Dual‐specificity tyrosine phosphorylation‐regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner

Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign‐body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion‐founder cells that fuse to fusion‐follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.     

SLP‐2 is required for stress‐induced mitochondrial hyperfusion

Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin‐related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress‐induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L‐OPA1, MFN1, and the mitochondrial inner membrane protein SLP‐2. In the absence of SLP‐2, L‐OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro‐survival response against stress.     

To mdivi‐1 or not to mdivi‐1: Is that the question?

The fission/division and fusion of mitochondria are fundamental aspects of mitochondrial biology. The balance of fission and fusion sets the length of mitochondria in cells to serve their physiological requirements. The fission of mitochondria is markedly induced in many disease states and in response to cellular injury, resulting in the fragmentation of mitochondria into dysfunctional units. The mechanism that drives fission is dependent on the dynamin related protein 1 (Drp1) GTPase. mdivi‐1 is a quinazolinone originally described as a selective inhibitor of Drp1, over other dynamin family members, and reported to inhibit mitochondrial fission. A recent study has challenged the activity of mdivi‐1 as an inhibitor of Drp1. This study raises serious issues regarding the interpretation of data addressing the effects of mdivi‐1 as reflective of the inhibition of Drp1 and thus fission. This commentary considers the evidence for and against mdivi‐1 as an inhibitor of Drp1 and presents the following considerations; (1) the activity of mdivi‐1 toward Drp1 GTPase activity requires further biochemical investigation, (2) as there is a large body of literature using mdivi‐1 in vitro with effects as predicted for inhibition of Drp1 and mitochondrial fission, reviewed herein, the evidence is in favor of mdivi‐1's originally described bioactivity, and (3) until the issue is resolved, experimental interpretations for the effects of mdivi‐1 on inhibition of fission in cell and tissue experiments warrants stringent positive controls directly addressing the effects of mdivi‐1 on fission. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1260–1268, 2017     

Mitochondrial ‘kiss‐and‐run’: interplay between mitochondrial motility and fusion–fission dynamics

Visualizing mitochondrial fusion in real time, we identified two classes of fusion events in mammalian cells. In addition to complete fusion, we observed transient fusion events, wherein two mitochondria came into close apposition, exchanged soluble inter‐membrane space and matrix proteins, and re‐separated, preserving the original morphology. Transient fusion exhibited rapid kinetics of the sequential and separable mergers of the outer and inner membranes, but allowed only partial exchange of integral membrane proteins. When the inner membrane fusion protein Opa1 level was lowered or was greatly elevated, transient fusions could occur, whereas complete fusions disappeared. Furthermore, transient fusions began from oblique or lateral interactions of mitochondria associated with separate microtubules, whereas complete fusions resulted from longitudinal merging of organelles travelling along a single microtubule. In contrast to complete fusion, transient fusions both required and promoted mitochondrial motility. Transient fusions were also necessary and sufficient to support mitochondrial metabolism. Thus, Opa1 expression and cytoskeletal anchorage govern a novel form of fusion that has a distinct function in mitochondrial maintenance.     

Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion‐to‐fusion transition

SNAREs fuse membranes in several steps. Trans‐SNARE complexes juxtapose membranes, induce hemifused stalk structures, and open the fusion pore. A recent penetration model of fusion proposed that SNAREs force the hydrophilic C‐termini of their transmembrane domains through the hydrophobic core of the membrane(s). In contrast, the indentation model suggests that the C‐termini open the pore by locally compressing and deforming the stalk. Here we test these models in the context of yeast vacuole fusion. Addition of small hydrophilic tags renders bilayer penetration by the C‐termini energetically unlikely. It preserves fusion activity, however, arguing against the penetration model. Addition of large protein tags to the C‐termini permits SNARE activation, trans‐SNARE pairing, and hemifusion but abolishes pore opening. Fusion proceeds if the tags are detached from the membrane by a hydrophilic spacer or if only one side of the trans‐SNARE complex carries a protein tag. Thus, both sides of a trans‐SNARE complex can drive pore opening. Our results are consistent with an indentation model in which multiple SNARE C‐termini cooperate in opening the fusion pore by locally deforming the inner leaflets.     

