Actin-rich lamellipodia-like protrusions contribute to the integrity of epithelial cell-cell junctions.

Metastasis-suppressor 1 (MTSS1) is a membrane-interacting scaffolding protein that regulates the integrity of epithelial cell-cell junctions and functions as a tumor suppressor in a wide range of carcinomas. MTSS1 binds phosphoinositide-rich membranes through its I-BAR domain and is capable of sensing and generating negative membrane curvature in vitro. However, the mechanisms by which MTSS1 localizes to intercellular junctions in epithelial cells and contributes to their integrity and maintenance have remained elusive. By carrying out EM and live-cell imaging on cultured Madin-Darby canine kidney cell monolayers, we provide evidence that adherens junctions of epithelial cells harbor lamellipodia-like, dynamic actin-driven membrane folds, which exhibit high negative membrane curvature at their distal edges. BioID proteomics and imaging experiments demonstrated that MTSS1 associates with an Arp2/3 complex activator, the WAVE-2 complex, in dynamic actin-rich protrusions at cell-cell junctions. Inhibition of Arp2/3 or WAVE-2 suppressed actin filament assembly at adherens junctions, decreased the dynamics of junctional membrane protrusions, and led to defects in epithelial integrity. Together, these results support a model in which membrane-associated MTSS1, together with the WAVE-2 and Arp2/3 complexes, promotes the formation of dynamic lamellipodia-like actin protrusions that contribute to the integrity of cell-cell junctions in epithelial monolayers.

GOLM1 depletion modifies cellular sphingolipid metabolism and adversely affects cell growth.

Golgi membrane protein 1 (GOLM1) is a Golgi-resident type 2 transmembrane protein known to be overexpressed in several cancers, including hepatocellular carcinoma (HCC), as well as in viral infections. However, the role of GOLM1 in lipid metabolism remains enigmatic. In this study, we employed siRNA-mediated GOLM1 depletion in Huh-7 HCC cells to study the role of GOLM1 in lipid metabolism. Mass spectrometric lipidomic analysis in GOLM1 knockdown cells showed an aberrant accumulation of sphingolipids, such as ceramides, hexosylceramides, dihexosylceramides, sphinganine, sphingosine, and ceramide phosphate, along with cholesteryl esters. Furthermore, we observed a reduction in phosphatidylethanolamines and lysophosphatidylethanolamines. In addition, Seahorse extracellular flux analysis indicated a reduction in mitochondrial oxygen consumption rate upon GOLM1 depletion. Finally, alterations in Golgi structure and distribution were observed both by electron microscopy imaging and immunofluorescence microscopy analysis. Importantly, we found that GOLM1 depletion also affected cell proliferation and cell cycle progression in Huh-7 HCC cells. The Golgi structural defects induced by GOLM1 reduction might potentially affect the trafficking of proteins and lipids leading to distorted intracellular lipid homeostasis, which may result in organelle dysfunction and altered cell growth. In conclusion, we demonstrate that GOLM1 depletion affects sphingolipid metabolism, mitochondrial function, Golgi structure, and proliferation of HCC cells.

Microscopic characterization reveals the diversity of EVs secreted by GFP-HAS3 expressing MCF7 cells.

We have shown the connection of hyaluronan synthesis activity with the enhanced shedding of extracellular vesicles, but detailed morphological analysis of those hyaluronan-induced EVs is still missing. In this study we utilized a comprehensive set of high-resolution imaging techniques to characterize in high detail the size and morphology of EVs originating from stable MCF7 breast cancer cell line and transiently transfected cells expressing GFP-HAS3. To avoid possible artefacts or loss of EVs resulting from the isolation process, special attention was paid to analysis of EVs in situ in monolayer and in 3D cultures. The results of this study show that GFP-HAS3 expressing MCF7 cells produce morphologically diverse EVs but also demonstrates the variation in results obtained with different experimental setup, which emphasizes the importance of comparison between different methods when interpreting the observations.

Functional, metabolic and transcriptional maturation of human pancreatic islets derived from stem cells.

