAT repeats, flexible arrays of amphiphilic helices found in many eukaryotic proteins, such as karyopherins and condensins, and discuss how these uniquely designed helical repeats might underlie dynamic protein-protein interactions and support cellular functions in crowded environments. We will make bold speculations on functional similarities between the action of HEAT repeats and intrinsically disordered regions (IDRs) in macromolecular phase separation. Potential contributions of HEAT-HEAT interactions, as well as cooperation between HEATs and IDRs, to mesoscale organelle assembly will be discussed.

ZO-1 interactions with F-actin and occludin direct epithelial polarization and single lumen specification in 3D culture.

Abstract: Epithelia within tubular organs form and expand lumens. Failure of these processes can result in serious developmental anomalies. Although tight junction assembly is crucial to epithelial polarization, the contribution of specific tight junction proteins to lumenogenesis is undefined. Here, we show that ZO-1 (also known as TJP1) is necessary for the formation of single lumens. Epithelia lacking this tight junction scaffolding protein form cysts with multiple lumens and are defective in the earliest phases of polarization, both in two and three dimensions. Expression of ZO-1 domain-deletion mutants demonstrated that the actin-binding region and U5-GuK domain are crucial to single lumen development. For actin-binding region, but not U5-GuK domain, mutants, this could be overcome by strong polarization cues from the extracellular matrix. Analysis of the U5-GuK binding partners shroom2, α-catenin and occludin showed that only occludin deletion led to multi-lumen cysts. Like ZO-1-deficiency, occludin deletion led to mitotic spindle orientation defects. Single lumen formation required the occludin OCEL domain, which binds to ZO-1. We conclude that ZO-1-occludin interactions regulate multiple phases of epithelial polarization by providing cell-intrinsic signals that are required for single lumen formation.

DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors.

Abstract: The DNA damage response (DDR) plays a central role in preserving genome integrity. Recently, we reported that the endoribonucleases DICER and DROSHA contribute to DDR activation by generating small non-coding RNAs, termed DNA damage response RNA (DDRNA), carrying the sequence of the damaged locus. It is presently unclear whether DDRNAs act by promoting the primary recognition of DNA lesions or the secondary recruitment of DDR factors into cytologically detectable foci and consequent signal amplification. Here, we demonstrate that DICER and DROSHA are dispensable for primary recruitment of the DDR sensor NBS1 to DNA damage sites. Instead, the accumulation of the DDR mediators MDC1 and 53BP1 (also known as TP53BP1), markers of secondary recruitment, is reduced in DICER- or DROSHA-inactivated cells. In addition, NBS1 (also known as NBN) primary recruitment is resistant to RNA degradation, consistent with the notion that RNA is dispensable for primary recognition of DNA lesions. We propose that DICER, DROSHA and DDRNAs act in the response to DNA damage after primary recognition of DNA lesions and, together with γH2AX, are essential for enabling the secondary recruitment of DDR factors and fuel the amplification of DDR signaling.

Role of cytonemes in Wnt transport.

Abstract: Wnt signaling regulates a broad variety of processes during embryonic development and disease. A hallmark of the Wnt signaling pathway is the formation of concentration gradients by Wnt proteins across responsive tissues, which determines cell fate in invertebrates and vertebrates. To fulfill its paracrine function, trafficking of the Wnt morphogen from an origin cell to a recipient cell must be tightly regulated. A variety of models have been proposed to explain the extracellular transport of these lipid-modified signaling proteins in the aqueous extracellular space; however, there is still considerable debate with regard to which mechanisms allow the precise distribution of ligand in order to generate a morphogenetic gradient within growing tissue. Recent evidence suggests that Wnt proteins are distributed along signaling filopodia during vertebrate and invertebrate embryogenesis. Cytoneme-mediated transport has profound impact on our understanding of how Wnt signaling propagates through tissues and allows the formation of a precise ligand distribution in the recipient tissue during embryonic growth. In this Commentary, we review extracellular trafficking mechanisms for Wnt proteins and discuss the growing evidence of cytoneme-based Wnt distribution in development and stem cell biology. We will also discuss their implication for Wnt signaling in the formation of the Wnt morphogenetic gradient during tissue patterning.

Multifunctional Abl kinases in health and disease.

Abstract: The Abelson tyrosine kinases were initially identified as drivers of leukemia in mice and humans. The Abl family kinases Abl1 and Abl2 regulate diverse cellular processes during development and normal homeostasis, and their functions are subverted during inflammation, cancer and other pathologies. Abl kinases can be activated by multiple stimuli leading to cytoskeletal reorganization required for cell morphogenesis, motility, adhesion and polarity. Depending on the cellular context, Abl kinases regulate cell survival and proliferation. Emerging data support important roles for Abl kinases in pathologies linked to inflammation. Among these are neurodegenerative diseases and inflammatory pathologies. Unexpectedly, Abl kinases have also been identified as important players in mammalian host cells during microbial pathogenesis. Thus, the use of Abl kinase inhibitors might prove to be effective in the treatment of pathologies beyond leukemia and solid tumors. In this Cell Science at a Glance article and in the accompanying poster, we highlight the emerging roles of Abl kinases in the regulation of cellular processes in normal cells and diverse pathologies ranging from cancer to microbial pathogenesis.