Syntaxin‐1 N‐peptide and Habc‐domain perform distinct essential functions in synaptic vesicle fusion

Among SNARE proteins mediating synaptic vesicle fusion, syntaxin‐1 uniquely includes an N‐terminal peptide (‘N‐peptide’) that binds to Munc18‐1, and a large, conserved H_abc‐domain that also binds to Munc18‐1. Previous in vitro studies suggested that the syntaxin‐1 N‐peptide is functionally important, whereas the syntaxin‐1 H_abc‐domain is not, but limited information is available about the in vivo functions of these syntaxin‐1 domains. Using rescue experiments in cultured syntaxin‐deficient neurons, we now show that the N‐peptide and the H_abc‐domain of syntaxin‐1 perform distinct and independent roles in synaptic vesicle fusion. Specifically, we found that the N‐peptide is essential for vesicle fusion as such, whereas the H_abc‐domain regulates this fusion, in part by forming the closed syntaxin‐1 conformation. Moreover, we observed that deletion of the H_abc‐domain but not deletion of the N‐peptide caused a loss of Munc18‐1 which results in a decrease in the readily releasable pool of vesicles at a synapse, suggesting that Munc18 binding to the H_abc‐domain stabilizes Munc18‐1. Thus, the N‐terminal syntaxin‐1 domains mediate different functions in synaptic vesicle fusion, probably via formation of distinct Munc18/SNARE‐protein complexes.     

Remodeling of peripheral nerve ensheathment during the larval‐to‐adult transition in Drosophila

Over the course of a 4‐day period of metamorphosis, the Drosophila larval nervous system is remodeled to prepare for adult‐specific behaviors. One example is the reorganization of peripheral nerves in the abdomen, where five pairs of abdominal nerves (A4–A8) fuse to form the terminal nerve trunk. This reorganization is associated with selective remodeling of four layers that ensheath each peripheral nerve. The neural lamella (NL), is the first to dismantle; its breakdown is initiated by 6 hours after puparium formation, and is completely removed by the end of the first day. This layer begins to re‐appear on the third day of metamorphosis. Perineurial glial (PG) cells situated just underneath the NL, undergo significant proliferation on the first day of metamorphosis, and at that stage contribute to 95% of the glial cell population. Cells of the two inner layers, Sub‐Perineurial Glia (SPG) and Wrapping Glia (WG) increase in number on the second half of metamorphosis. Induction of cell death in perineurial glia via the cell death gene reaper and the Diptheria toxin (DT‐1) gene, results in abnormal bundling of the peripheral nerves, suggesting that perineurial glial cells play a role in the process. A significant number of animals fail to eclose in both reaper and DT‐1 targeted animals, suggesting that disruption of PG also impacts eclosion behavior. The studies will help to establish the groundwork for further work on cellular and molecular processes that underlie the co‐ordinated remodeling of glia and the peripheral nerves they ensheath. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1144–1160, 2017     

Probing the disulfide folding pathway of insulin‐like growth factor‐I

The crucial step of folding of recombinant proteins presents serious challenges to obtaining the native structure. This problem is exemplified by insulin‐like growth factor (IGF)‐I which when refolded in vitro produces the native three‐disulfide structure, an alternative structure with mispaired disulfide bonds and other isomeric forms. To investigate this phenomenon we have examined the refolding properties of an analog of IGF‐I which contains a 13‐amino acid N‐terminal extension and a charge mutation at position 3 (Long‐ [Arg³]IGF‐I). Unlike IGF‐I, which yields 45% of the native structure and 24% of the alternative structure when refolded in vitro, Long‐[Arg³]GF‐I yields 85% and 10% of these respective forms. To investigate the interactions that affect the refolding of Long‐[Arg³]IGF‐I and IGF‐I, we acid‐trapped folding intermediates and products for inclusion in a kinetic analysis of refolding. In addition to non‐native intermediates, three native‐like intermediates were identified, that appear to have a major role in the in vitro refolding pathway of Long‐[Arg³]IGF‐I; a single‐disulfide Cys¹⁸–Cys⁶¹ intermediate, an intermediate with Cys¹⁸–Cys⁶¹ and Cys⁶–Cys⁴⁸ disulfide bonds and another with Cys¹⁸–Cys⁶¹ and Cys⁴⁷–Cys⁵² disulfide bonds. Furthermore, from our kinetic analysis we propose that the Cys¹⁸‐Cys⁶¹, Cys⁶‐Cys⁴⁸ intermediate forms the native structure, not by the direct formation of the last (Cys⁴⁷‐Cys⁵²) disulfide bond, but by rearrangement via the Cys¹⁸–Cys⁶¹ intermediate and a productive Cys¹⁸–Cys⁶¹, Cys⁴⁷–Cys⁵² intermediate. In this pathway, the last disulfide bond to form involves Cys⁶ and Cys⁴⁸. Finally, we apply this pathway to IGF‐I and conclude that the divergence in the in vitro folding pathway of IGF‐I is caused by non‐native interactions involving Glu³ that stabilize the alternative structure. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 693–703, 1999.     