Transplantation of pancreatic islet cells derived from human pluripotent stem cells is a promising treatment for diabetes. Despite progress in the generation of stem-cell-derived islets (SC-islets), no detailed characterization of their functional properties has been conducted. Here, we generated functionally mature SC-islets using an optimized protocol and benchmarked them comprehensively against primary adult islets. Biphasic glucose-stimulated insulin secretion developed during in vitro maturation, associated with cytoarchitectural reorganization and the increasing presence of alpha cells. Electrophysiology, signaling and exocytosis of SC-islets were similar to those of adult islets. Glucose-responsive insulin secretion was achieved despite differences in glycolytic and mitochondrial glucose metabolism. Single-cell transcriptomics of SC-islets in vitro and throughout 6 months of engraftment in mice revealed a continuous maturation trajectory culminating in a transcriptional landscape closely resembling that of primary islets. Our thorough evaluation of SC-islet maturation highlights their advanced degree of functionality and supports their use in further efforts to understand and combat diabetes.

Myosin-X and talin modulate integrin activity at filopodia tips.

Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin regulation in filopodia are poorly defined. Here, we report that active integrins accumulate at the tip of myosin-X (MYO10)-positive filopodia, while inactive integrins are uniformly distributed. We identify talin and MYO10 as the principal integrin activators in filopodia. In addition, deletion of MYO10's FERM domain, or mutation of its ß1-integrin-binding residues, reveals MYO10 as facilitating integrin activation, but not transport, in filopodia. However, MYO10's isolated FERM domain alone cannot activate integrins, potentially because of binding to both integrin tails. Finally, because a chimera construct generated by swapping MYO10-FERM by talin-FERM enables integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.

A Na,K-ATPase-Fodrin-Actin Membrane Cytoskeleton Complex is Required for Endothelial Fenestra Biogenesis.

Fenestrae are transcellular plasma membrane pores that mediate blood-tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin-fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the formation of a submembrane cytoskeletal signalling complex.

Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage.

Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy.

BACKGROUND: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality. OBJECTIVE: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome. METHODS: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed. RESULTS: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages. CONCLUSION: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.

Mitochondrial stress response triggered by defects in protein synthesis quality control.

Mitochondria have a compartmentalized gene expression system dedicated to the synthesis of membrane proteins essential for oxidative phosphorylation. Responsive quality control mechanisms are needed to ensure that aberrant protein synthesis does not disrupt mitochondrial function. Pathogenic mutations that impede the function of the mitochondrial matrix quality control protease complex composed of AFG3L2 and paraplegin cause a multifaceted clinical syndrome. At the cell and molecular level, defects to this quality control complex are defined by impairment to mitochondrial form and function. Here, we establish the etiology of these phenotypes. We show how disruptions to the quality control of mitochondrial protein synthesis trigger a sequential stress response characterized first by OMA1 activation followed by loss of mitochondrial ribosomes and by remodelling of mitochondrial inner membrane ultrastructure. Inhibiting mitochondrial protein synthesis with chloramphenicol completely blocks this stress response. Together, our data establish a mechanism linking major cell biological phenotypes of AFG3L2 pathogenesis and show how modulation of mitochondrial protein synthesis can exert a beneficial effect on organelle homeostasis.

FAM92A1 is a BAR domain protein required for mitochondrial ultrastructure and function.

Mitochondrial function is closely linked to its dynamic membrane ultrastructure. The mitochondrial inner membrane (MIM) can form extensive membrane invaginations known as cristae, which contain the respiratory chain and ATP synthase for oxidative phosphorylation. The molecular mechanisms regulating mitochondrial ultrastructure remain poorly understood. The Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of diverse cellular processes related to membrane remodeling and dynamics. Whether BAR domain proteins are involved in sculpting membranes in specific submitochondrial compartments is largely unknown. In this study, we report FAM92A1 as a novel BAR domain protein localizes to the matrix side of the MIM. Loss of FAM92A1 caused a severe disruption to mitochondrial morphology and ultrastructure, impairing organelle bioenergetics. Furthermore, FAM92A1 displayed a membrane-remodeling activity in vitro, inducing a high degree of membrane curvature. Collectively, our findings uncover a role for a BAR domain protein as a critical organizer of the mitochondrial ultrastructure that is indispensable for mitochondrial function.

Secretion of Tau via an Unconventional Non-vesicular Mechanism.

Tauopathies are characterized by cerebral accumulation of Tau protein aggregates that appear to spread throughout the brain via a cell-to-cell transmission process that includes secretion and uptake of pathological Tau, followed by templated misfolding of normal Tau in recipient cells. Here, we show that phosphorylated, oligomeric Tau clusters at the plasma membrane in N2A cells and is secreted in vesicle-free form in an unconventional process sensitive to changes in membrane properties, particularly cholesterol and sphingomyelin content. Cell surface heparan sulfate proteoglycans support Tau secretion, possibly by facilitating its release after membrane penetration. Notably, secretion of endogenous Tau from primary cortical neurons is mediated, at least partially, by a similar mechanism. We suggest that Tau is released from cells by an unconventional secretory mechanism that involves its phosphorylation and oligomerization and that membrane interaction may help Tau to acquire properties that allow its escape from cells directly through the plasma membrane.

OSBP-related protein-2 (ORP2): a novel Akt effector that controls cellular energy metabolism.

ORP2 is a ubiquitously expressed OSBP-related protein previously implicated in endoplasmic reticulum (ER)-lipid droplet (LD) contacts, triacylglycerol (TG) metabolism, cholesterol transport, adrenocortical steroidogenesis, and actin-dependent cell dynamics. Here, we characterize the role of ORP2 in carbohydrate and lipid metabolism by employing ORP2-knockout (KO) hepatoma cells (HuH7) generated by CRISPR-Cas9 gene editing. The ORP2-KO and control HuH7 cells were subjected to RNA sequencing, analyses of Akt signaling, carbohydrate and TG metabolism, the extracellular acidification rate, and the lipidome, as well as to transmission electron microscopy. The loss of ORP2 resulted in a marked reduction of active phosphorylated Akt(Ser473) and its target Glycogen synthase kinase 3ß(Ser9), consistent with defective Akt signaling. ORP2 was found to form a physical complex with the key controllers of Akt activity, Cdc37, and Hsp90, and to co-localize with Cdc37 and active Akt(Ser473) at lamellipodial plasma membrane regions, in addition to the previously reported ER-LD localization. ORP2-KO reduced glucose uptake, glycogen synthesis, glycolysis, mRNA-encoding glycolytic enzymes, and SREBP-1 target gene expression, and led to defective TG synthesis and storage. ORP2-KO did not reduce but rather increased ER-LD contacts under basal culture conditions and interfered with their expansion upon fatty acid loading. Together with our recently published work (Kentala et al. in FASEB J 32:1281-1295, 2018), this study identifies ORP2 as a new regulatory nexus of Akt signaling, cellular energy metabolism, actin cytoskeletal function, cell migration, and proliferation.

Extracellular Lipids Accumulate in Human Carotid Arteries as Distinct Three-Dimensional Structures and Have Proinflammatory Properties.

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

NOGO-A/RTN4A and NOGO-B/RTN4B are simultaneously expressed in epithelial, fibroblast and neuronal cells and maintain ER morphology.

Reticulons (RTNs) are a large family of membrane associated proteins with various functions. NOGO-A/RTN4A has a well-known function in limiting neurite outgrowth and restricting the plasticity of the mammalian central nervous system. On the other hand, Reticulon 4 proteins were shown to be involved in forming and maintaining endoplasmic reticulum (ER) tubules. Using comparative transcriptome analysis and qPCR, we show here that NOGO-B/RTN4B and NOGO-A/RTN4A are simultaneously expressed in cultured epithelial, fibroblast and neuronal cells. Electron tomography combined with immunolabelling reveal that both isoforms localize preferably to curved membranes on ER tubules and sheet edges. Morphological analysis of cells with manipulated levels of NOGO-B/RTN4B revealed that it is required for maintenance of normal ER shape; over-expression changes the sheet/tubule balance strongly towards tubules and causes the deformation of the cell shape while depletion of the protein induces formation of large peripheral ER sheets.

Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor.

Endocytosis controls many functions including nutrient uptake, cell division, migration and signal transduction. A clathrin- and caveolin-independent endocytosis pathway is used by important physiological cargos, including interleukin-2 receptors (IL-2R). However, this process lacks morphological and dynamic data. Our electron microscopy (EM) and tomography studies reveal that IL-2R-pits and vesicles are initiated at the base of protrusions. We identify the WAVE complex as a specific endocytic actor. The WAVE complex interacts with IL-2R, via a WAVE-interacting receptor sequence (WIRS) present in the receptor polypeptide, and allows for receptor clustering close to membrane protrusions. In addition, using total internal reflection fluorescent microscopy (TIRF) and automated analysis we demonstrate that two timely distinct bursts of actin polymerization are required during IL-2R uptake, promoted first by the WAVE complex and then by N-WASP. Finally, our data reveal that dynamin acts as a transition controller for the recruitment of Arp2/3 activators required for IL-2R endocytosis. Altogether, our work identifies the spatio-temporal specific role of factors initiating clathrin-independent endocytosis by a unique mechanism that does not depend on the deformation of a flat membrane, but rather on that of membrane protrusions.

Indications of lymphatic endothelial differentiation and endothelial progenitor cell activation in the pathology of proliferative diabetic retinopathy.

PURPOSE: Proliferative diabetic retinopathy (PDR) is characterized by ischaemia- and inflammation-induced neovascularization, but the pathological vascular differentiation in PDR remains poorly characterized. Here, endothelial progenitor and growth properties, as well as potential lymphatic differentiation, were investigated in the neovascular membrane specimens from vitrectomized patients with PDR. METHODS: The expression of pan-endothelial CD31 (PECAM-1), ETS-related gene (ERG), α-smooth muscle actin (α-SMA), and stem/progenitor cell marker CD117 (c-kit) and cell proliferation marker Ki67 was investigated along with the markers of lymphatic endothelial differentiation (vascular endothelial growth factor receptor (VEGFR)-3; prospero-related homeobox gene-1 (Prox-1), lymphatic vessel endothelial receptor [LYVE)-1 and podoplanin (PDPN)] by immunohistochemistry. Lymphocyte antigen CD45 and pan-macrophage marker CD68 were likewise investigated. RESULTS: All specimens displayed CD31, ERG and α-SMA immunoreactivity in irregular blood vessels. Unexpectedly, VEGFR3 and Prox-1 lymphatic marker positive vessels were also detected in several tissues. Prox-1 was co-expressed with CD117 in lumen-lining endothelial cells and adjacent cells, representing putative endothelial stem/progenitor cells and pro-angiogenic perivascular cells. Immunoreactivity of CD45 and CD68 was detectable in all investigated diabetic neovessel specimens. PDPN immunoreactivity was also detected in irregular lumen-forming structures, but these cells lacked CD31 and ERG that mark blood and lymphatic endothelium. CONCLUSIONS: Although the inner part of human eye is physiologically devoid of lymphatic vessels, lymphatic differentiation associated with endothelial stem/progenitor cell activation may be involved in the pathogenesis of human PDR. Further studies are warranted to elucidate whether targeting lymphatic factors could be beneficial in the treatment of patients with the sight-threatening forms of DR.

ER sheet persistence is coupled to myosin 1c-regulated dynamic actin filament arrays.