Rotaviral nonstructural protein 4 triggers dynamin‐related protein 1‐dependent mitochondrial fragmentation during infection

Dynamic equilibrium between mitochondrial fission and mitochondrial fusion serves as an important quality control system within cells ensuring cellular vitality and homeostasis. Viruses often target mitochondrial dynamics as a part of their obligatory cellular reprogramming. The present study was undertaken to assess the status and regulation of mitochondrial dynamics during rotavirus infection. Distinct fragmentation of mitochondrial syncytia was observed during late hours of RV (SA11, Wa, A5‐13) infection. RV nonstructural protein 4 (NSP4) was identified as the viral trigger for disrupted mitochondrial morphology. Severance of mitochondrial interconnections was found to be a dynamin‐related protein 1 (Drp1)‐dependent process resulting synergistically from augmented mitochondrial fission and attenuated mitochondrial fusion. Cyclin‐dependent kinase 1 was subsequently identified as the cellular kinase responsible for fission‐active Ser616 phosphorylation of Drp1. In addition to its positive role in mitochondrial fission, Drp1 also resulted in mitochondrial translocation of E3‐ubiquitin ligase Parkin leading to degradation of mitochondrial fusion protein Mitofusin 1. Interestingly, RV‐NSP4 was found to interact with and be involved in recruiting fission‐active pool of Serine 616 phosphoDrp1 (Ser616 pDrp1) to mitochondria independent of accessory adaptors Mitochondrial fission factor and Fission protein 1 (Fis1). Inhibition of either Drp1 or Ser616 pDrp1 resulted in significant decrease in RV‐NSP4‐induced intrinsic apoptotic pathway. Overall, this study underscores an efficient strategy utilised by RV to couple apoptosis to mitochondrial fission facilitating dissemination of viral progeny.     

Re‐examining how Munc13‐1 facilitates opening of syntaxin‐1

Munc13‐1 is crucial for neurotransmitter release and, together with Munc18‐1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin‐1, SNAP‐25, and synaptobrevin. Assembly starts with syntaxin‐1 folded into a self‐inhibited closed conformation that binds to Munc18‐1. Munc13‐1 is believed to catalyze the opening of syntaxin‐1 to facilitate SNARE complex formation. However, different types of Munc13‐1‐syntaxin‐1 interactions have been reported to underlie this activity, and the critical nature of Munc13‐1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13‐1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin‐1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13‐1 fragments, even though binding of this linker region to Munc13‐1 is barely detectable. Conversely, the syntaxin‐1 SNARE motif clearly binds to Munc13‐1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13‐1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13‐1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin‐1 via interactions with the linker.     

Directed self‐immobilization of alkaline phosphatase on micro‐patterned substrates via genetically fused metal‐binding peptide

Current biotechnological applications such as biosensors, protein arrays, and microchips require oriented immobilization of enzymes. The characteristics of recognition, self‐assembly and ease of genetic manipulation make inorganic binding peptides an ideal molecular tool for site‐specific enzyme immobilization. Herein, we demonstrate the utilization of gold binding peptide (GBP1) as a molecular linker genetically fused to alkaline phosphatase (AP) and immobilized on gold substrate. Multiple tandem repeats (n = 5, 6, 7, 9) of gold binding peptide were fused to N‐terminus of AP (nGBP1‐AP) and the enzymes were expressed in E. coli cells. The binding and enzymatic activities of the bi‐functional fusion constructs were analyzed using quartz crystal microbalance spectroscopy and biochemical assays. Among the multiple‐repeat constructs, 5GBP1‐AP displayed the best bi‐functional activity and, therefore, was chosen for self‐immobilization studies. Adsorption and assembly properties of the fusion enzyme, 5GBP1‐AP, were studied via surface plasmon resonance spectroscopy and atomic force microscopy. We demonstrated self‐immobilization of the bi‐functional enzyme on micro‐patterned substrates where genetically linked 5GBP1‐AP displayed higher enzymatic activity per area compared to that of AP. Our results demonstrate the promising use of inorganic binding peptides as site‐specific molecular linkers for oriented enzyme immobilization with retained activity. Directed assembly of proteins on solids using genetically fused specific inorganic‐binding peptides has a potential utility in a wide range of biosensing and bioconversion processes. Biotechnol. Bioeng. 2009;103: 696–705. © 2009 Wiley Periodicals, Inc.     