The endoplasmic reticulum (ER) comprises a dynamic three-dimensional (3D) network with diverse structural and functional domains. Proper ER operation requires an intricate balance within and between dynamics, morphology, and functions, but how these processes are coupled in cells has been unclear. Using live-cell imaging and 3D electron microscopy, we identify a specific subset of actin filaments localizing to polygons defined by ER sheets and tubules and describe a role for these actin arrays in ER sheet persistence and, thereby, in maintenance of the characteristic network architecture by showing that actin depolymerization leads to increased sheet fluctuation and transformations and results in small and less abundant sheet remnants and a defective ER network distribution. Furthermore, we identify myosin 1c localizing to the ER-associated actin filament arrays and reveal a novel role for myosin 1c in regulating these actin structures, as myosin 1c manipulations lead to loss of the actin filaments and to similar ER phenotype as observed after actin depolymerization. We propose that ER-associated actin filaments have a role in ER sheet persistence regulation and thus support the maintenance of sheets as a stationary subdomain of the dynamic ER network.

Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells.

The endoplasmic reticulum (ER) is both structurally and functionally complex, consisting of a dynamic network of interconnected sheets and tubules. To achieve a more comprehensive view of ER organization in interphase and mitotic cells and to address a discrepancy in the field (i.e., whether ER sheets persist, or are transformed to tubules, during mitosis), we analyzed the ER in four different mammalian cell lines using live-cell imaging, high-resolution electron microscopy, and three dimensional electron microscopy. In interphase cells, we found great variation in network organization and sheet structures among different cell lines. In mitotic cells, we show that the ER undergoes both spatial reorganization and structural transformation of sheets toward more fenestrated and tubular forms. However, the extent of spatial reorganization and sheet-to-tubule transformation varies among cell lines. Fenestration and tubulation of the ER correlates with a reduced number of membrane-bound ribosomes.

Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism.

The actin cytoskeleton plays a fundamental role in various motile and morphogenetic processes involving membrane dynamics. We show that actin-binding proteins MIM (missing-in-metastasis) and IRSp53 directly bind PI(4,5)P(2)-rich membranes and deform them into tubular structures. This activity resides in the N-terminal IRSp53/MIM domain (IMD) of these proteins, which is structurally related to membrane-tubulating BAR (Bin/amphiphysin/Rvs) domains. We found that because of a difference in the geometry of the PI(4,5)P(2)-binding site, IMDs induce a membrane curvature opposite that of BAR domains and deform membranes by binding to the interior of the tubule. This explains why IMD proteins induce plasma membrane protrusions rather than invaginations. We also provide evidence that the membrane-deforming activity of IMDs, instead of the previously proposed F-actin-bundling or GTPase-binding activities, is critical for the induction of the filopodia/microspikes in cultured mammalian cells. Together, these data reveal that interplay between actin dynamics and a novel membrane-deformation activity promotes cell motility and morphogenesis.

Phosphorylation of the potyvirus capsid protein by protein kinase CK2 and its relevance for virus infection.

We reported previously that the capsid protein (CP) of Potato virus A (PVA) is phosphorylated both in virus-infected plants and in vitro. In this study, an enzyme that phosphorylates PVA CP was identified as the protein kinase CK2. The alpha-catalytic subunit of CK2 (CK2alpha) was purified from tobacco and characterized using in-gel kinase assays and liquid chromatography-tandem mass spectrometry. The tobacco CK2alpha gene was cloned and expressed in bacterial cells. Specific antibodies were raised against the recombinant enzyme and used to demonstrate the colocalization of PVA CP and CK2alpha in infected tobacco protoplasts. A major site of CK2 phosphorylation in PVA CP was identified by a combination of mass spectrometric analysis, radioactive phosphopeptide sequencing, and mutagenesis as Thr-242 within a CK2 consensus sequence. Amino acid substitutions that affect the CK2 consensus sequence in CP were introduced into a full-length infectious cDNA clone of PVA tagged with green fluorescent protein. Analysis of the mutant viruses showed that they were defective in cell-to-cell and long-distance movement. Using in vitro assays, we demonstrated that CK2 phosphorylation inhibited the binding of PVA CP to RNA, suggesting a molecular mechanism of CK2 action. These results suggest that the phosphorylation of PVA CP by CK2 plays an important regulatory role in virus infection.