Pairwise Variable Selection for High‐Dimensional Model‐Based Clustering

Summary Variable selection for clustering is an important and challenging problem in high‐dimensional data analysis. Existing variable selection methods for model‐based clustering select informative variables in a “one‐in‐all‐out” manner; that is, a variable is selected if at least one pair of clusters is separable by this variable and removed if it cannot separate any of the clusters. In many applications, however, it is of interest to further establish exactly which clusters are separable by each informative variable. To address this question, we propose a pairwise variable selection method for high‐dimensional model‐based clustering. The method is based on a new pairwise penalty. Results on simulated and real data show that the new method performs better than alternative approaches that use ?₁ and ?_∞ penalties and offers better interpretation.     

Activation loop phosphorylation regulates B‐Raf in vivo and transformation by B‐Raf mutants

Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B‐Raf signaling in vivo. Here, we generated a conditional knock‐in mouse allowing the expression of the B‐Raf^AVKA mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase‐impaired protein, the Braf^AVKA allele does not phenocopy the lethality of Braf‐knockout or paradoxically acting knock‐in alleles. However, Braf^AVKA mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B‐Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B‐Raf^AVKA. Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non‐V600E B‐Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP‐competitive inhibitors.     

Plant‐expressed Fc‐fusion protein tetravalent dengue vaccine with inherent adjuvant properties

Dengue is a major global disease requiring improved treatment and prevention strategies. The recently licensed Sanofi Pasteur Dengvaxia vaccine does not protect children under the age of nine, and additional vaccine strategies are thus needed to halt this expanding global epidemic. Here, we employed a molecular engineering approach and plant expression to produce a humanized and highly immunogenic poly‐immunoglobulin G scaffold (PIGS) fused to the consensus dengue envelope protein III domain (cEDIII). The immunogenicity of this IgG Fc receptor‐targeted vaccine candidate was demonstrated in transgenic mice expressing human FcγRI/CD64, by induction of neutralizing antibodies and evidence of cell‐mediated immunity. Furthermore, these molecules were able to prime immune cells from human adenoid/tonsillar tissue ex vivo as evidenced by antigen‐specific CD4⁺ and CD8⁺ T‐cell proliferation, IFN‐γ and antibody production. The purified polymeric fraction of dengue PIGS (D‐PIGS) induced stronger immune activation than the monomeric form, suggesting a more efficient interaction with the low‐affinity Fcγ receptors on antigen‐presenting cells. These results show that the plant‐expressed D‐PIGS have the potential for translation towards a safe and easily scalable single antigen‐based tetravalent dengue vaccine.     

Stress‐induced OMA1 activation and autocatalytic turnover regulate OPA1‐dependent mitochondrial dynamics

The dynamic network of mitochondria fragments under stress allowing the segregation of damaged mitochondria and, in case of persistent damage, their selective removal by mitophagy. Mitochondrial fragmentation upon depolarisation of mitochondria is brought about by the degradation of central components of the mitochondrial fusion machinery. The OMA1 peptidase mediates the degradation of long isoforms of the dynamin‐like GTPase OPA1 in the inner membrane. Here, we demonstrate that OMA1‐mediated degradation of OPA1 is a general cellular stress response. OMA1 is constitutively active but displays strongly enhanced activity in response to various stress insults. We identify an amino terminal stress‐sensor domain of OMA1, which is only present in homologues of higher eukaryotes and which modulates OMA1 proteolysis and activation. OMA1 activation is associated with its autocatalyic degradation, which initiates from both termini of OMA1 and results in complete OMA1 turnover. Autocatalytic proteolysis of OMA1 ensures the reversibility of the response and allows OPA1‐mediated mitochondrial fusion to resume upon alleviation of stress. This differentiated stress response maintains the functional integrity of mitochondria and contributes to cell survival.     

Scale‐up of hydrophobin‐assisted recombinant protein production in tobacco BY‐2 suspension cells

Plant suspension cell cultures are emerging as an alternative to mammalian cells for production of complex recombinant proteins. Plant cell cultures provide low production cost, intrinsic safety and adherence to current regulations, but low yields and costly purification technology hinder their commercialization. Fungal hydrophobins have been utilized as fusion tags to improve yields and facilitate efficient low‐cost purification by surfactant‐based aqueous two‐phase separation (ATPS) in plant, fungal and insect cells. In this work, we report the utilization of hydrophobin fusion technology in tobacco bright yellow 2 (BY‐2) suspension cell platform and the establishment of pilot‐scale propagation and downstream processing including first‐step purification by ATPS. Green fluorescent protein‐hydrophobin fusion (GFP‐HFBI) induced the formation of protein bodies in tobacco suspension cells, thus encapsulating the fusion protein into discrete compartments. Cultivation of the BY‐2 suspension cells was scaled up in standard stirred tank bioreactors up to 600 L production volume, with no apparent change in growth kinetics. Subsequently, ATPS was applied to selectively capture the GFP‐HFBI product from crude cell lysate, resulting in threefold concentration, good purity and up to 60% recovery. The ATPS was scaled up to 20 L volume, without loss off efficiency. This study provides the first proof of concept for large‐scale hydrophobin‐assisted production of recombinant proteins in tobacco BY‐2 cell suspensions.     

Enhancing epi‐cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi‐cedrol synthase

Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi‐cedrol synthase (AaECS). The stop codon at the N‐terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS‐ECS produced sesquiterpene 8‐epi‐cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The K_m values for FPPS‐ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi‐cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi‐cedrol in fermentation medium. The strain having fusion plasmid (pET32a‐FPPS‐ECS) produced 1.084 ± 0.09 mg/L epi‐cedrol, while the strain harbouring mixed plasmid (pRSETB‐FPPS and pET28a‐ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I‐TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N‐ C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.     

HDAC6 controls autophagosome maturation essential for ubiquitin‐selective quality‐control autophagy

Autophagy is primarily considered a non‐selective degradation process induced by starvation. Nutrient‐independent basal autophagy, in contrast, imposes intracellular QC by selective disposal of aberrant protein aggregates and damaged organelles, a process critical for suppressing neurodegenerative diseases. The molecular mechanism that distinguishes these two fundamental autophagic responses, however, remains mysterious. Here, we identify the ubiquitin‐binding deacetylase, histone deacetylase‐6 (HDAC6), as a central component of basal autophagy that targets protein aggregates and damaged mitochondria. Surprisingly, HDAC6 is not required for autophagy activation; rather, it controls the fusion of autophagosomes to lysosomes. HDAC6 promotes autophagy by recruiting a cortactin‐dependent, actin‐remodelling machinery, which in turn assembles an F‐actin network that stimulates autophagosome–lysosome fusion and substrate degradation. Indeed, HDAC6 deficiency leads to autophagosome maturation failure, protein aggregate build‐up, and neurodegeneration. Remarkably, HDAC6 and F‐actin assembly are completely dispensable for starvation‐induced autophagy, uncovering the fundamental difference of these autophagic modes. Our study identifies HDAC6 and the actin cytoskeleton as critical components that define QC autophagy and uncovers a novel regulation of autophagy at the level of autophagosome–lysosome fusion.     

‘Porosome’ discovered nearly 20 years ago provides molecular insights into the kiss‐and‐run mechanism of cell secretion

Secretion is a fundamental cellular process in living organisms, from yeast to cells in humans. Since the 1950s, it was believed that secretory vesicles completely merged with the cell plasma membrane during secretion. While this may occur, the observation of partially empty vesicles in cells following secretion suggests the presence of an additional mechanism that allows partial discharge of intra‐vesicular contents during secretion. This proposed mechanism requires the involvement of a plasma membrane structure called ‘porosome’, which serves to prevent the collapse of secretory vesicles, and to transiently fuse with the plasma membrane (Kiss‐and‐run), expel a portion of its contents and disengage. Porosomes are cup‐shaped supramolecular lipoprotein structures at the cell plasma membrane ranging in size from 15 nm in neurons and astrocytes to 100–180 nm in endocrine and exocrine cells. Neuronal porosomes are composed of nearly 40 proteins. In comparison, the 120 nm nuclear pore complex is composed of >500 protein molecules. Elucidation of the porosome structure, its chemical composition and functional reconstitution into artificial lipid membrane, and the molecular assembly of membrane‐associated t‐SNARE and v‐SNARE proteins in a ring or rosette complex resulting in the establishment of membrane continuity to form a fusion pore at the porosome base, has been demonstrated. Additionally, the molecular mechanism of secretory vesicle swelling, and its requirement for intra‐vesicular content release during cell secretion has also been elucidated. Collectively, these observations provide a molecular understanding of cell secretion, resulting in a paradigm shift in our understanding of the secretory process.